The Foundations of Virology

Discoverers and Discoveries

Inventors and Inventions
Developers and Technologies

Frederick A. Murphy

The Foundations of Virology

Discoverers and Discoveries
Inventors and Inventions
Developers and Technologies
Frederick A. Murphy
University of Texas Medical Branch
Galveston, Texas, USA

2014

The Foundations of Virology

Discoverers and Discoveries, Inventors and Inventions, Developers and Technologies
Copyright 2011/2014 by Frederick A. Murphy
Key references pertaining to discovery, development and innovation are provided on each page, using the style of PubMed. A Further Reading list is
included at the end of the book.
The images contained in this book were obtained from (1) my own image collection, (2) image collections of colleagues, (3) public Internet sites, and
(4) private image archives and library collections (each with permission, cited with the image). It was found that sources cited on public Internet sites
were very often inaccurate, or secondary, tertiary, in daisy-chain fashion, never getting to the proper original source. Many of my images (mostly
electron micrographs) found on Internet sites suffer the same failings wrong attribution, inaccurate legends, misleading colorizing and other
manipulations, etc. If I receive objections to the inclusion of any images, I will remove them.
This book is meant for educational purposes only.
This book goes with a PowerPoint slide set (~950 slides) which is on a website at the University of Texas Medical Branch / Galveston. The link to the
slides is on the second page of the website: http://www.utmb.edu/virusimages/
This book was composed using Adobe InDesign CS5 (.indd files). In this program all image files are linked; therefore, the highest resolution original
image files (.tif, .jpg) are only available directly from me I would be please to share them, using email or if files are too large, Dropbox or CDs/
DVDs. The camera-ready products of InDesign files are converted to special Adobe Acrobat files (.pdf ), small file and press quality versions of
which are also available for online and hard-copy printing. This book was composed using the Adobe Warnock Pro typeface set.
Frederick A Murphy
University of Texas Medical Branch
Department of Pathology, Route 0609
301 University Boulevard
Galveston, TX 77555-0609 USA
(Email) famurphy@utmb.edu
First Published April 2012
Revision 2013
Revision 2014, made possible by a fellowship at the
Stellenbosch Institute for Advanced Study (STIAS)
Wallenberg Research Centre at Stellenbosch University
Stellenbosch, South Africa
Printed in the United States of America
ISBN 978-0-7414-7365-3 / 0-7414-7365-8

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Dedication

This work is dedicated to my beloved wife, Irene.

Foreword

If I have seen further it is by standing on the shoulders of giants.

Isaac Newton in a letter to Robert Hooke, 1676
A great many highly creative scientists take it quite for granted that an
interest in the history of science is a sign of failing powers. The history of
science does not often interest the scientist as science.

Peter Medawar, 1968
The foundation of the medical and veterinary virology/viral disease sciences predates
the concept of the specificity of disease causation and is heavily dependent upon
initial discoveries about bacteria and bacterial diseases. Upon a broad and venerable
foundation, Louis Pasteur established the microbiologic/virologic/infectious disease
sciences, first in 1857 by discovering the specificity of microbial fermentations
(wine, beer, cheese), then in 1865 by extending the concept to infectious diseases of
silkworms, and finally between 1877 and 1895 by extending the concept to human
and animal diseases. His early infectious disease work centered on septic war wounds;
he then turned to anthrax and other bacterial diseases, and lastly to the viral disease
rabies. In each instance, he moved quickly from studies aimed at discovering the
causative agent to the development of specific intervention. In 1885, Pasteur gave the
first rabies vaccine to a boy, Joseph Meister, who had been bitten severely by a rabid
dogthat day marked the opening of the modern era of infectious disease science
aimed at disease prevention and control. Pasteur was joined by Robert Koch, who
discovered the causative agents of tuberculosis and cholera and contributed much to
the development of laboratory methods in bacteriology. Koch also worked on several
diseases which others eventually showed were caused by viruses. As a result of the
work of Pasteur, Koch and others, the identification of the causative agents of many
important human and domestic animal diseases proceeded at breakneck pace around
the turn of the twentieth century.
From the foundation laid by Pasteur, Koch and their colleagues, others extended the
breadth and depth of the infectious disease sciences in many ways. There are many
names to be remembered. This book is an attempt to remember the discoverers
and their discoveries, inventors and their inventions, and developers and their
technologies, the large and small breakthroughs that were seminal in the advance of
medical and veterinary virology.
The content of the book is completely arbitrary, based only upon my decisions as to
what to include and what to leave out. Several factors guided these decisions: (1) It
was clear from the beginning that this would be a book about the viruses, not the
diseases they causethe latter would be an incredibly more daunting challenge, with
so many key discoveries lost in time and in the vernacular of many, many languages.
(2) It was also clear from the beginning that it would not be possible for me to expand
coverage of plant, insect/invertebrate and bacterial (bacteriophage) virologyI do
not have the career perspective nor the experience. (3) It was also clear that the book
should not be limited simply to human pathogenic viruses per se; many important
veterinary and zoonotic pathogens are included because they have always been part
of the context in which human viruses are studied, and because it is the zoonotic and

species-jumping viruses from which most of the new, emerging and re-emerging
human infectious diseases derive. Further, in the era of the founding of virology there
was exceptional crossover between human and veterinary virology, much more than
is seen todaythere is value in being reminded of this. (4) It was also clear that the
book should not be limited to the type viruses of families and genera; rather it seemed
important to include as many as possible of the important and interesting pathogens
that are the focus of contemporary medical and veterinary virology and comparative
virology research. (5) In many instances I have included several entries for a given
virus, for example some from the era when the classification of an infectious agent
as a virus was based solely upon its ultrafilterability, and some from the modern era,
when the virus has been described by more definitive methods. (6) The discovery/
development of the great attenuated viruses used in vaccines is included, but in
condensed forma comprehensive listing here would have to include the many
independently derived vaccines and substrates, clearly the subject for a different book.
(7) The arboviruses and hemorrhagic fever viruses proved to be a special problem
because of their profusion, so a selective approach was taken in order to emphasize
those viruses causing major human and veterinary diseases and the initial discoveries
of viruses representing major taxait will be necessary for someone to produce
a separate book to give due credit to all the deserving discoverers, inventors and
developers in this field. (8) There are topics that are grossly underrepresented, HIV/
AIDS being the most obviousdespite the difficulties caused by controversies and the
sheer mass of the topic, an effort will be made to rectify this matter in future editions.
(9) The attempt to include inventors and their inventions, and developers and their
technologies proved especially difficultthe crucial role of these in the advance of
the virology/viral disease sciences and their infrastructure is acknowledged, but in
many cases breakthroughs derive to commercial corporations rather than individuals
and credit is difficult to attribute. (10) The book may fail in not capturing all the
excitement, the romance, the Eureka, behind each entryonly a full article, or in
some cases a full book, on each discovery, invention and development would do
justice to the story behind each entry. (11) It will be evident that my first love is for the
images, the photographs and graphics, rather than the text. The book has more than
1,800 images/graphics. No apology offered! (12) The book must be seen as a workin-progressit is meant to be used, to be modified, and changed to meet various
purposessuch is the power of electronic publishing. Toward this end, a PowerPoint
slide set containing the images, but little text is on my websitethe ease in updating
the slides will facilitate the production of future editions of the book. (13) The book
inevitably contains errorsthese are my responsibility. I will collect communications
about such errors for correction of the next edition.
Everyone, especially every virologist, who turns the pages of this book will come away
with a different summary thought, a different personal perspective. For me, one initial
thought is that I have been part of a very large, long-running scholarly enterprise,
whether I understood this all along or not, and that everyone who has contributed to
this enterprise, Nobel laureate or technologist, has filled only a very small niche in the
science-space-time continuum. Everyone has been a small fish in a very big pond, but
it is a grand pond.

Frederick A. Murphy
2012/2014

Many colleagues responded to my inquiries about this project, in every case with incredible enthusiasmit must
be that this subject, the founding of virology and the viral disease sciences, really touches a common chord. The
contributions of the following people are gratefully acknowledged:
Arstila, Pertti
Barin, Francis
Beaty, Barry
Berger, Ronald
Berns, Kenneth
Bettinger, George
Biggs, Peter
Bittle, James
Blair, Carol
Bloom, Marshall
Bollas, Kristin
Boschung, Urs
Boulton, Clare
Buchmeier, Michael
Burk, Janet
Burrell, Christopher
Calisher, Charles
Carmichael, Leland
, .
Coimbra, Terezinha L.M.
Compans, Richard
Coto, Celia
Coulson, Barbara
Cox, Francis
Degueurce, Christophe
DeMartini, James
Dermody, Terence
Diers, Bailey
Estes, Mary
Fagan, Kelvin
Fenner, Frank
French, Gary
Gaza, Judy
Gelb, Jack
Gelderblom, Hans
Gerin, John
Gibbs, Adrian
Goldberg, Michael

Gonzalez, Jean-Paul
Grafman, Eric
Greenberg, Harry
Greifenstein, Charles
Haddow, Andrew
Haire, Kevlin
Hardy, Anne
Heilman, Carole
Hirsch, Martin
Holland, John
Horzinek, Marian
Ivanova, Olga
Jeggo, Martyn
Johnson, Karl
Joklik, Bill
Jozan, Martine
Kaplan, Bruce
Klenk, Hans-Dieter
Knowles, James
Knowles, Wendy
Ksiazek, Thomas
Kurata, Takeshi
Laemmli, Ulrich
Larghi, Oscar
Lefkowitz, Elliot
Lipton, Howard
Luciw, Paul
MacFarlane, Ross
Mackenzie, Jan
Mackenzie, John
MacLachlan, Jim
Madeley, Richard
Madin, Larry
Mahieux, Renaud
Mahy, Brian
Mialot, Jean-Paul
Mcleod, William
McMinn, Peter

Mercurio, Jennifer
Mettenleiter, Thomas
Mims, Cedric
Mocarski, Ed
Monath, Thomas
Morens, David
Morgan, Robin
Moussatch, Nissin
Nathanson, Neal
Neumann-Haefelin, Dieter
Nimtz, Kelly
Norrby, Erling
Okamoto, Hiroaki
Oldstone, Michael
Olsen, Anders
Osterrieder, Klaus
Palmarini, Massimo
Palmenberg, Ann
Parrish, Colin
Peters, C.J.
Philpott, Howard
Popov, Vsevolod
Preisler, Don
Quiroz, Baudilio
Rizzetto, Mario
Rose, Kenneth
Roveland-Brenton, Blythe
Rowlands, David
Rupprecht, Charles
Rybicki, Edward
Sabattini, Marta
Sawicki, Dorothea
Schilgerius, Silvia
Schmaljohn, Connie
Schreiber, Tiffany Monet
Shadrin, Olga
Shatkin, Aaron
Sipe, Jean

Skehel, John
Smith, Jean
Smith, Ralph
Smith, Roger
Sderqvist, Thomas
Spamer, Earle
Staeheli, Peter
Stapleton, Darwin
Steck, Marianne
Studdert, Michael
Sudia, Daniel
Talasek, J.D.
Taylhardat, Leonardo
Teloh, Mary
Terquem, Micheline
Tesh, Robert
Thiel, Heinz-Juergen
Timoney, Peter
Toma, Bernard
Torres, Alfonso
Traum, Jeffrey
van der Groen, Guido
van Regenmortel, Marc
Vasilakis, Nikolaos
Villarreal, Luis
Wandeler, Alex
Weaver, Scott
Webster, Leslie III
Webster, Rob
Wimmer, Eckard
Witkowski, Jan
Woodall, John
Wooley, Dawn
Wunner, William
Wyllie, Andrew
Yamanouchi, Kazuya
Yanagihara, Ric
Zaki, Sherif

Table of Contents
TITLE
DEDICATION
FOREWORD
ACKNOWLEDGMENTS
TABLE OF CONTENTS
DATE DISCOVERER/DEVELOPER/INVENTOR

PAGE

~400BCE Hippocrates father of medicine, important epidemiologic observations

6
1224 Mesopotamian medicine rabies, the oldest documented disease of humans
7
1546 Girolamo Fracastoro proposal that epidemic diseases are contagious, first germ theory
8
1595 Sacharias & Hans Jansen development of the first compound microscope
9
1660 Founding of the Royal Society
10
1665 Robert Hooke first microscopic observation of a microorganism, a mold, genus Mucor 11
1668 Anton van Leeuwenhoek invention of (single lens) microscope, discovery of bacteria
12
1718 Lady Mary Wortley Montagu introduction of variolation to western countries
13
1735 Carolus Linnaeus development of hierarchical system for classification of all organisms
14
1765 Lazzaro Spallanzani first experiments refuting the theory of spontaneous generation
15
1774 Benjamin Jesty vaccination against smallpox using material from cowpox lesions
16
1789 Edward Jenner vaccination against smallpox using material from cowpox lesions
17
1793 John Hunter, Pierre-Victor Galtier, Georg Zinke, others experimental pathology
20
1793 Benjamin Rush account of the great yellow fever epidemic in Philadelphia, 1793
22
1798 Founding of the U.S. Public Health Service, first as the Marine Hospital Service
24
1805 John Pintard, others founding of New York City Department of Health, first in the U.S.
25
1830 John Dollond, Ernst Abbe, others advances in microscopes, achromatic lens, condenser
26
1835 Charles Chevalier, others development of microtome, key to cellular biology/pathology
27
1835 Theodor Schwann, Matthias Schleiden concept that organisms are composed of cells
28
1838 John Snow, John Graunt, William Farr, others founding of medical epidemiology
29
1840 Friedrich Gustav Jakob Henle father of the germ theory, and the Henle-Koch Postulates
30
1841 William Henderson discovery of the inclusion body of molluscum contagiosum virus
31
1843 Ignaz Semmelweis, Oliver Wendell Holmes, Joseph Lister hygiene/disinfection
32
1844 Alfred Donn, Lon Foucault, others development of photomicroscopy 34
1844 The projection microscope and the beginnings of biomedical illustration media
35
1846 Peter Panum measles in the Faroe Islands, the seminal study in viral disease epidemiology
36
1857 Louis Pasteur father of microbiology, father of virology
37
1858 Rudolf Virchow father of cellular (microscopic) pathology and comparative pathology
39
1859 Charles Darwin father of evolutionary science: common descent, natural selection
40
1863 Abraham Lincoln, others founding of the U.S. National Academy of Sciences
43
1863 Casimir Davaine first association of a specific infectious organism and a specific disease
44
1865 Gregor Mendel father of genetics
45
1865 Claude Bernard seminal book, Introduction to the Study of Experimental Medicine
46
1868 Johan-Friedrich Miescher discovery and characterization of nucleic acids
47
1874 William Osler father of modern scholarly medicine
48
1876 Robert Koch along with Louis Pasteur, the founder of microbiology
49
1877 Patrick Manson, Charles Laveran, Ronald Ross, Giovanni Grassi etiology of malaria
50
1879 Stanford Chaille father of hygiene and health education in the United States
51
1690 Yellow fever epidemics in the United States
52
1881 Carlos Finlay initial experiments on the mode of transmission of yellow fever virus
53
1883 Elie Metchnikoff, Paul Ehrlich founding of immunology, hematology, chemotherapy
54
1884 Louis Pasteur, Emile Roux, Charles Chamberland, Louis Thuillier rabies vaccine
54
1884 Charles Chamberland development of porcelain ultrafilter, key to discovery of viruses
57
1884 Charles Chamberland development of the autoclave
59
1886 Adolf Mayer the concept of transmissibility, earliest concept of an ultrafilterable virus
60
1887 John Buist, Edmund Cowdry elementary & inclusion bodies of vaccinia & variola viruses
61
1887 Joseph Kinyoun founding of the National Institutes of Health
63
1887 Louis Pasteur, colleagues founding of the Institut Pasteur
64
1892 Hermann Biggs New York City Department of Health microbiology laboratory
65
1892 George Sternberg discovery of virus neutralization (vaccinia virus)
66
1893 Theobald Smith, Cooper Curtice proof of arthropod transmission of disease, Texas fever
67
1892 Dmitry Ivanovsky discovery of tobacco mosaic virus
69
1898 Martinus Beijerinck discovery of tobacco mosaic virus
70

1898 Friedrich Loeffler, Paul Frosch discovery of foot-and-mouth disease virus (the first virus)
71
1898 Giuseppe Sanarelli discovery of myxoma virus (the first poxvirus)
75
1898 Henry Rose Carter discovery of yellow fever virus mosquito extrinsic incubation period
76
1900 Walter Reed, colleagues discovery of yellow fever virus (the first human virus)
77
1900 John McFadyean discovery of African horse sickness virus (the first orbivirus)
84
1900 Hugo de Vries founding of genetics, rediscovery of Mendels work
85
1901 John D. Rockefeller Sr. founding of the Rockefeller Institute for Medical Research
86
1901 Eugenio Centanni, others discovery of fowl plague virus (the first orthomyxovirus)
88
1902 Aladr Aujeszky discovery of pseudorabies virus (the first herpesvirus)
89
1902 Amde Borrel discovery of sheeppox virus
90
1902 Maurice Nicolle, Mustafa Adil Bey discovery of rinderpest virus (the first morbillivirus)
91
1902 James Spreull, Arnold Theiler discovery of bluetongue viruses
92
1903 Emil De Schweinitz, Marion Dorset classical swine fever virus (the first pestivirus)
93
1903 Adelchi Negri discovery of the rabies inclusion bodythe Negri body
94
1903 Paul Remlinger, Riffat-Bay, Alfonso di Vestea discovery of rabies virus (first rhabdovirus)
96
1903 Arne Tiselius development of electrophoresis
98
1904 Henri Valle, Henri Carr discovery of equine infectious anemia virus (the first retrovirus)
99
1904 William Gorgas control of yellow fever, allowing construction of the Panama Canal
100
1905 Henri Carr, Patrick Laidlaw, George Dunkin discovery of canine distemper virus
103
1905 Adelchi Negri, Paul Remlinger, others proof that variola and vaccinia are viruses
104
1907 Ross Harrison development of first cell cultures (nerve fibers from frog embryo tissues)
105
1907 Percy Ashburn Charles Craig discovery of dengue viruses
106
1907 Giuseppe Ciuffo discovery of human papillomaviruses (the first papillomaviruses)
107
1908 Vilhelm Ellerman, Oluf Bang discovery of avian leukemia virus (the first leukemia virus)
108
1909 Karl Landsteiner, Erwin Popper discovery of polioviruses (the first enteroviruses)
109
1909 Constantin Levaditi, Karl Landsteiner poliovirus in non-nervous system tissues
110
1909 Robert Doerr, K. Franz, S. Taussig discovery of sandfly fever viruses (first phleboviruses)
111
1910 Thomas Hunt Morgan discovery of the nature and role of the chromosome
112
1911 John Anderson, Joseph Goldberger discovery of measles virus
113
1911 Peyton Rous, Joseph Beard discovery of Rous sarcoma virus (first solid tumor virus)
115
1911 George de Hevesy, others development of radioisotopic labeling
116
1911 David Semple development of stable inactivated rabies vaccine
117
1912 Alexis Carrel, others development of many cell and tissue culture methods
118
1912 Hugh and Mary Maitland, Harry Eagle development of cell and tissue culture methods
120
1913 Edna Steinhardt, Clara Israeli, Robert Lambert first cultivation of a virus in cell culture
121
1914 Alfred Hess, Y Hiro, S Tasaka discovery of rubella virus (the only rubivirus)
124
1916 Development of trypsinization for the dispersal of cells in culture
125
1916 Frederick Twort, Flix dHerelle discovery of bacteriophages
126
1917 Robert Montgomery Nairobi sheep disease virus (first nairovirus, first tick-borne virus)
127
1918 Influenza pandemic, 40-100 million deaths worldwide
128
1918 Charles Nicolle, Charles Lebailly, Ren Dujarric de la Rivire discovery of influenza virus
130
1918 Anton Breinl, John Cleland, Eric French discovery of Murray Valley encephalitis virus
132
1919 Arnold Lwenstein discovery of herpes simplex virus 1
133
1920 Gaston Ramon, others improvements in many early vaccine technologies
134
1921 Robert Montgomery discovery of African swine fever virus (the only asfarvirus)
135
1921 Hans Creutzfeldt, Alfons Jakob initial description of Creutzfeldt-Jakob disease
136
1923 Theodor Svedberg development of the ultracentrifuge
137
1925 Florence Sabin the role of monocytes and other WBCs in viral/bacterial infections
138
1925 Helmut Ruska, Thomas Weller, others discovery of varicella-zoster virus
139
1925 Henri Valle Otto Waldmann Herman Frenkel foot-and-mouth disease vaccines
140
1926 Sinclair Lewis, Paul De Kriuf Arrowsmith, Microbe Hunters, books inspiring career choice
141
1926 Peter Olitsky, Jacob Traum, William Cotton discovery of vesicular stomatitis viruses
142
1926 Frederik Kraneveld, Thomas Doyle discovery of Newcastle disease virus (first paramyxovirus) 143
1927 Martin Frobisher Jr., J. Howard Brown bacteriophages and bacterial host pathogenicity
144
1927 Thomas Rivers publication of the paper that established virology as a distinct science
145
1928 Thomas Rivers publication of the first major virology book, Filterable Viruses 147
1928 Rockefeller Foundation Virus Laboratory (RFVL) and its international research programs
148
1928 Fred Griffith transformation in bacteria, seminal to development of molecular genetics
149
1928 Rebecca Lancefield, Edwin Lennette, others founding of viral disease diagnostics
150
1928 Adrian Stokes, Noel Hudson transmission of yellow fever virus to rhesus macaques
151
1928 Constant Mathis, Andrew Sellards transmission of yellow fever virus to rhesus macaques
152
1928 Jean-Louis Verge, J. Christofferoni feline panleukopenia virus (the first parvovirus)
153
1929 Stefan Nicolau, Ian Galloway discovery of Borna disease virus (the first bornavirus)
154
1930 Robert Green, Newell Ziegler infectious canine hepatitis virus (the first adenovirus)
155

1930 Leslie Webster, Charles Armstrong, Max Theiler development of the mouse for virology
157
1930 Richard Shope, Paul Lewis discovery of swine influenza virus
159
1930 Karl Meyer, Beatrice Howitt western equine encephalitis virus (the first alphavirus)
160
1931 William Elford, Christopher Andrewes graded collodion membranes for virion size
162
1931 Alice Woodruff, Ernest Goodpasture embryonating hens eggs as host for viruses
163
1931 Frank Macfarlane Burnet, Herald Cox embryonating hens eggs as host for viruses
164
1931 Robert Daubney, J. Richard Hudson, Percy Garnham discovery of Rift Valley fever virus
165
1932 John Bittner, Clarence Little Jackson Laboratory mouse mammary tumor virus
168
1932 Frederick Gay, Albert Sabin, Margaret Holden, Arthur Wright discovery of B virus
169
1933 Wilson Smith, Christopher Andrewes, Patrick Laidlaw ferret model, etiology of influenza
170
1933 Richard Shope, Peyton Rous, Joseph Beard discovery of rabbit (Shope) papillomavirus
171
1933 Ernst Ruska, Max Knoll invention of the transmission electron microscope
172
1933 Ralph Muckenfuss, Leslie Webster, Charles Armstrong St. Louis encephalitis virus
174
1933 Roberto Franco, Fred Soper, others discovery of the sylvan/jungle yellow fever cycle
176
1933 William Dimmock, Elvis Doll equine abortion virus, equine rhinopneumonitis virus
177
1934 Claud Johnson, Ernest Goodpasture discovery of mumps virus
178
1934 Arnold Beckman invention of the electronic pH meter
179
1934 Malcolm Merrill, Charles Lacaillade, Carl Ten Broeck eastern equine encephalitis virus
180
1934 Michitomo Hayashi, Shiro Kasahara, others discovery of Japanese encephalitis virus
181
1935 Wendell Stanley purification and crystallization of tobacco mosaic virus
182
1935 Max Theiler, Hugh Smith development of yellow fever vaccine
183
1935 Max Knoll, Manfred Ardenne, Vladimir Zworykin, Charles Oatley scanning microscope
184
1936 Frederick Bawden, Norman Pirie tobacco mosaic virus is comprised of RNA/protein
185
1936 Jean Cuill, Paul-Louis Chelle transmission of scrapie prion using ultrafiltered tissue
186
1936 Charles Armstrong, Thomas Rivers, Erich Traub lymphocytic choriomeningitis virus
187
1936 Peyton Rous, Joseph Beard induction of carcinomas in other species by papillomavirus
188
1936 Jacob Traum discovery of vesicular exanthema virus of swine (the first calicivirus)
189
1936 Theodosius Dobzhansky publication of Genetics and the Origin of Species 190
1937 Fred Beaudette, Charles Hudson avian infectious bronchitis virus (the first coronavirus)
191
1937 John Bernal, Dorothy Hodgkin, others X-ray crystallography of tobacco mosaic virus
192
1937 Thomas Rivers criteria for proof of viral disease causation: Koch postulates revisited
193
1937 Lev Zilber, Mikhail Chumakov, Elizabeth Levkovich tick-borne encephalitis virus
194
1937 Elizabeth Wollman, Eugne Wollman, Augustus Doermann the eclipse period
195
1938 G. Krber, Lowell Reed, Hugo Muench methods for estimating fifty percent endpoints
196
1938 Vladimir Kubes, Francisco Rios discovery of Venezuelan equine encephalitis virus
197
1938 Robert Doerr, Curt Hallauer the major virology book, Handbuch der Virusforschung 198
1938 Bodo von Borries, Ernst Ruska, Helmut Ruska first electron micrographs of viruses
199
1939 First international virology journal, Archiv fr die gesamte Virusforschung 200
1939 Charles Armstrong adaptation of poliovirus to cotton rats and mice
201
1939 Paul Mller discovery of insecticidal quality of DDT for control of vector-borne diseases
203
1939 Emory Ellis, Max Delbrck one-step growth experiment (single burst experiment)
204
1940 Kenneth Smithburn, Thomas Hughes, Alexander Burke, John Paul West Nile virus
205
1940 Ernst Mayr, others the conceptual basis of the modern evolutionary synthesis
206
1940 Barbara McClintock development of the concept of transposable elements, transposons
207
1940 Katherine Sanford, Wilton Earle development of the L cell line and L929 cloned cell line
208
1940 Keith Porter, others description of fine structure / biochemistry of cellular organelles
209
1940 Presidential Medal of Freedom, National Medal of Science, Copley Medal of Royal Society
210
1940 Albert Claude, Christian de Duve, George Palade structure / biochemistry of organelles
211
1941 George Hirst influenza hemagglutinin, hemagglutination-inhibition, neuraminidase
212
1941 Norman Gregg discovery of congenital abnormalities caused by rubella virus infection
213
1941 William Hammon, William Reeves natural history of arboviruses in vector arthropods
214
1941 Arnold Beckman development of the ultraviolet spectrophotometer (the Beckman DU)
215
1941 Thomas Anderson discovery of the amazing variety of bacteriophage morphologies
216
1942 Chen-Hsiang Huang development of the quantitative virus neutralization assay
217
1942 Joseph Mountin, others founding of U.S. Centers for Disease Control and Prevention
218
1942 Albert Coons development of immunofluorescence labeling methods
220
1943 U.S. Army, Camp Detrick military high-containment virology laboratories
221
1944 Kenneth Smithburn, Alexander Haddow discovery of Semliki Forest virus
222
1944 Oswald Avery, Colin MacLeod, Maclyn McCarty DNA as the material of inheritance
223
1944 Erwin Schrdinger book, What is Life? motivating scientists toward molecular biology
224
1944 Fred MacCallum, Saul Krugman, others separation of hepatitis A and hepatitis B
225
1945 Max Delbrck, Salvador Luria, others founding of Phage Course at Cold Spring Harbor
226
1945 Thomas Francis, George Hirst, Fred Davenport, others influenza vaccines
227
1945 Mikhail Chumakov, Ghislain Courtois, others Crimean-Congo hemorrhagic fever virus
228

1946 Joshua Lederberg, Edward Tatum discovery of genetic recombination in bacteria

229
1946 Max Delbrck discovery of genetic recombination in viruses (bacteriophage)
231
1946 Kenneth Smithburn, Alexander Haddow, Alexander Mahaffy Bunyamwera virus
232
1946 Lloyd Florio, Mabel Miller, Edward Mugrage Colorado tick fever virus (first coltivirus)
233
1947 Frank Macfarlane Burnet, Alfred Gottschalk, Ernst Klenk viral receptors on target cells
234
1947 Arnold Beckman development of the Spinco Model E analytical ultracentrifuge
235
1947 Mettler Instruments AG development of first single-pan precision analytical balance
236
1948 Frank Fenner founding of viral pathogenesis research (ectromelia virus in mice)
237
1948 Frank Fenner, Francis Ratcliff, Ian Marshall, Gwen Woodruff co-evolution of virus and host
238
1948 Gilbert Dalldorf, Grace Sickles discovery of Coxsackieviruses
239
1948 Thomas Rivers, Frank Horsfall, Igor Tamm book, Viral and Rickettsial Infections of Man 240
1948 Gueh-djen Hsiung, Phillip Gardner, Pekka Halonen application of viral diagnostics
241
1949 Edward Pickels development of the Spinco Model L preparative ultracentrifuge
242
1949 John Enders, Thomas Weller, Frederick Robbins cell culture for polio, measles
243
1949 Andr Lwoff, Louis Siminovitch, Niels Kjeldgaard lysogeny / induction (bacteriophage)
244
1950 Erwin Chargaff discovery in DNA that number of molecules of A+T equals that of G+C
245
1950 Cedric Mims development of immunofluorescence in viral pathogenesis research
246
1951 George Gey HeLa cell line (human cervical adenocarcinoma)
247
1951 Ludwik Gross, Charlotte Friend, others murine leukemia and lymphoma viruses
248
1951 Esther Lederberg discovery of the bacteriophage
249
1951 Linus Pauling, John Kendrew, Max Perutz structure of proteins (X-ray crystallography)
250
1951 Preben & Herdis von Magnus, Alice Huang, David Baltimore defective interfering viruses
251
1951 Myron Brakke development of rate zonal density gradient centrifugation
252
1952 Alfred Hershey, Martha Chase biological proof of DNA as the material of inheritance
253
1952 Wendell Stanley establishment of Virus Laboratory at University of California Berkeley
254
1952 Joshua Lederberg, Norton Zinder transduction: DNA transfer by bacteriophage
255
1952 Karl Maramorosch discovery that some viruses can replicate in both plants and insects
256
1952 Renato Dulbecco, Marguerite Vogt virus quantification by plaque assay
257
1952 Seymour Cohen DNA of some bacteriophages contains 5-hydroxymethylcytosine
258
1953 Wallace Rowe, Robert Huebner, others discovery of human adenoviruses
259
1953 James Watson, Francis Crick, Maurice Wilkins, Rosalind Franklin the structure of DNA
260
1953 James Murphy, Frederik Bang discovery of virus release by budding at the cell surface
262
1953 Nakao Ishida discovery of Sendai virus (murine parainfluenza virus 1)
263
1953 Rupert Billingham, Peter Medawar, Leslie Brent discovery of immunological tolerance
264
1953 Wilhelm Bernhard, Albert Dalton morphogenesis / classification of retroviruses
265
1953 Albert Sabin discovery of orthoreoviruses (the first reoviruses)
266
1954 Renato Dulbecco, Marguerite Vogt concept of the latent period, eclipse period
267
1954 Jonas Salk, Julius Youngner, Pierre Lpine, Thomas Francis inactivated polio vaccine
268
1954 Margaret Smith discovery of murine cytomegalovirus
269
1954 Bjrn Sigurdsson concept of slow viruses (e.g., maedi-visna virus, scrapie prion)
270
1955 Gyula Taktsy invention of the microtiter system for virologic and serologic assays
271
1955 George Hirst, Peter Wildy, Lloyd Kozloff, Robert Wagner, others major virology journals
272
1955 Richard Taylor, Herbert Hurlbut, Telford Work, others discovery of Sindbis virus
273
1955 Heinz Fraenkel-Conrat, Robley Williams reconstitution of tobacco mosaic virus
274
1955 Seymour Benzer, Paul Berg, Maxine Singer evolving definitions of the gene
275
1955 Werner Schfer, Svonimir Dinter discovery that fowl plague virus is an influenza virus
276
1956 J. Anthony Morris, Robert Chanock respiratory syncytial virus (the first pneumovirus)
277
1956 William Pelon, WH Price, William Mogabgab discovery of human rhinoviruses
278
1956 Alfred Gierer, Heinz Fraenkel-Conrat infectivity of viral RNA (tobacco mosaic virus)
279
1956 Stewart Madin, Charles York, Delbert McKercher infectious bovine rhinotracheitis virus
280
1956 R.William Ross discovery of chikungunya virus
281
1956 Robert Chanock discovery of human parainfluenza viruses
282
1956 Margaret Smith, Thomas Weller, Wallace Rowe discovery of human cytomegalovirus
283
1956 John Gorham, James Henson, Robert Leader, Frank Dixon Aleutian disease of mink virus
284
1957 Niels Jerne, David Talmage, Frank Macfarlane Burnet clonal selection in immunity
285
1957 Marvin Minsky development of the confocal microscope
286
1957 Alick Isaacs, Jean Lindenmann discovery of interferons
287
1957 Telford Work, F.R. Rodriguez, Pravin Bhatt discovery of Kyasanur Forest disease virus
288
1957 Elvis Doll, John Bryans, William McCollum equine arteritis virus (the first arterivirus)
289
1957 James Gillespie, K.M. Lee, others discovery of bovine viral diarrhea virus
290
1957 Matthew Meselson, Frank Stahl, Jerome Vinograd density gradient ultracentrifugation
291
1958 Matthew Meselson, Frank Stahl the semi-conservative mode of replication of DNA
292
1958 Howard Temin, Harry Rubin focus assay for Rous sarcoma virus, neoplastic transformation
293
1958 Sarah Stewart, Bernice Eddy discovery of murine polyoma virus (the first polyomavirus)
294

1958 Robert Kissling, Robert Goldwasser rabies immunofluorescence diagnostic method

295
1958 Armando Parodi, Ignacio Pirosky, colleagues Junin virus (Argentine hemorrhagic fever)
296
1958 Denis Burkitt description of Burkitts lymphoma in African children
297
1958 Gabriele Zu Rhein, Duard Walker progressive multifocal leukoencephalopathy, JC virus
298
1959 James Gowans, Peter Medawar discovery of lymphocyte recirculation
299
1959 Albert Sabin, Herald Cox, Hilary Koprowski, others attenuated live-virus polio vaccine
300
1959 Lawrence Kilham, others discovery of murine parvoviruses
301
1959 Sydney Brenner Robert Horne invention of negative contrast electron microscopy
302
1959 Albrecht Kleinschmidt invention of electron microscopic visualization of viral DNA
304
1959 Gerald Edelman, Rodney Porter, Alfred Nisonoff structure of antibody molecules
305
1959 Robert Sinsheimer that a virus may have a single stranded DNA genome (X174)
306
1959 Rosalyn Yalow, Solomon Berson development of radioimmunoassays (RIAs)
307
1959 Arthur Kornberg, Severo Ochoa mechanisms in the replication of DNA and RNA
308
1959 William Prusoff development of the first antiviral drug, 5-iodo-2-deoxyuridine (IUdR)
309
1959 William Reeves, others founding of American Committee on Arthropod-Borne Viruses
310
1960 Walter Plowright development of attenuated-live virus rinderpest vaccine
311
1960 Benjamin Sweet, Maurice Hilleman discovery of SV40 virus
312
1960 Vernon Riley, Kenneth Rowson discovery of lactate dehydrogenase-elevating virus
313
1960 Wayne Thompson, Bernard Kalfayan, Ralph Anslow discovery of La Crosse virus
314
1960 Frank Dixon, others founding of immunopathology, viruses in immune complex diseases
315
1960 Cedric Mims, Neal Nathanson, others the modern era of viral pathogenesis research
316
1960 Robert Merrifield solid phase synthesis technology (polypeptides, oligonucleotides)
317
1961 Franois Jacob, Jacques Monod, Andr Lwoff genetic control of enzyme / virus synthesis
318
1961 Sydney Brenner, Franois Jacob, Matthew Meselson discovery of messenger RNA
320
1961 Jacques Miller discovery of the role of the thymus in cellular immunity
321
1961 Michael Stoker, Ian Macpherson development of BHK-21 cell line
322
1961 Francis Crick, Sydney Brenner, others the triplet coding strategy of DNA
323
1961 John Cairns discovery of the length of the circular DNA of the bacteriophage T2
324
1961 U.S. Communicable Disease Center Morbidity and Mortality Weekly Report (MMWR)
325
1961 Marshall Nirenberg, Har Khorana, Robert Holley deciphering of the genetic code
326
1961 Jordi Casals, Richard Taylor, Robert Shope, Charles Calisher arbovirus classification
327
1962 Yosihiro Yasumura, Yosio Kawakita establishment of Vero cell line
328
1962 Lizbeth Kraft discovery of mouse hepatitis virus
329
1962 Cold Spring Harbor Symposium: Basic Mechanisms in Animal Virus Biology 330
1962 Andr Lwoff, Robert Horne, Paul Tournier classification of viruses
331
1962 Aaron Klug, Donald Caspar the principles of the icosahedral structure of viruses
332
1962 John Trentin, colleagues the induction of tumors in hamsters by human adenoviruses
333
1962 Albert Cosgrove, Hiram Lasher infectious bursal disease virus (the first birnavirus)
334
1962 Norman Anderson invention of rate-zonal ultracentrifugation
335
1962 Karl-Eduard Schneweis, Walter Dowdle, Andr Nahmias differentiation of HSV 1 and 2
336
1962 Robert Webster, Graeme Laver the link between avian and human influenza viruses
337
1962 Osamu Shimomura, Martin Chalfie, Roger Tsien green fluorescent protein
338
1963 Peter Gomatos, Igor Tamm discovery of double-stranded RNA in a virus (reovirus)
339
1963 John Enders, Thomas Peebles, Samuel Katz, Kevin McCarthy measles vaccine
340
1963 Maurice Hilleman, others development, commercialization of pediatric viral vaccines
341
1963 Wilbur Downs, Charles Anderson, Leslie Spence discovery of Tacaribe virus
342
1963 Ralph Doherty, Robert Shope, colleagues discovery of Ross River virus
343
1963 Seiichi Matsumoto, Kaneatsu Miyamoto morphogenesis of rabies virus, Negri body
344
1964 Michael Epstein, Bert Achong, Yvonne Barr Epstein-Barr virus and Burkitts lymphoma
345
1964 William Jarrett, colleagues discovery of feline leukemia virus
346
1964 Karl Johnson, Patricia Webb, others Machupo virus, Bolivian hemorrhagic fever
347
1965 Michel Bouteille, John Sever, others etiology of subacute sclerosing panencephalitis
348
1965 Baruch Blumberg, Harvey Alter, others discovery of Australia antigen, hepatitis B virus
349
1965 David Tyrrell, June Almeida, others discovery of human coronaviruses
350
1965 Wayne Atchison, Joseph Melnick, colleagues discovery of adeno-associated viruses
351
1965 Robin Valentine, Helio Pereira, Erling Norrby adenovirus structural elements
352
1965 Bernard Roizman seminal studies of the molecular biology of herpes simplex virus
353
1965 Shinpei Kasakura, Kendall Smith, others discovery of the first cytokine, IL-2
354
1965 Leonard Hayflick development of human diploid cell strains (WI-1 through WI-44)
355
1966 Mark Ptashne, Walter Gilbert identification of repressor genes (lac operon)
356
1966 Peter Wildy, Frank Fenner, others International Committee on Taxonomy of Viruses
357
1966 Carleton Gajdusek, Clarence Gibbs, William Hadlow kuru prion in non-human primates
359
1966 Eugene Buynak, Maurice Hilleman development of mumps vaccine
360
1966 Stanley Plotkin, Paul Parkman, Harry Meyer, Maurice Hilleman development of rubella vaccine 361

1967 Rudolf Siegert, Werner Slenczka, Robert Kissling, Frederick Murphy Marburg virus
362
1967 U.S. Communicable Disease Center civilian high-containment virology laboratory
363
1967 Joseph Kates, Brian McAuslan viral DNA-dependent RNA polymerase (vaccinia virus)
364
1967 Peter Biggs, Anthony Churchhill, Ben Burmester, others Mareks disease virus
365
1967 Jacob Maizel Jr., Ulrich Laemmli SDS polyacrylamide gel electrophoresis of proteins
366
1967 Barnard van der Westhuizen, Yuji Inaba, Yoshio Tanaka bovine ephemeral fever virus
367
1968 Aaron Shatkin, Jean Sipe discovery of dsRNA-dependent RNA polymerase (reovirus)
368
1968 Werner Henle, Gertrude Henle Epstein-Barr virus and infectious mononucleosis
369
1968 Peter Wildy, Joseph Melnick, others the First International Congress for Virology
370
1968 Werner Arber, Hamilton Smith, Daniel Nathans, others restriction endonucleases
373
1969 Robert Huebner, George Todaro development of the viral oncogene hypothesis
374
1969 Sonja Buckley, Jordi Casals-Ariet discovery of Lassa virus
375
1969 Charles Mebus, Marvin Twiehaus, others discovery of bovine rotavirus (first rotavirus)
376
1970 Irvine Page, others Institute of Medicine of the U.S. National Academy of Sciences
377
1970 Robert Shope, Frederick Murphy, others rabies-like viruses (the genus Lyssavirus)
378
1970 Frank Fenner, David White book, Medical Virology 379
1970 David Baltimore, Alice Huang RNA-dependent RNA polymerase in VSV
380
1970 Howard Temin, David Baltimore, colleagues reverse transcriptase of retroviruses
381
1970 Roland Robins, Joseph Witkowski, colleagues ribavirin, broad spectrum antiviral drug
382
1970 Joseph Kates James Darnell Jr., others discovery of the 3 poly(A) tail on viral mRNAs
383
1970 Walter Plowright, Robert Tesh, Douglas Watts, others transovarial transmission
384
1970 Pekka Halonen, others linkage of rapid virus diagnostics to clinical care
385
1970 Seymour Kalter, Richard Heberling development of non-human primate virology
386
1970 Janet Butel, Keerti Shah controversy whether SV40 virus (vaccines) causes brain tumors
387
1970 Gertrude Elion, George Hitchings, others rational drug design, acyclovir / her pes
388
1971 Eva Engvall, Bauke van Weemen, colleagues enzyme-linked immunoassays
389
1971 David Baltimore system of virus categorization based on the replicative path to mRNA
390
1971 Theodore Diener discovery of viroids (infectious naked RNA molecules)
391
1971 Sylvia Gardner, Anne Field, colleagues discovery of BK virus, kidney / bladder disease
392
1971 Donald Carey, Jin-Won Song, Robert Shope, Ric Yanagihara Thottapalayam virus
393
1971 Bert Achong, Michael Epstein, Thomas Folks simian foamy virus infection in humans
394
1971 George Baer, Franz Steck, Alex Wandeler oral vaccination of wildlife against rabies
395
1972 Albert Kapikian, colleagues discovery of Norwalk virus (the first human calicivirus)
396
1972 Paul Berg, Dale Jackson, Robert Symons development of recombinant-DNA technology
397
1973 Stanley Cohen, Herbert Boyer application of recombinant DNA technology
398
1973 Paul Berg, others Asilomar conferences: Biohazards in Biological Research 399
1973 Stephen Feinstone, Albert Kapikian, Robert Purcell discovery of hepatitis A virus
400
1973 Ruth Bishop, Ian Holmes, Thomas Flewett, Albert Kapikian, others human rotaviruses
401
1973 Joseph Sambrook, others agarose gel electrophoresis of DNA
402
1973 Ralph Steinman, Zanvil Cohn discovery of dendritic cells
403
1973 Councilman Morgan, Ari Helenius, others variety and complexity of virus entry mechanisms 404
1973 Daniel Nathans, others restriction enzyme map of a viral genome (SV40 virus)
405
1974 Peter Doherty, Rolf Zinkernagel how immune system recognizes virus-infected cells
406
1974 Georges Khler, Csar Milstein development of monoclonal antibodies
407
1974 Vigdis Torsvik, Ian Dundas, Allen Wais, others viruses of Archaea, odd morphologies
408
1974 Harald zur Hausen association of human papillomaviruses and cervical carcinoma
409
1974 Ilse Tischer, Hans Gelderblom discovery of porcine circovirus (the first circovirus)
410
1975 Yvonne Cossart, Anne Field, colleagues parvovirus B-19, fifth disease and aplastic crisis
411
1975 C. Richard Madeley, colleagues discovery of human astrovirus 1 (the first astrovirus)
412
1975 Aaron Shatkin, Bernard Moss, colleagues discovery of 5 cap on messenger RNAs
413
1975 Edwin Southern development of southern blotting
414
1976 Karl Johnson, Patricia Webb, Frederick Murphy, Guido Van der Groen Ebola virus
415
1976 J. Michael Bishop, Harold Varmus the cellular origin of retroviral oncogenes
417
1976 Rudolf Rott, Christoph Scholtissek optimum gene constellation determines virulence
418
1976 Alfred Evans criteria for proof of viral disease causation: Henle-Koch postulates again
419
1976 U.S. Government threat of swine flu epidemic, national emergency vaccination program
420
1976 Walter Fiers, Frederick Sanger sequencing of viral genomes (phages MS2 and 174)
421
1977 Phillip Sharp, Richard Roberts, colleagues RNA splicing and split genes (adenovirus)
422
1977 D.A. Henderson, Isao Arita, Frank Fenner, many others global eradication of smallpox
423
1977 Walter Gilbert, Frederick Sanger development of rapid DNA sequencing technology
424
1977 Mario Rizzetto, John Gerin, colleagues hepatitis delta virus (the only deltavirus)
425
1977 Scott Halstead immune enhancement in dengue hemorrhagic fever / shock syndrome
426
1977 Carl Woese, colleagues The Tree of Life, phylogeny based on mRNA gene sequences
427
1978 Ho Wang Lee, Pyung Woo Lee, Karl Johnson Hantaan virus, HFRS
428

1978 Leland Carmichael, Max Appel discovery / characterization of canine parvovirus

429
1978 Charles Weissmann, colleagues first reverse genetics experiment (bacteriophage Q)
430
1978 Anthony Waterson, Lise Wilkinson book, An Introduction to the History of Virology 431
1978 Steven Harrison, James Hogle, others structure of viruses (poliovirus, rhinovirus)
432
1978 David Sabatini, Enrique Rodriguez-Boulan, Richard Compans directional budding
433
1978 Wolf Szmuness, Palmer Beasley, others HBV infection and hepatocellular carcinoma
434
1979 Lynn Enquist, colleagues cloning viral DNA fragments into phage vector
435
1979 David Lane, Arnold Levine, others p53 tumor suppressor protein in SV40 infected cells
436
1979 George Stark, others western blotting: transfer of proteins from gels, antibody detection
437
1979 Paul Gibbs, William Taylor peste des petits ruminants virus, a distinct morbillivirus
438
1980 Robert Swanson, Herbert Boyer Genentech, first biotech company listed on NYSE
439
1980 Bernard Poiesz, Robert Gallo, Mitsuaki Yoshida, David Golde, others HTLV 1 & 2
440
1980 Walter Dowdle, Frederick Murphy, James Hughes National Center for Infectious Diseases
441
1980 Elizabeth Williams, Stuart Young chronic wasting disease of deer / elk is a prion disease
442
1980 Thomas Cech, Sidney Altman, colleagues catalytic properties of RNA, ribozyme concept
443
1980 Gary Larson The Far Side, a fixture in labs everywhere
444
1981 MMWR - Pneumocystis carinii pneumonia in Los Angeles, Kaposi sarcoma in NY AIDS
445
1981 Vincent Racaniello, David Baltimore, Eckard Wimmer infectious clone of poliovirus
447
1981 Gerd Binnig, Heinrich Rohrer development of scanning tunneling electron microscope
448
1981 Don Wiley, John Skehel, Ian Wilson, others structure of influenza virus hemagglutinin
449
1981 Robert Weinberg, Michael Wigler cloning of first human tumor-derived oncogene, H-ras
450
1981 Bill Joklik, Harry Ginsberg, others founding of the American Society for Virology
451
1981 William Rutter, Pablo Valenzuela, colleagues first recombinant human vaccine, HBsAg
453
1982 Stanley Prusiner concept of the prion etiologic agents of spongiform encephalopathies
454
1982 Walter Goad, others development of GenBank
455
1982 Michael Oldstone, colleagues concept of viral damage to cellular homeostasis
456
1982 Gerald Woode, Marian Horzinek, Graham Beards bovine, equine, human toroviruses
457
1983 Mikhail Balayan discovery of hepatitis E virus (the first hepevirus)
458
1983 Francoise Barr-Sinoussi, Luc Montagnier, Jean-Claude Chermann discovery of HIV1
459
1983 Marvin Carruthers, Applied Biosystems, Inc. commercial DNA synthesis technology
460
1983 Andrew Murray, Jack Szostack yeast artificial chromosome, cloning large DNA fragments
461
1983 Bernard Moss, Enzo Paoletti recombinant vaccinia virus as vector for viral vaccines
462
1984 Marc Adrian, Jacques Dubochet, others cryo-electron microscopic analysis of virus structure 463
1985 Kary Mullis, colleagues, Cetus, Perkin-Elmer polymerase chain reaction (PCR)
464
1985 Julio Maiztegui, Julio Barrera Oro, George French Argentine hemorrhagic fever vaccine
465
1985 Bernard Fields, others publication of Fields Virology, now six editions
466
1985 bioMrieux, Bio-Rad Genetic Systems, Abbott first FDA approved HIV antibody EIAs
467
1985 Norman Letvin, Ronald Desrosiers, colleagues simian immunodeficiency viruses (SIVs)
468
1985 Francis Barin, Phyllis Kanki, Max Essex, colleagues human immunodeficiency virus 2
469
1985 Alec Jeffreys development of DNA fingerprinting, single nucleotide polymorphisms
470
1985 Carole Richardson bovine spongiform encephalopathy in the United Kingdom
471
1985 Saul Kit, colleagues development of first recombinant live-virus vaccine (pseudorabies)
472
1981 Development of modern viral vaccinology based upon innovative technologies
473
1986 Niels Pedersen, Janet Yamamoto, colleagues discovery of feline immunodeficiency virus
474
1986 Leroy Hood, Lloyd Smith, Applied Biosystems automated DNA sequencing
475
1987 Frank Fenner, others publication of book, Veterinary Virology (currently four editions)
476
1987 Marty St. Clair, Samuel Broder, Burroughs Wellcome/NCI first anti-HIV drug (AZT)
477
1988 Mario Capecchi, Oliver Smithies, Martin Evans knockout, genetically manipulated mice
478
1988 Koichi Yamanishi, Takeshi Kurata, Philip Pellett, Carlos Lopez human herpesvirus 6B
479
1988 Eckard Wimmer, Nahum Sonnenberg internal ribosomal entry sites in virus mRNA
480
1988 National Library of Medicine National Center for Biotechnology Information (NCBI)
481
1988 H. Fred Clark, Albert Kapikian, Richard Ward development of human rotavirus vaccines
482
1988 Albert Osterhaus, colleagues phocine distemper virus, marine mammal morbilliviruses
483
1988 Manfred Eigen, others modern concepts of origin of viruses, virus evolution, quasispecies
484
1988 Helio Pereira, Thomas Flewett, colleagues discovery of picobirnaviruses
487
1988 Thomas Brock discovery of Taq polymerase
488
1989 Michael Houghton, Qui-Lim Choo, George Kuo, Daniel Bradley hepatitis C virus
489
1989 Stephen Fodor, Patrick Brown, others development of microarray technology
490
1989 Marc van Regenmortel definition of virus species
491
1989 ivind Bergh, Curtis Suttle, Craig Venter, others high concentrations of viruses in ocean
492
1989 Ian Lipkin, colleagues identification of a virus in clinical specimens with only molecular tools 493
1990 International Human Genome Sequencing Consortium Human Genome Project
494
1990 Nitza Frenkel discovery of human herpesvirus 7 (HHV7)
495
1990 Cathrinus Terpstra, others porcine reproductive and respiratory syndrome virus
496

1990 Systems Biology / Systems Virology as a paradigm shift in viral disease research
497
1990 Development of viral phylogenetics, phylogeography, phylodynamics 498
1991 Tim Berners-Lee development of the World Wide Web (WWW)
499
1991 Craig Venter, Hamilton Smith (TIGR), others shotgun cloning/sequencing methods
500
1991 Gregory Reyes, Michael Wigler, others novel techniques to find uncultivable viruses
501
1991 Rosalba Salas, Robert Tesh, others Guanarito virus, Venezuelan hemorrhagic fever
502
1992 Jean Smith, Charles Rupprecht, others rabies virus genotyping, mapping of variants
503
1992 Joshua Lederberg, Stephen Morse, others concept of New and Emerging Infectious Diseases 504
1993 Stephen Morse, others Program for Monitoring Emerging Diseases (ProMED)
506
1993 Stuart Nichol, C.J. Peters, Thomas Ksiazek, others Sin Nombre virus, HPS
507
1994 Robert Webster, Alan Granoff, Brian Mahy, M. van Regenmortel Encyclopedia of Virology 508
1994 Yuan Chang, Patrick Moore, colleagues herpesvirus 8, Kaposi sarcoma herpesvirus
509
1994 Karl-Klaus Conzelmann, colleagues reverse genetics for negative-strand RNA viruses
510
1994 Terezinha Lisieux Moraes Coimbra, others Sabi virus and Brazilian hemorrhagic fever
511
1995 Keith Murray, Peter Hooper, Alex Hyatt, colleagues Hendra virus and reservoir host bats
512
1995 Richard Preston publication of the influential book, The Hot Zone 513
1996 Robert Will, James Ironside, John Collinge BSE prion, variant Creutzfeldt-Jakob disease
514
1996 David Fredricks, David Relman sequenced-based criteria for causation of viral diseases
515
1996 Martin Hirsch, David Ho, Thomas Merigan, others HAART therapy for AIDS
516
1996 Jules Hoffmann, Bruce Beutler, others discovery of Toll genes / Toll-like receptors
517
1996 Hans Kuypers, Gabriella Ugolini, others viruses as neuronal pathway tracers
518
1996 National Center for Biotechnology Information development of PubMed
519
1997 Robert Webster, others highly pathogenic H5N1 influenza virus, lethal disease
520
1997 Tsutomu Nishizawa, Hiroaki Okamoto, colleagues torque teno virus, anelloviruses
521
1997 Luminex Corporation, others development of high throughput viral diagnostics instruments 522
1998 Andrew Fire, Craig Mello RNA interference (RNAi, RNA silencing by dsRNA)
523
1998 Larry Page, Sergey Brin development of Google and Internet-based viral disease surveillance 524
1998 Nancy Cox, Yoshihiro Kawaoka, Robert Webster molecular basis of influenza virulence
525
1999 Kaw Bing Chua, Kenneth Lam, William Bellini, T Ksiazek, colleagues discovery of Nipah virus 526
1999 Deborah Asnis, Marcelle Layton, W. Ian Lipkin, Robert Lanciotti West Nile virus in U.S.
527
2001 Albert Osterhaus, Bernadette van den Hoogen, colleagues human metapneumovirus
528
2001 U.S. Government anthrax bioterrorism events, large biodefense research programs
529
2001 Foot-and-mouth disease epidemic in U.K., destruction of large numbers of livestock
530
2002 Eckard Wimmer, colleagues synthesis of poliovirus complementary DNA
531
2002 Joseph DeRisi, W. Ian Lipkin microarray technology for identification of viruses
532
2002 Ian Frazer, Sanofi Pasteur, Merck, GlaxoSmithKline papillomavirus vaccine
533
2002 Erling Norrby papers and book on awarding of the Nobel Prizes pertinent to virology
534
2003 Peter Walsh, Eric Leroy, others Ebola epidemics in endangered great apes
535
2003 International Human Genome Project consensus sequence of the human genome
536
2003 Carlo Urbani, Malik Peiris, Leo Poon, others SARS coronavirus and global epidemic
537
2003 Bernard La Scola, Didier Raoult, others discovery of mimivirus, the largest virus known
538
2003 Lawrence Livermore National Laboratory Autonomous Pathogen Detection System
539
2005 Eric Leroy, Jonathan Towner, others reservoir hosts of Ebola / Marburg viruses are bats
540
2005 Jeffery Taubenberger, Terrence Tumpey, others 1918 influenza virus sequenced
541
2005 Renaud Mahieux, Antoine Gessain discovery of human T lymphotropic viruses 3 and 4
542
2005 Takaji Wakita, Charles Rice, others in vitro replication of hepatitis C virus
543
2005 Jonathan Rothberg, 454 Life Sciences (Roche) development of 454 pyrosequencing
544
2005 Founding of the European Centre for Disease Prevention and Control (ECDC)
545
2005 Tobias Allander, colleagues discovery of human bocavirus (parvovirus)
546
2006 Laura Kahn, Bruce Kaplan, Thomas Monath One Health / One Medicine Initiative
547
2007 Massimo Pigliucci, Gerd Mller, others the Extended Evolutionary Synthesis
548
2007 Tobias Allander, David Wang, Yuan Chang, others human polyomaviruses KI, WU, MC
549
2008 Bernard La Scola, Didier Raoult, others discovery of first virophage, Sputnik
550
2010 FAO Global Rinderpest Eradication Programme global eradication of rinderpest
551
2011 Berndt Hoffmann, Martin Beer, Thomas Mettenleiter, colleagues Schmallenberg virus
552
2012 Ali Mohamed Zaki, Ron Fouchier, W. Ian Lipkin MERS coronavirus
553
2014 Chantal Abergel, Jean-Michel Claverie, colleagues pandoraviruses and pithovirus sibericum
554
2018 WHO Global Polio Eradication Initiative planned global eradication of poliomyelitis
555
FURTHER READING
SOME MAJOR IMAGE LIBRARIES
INDEX

Hippocrates of Kos II
(~460 BCE - ~370 BCE)
Hippocrates is considered the father of modern medicine, still influencing, 25 centuries after his time, various aspects of medical
and veterinary practice and biomedical research. His works (Corpus Hippocraticum, Hippocratic Collection) include references to
infectious diseases ranging from the nature of infection, hygiene, epidemiology, and the immune response, to detailed descriptions of
syndromes such as mumps, herpes, smallpox, hepatitis (jaundice), the common cold, pneumonia, tuberculosis, malaria and tetanus.

~400BCE Hippocrates father of medicine, important epidemiologic and clinical observations of many diseases
page 6

Man Bitten by Mad Dog

Illustration from an Arabic translation of the Materia

Medica, Baghdad, Iraq, 1224.
Freer Gallery of Art, Smithsonian Institution,
Washington, DC.
Rabies (derived from the Sanskrit, rabhas meaning to
do violence) was first described in the 23rd century BCE in
the Eshuma Code of Babylonit is the oldest documented
infectious disease of humans.
Other ancient scholars in Mesopotamia, China, Greece, Rome,
and India described its clinical signs and symptoms. In the first
century CE, Celsus and Galen described treatment, including
excision of the bitten tissue and cauterization of the wound with
a hot iron.
In 1546 the Italian physician, Fracastoro, published The
Incurable Wound, a treatise describing clinical rabies in humans:
The patient can neither stand nor lie down, like a mad man he
flings himself hither and thither, tears his flesh with his hands,
and feels intolerable thirst. This is the most distressing symptom,
for he so shrinks from water and all liquids. He would rather die
than drink or be brought near to water. It is then he bites other
persons, foams at the mouth, his eyes look twisted, and finally he
becomes exhausted and painfully breathes his last.

[from Smithsonian Institution, used with permission]

Rabid dog attacking a calf. Relief from the ruin of a 4th

century Roman settlement at Neumagen-Dhron near Trier
(Rheinland-Pfalz), Germany [from Marian Horzinek]

1224 Mesopotamian medicine rabies, the oldest documented infectious disease of humans and domestic animals

page 7

Girolamo Fracastoro (1478-1553)

(Hieronymus Fracastorius)

In 1546, Fracastoro proposed that epidemic diseases are caused by transferable

tiny particles or spores that can transmit infection by direct or indirect
contact or even without contact over long distances. I call fomites [from
the Latin fomes, meaning tinder] such things as clothes, linen, etc., which
although not themselves corrupt, can nevertheless foster the essential seeds
of the contagion and thus cause infection. His theory remained influential for
nearly three centuries, before being displaced by the germ theory of Pasteur
and Koch.

1546 Girolamo Fracastoro proposal that epidemic diseases are contagious, leading to the germ theory

page 8

Sacharias Jansen was a Dutch spectacle-maker credited with inventing, or contributing advances
towards the invention of, the telescope. In 2008, the Netherlands commemorated the 400th
anniversary of the telescope, honoring Jansen as one of the two possible inventors, the other being
his neighbor Hans Lippershey.
Jansen is credited with inventing the first compound microscope, with the help of his father, Hans,
in 1595. Jansens life was documented very well before WWII. Many of the archives were destroyed
by a bombardment during the Nazi invasion of the Netherlands. If there had not been earlier
studies, we would not know anything of Jansens life, since all primary files were lost in the fires
following the bombardment.
It must be said that early compound
(at least two lenses) microscopes
were only capable of magnifications
of about x20-30, less than van
Leeuwenhoeks first simple (single
lens) microscope. But, the quest to
see things smaller than visible to the
naked eye had begun.
Marker at the site of the Sacharias Jansen (Zacharias
Janse) house, Middleburg, The Netherlands
[Uitvinder der Verrekkers Inventor of the Telescope]

Sacharias Jansen (~1580-~1632)

(Zacharias Janssen)

Jansens microscope consisted of three sliding tubes with lenses fixed in the ends. The
eyepiece lens was bi-convex and the objective lens was plano-convex, an advanced compound
design for the time. Focusing was achieved by sliding the draw tube in or out while
observing the specimen. No early models of Jansen microscopes have survived, but there are
reproductions, including this one made in the 1890s.

1595 Sacharias & Hans Jansen development of the first compound microscope

page 9

The origins of the Royal Society go back to the mid-1640s when

groups of physicians and natural philosophers met at several locations, including Gresham College in London, forming an invisible
college, that is, a venue for promoting knowledge of the natural
world through observation and experiment. Topics for discussion
included physic, anatomy, geometry, astronomy and navigational aids, circulation of blood, etc. They were influenced by the
new science, that is, science grounded in testing ideas through
experiments, as earlier promoted by Francis Bacon (1561-1626).
In 1660, a committee of 12 announced the formation of a College
for the Promoting of Physico-Mathematical Experimental Learning, which would meet weekly. The first meeting was attended
by Robert Boyle (1627-1691), John Wilkins (1614-1672)., Robert
Moray (1608/9-1673), William, Viscount Brouncker (1620-1684)
and Christopher Wren (1632-1723), then the Gresham Professor of Astronomy, who presented a lecture. A royal charter was
signed in 1662 creating the Royal Society of London, with Lord
Brouncker serving as the first President. Robert Hooke was appointed as curator of experiments. The Societys early weekly
meetings included experiments performed first by Hooke and
then by Denis Papin (who invented the centrifuge). The Society
found accommodation at Gresham College and rapidly began to
acquire a library, a museum and a book publishing enterprise (the
first two books, published in 1661, were John Evelyns Sylva and
Robert Hookes Micrographia). In the presidency of Isaac Newton
from 1703 to 1727, the society became a dominant influence in
in Britain and farther afield. Earlier, Newtons Principia
Francis Bacon (1561-1626) science
had been published with the societys imprimatur. The Society
Wenceslaus Hollars frontispiece engraving in Thomas Sprats History of the Royal Society (1666). On one moved during Newtons presidency, and again in 1780 to premises
side is represented a library, and on the table lie the statutes, the journals, and the mace of the Royal Society; on at Somerset House through an arrangement made by Joseph Banks
the opposite side are suspended numerous instruments. The bust of Charles II, the Societys first patron, is about who had become President in 1778 and was to remain so until his
to be crowned by a symbolical angel representing Fame. Viscount Brouncker, the first president of the Society, at death in 1820.
the left, points to the Latin inscription, CAROULUS II SOCIETATIS REGALIS AUTHOR ET PATRONUS. At The Societys motto is Nullius in verba, which roughly translates
the right is Francis Bacon; at his feet is the legend ARTIUM INSTAURATOR (promoter or renewer of practical as take no theory on trust. It expressed the determination of
science), which at once reminds us of his great vision for the future, a complete division and systematic
the members to stand up to the domination of scientific ideas by
classification of all sciences, as begun in Bacons neverauthority and historical dogma and to verify all long-standing trufinished publication, the Instauratio Magma. Bacons
isms by direct observation and experiment. From the beginning,
influential emphasis of inductive logic in science was
membership in the society was by election, although the criteria
the first iteration of the scientific method, which later
for election were vague and the vast majority of members were not
changed to rely on deductive logic, with the testing of
professional scientists. From the outset the society aspired to comhypotheses by experimentation and observation, and
bine the role of research institute with that of clearinghouse for
with the use of controls.
knowledge and forum for arbitration, the latter two functions beGresham College, London, the first home
of the Royal Society, 1660-1710

coming dominant later on. A key development was the establishment in 1665 of a periodical, the Philosophical Transactions, which
today is the oldest scientific journal in continuous publication.

1660 Robert Boyle, William, Viscount Brouncker, Christopher Wren, others founding of the Royal Society

page 10

Robert Hooke (1635-1703)

Robert Hooke is one of the most neglected important natural philosophers of all
time. He was the inventor of, among other things, the iris diaphragm in cameras, the
universal joint used in motor vehicles, the balance wheel in watches, and he was the
originator of the word cell in biology He was Surveyor of the City of London after
the Great Fire of 1666, and was also an architect, experimenter and astronomer.
And, he was one of the inventors of the compound microscope. [Sacharias and Hans
Jansen in Holland built the first compound microscope around 1595, but their work
was little appreciated outside Holland.] Hookes observations received widespread
acclaim, since they were published in his remarkable book, Micrographia, in 1665.
There are no incontrovertible images of Robert Hooke the formulaic image here is
from the Hooke memorial window, St. Helens Church, Bishopsgate, London, where
Hooke was buried (and which was destroyed in an IRA bombing). Hooke served
for many years as Curator of Experiments for the Colledge for the Promoting of
Physico-Mathematicall Experimentall Learning and then its successor, the Royal
Society. As such he resided at its site in Gresham College, Bishopsgate. After Hooke
died, Isaac Newton became president of the Royal Society, and it fell to him to move
to new headquarters. Among the many items to be relocated was Robert Hookes
portrait during the move the portrait simply disappeared.

Microscopic view of a hairy mould colony by

Robert Hooke, 1665.
Published in Micrographia.

This image was the first published depiction of a

microorganism. The reproductive structures (sporangia)
are characteristic of the genus Mucor. Sporangia in
different stages of maturation are identified by letters
A, B, C, D.

Robert Hookes
compound microscope, 1665,
and lighting system (a brine-filled glass globe
placed between the light source and the microscope
to focus the light). As published in Micrographia.

1665 Robert Hooke first microscopic observation of a microorganism, a mold, genus Mucor

page 11

Anton van Leeuwenhoek

(1632-1723)
van Leeuwenhoeks Discoveries
Using his simple microscopes, van Leeuwenhoek
discovered, protozoa, Giardia, spermatozoa, rotifers,
hydra and volvox. He made observations of blood cells,
the nucleus of fish erythrocytes, capillaries and lymphatics,
but perhaps his greatest discovery was of bacteria. In 1674
he wrote: Last winter while being sickly and nearly unable
to taste, I examined the appearance of my tongue, which
was very furred This led him to examine an ox tongue
with his microscope, and he saw that there were very fine
pointed projections (taste buds) which were composed of
very small globules (bacteria). Separately, he was curious
why pepper and other spices (ginger, nutmeg and cloves) had such potent tastes. He examined pepper
water, an infusion that had been sitting for three weeks he was astonished to see many very small
organisms that he called animalcules (again, bacteria, typically 1-2m in diameter). He then looked for
animalcules in other places such as in plaque on his teeth: I have mixed it with clean rain water, in which
there were no animalcules, and I almost always saw with great wonder that there were many very little
animalcules, very prettily amoving.

van Leeuwenhoeks Microscopes

van Leeuwenhoek constructed hundreds of microscopes
he sent many to the Royal Society and left 247 completed
microscopes (only nine are known to have survived; one sold
in 2009 for $459,000) and 172 additional mounted lenses
actually, they were not microscopes but simple magnifying
lenses. To make a lens, he drew out a small soda lime glass
rod in a flame, creating a long glass whisker. By reinserting the
end of a whisker into the flame, he created a very small, highquality glass sphere. These spheres became the lenses of his
microscopes, with the smallest spheres providing the highest
magnifications. The lenses were of exceptional optical quality
and had magnifying powers ranging from x50 to x275 (possibly
up to x500). His microscopes consisted of a single spherical lens
mounted between two metal plates as seen in the illustration.
The short (about 1 millimeter) focal lengths of the lenses would
have necessitated placing the eye almost in contact with the
lens.

1668 Anton van Leeuwenhoek invention of the simple (single lens) microscope, discovery of bacteria
page 12

Variolation (also known as

Inoculation) was introduced to the
west by Lady Mary Wortley Montagu,
who witnessed it being practiced by
physicians in Constantinople, and
was greatly impressed: she had lost
a brother to smallpox and her own
famous beauty had been marred by a
bout with the disease. She was eager
to spare her children similar suffering,
and had them inoculated while in
Turkey. On her return to London,
she enthusiastically promoted the
procedure, but encountered a great
deal of resistance from the medical
establishment both because it was
an Oriental process and because
of her gender. However, the British
royal family had their own children
inoculated, furthering acceptance.

Lady Mary Wortley Montagu (1689-1762)

She wrote: I am going to tell you a

thing, that will make you wish yourself
here [Turkey]. The small-pox, so
fatal, and so general amongst us, is
here entirely harmless. There is a
set of old women, who make it their
business to perform the operation...;
they make parties for this purpose...;
the old woman comes with a nut-shell
full of the matter of the best sort of
small-pox. She opens that vein which
you offer to her and with a needle
puts into the vein as much matter as
can lie upon the head of her needle.
The children or young patients are in
perfect health to the eighth day. Then
the fever begins to seize them, and
they keep their beds for two days.... I
am well satisfied of the safety of this
experiment, since I intend to try it on
my dear little son. I am patriot enough
Ivory and boxwood variolator/vaccinator, ~1800
to take the pains to bring this useful
invention into fashion in England...

1718 Lady Mary Wortley Montagu introduction of variolation to western countries

page 13

Carolus Linnaeus (1707-1778)

Carolus Linnaeus, is called the Father of Taxonomy, for his system for naming, ranking, and classifying organisms that is still in wide use today (with many changes). The first
edition of Systema Naturae was printed in the Netherlands in 1735. It was a twelve page work. By the time it reached its 10th edition (1758), the multivolume work included
4,400 species of animals and 7,700 species of plants. In it, the unwieldy names in use at the time were supplemented with concise and now familiar binomials, composed of
the generic name, followed by a specific epithet. Higher taxa were constructed and arranged in a simple manner based on shared characters genera were grouped into orders,
orders into classes, and classes into kingdoms. In 1939, Francis O. Holmes first tried to extend the Linnaean sytsem to the viruses: he divided the viruses into three groups
under one order, the Virales the Phaginae (bacteriophage), Phytophaginae (plant viruses) and Zoophaginae (animal viruses). He extended his notion as a supplement to the
1948 edition of Bergeys Manual of Determinative Bacteriology. However, for the most part his taxa were based on clinical signs and pathology in animal hosts and lesions
in infected plants, not on properties of the viruses themselves. Even in 1948 this was not considered appropriate. Then, as described later in this book, in 1962 Andr Lwoff,
Robert Horne and Paul Tournier developed the first comprehensive virus classification system based on virion properties except for its Latinized nomenclature, it is the basis
for the current system managed by the International Committee on Taxonomy of Viruses.
Holmes FO, Proposal for extension of the binomial system of nomenclature to include viruses. Phytopathology 1939;29:431-436

1735 Carolus Linnaeus development of the hierarchical system for the classification of all organisms
page 14

Spallanzanis great experiment

refuting spontaneous generation
The debate over spontaneous generation continued for centuries. In 1745, John Needham,

an English clergyman, did what he considered the definitive experiment. Everyone knew
that boiling killed microorganisms, so he boiled chicken broth, put it into a flask, sealed it,
and waited - sure enough, microorganisms grew. Needham claimed victory for spontaneous
generation.
The Italian priest, Lazzaro Spallanzani, was not convinced, and he suggested that perhaps the
microorganisms had entered the broth from the air after the broth was boiled, but before it was
sealed. To test his theory, he placed the chicken broth in a flask, sealed the flask, drew off the
air to create a partial vacuum, then boiled the broth. No microorganisms grew. Proponents of
spontaneous generation argued that Spallanzani had only proven that spontaneous generation
could not occur without air.

Lazzaro Spallanzani (1729-1799)

The theory of spontaneous generation was finally laid to rest in 1859 by Louis Pasteur. The
French Academy of Sciences sponsored a contest for the best experiment either proving or
disproving spontaneous generation. Pasteurs winning experiment was a variation of the
methods of Needham and Spallanzani. He boiled meat broth in a flask, heated the neck of the
flask in a flame until it became pliable, and bent it into the shape of an S. Air could enter the
flask, but airborne microorganisms could not - they would settle by gravity in the neck. As
Pasteur had expected, no microorganisms grew. When Pasteur tilted the flask so that the broth
reached the lowest point in the neck, where any airborne particles would have settled, the broth
rapidly became cloudy with microbes. Pasteur had both refuted the theory of spontaneous
generation and convincingly demonstrated that microbes are everywhere even in the air.

1765 Lazzaro Spallanzani first experiments refuting the theory of spontaneous generation

page 15

In recent years, there has been a growing recognition that Benjamin Jesty was among the first to
vaccinate against smallpox. In fact, the use of cowpox materials (lymph, scabs) was rather widely
known among country physicians in 18th century England. When smallpox occurred in Jestys
locality in 1774, he was determined to protect his family. Jesty used lesion material from cattle
suffering from cowpox, transferring the material with a small lancet to the arms of his wife and
two sons. The vaccinees remained free of smallpox, although they were exposed on numerous
occasions in later life. However, this does not diminish Edward Jenners accomplishments. It was
Jenners relentless promotion and devoted research of vaccination that led the way to the control
of smallpox globally.

Benjamin Jesty (1737-1816)

1774 Benjamin Jesty vaccination against smallpox using material from cowpox lesions
page 16

Edward Jenner Vaccinating James Phipps, 1796

Edward Jenner (1749-1823)

painting by Gaston Melingue, 1879, Academie Nationale de Medecine, Paris

In Jenners time, smallpox was greatly feared, as one-third of those who contracted the disease died, and those
who survived were often badly disfigured. Noting the common observation that milkmaids did not generally
get smallpox, Jenner theorized that contact with the lesions of cowpox on the teats of infected cows protected
them from smallpox. In 1796, Jenner tested his hypothesis by inoculating James Phipps, an eight year old boy
(the son of Jenners gardener), with material from cowpox lesions on the hand of Sarah Nelmes, a milkmaid
who had been infected with cowpox from a cow named Blossom. This produced a fever and some uneasiness
in James Phipps, but no great illness. Later, he injected the boy several times with material from smallpox
lesions no disease followed and he went on to test his method successfully on a series of 23 subjects.
[There is no mention whether Jenner knew that smallpox and cowpox material had been used for some years
to prevent smallpox.] Jenners unique contribution was not that he inoculated a few persons with cowpox
virus, but that he then proved they were immune to smallpox. The medical establishment, cautious as ever,
considered his findings for some time before accepting them, but in time vaccination became universal, even
leading to the eradication of smallpox globally the first human disease to be eradicated globally.

1796 Edward Jenner application of cowpox virus for vaccination against smallpox

Edward Jenner
An Inquiry into the Causes and
Effects of th e Variol Vaccin
(London: Sampson Low, 1798)

page 17

Edward Jenner vaccinating his son, who is held by his wife, while the maid prepares for her vaccination
Water-colored engraving by Jean Claude Manigaud (1825-?) from a painting by Edouard Jean Conrad Hamman (1819-1888). Wellcome Images.

1796 Edward Jenner application of cowpox virus for vaccination against smallpox
page 18

The Cow-Pock-or-the Wonderful Effects of the New Inoculation!-vide. the Publications of ye Anti-Vaccine Society.

Print (hand colored engraving) published in 1802 by H. Humphrey, St. Jamess Street, London. The British satirist James Gillray (1756-1815) caricatured a vaccination scene at
the Smallpox & Inoculation Hospital at St. Pancras, London, showing Edward Jenner vaccinating a frightened young women, and cows emerging from different parts of peoples
bodies. The cartoon was inspired by the controversy over inoculating against smallpox. The inoculation agent, cowpox vaccine, was rumored to have the ability to sprout cowlike appendages. A serene Edward Jenner stands amid the crowd. A boy next to Jenner holds a container labeled VACCINE POCK hot from ye COW; papers in the boys pocket
are labeled Benefits of the Vaccine. The tub on the desk next to Jenner is labeled OPENING MIXTURE. The painting on the wall depicts worshippers of the Golden Calf.

1796 Edward Jenner application of cowpox virus for vaccination against smallpox

page 19

In 1793, John Hunter (1754-1809) and

Samuel Argent Bardsley (1764-1851) each
published papers suggesting experiments to
determine the mode of rabies transmission,
including the inoculation of dogs and other
species with saliva from rabid dogs and even
from an hydrophobic human patient. The
inoculated animals were to be observed,
recording progress of the disease, and of the
length of time beyond which excision of the
tissue around the inoculation site could no
longer prevent development of the disease.
In 1804, Georg Gottfried Zinke (1771-1813),
carried out all the experiments suggested by
Hunter except for using saliva from a human
patient he successfully produced rabies in
healthy dogs, cats and rabbits by inoculating
saliva from rabid dogs. The experiment with
saliva from a human case was carried out in
1821 by Franois Magendie (1783-1855). At
the same time William Youatt (1776-1847)
showed that the rabbit was a particularly
useful experimental animal.

Pierre-Victor Galtier
(1846-1908)

John Hunter (1754-1809)

[There were two nearly contemporary John
Hunters, the other the famous surgeon and
anatomist (1728-1793), who was a mentor and
colleague of the John Hunter cited here.]

Plaque honoring Galtier, Langogne, France

In 1879, Pierre-Victor Galtier (1846-1908),

professor at the Ecole Vtrinaire de Lyon,
reintroduced the rabbit into rabies research
he transmitted rabies to rabbits and from
rabbit to rabbit. Pasteur was aware of the
work of Galtier before he started his work on
rabies in 1880 he had visited Lyon, where
Jean-Baptiste Auguste Chauveau (1827-1917)
had introduced him to Galtiers publications.
Galtier received many honors for his work
and was nominated for a Nobel Prize in 1908,
but he died a few months before the Nobel
Commissions decision.
Wilkinson L. The development of the virus
concept as reflected in corpora of studies
on individual pathogens. 4. Rabies two
millennia of ideas and conjecture on the
aetiology of a virus disease. Medical History
1977;21:15-31.

1793 John Hunter, Pierre-Victor Galtier, Georg Zinke, others beginnings of experimental pathology
page 20

In 1804, Georg Gottfried Zinke (1771-1813) first transmitted rabies from a rabid dog to a normal one, and from
a dog to a rabbit and a hen, by injection of saliva. This proved that the disease was infectious.
By 1826, Franz Christian Karl Krugelstein (1779-1864) wrote a full account of rabies, with a bibliography of
300 entries. But the nature of species susceptibility was unclear until Pierre-Victor Galtier demonstrated the
transmission from dog to rabbit to rabbit, in series. He then immunized sheep by inoculating rabid saliva
intravenously. This did not produce the disease, but interestingly, protected the animals from the effects of a
subsequent inoculation. His work aroused the interest of Louis Pasteur who with Charles Chamberland, Emile
Roux and Louis Thuillier wrote the first of their papers in 1881, heralding the beginning of Pasteurs studies on
rabies. It first lodges and multiplies in the spinal cord and brain.
Zinke GG. Neue ansichten der hundswuth, ihrer ursachen und folgen, nebst einer sichern behandlungsart der von tollen
thieren gebissenen menschen. Jena: CE Gabler; 1804.
Krugelstein FCK. Die geschichte der hundswuth und der wasserscheu und deren behandlung. Gotha: In der Henningsschen
Buchhandlung; 1826.
Galtier P-V. tudes sur la rage. Ann Med Vet. 1879;28:627-639.
Galtier V. Les injections de virus rabique dans le torrent circulatoire ne provoquent pas lclosion de la rage et semblant
confrer limmunit. La rage peut tre transmise par lingestion de la matire rabique. C R Acad Sci (Paris) 1881;93:284-285.
Wilkinson L. The development of the virus concept as reflected in corpora of studies on individual pathogens. 4. Rabies
two millennia of ideas and conjecture on the aetiology of a virus disease. Medical History 1977;21:15-31.

Neue Ansichten der Hundswuth, ihrer Ursachen und Folgen,

nebst einer sichern Behandlungsart der von tollen Thieren
gebissenen Mensch. [New views of rabies, its causes and
consequences, in addition to a safe treatment for humans
bitten by animals] Georg Gottfried Zinke.
Published by C.E. Gabler, Jena, 1804.

Emile Roux trephining a rabbits skull for rabies research,

while Louis Pasteur and Louis Thuillier observe

1793 John Hunter, Pierre-Victor Galtier, Georg Zinke, others beginnings of experimental pathology

page 21

Philadelphias yellow fever epidemic of 1793 was among the largest in U.S. history,
with nearly 5,000 deaths (10% of the population). As the number of deaths began
to climb, 20,000 citizens fled to the countryside, including President George
Washington, Thomas Jefferson, and other members of the fledgling federal
government. Another severe epidemic occurred in 1798 by this time, the
advantages of flight were clear all but 7,000 residents left the city.
Benjamin Rush believed that yellow fever was caused by unsanitary conditions,
especially those of the docks, sewage system, and rotting vegetables such as rotting
coffee on the Arch Street wharf. He concluded that the illness was not transmitted
from human to human but by putrid exhalations in the atmosphere. He also
recognized that weather played a part. Though many people wanted to blame newly
arrived Saint Domingue revolution refugees, Rush instead blamed the sanitary
conditions of the city and implored residents to clean up the city so as to not entrail
the disease upon future generations.

1793 Benjamin Rush account of the great yellow fever epidemic in Philadelphia, 1793
page 22

Benjamin Rush (1746-1813), at age 37

by Charles Willson Peale, 1783

[Winterthur Museum, used with permission]

1793 Benjamin Rush account of the great yellow fever epidemic in Philadelphia, 1793

page 23

Establishment of the U.S. Public Health Service [first as the

Marine Hospital Service (1798-1902), then the Public Health
and Marine Hospital Service (1902-1912), and finally as the
Public Health Service (1912-present)]

1798 Founding of the U.S. Public Health Service, first as the Marine Hospital Service
page 24

John Pintard (1759-1844)

At The Gates: Our Safety Depends

Upon Official Vigilance

Harpers Weekly, Sept 5, 1885. The specters of cholera,

yellow fever, and smallpox recoil in fear as their way
through the Port of New York is blocked by a barrier on
which is written quarantine and by an angel holding a
sword and shield on which is written cleanliness.

Stephen Smith (1823-1922)

The kind of assisted emigrant we can not afford to admit.

Puck, 1883

In 1793, when a yellow fever epidemic killed 5,000 people in Philadelphia, doctors in New York organized a committee to prevent ships from Philadelphia from entering
New York harbor. These quarantine efforts may have helped keep the disease away that year but in 1795, 1799, and 1803, yellow fever felled thousands in New York. These
epidemics lent weight to the view that sanitary measures, and not just quarantine, were needed to curb the disease. The citys Common Council determined that it needed
greater authority over local sanitation, so in 1805 it passed an ordinance to create the first New York City Board of Health. John Pintard (1759-1844) began collecting mortality
statistics for New York in 1802, and two years later, the city appointed him its first City Health Inspector to keep official mortality statistics. In the 1805 City Inspectors Report,
he wrote that the public health system was designed to furnish data for reflection and calculation, in order that the awful malady of yellow fever could one day become
more controllable and less mortal. That year, there had been 600 reported cases and 262 deaths from yellow fever. This Board of Health eventually failed and had to be reestablished in 1866. This was led by Stephen Smith (1823-1922); again, it was the first such public health agency in the U.S. Smith later founded the American Public Health
Association.

1805, 1866 John Pintard, others founding of the New York City Department of Health, the first in the U.S.

page 25

John Dollond
(1706-1761)

Giovanni Battista Amici

(1786-1863)

Joseph Jackson Lister

(1786-1869)

(the father of Joseph Lister)

Ernst Karl Abbe

(1840-1905)

Early microscopes had severely limited resolution caused by spherical and chromatic aberration the understanding of cells and cellular
pathology could not have occurred without improvements. Such improvements started in the 1730s with Chester Moore Hall, George
Bass, John Dollond and in the early 1800s with Giovanni Battista Amici and Vincent and Charles Chevalier who dealt with chromatic
aberration. The solution was to use a doublet lens, composed of two lenses made from glasses with different refractive indices so that
red and blue light waves were focused at the same point. In 1830, Joseph Jackson Lister solved the problem of spherical aberration (light
bends at different angles depending on where it hits the lens) by placing lenses at precise distances from each other. In 1866 Carl Zeiss
recruited Ernst Abbe, who laid out the mathematical framework of what would become the modern computational optics approach, and
who invented a condenser that controlled the diameter of the beam of incoming light and focused it on the specimen. Combined, these
developments allowed the manufacture of microscopes that could magnify 500 times with a very high quality image.

Lister type microscope

~1830
1830> John Dollond, Ernst Abbe, others advances in microscopes, achromatic lenses, Abbe condenser, light sources
page 26

Charles Chevalier
(1804-1859)

Wilhelm His
(1831-1904)

Rudolph Jung
(1845-1900)

Charles Sedgwick Minot

(1852-1914)

The first microtome was devised around 1790;

it consisted of a wooden device with a berth to
accommodate the specimen and a flat surface to slide
a razor against the specimen surface. The term
microtome was given to these instruments by Charles
Chevalier, who along with Johannes Purkinje (17871869), perfected the instrument around 1835. Finally,
starting in 1865, precision mechanical devices were
developed and used by Wilhelm His, Charles Sedgwick
Minot, Adam Pfeiffer, and others. They consisted of a
metal stage holding a paraffin or collodion tissue block
and a mechanical swing holding the blade in precise
alignment. Rudolph Jung, a precision engineer and the
founder of Leitz, together with the pathologist Rudolf
Thoma (1847-1923) developed the first commercially
produced microtome. Carl Zeiss (1816-1888) was also
making microtomes by the 1870s. The famous Cambridge
rocking arm microtome, designed by Horace Darwin
(1851-1928; son of Charles) in 1885, was produced until
recently.

1835 Charles Chevalier, others development of the microtome, key to the founding of cellular biology/pathology

page 27

Theodor Schwann (1810-1882)

Matthias Schleiden (1804-1881)

In 1838, Theodor Schwann and Matthias Schleiden were enjoying after-dinner coffee and talking about their studies on cells. When Schwann heard Schleiden describe
plant cells with nuclei, he was struck by their similarity to cells he had observed in animal tissues. The two scientists went immediately to Schwanns lab to look at his slides.
Schwann published his book on animal and plant cells the next year, a treatise devoid of acknowledgments of anyone elses contribution, including Schleidens. He made three
conclusions: (1) The cell is the unit of structure, physiology, and organization in living things. (2) The cell retains a dual existence as a distinct entity and a building block in the
construction of organisms. (3) Cells form by free-cell formation, similar to the formation of crystals (spontaneous generation) [Not true! The correct interpretation of cell
formation by division was finally enunciated in Rudolph Virchows 1858 epigram: Omnis Cellula e Cellula (every cell originates from another existing cell like it).]

1835 Theodor Schwann, Matthias Schleiden, others development of concept that organisms are composed of cells
page 28

John Snow (1813-1858)

The Broad Street Pump

John Graunt, William Farr, Louis-Ren Villerm, Peter Ludwig Panum, Mitchell Pruden, Hermann Biggs, and others are recognized for their
varied contributions to the founding of medical epidemiology, but it is John Snow who is most often given the title Father of Epidemiology.
This title stems from his two classic studies of the London cholera epidemic of 1853-1855. Snows first study concerned the Broad Street pump
outbreak of 1854, which killed more than 600 people in the Soho region of London. He used skilled reasoning, interviews with inhabitants,
graphs, and the now famous spot map locating cases to demonstrate the impact of contaminated water coming from the Broad Street pump.
His second study compared London neighborhoods receiving water from two different water companies. One company relied on inlets on the
upper River Thames, away from urban pollution, and the other company relied on inlets in the heart of London, where sewage contamination
was apparent. Snow compared the effect of contaminated water in two nearly equivalent populations, and suggested intervention strategies
to control the epidemic. His ideas and observations, including innovative disease maps, were published in his book, On the Mode of
Communication of Cholera (1849 and 1855). [The causative agent, Vibrio cholerae, was initially discovered in 1854 by Filippo Pacini, but his
discovery was not widely known until Robert Koch rediscovered it thirty years later.]
University of California Los Angeles, School of Public Health, Department of Epidemiology, John Snow: http://www.ph.ucla.edu/epi/snow.html

1838 John Snow, John Graunt, William Farr, others founding of medical epidemiology

page 29

Friedrich Gustav Jakob Henle

(1809-1885)

Friedrich Gustav Jakob Henle, anatomist,

pathologist, and eminent scholar, was
an important figure in the development
of modern medicine. For example, he is
credited with the discovery of the loop
of Henle in the kidney. Henle developed
the concepts of contagium vivum and
contagium animatum [Von den Miasmen
und Kontagien (On Miasma and
Contagia)] in 1840, following the ideas of
Girolamo Fracastoro and Agostino Bassi
(who discovered that the muscardine
disease of silkworms was caused by a
living, very small, parasitic organism, a
fungus that would be named Beauveria
bassiana in his honor, and who in 1844
extended his findings to notions that
microscopic organisms also caused
human diseases). Henle thus laid the
groundwork for our understanding that
microorganisms are the cause of many
diseases. He did not find any bacteria
as etiologic agents this was achieved
by his student Robert Koch and others
including Louis Pasteur. Henle and
Koch, together with Friedrich Loeffler,
developed the fundamental rules for
proving the etiologic association between
an infectious agent and a specific disease:
the Henle-Loeffler-Koch Postulates.

Sculpture of The Loop of Henle

Honoring Henle at
der Universitt Gttingen

1840 Friedrich Gustav Jakob Henle father of the germ theory, development of the Henle-Loeffler-Koch Postulates

page 30

Molluscum contagiosum

Skin lesion filled with poxvirus

thin section electron microscopy

Molluscum contagiosum lesion,

skin biopsy
H&E

In 1841, William Henderson (1810-1872), a pathologist at the Royal Infirmary of Edinburgh, wrote in the Edinburgh Medical & Surgical Journal about molluscum
contagiosum, which to him appeared to be among the rarest of cutaneous diseases. Within a transverse section of the wall of a lesion, he described and illustrated the
microscopic appearance of globular bodies as follows:
the wall consists of parallel cells projecting toward the interior cavity from an investing tegument or cyst of much delicacy. Within their parallel cells are exhibited small
globular bodies in considerable abundance, seen the most distinctly toward the inner extremities of the parallel cells. It is of these globular bodies that the atheromatous
matter which can be squeezed from the tubercles chiefly consists. The globules vary in size and contain nucleoli, some of which appear also in the fluid in a separate state.
These globular bodies are from the 1400th to the 3000th part of an inch in diameter.
In that same year, 1841, Dr. Robert Paterson (1814-1889), a physician of the Leith Dispensary, also found, microscopically, globular bodies within lesions of molluscum
contagiosum. This was the first description of virus-infected cells, a forerunner of the science of viral pathology.
Henderson W. Notice of the molluscum contagiosum. Edinburgh Medical & Surgical Journal 1841;56:213-218 and 279-288.

1841 William Henderson discovery of the inclusion body of molluscum contagiosum virus

page 31

Ignaz Philipp Semmelweis

(1818-1865)

Oliver Wendell Holmes, Sr.

(1809-1894)

Joseph Lister
(1827-1912)

In 1843, Oliver Wendell Holmes, Sr. investigated the circumstances surrounding puerperal (or childbed) fever and concluded that it was transmitted from patient to patient by
doctors, nurses and midwives, via their hands and clothing. He advocated the washing of hands with strong soap and chlorinated lime solution. His essay on the subject took
a strong line against opinions then prevailing (Holmes OW. The contagiousness of puerperal fever. New England Quarterly Journal of Medicine and Surgery 1842-1843;1:503530). Ignaz Philipp Semmelweis independently discovered at about the same time that the incidence of puerperal fever in obstetrical clinics could be drastically cut by the use
of hand washing with chlorinated lime solution. Puerperal fever was common in mid-19th-century hospitals, with mortality rates of 10-35%. Semmelweis worked in the Vienna
General Hospitals First Obstetrical Clinic, where doctors wards had three times the mortality rates of midwives wards. He published a book, The Etiology, Concept and
Prophylaxis of Childbed Fever, in which controlled quantitative studies of the value of disinfection were included. Joseph Lister was concerned about frequently fatal wound
infections that followed surgical procedures, and, in search of solutions to this problem, he studied the work of European bacteriologists, notably Louis Pasteur. Lister thought
that bacteria caused postoperative infections, and although the connection between bacteria and infection had not been confirmed at that time, he understood that bacteria
could be killed by antiseptics. He came up with the idea of using carbolic acid for preoperative cleansing of his and his assistants hands. He also sprayed carbolic acid in the
air in the operating room and on the surgical incision site. His dramatic beneficial results were reported in The Lancet in 1867 (Lister J. Illustrations of the antiseptic system of
treatment in surgery. The Lancet 1867;2:668), and soon transformed the practice of surgery into a relatively safe procedure for many conditions including the management of
childbirth.

1843 Ignaz Semmelweis, Oliver Wendell Holmes, Joseph Lister development of methods of hygiene/disinfection
page 32

Lister spraying carbolic acid over a surgical incision site:

While he was a professor of surgery at the University of
Glasgow, Lister became aware of the work of Louis Pasteur,
showing that microbial growth could occur under anaerobic
conditions. Pasteur suggested three methods to eliminate the
microorganisms responsible for gangrene: filtration, exposure
to heat, or exposure to chemical solutions it was the latter
that interested Lister. He confirmed Pasteurs conclusions with
his own experiments, which led him to develop antiseptic
techniques for wounds. Carbolic acid (phenol) had been in
use as a means of deodorizing sewage, so Lister tested it,
using spraying devices for surgical incision sites, and soaking
dressings with it. He found that the carbolic acid solution
remarkably reduced the incidence of gangrene. He published
a series of articles on the Antiseptic Principle of the Practice
of Surgery in 1867, immediately influencing medical/surgical
practice everywhere.

Ignaz Philipp Semmelweis: In the 1840s, puerperal or childbirth fever, a bacterial

infection occurring after childbirth, killed up to 30% of women who gave birth in
hospitals. Women who gave birth at home were relatively unaffected. Semmelweis, an
assistant professor on the maternity ward of the Vienna General Hospital observed that
women examined by student doctors who had not washed their hands had very high death
rates. When a colleague who had received a scalpel cut died of infection, he concluded
that puerperal fever was septic and contagious. He ordered students to wash their hands
with chlorinated lime before examining patients; as a result, the maternal death rate was
reduced from 12% to 1% in 2 years. Shortly afterward, as a professor of obstetrics at the
University of Pest Hospital, he enforced antiseptic practices and reduced the death rate
from puerperal fever to 0.85%.

1843 Ignaz Semmelweis, Oliver Wendell Holmes, Joseph Lister development of methods of hygiene/disinfection

page 33

In 1839, Louis Daguerre (1787-1851) presented

to the French Academy of Sciences his invention
of what became the first practical photographic
process, the daguerreotype. Alfred Franois Donn
(1801-1878), a member of the Faculty of Medicine
and Charit Hospital, Paris, saw this and resolved
to incorporate images seen in his microscope
into his lectures. It became his style to lecture
while showing daguerreotype illustrations. He
and his medical-student assistant, Lon Foucault
(1819-1868), who later became a famous physicist, published a book and atlas entitled Cours de
microscopie complementaire des etudes medicales,
anatomie microscopique et physiologie des fluides de leconomie (1844); and Atlas du Cours de
microscopie excute daprs nature, au microscopedaguerrotype (1845) Paris: Baillire. The images
in the atlas are engravings made from the original
daguerreotypes. Donn stated, My lectures have
contributed to making the importance of the
microscope understood and to gaining the interest and confidence it merits. Donne was also the
discoverer of Trichomonas vaginalis and the first to
describe leukemia histologically. At the time there
was a peculiarly obstructive bloc of physicians who
opposed any innovation arising outside the established system. They raised profound arguments
against new inventions, including the microscope.
It took years for teachers and clinicians to recognize the potential of the microscope. Nevertheless, at the same time important improvements in
instruments facilitated new investigations and the
use of photographic images in research spread. No
image of Donns photomicroscope has survived,
but soon instruments similar to it appeared in
several countries. Parallel developments were
important: tissue fixation and sectioning, histological stains, and most importantly achromatic lenses
(controlling spherical and chromatic abberations
and greatly improving resolution). By the time the
concept of virus was developed at the turn of the
20th century, the technologies and scholarly bases
for studies of viral pathology and pathogenesis
advanced in concert with further developments in
microscopy and photography.

Nucleated red blood cells,

frog, daguerreotype, 1844
Daguerreotype plates were made of
copper with a coating of silver; in the
dark they were exposed to iodine,
bromine and/or chlorine fumes.
The silver halide which formed was
light sensitive. After expsure in the
Alfred Franois Donn (1801-1878)
camera, the plate with its latent image
an early daguerreotype with his daughter
was developed over mercury vapor,
making the silver recrystalize where
light had fallen, forming a fine image.
The image was fixed in a solution of
sodium thiosulfate and sealed under
glass to prevent tarnishing. Original
daguerreotype images are not impres- Photographic
microscopic
sive; they lack contrast and the apcamera
pearance of the image depends upon
London
the angle of reflected light. However,
1857
when originals are digitally scanned
and Photoshopped, the resulting images can be remarkable.

Arthur E. Smith focusing an image on the 10x12 inch camera screen (a very fine diffraction grating). A cord and
pulley system is used to adjust the fine focus adjustment screw. From Nature Through the Microscope and Camera,
by Richard Kerr with 65 photomicrographs by Arthur E. Smith. London: The Religious Tract Society; 1909. p. 1-196.

1844 Alfred Donn, Lon Foucault, others development of photomicroscopy

page 34

Lon Foucault (1819-1868)

Projecting images goes back to the camera obscura in ancient times. Eventually, it was improved by the use of a biconvex lens in the light path. Daniel Fahrenheit (1686-1736) and
separately Johann Lieberkhn (1711-1756) were first to integrate a mirror and a projection
device into a camera obscura, producing the first practical solar microscope (solar referring to the light source). Structurally similar to a microscope, the solar microscope was
a projector that required very intense light for good resolution. The horizontal instrument
was placed at a window along with a mirror that collected and reflected incident sunlight.
The resulting image was enlarged and could be projected a considerable distancein most
instances such projected images were used for entertainment (imagine a flea magnified to
be six foot long!). In this era, if sunlight was not available, gas lamps or other light sources
were used whether safe or not. Several new technologies came together in the 1840s to
change all this: (1) the invention of the carbon arc as a very bright light source; (2) the
development of more powerful and cheaper batteries; (3) the development of achromatic
lenses, which greatly improved image resolution (projected images are particularly degraded by chromatic aberration); (4) the development of aniline dye staining to add contrast to transparent specimens; and (5) the continuing advance of the biological sciences
per se and the concept of cellular life in particular.
When he first started giving his course at the Faculty of Medicine in Paris, Alfred Franois
Donn (1801-1878) (see page on photomicroscopy, 1844) would invite students to use a
few microscopes that he had set up. However, when attendance reached more than 100,
the need for a projection microscope became acute. Starting in 1843, Donns assistant,
Lon Foucault (1819-1868) adapted a carbon arc light source to a projecting microscope.
Progress was rapid, and within months they presented their instrument to the French
Academy of Sciences. They stated that their goal had not been to construct a research
instrument, but to put scientific results before the eyes of the public. A favorite demonstration was to project the brilliant spectacle of the circulation of blood in the tongue
of a frog. Ground-breaking as they were, their projected images and their book and atlas
did not captivate the medical world and were rapidly forgotten. It has been stated that the
work would have had much greater influence had they been full members of the powerful academic medical elite. The first large-scale and effective use of medical micrographs
came two decades later when U.S. Army Lieutenant-Colonel J. J. Woodward (1833-1884)
distributed photomicrographs of clinical specimens widely. Woodwards photomicrographs caused a sensation when exhibited at the U.S. Centennial Exposition in Philadelphia in 1876.

In his excellent review of this subject, William Tobin noted, We can see examples of the
1. Photo-electric projecting microscope of
crucial interplay between research and teaching in the photo-electric microscope and
Foucault and Donn, 1843; current through
atlas [of Alfred Donn and Lon Foucault], both of which harnessed recent technologithe carbon arc was controlled by dipping
cal developments to provide improved methods of medical illustration for educational
graphite electrodes to different depths in
purposes. I need only whisper the word PowerPoint to make it clear that this interconthe battery acid.
nection remains as pertinent today as it was 160 years ago. PowerPoint was developed in 2. Donn and Foucault images of human red
blood cells, engravings of daguerreotypes.
1987 by Forethought Inc. and bought by Microsoft in 1990. By 2012, it claimed ~95% of
the global presentation software market, with installations on at least 1 billion computers. 3. Gas fired projector with foot bellows, 1868.
Tobin W. Alfred Donn and Lon Foucault: The first applications of electricity and photography to medical illustration. J Vis Commun Med. 2006;29:6-13.

4. Leitz electric projection microscope, 1903.

5. Leitz 35mm slide projector, 1950s.
6. PowerPoint 1.0, Mac, 1987.

1844 Alfred Donn, Lon Foucault the projection microscope and the beginnings of biomedical illustration media

page 35

In 1846, Peter Panum, a 26-year-old physician just out of medical school. not having completed his hospital
training, and 25-year-old physician August Henrik Manicus (1821-1850) were sent by the Danish government
to investigate a measles epidemic in the Faroe Islands (Danish territory, half way between Norway and Iceland).
They stayed for five months. Panums 116 page report, Observations Made During the Epidemic of Measles on
the Faroe Islands in the Year 1846, is one of the classics of medical science, and is the seminal study in viral disease epidemiology. We often think that modern understanding of the infectious diseases originated in the work
of Pasteur and Koch, but the context was set beforehand by scholarly clinicians like Panum, whose early career
predated the general acceptance of the germ theory. Panums study provided a convincing natural example to
contrast with the enteric diseases, cholera and typhoid, that were still thought to derive from miasmas. If ever
there was an example of Pasteurs aphorism, Chance favors only the prepared mind, this was it.
There had been no cases of measles in the Faroe Islands for 65 years, but in 1846 there were more than 6,000
cases among the 7,782 inhabitants (The first of the Faroese to be attacked by measles was a carpenter He left
Copenhagen on March 2nd and arrived 8 days later in Thorshavn; on the way he felt perfectly well, but in early
April he came down with measles. Shortly before leaving Copenhagen he visited several persons who were ill
with measles). In villages where the epidemic occurred, the attack rate was ~95%, and the mortality rate 2.8%.
This was the ultimate virgin soil epidemic. Panum wrote, The circumstances under which the disease was
observed were so favorable that similar ones were rarely, if ever presented to an observer. The population of the
islands was separated in coastal valleys of 17 islands, each small village isolated, such that Panum could study
each independentlyhe obtained complete demographic and disease data for 52 sites. His background description of the islands was strikingit is hard to imagine a less hospitable place to live.

Peter Ludvig Panum (1820-1885)

Panum established the incubation period of the disease as 13-14 days (The isolated situation of the villages,
and their limited intercourse with each other, made it possible in many, in fact in most cases, to ascertain where
and when the person who first fell ill had been exposed to the infection, and to prove that the contagion could
not have affected him either before or after the day stated). He showed that transmission generally traced
to a primary case in the early rash stage of infection; no cases were traced to exposure after the disappearance
of the rash. He described clear evidence of long-lasting immunity; he interviewed 98 older persons who had
been infected previously, none of whom were affected in 1846. Panum had no idea of the nature of the etiologic
agent, but he was properly skeptical: If among 6,000 cases, of which I saw and treated 1,000 myself, there was
not a single one in which it was justifiable to attribute the affection to a miasmatic origin, while at the same time
it was everywhere clear that the disease had spread from man to man and from village to village by means of the
contagium, then one is certainly justified at least to doubt very much the miasmatic nature of the disease.
Panum considered means of prevention, even though so much about measles epidemiology was unknown; he
understood that quarantine could not work in urban centers such as Copenhagen, but he thought that such
would work in the Faroe Islands. He hypothesized that the same isolation that generally protected the islanders
also led to increased mortality when disease did reach the islands. He wrote, It is obvious, then, that prophylactic measures against the introduction and spread of foreign diseases are of very great importance in such places,
where they can be put into execution. In later years Panum became one of the leading figures in Scandinavian
medicine. He became professor of physiology, medical chemistry, and pathology in Kiel in 1852, and later filled
a similar post in Copenhagen. He was a pioneer in laboratory-based physiology and pathology research and
made notable contributions to medical education.

Panum PL. Observations made during the epidemic of measles on the Faroe Islands in the year 1846. Published
in Copenhagen by Bibliothek for Laeger, in Danish in 1846 and in German in 1847. Reprinted by the Delta
Omega Society, 1940, and distributed by the American Public health Association, New York.
Map from Panums report (http://www.deltaomega.org/documents/PanumFaroeIslands.pdf )

1846 Peter Panum The 1846 epidemic of measles in the Faroe Islands, the seminal study in viral disease epidemiology
page 36

Louis Pasteur
(1822-1895)
Louis Pasteur established the microbiologic/virologic/infectious
disease sciences, first in 1857 by discovering the specificity of microbial
fermentations (wine, beer, cheese), then in 1865 by extending the
concept to infectious diseases of silkworms, and finally between 1877
and 1895 by extending the concept to animal and human infectious
diseases. His early infectious disease work centered on septic war
wounds; he then turned to anthrax and other bacterial diseases,
and lastly to rabies. In each instance, he moved quickly from studies
aimed at discovering the causative agent to the development of
specific intervention. Clearly, Pasteur deserves his title of Father of the
Microbiologic/Virologic/Infectious Disease Sciences.

1857> Louis Pasteur father of microbiology, father of virology, father of the infectious disease sciences

page 37

Jubil de Pasteur a la Sorbonne 27 December 1892 [Painting by Jean Andr Rixens (1846-1924)] Pasteurs seventieth birthday was the occasion of a national

holiday. The celebration was held in the great hall of the Sorbonne. Pasteur entered on the arm of the President of the Republic, Sadi Carnot. Both were wearing the grand cordon of the Lgion dHonneur. Pasteur was greeted by an immense ovation; he was too weak to speak to the delegates who had gathered from all over the world, so his address
was read by his son: Gentlemen, you bring me the greatest happiness that can be experienced by a man whose invincible belief is that science and peace will triumph over ignorance and war. Have faith that in the long run the future will belong not to the conquerors but to the saviors of mankind. The painting shows Joseph Lister coming forward
to greet his old friend. He said: It is my great privilege to convey to you, tributes, thanks and respect from all involved in medicine and surgery; it is true to say that, of all people
in the world today, medical sciences owe you the most. For centuries, infectious diseases have been shrouded, as it were under a dark curtain. In discovering the microbial origin of
disease you have raised that dark curtain!

1857> Louis Pasteur father of microbiology, father of virology, father of the infectious disease sciences
page 38

Die Cellularpathologie (1858)

English translation (1860)
Virchow R. Die cellularpathologie in
ihrer begrundung auf physiologische und
pathologische gewebelehre. Berlin: August
Hirschwald; 1858.
Virchow RLK. Cellular pathology. London:
John Churchill; 1860.

Rudolf Ludwig Karl Virchow (1821-1902)

Virchow is credited with many important advances in medical science: he was the first to accept the work of Robert Remak who showed that the origin of cells was the division
of preexisting cells (Virchows 1858 epigram: Omnis Cellula e Cellula (every cell originates from another existing cell like it.). Virchow founded the fields of cellular pathology
and comparative pathology, always urging the use of the microscope (think microscopically). Virchow astutely observed the interdependence of animal and human diseases
and noted that each relies on a common pool of medical and scientific knowledge this led him to coin the term zoonosis to describe diseases transmitted from animals to
humans. He stated, Between animal and human medicine there is no dividing line nor should there be. The object is different but the experience obtained constitutes the basis
of all medicine.

1858> Rudolf Virchow father of cellular (microscopic) pathology and comparative pathology

page 39

HMS Beagle in Sydney Harbor

On the voyage of HMS Beagle (1831 to 1836) to carry out surveys to produce Admiralty
navigation charts of the coastline of South America and Australia, Charles Darwin, then
22 years of age, as guest naturalist, spent as much time as possible exploring on land; this
experience provided the basis for ideas which he developed into his theory of evolution by
natural selection. HMS Beagle painting by Ron Scobie.
Darwin: In October 1838, that is, fifteen months after I had begun my systematic enquiry, I happened
to read for amusement Malthus on Population, and being well prepared to appreciate the struggle
for existence which everywhere goes on from long-continued observation of the habits of animals and
plants, it at once struck me that under these circumstances favourable variations would tend to be
preserved, and unfavourable ones to be destroyed. The result of this would be the formation of new
species. Here, then, I had at last got a theory by which to work...

Charles Robert Darwin (1809-1882)

Watercolor, painted by
George Richmond in the late 1830s

[Darwin Museum at Down House, Downe, used with permission]

Darwins book was half completed when, in 1858, he received a paper from Alfred Russell
Wallace (1823-1918) describing natural selection. Shocked that he had been forestalled,
Darwin sent it on to Charles Lyell, suggesting that it be sent to any journal that Wallace chose.
Lyell and Joseph Hooker decided on a joint presentation at the Linnean Society: there was
little immediate attention to the presentation. Darwin then struggled for thirteen months to
produce his big book, and Lyell arranged to have it published. On the Origin of Species proved
unexpectedly popular, with the entire stock of 1,250 copies subscribed when it went on sale in
1859. In his book, Darwin set out one long argument providing his bases for what has been
called the single best idea anyone has ever had.

1859 Charles Darwin father of evolutionary science, concepts of common descent, gradualism, natural selection
page 40

Charles Robert Darwin (1809-1882)

Cropped version of a portrait from 1881 by John Collier (1850-1934), painted in 1883.
Darwins son, Erasmus, stated: The picture is a replica of the one in the rooms in the
Linnaean Society and was made by Collier after the original. I took some trouble about it
and as a likeness it is an improvement on the original.
[National Portrait Gallery, London, used with permission]

In mid-July 1837 Darwin started his B

notebook on Transmutation of Species, and
on page 36 wrote, I think. This was his first
evolutionary tree.

1859 Charles Darwin father of evolutionary science, concepts of common descent, gradualism, natural selection

page 41

When Charles Darwins On the Origin of Species went to press in 1859, viruses had
yet to be discovered it would be another 40 years before their discovery, and a
century before virology research would provide a clear understanding of their genetic
make-up, how they replicate and how they cause disease. From our contemporary
perspective, perhaps the most unexpected finding has been the discovery of the
degree to which viruses have been an evolutionary force, as pathogens affecting
host (human and animal) evolution and as agents of horizontal transfer of genetic
information (e.g., via mobile elements, transposons) and as such major contributors to
the variation upon which natural selection works.
The microbial world offers the most direct evidence of natural selection. The mass
of evidence of extraordinary biological diversity of the microbes and the viruses,
at molecular, morphologic and phenotypic levels, has continued to increase,
exponentially. Viruses have proven to be wonderful subjects for studying the forces
and outcomes of genetic variation: competition, selection bottleneck events, etc.
Their very rapid rate of mutation has been the substrate for amazing discoveries in
genetics, genomics, functional genomics, etc. In some ways, this understanding of
the mechanisms of variation has not been equaled by our understanding of the forces
driving natural selection per se we tend to oversimplify this, and evolutionary
biologists tend to ignore haploid organisms (bacteria) and viruses which replicate
clonally. Many discoveries described later in this volume illustrate great progress,
and yet more mystery and more of a sense of complexity, in arriving at the point of
satisfying our understanding of virus ecology and viral disease pathogenesis and
pathophysiology.

A typical caricature
of Darwin,
The Hornet,
a satirical magazine,
1871

One Long Argument: Charles Darwin and the Genesis of Modern Evolutionary
Thought. Ernst Mayr, Harvard University Press, 1993:
Ernst Mayr was a force in defining and elaborating Darwinism. One of his criticisms
of scholarly naysayers was that they did not deal with each of Darwins ideas in logical
fashion, but rather confused one for another. Mayrs way of focusing arguments was
to dissect Darwins conceptual framework of evolution into five parts, only the whole
worthy of being called Darwins Theory:
1. Evolution as such. Living organisms [and viruses] are not constant or recently
created or perpetually cycling, but rather are forever changing.
2. Common descent. All groups of organisms, including animals, plants, and
microorganisms, are descended from a common ancestor, ultimately going back
to a single origin of life on earth.
3. Multiplication of species. The origin of the enormous organic diversity seen on
earth stems from the multiplication of species, either by splitting of species into
daughter species or by budding, that is, by the establishment of geographically
isolated founder populations that evolve into new species.
4. Gradualism. Evolutionary change takes place through the gradual change of
populations and not by the sudden (saltational) production of new species.
5. Natural selection. Evolutionary change comes about through the abundant
production of genetic variation in every generation. Few variants survive; those
that do are better adapted by virtue of their combination of inherited characters,
and these variants give rise to succeeding generations.

Hugo Rheinholds
Affe mit Schdel
(Ape with Skull),
1893

1859 Charles Darwin father of evolutionary science, concepts of common descent, gradualism, natural selection
page 42

President Abraham Lincoln Signs the Charter of the U.S. National Academy of Sciences

Artist: Albert Herter. This painting, hanging in the U.S. National Academy of Sciences Board Room, depicts President Lincoln signing the charter, on March 3rd,
1863. In his apocryphal scene the Academy founders look on. Founding Members (left to right): Benjamin Pierce, Alexander Dallas Bache (first president of the
Academy), Joseph Henry, Louis Agassiz, President Lincoln, Senator Henry Wilson, Admiral Charles Henry Davis, and Benjamin Apthorp Gould
[from U.S. National Academy of Sciences, used with permission]

The National Academy of Sciences was created to investigate, examine, experiment, and report upon any subject of science or art whenever called upon to do so by any
department of the government. The demands of the Civil War (1861-1865) were conducive to the formation of a scientific consulting body, especially to advise on new
weapons. A huriedly written bill for the incorporation of the National Academy of Sciences and naming 50 charter members was signed by President Lincoln the same day
that it was passed by the Congress. Although hailed as a great step forward in government recognition of the role of science in American civilization, the National Academy
of Sciences did not solve the problems facing a nation in its Civil War, nor did it centralize American scientific efforts. However, over the years it developed into the most
influential body of its kind and extended its reach into all aspects of science and society.

1863 Abraham Lincoln, others founding of the U.S. National Academy of Sciences

page 43

Pierre-Franois-Olive Rayer
(1793-1867)

Casimir Davaine (1812-1882)

In 1850, Casimir Davaine, along with French dermatologist Pierre Franois Olive Rayer (1793-1867),
discovered a certain microorganism in the blood of diseased and dying sheep. In the diseased blood,
Rayer and Davaine observed the bacillus which is now known as Bacillus anthracis, the causative
bacterium of anthrax. Soon afterwards, Rayer published an essay on anthrax, which contained the first
description of Bacillus anthracis.
In 1863, Davaine demonstrated that the anthrax bacillus could be directly transmitted from one animal
to another. He was able to identify the causative organism but was unaware of its true etiology. Later,
Robert Koch did considerable research on Bacillus anthracis, and discovered its ability to produce
resting spores that could stay alive in the soil for a long period of time and serve as a continuing
source of infection.

1863 Casimir Davaine first association of a specific infectious organism with a specific disease
page 44

Mendels first experiment

Gregor Mendel was an Augustinian priest, who gained posthumous fame as the founder
of the science of genetics for his study of the inheritance of certain traits in pea plants.
Mendel showed that the inheritance of these traits follows particular laws, which were later
named after him. The significance of Mendels work was not recognized until the turn of the 20th
century, when the his laws were independently rediscovered.
Mendel conducted his study in his monasterys two hectare garden; between 1856 and 1863
Mendel cultivated and tested some 29,000 pea plants. His experiments led him to make two
generalizations, the Law of Segregation and the Law of Independent Assortment, which later
became known as Mendels Laws of Inheritance.
Mendel did read his paper, Experiments on Plant Hybridization, at two meetings in Brnn,
Moravia in 1865 and published his work in 1866 in the Proceedings of the Natural History
Society of Brnn, but it had little impact and was cited only three times during the next thirtyfive years. Mendels work was re-discovered in 1900 (see Hugo de Vries ).

1865 Gregor Mendel father of genetics

Gregor Johann Mendel (1822-1884)

page 45

Claude Bernard (1813-1878) was one of the most distinguished

scientists of the nineteenth century, and arguably the most
influential on subsequent generations. His work and especially
his teaching and writing led to the modern form of experimental medicine. In his major discourse on the scientific method,
An Introduction to the Study of Experimental Medicine (1865),
he described what we now call the scientific method. The
book was translated into many languages and has been used
in courses in biology and the philosophy of science ever since.
He espoused the importance of observation and controlled
experimentation, but also saw the importance of imagination,
intuition and instinct. He described how an observation had
to be followed by an hypothesis, and then a systematic series
of experiments to either support or disprove the hypothesis.
This was at a time when the metaphysics of Galen and other
ancient philosophers still dominated medical practice. Bernard
became the most famous experimental scientist of his day and
used his prominence to establish the first concept of evidencebased medicine. He stated: The complete scientist is one who
masters both theory and experimental practice: (1) he observes
a fact; (2) he conceives an idea with reference to this fact; (3)
on the basis of this idea he pursues a line of reasoning, plans
an experiment and imagines and organizes its material conditions; (4) this experiment produces more phenomena that shall
be subjected to observation and so on. In a sense, the scientists
mind is always between two observations: one is the starting
point of the reasoning, the other its conclusion.
Bernards ideas may be organized as follows: (1) Distinguishing
The Lesson of Claude Bernard by Lon Augustin Lhermitte (1844-1925), painted in 1889,
the known from the unknown (In the area of science that is still
Acadmie
Nationale de Mdicine. Bernard, the central figure wearing an apron, is surrounded
obscure and unknown the great [scientists] are recognized: They
by
some of his most famous students in the laboratory at the College de France
are marked by ideas which light up phenomena hiterto obscure
and carry science forward.); (2) Distinguishing ideas stemming from authority from those from observation and experimentation (When we meet a fact which contradicts
a prevailing theory, we must accept the fact and abandon the theory, even when the theory is supported by great names and generally accepted.); (3) Distinguishing inductive versus deductive reasoning (Experimental science is a constant interchange between theory and fact, induction and deductionthey are never truly separate. Induction:
reasoning from the particular to the general; deduction: reasoning from the general to the particular); (4) Distinguishing cause from effect (The scientist tries to determine the
relation of cause and effect. This is true for all sciences: the goal is to connect a natural phenomenon with its immediate causebefore that we have only groping and empiricism.); (5) Distinguishing verification from disproof (Theories are only hypotheses, verified by more or less numerous factsthose verified by the most facts are the best, but
even then they are never final, never to be absolutely believedwe must always try to disprove our own theories.); (6) Understanding determinism and averages (statistics)(In
the study of disease, the real and effective cause must be constant and determined, that is, unique; anything else would be a denial of science in medicine Sometimes averages
do not give the kind of information needed to save lives.); and (7) Understanding the many qualities that characterize physicians and scientists who favor their own ideas over
all others, no matter what, and stand in the way of real progress (A man of science rises ever, in seeking truth; and if he never finds it in its wholeness, he discovers nevertheless significant fragments; and these fragments of universal truth are precisely what constitutes science).
Abstracted from: Normandin S. Claude Bernard and An Introduction to the Study of Experimental Medicine: Physical Vitalism, Dialectic, and Epistemology. J Hist Med Allied
Sci. 2007;62:495-528.

1865 Claude Bernard publication of the seminal book, Introduction to the Study of Experimental Medicine
page 46

Ludwig Karl Martin Leonhard

Albrecht Kossel (1853-1927)

Johan-Friedrich Miescher (1844-1895)

Johan-Friedrich Miescher isolated various

phosphate-rich chemicals, which he called nuclein
(now nucleic acids), from the nuclei of white blood
cells in 1869 at Felix Hoppe-Seylers laboratory
at the University of Tbingen, Germany, thereby
paving the way for the identification of DNA as
the carrier of inheritance. The significance of the
discovery, first published in 1871, was not at first
apparent. Miescher and his students researched
much of the basic chemistry of the nucleic acids but
their function remained unknown. The importance
of Mieschers discovery was not apparent until
Albrecht Kossel carried out research on the
chemical structure of nuclein (i.e., DNA).

1868 Johan-Friedrich Miescher discovery and characterization of nucleic acids

page 47

Physician
Pathologist
Comparative Pathologist
Clinician
Teacher
Diagnostician
Bibliophile
Historian
Classicist
Essayist
Author
The Renaissance Man

William Osler (1849-1919)

Osler is credited with coining the term One Medicine to encompass the relationship
between human and veterinary medicine. Oslers philosophy and his most famous
work, The Principles and Practice of Medicine, did much to change the teaching and
practice of medicine from its historic didactic base to a clinical base, where students
learned from seeing and talking to patients. He established the first medical residency
program, and is credited with developing the concept of rounds. His approach changed
the teaching and practice of veterinary medicine in similar fashion.

Osler conducting Grand Rounds,

Johns Hopkins School of Medicine, 1900

1874 William Osler father of modern scholarly medicine

page 48

Koch in Africa, ~1904, seeking the cause of rinderpest

Robert Koch is credited with the first incontrovertible proof that a microorganism
can cause disease (Bacillus anthracisanthrax). After Casimir Davaine showed the
direct transmission of the anthrax bacillus between cows, Koch studied anthrax more
closely. He invented methods to purify the bacillus from blood and grow pure cultures.
He found that, while the anthrax bacillus could not survive outside a host for long,
it produces endospores that survive for many years. He developed bacterial staining
techniques, bacterial liquid and solid growth media (and agar plates thanks to the
advice of Angelina and Walther Hesse), and he developed the Petri dish, named after
its inventor, his assistant Julius Richard Petri. With these techniques, he was able to
discover the bacterium causing tuberculosis (Mycobacterium tuberculosis) in 1882.

Heinrich Hermann Robert Koch (1843-1910)

With Friedrich Gustav Jakob Henle and Friedrich Loeffler, in 1884, he developed the
Henle-Loeffler-Koch Postulates (Kochs Postulates) outlining the criteria required to
establish a causal relationship between a microbe and a disease:
1. The microorganism must be found in abundance in all organisms suffering from
the disease, but should not be found in healthy animals. 2. The microorganism must
be isolated from a diseased organism and grown in pure culture. 3. The cultured
microorganism should cause disease when introduced into a healthy organism. 4. The
microorganism must be reisolated from the inoculated, diseased experimental host and
identified as being identical to the original specific causative agent.

1876 Robert Koch along with Louis Pasteur, the founders of microbiology

page 49

Patrick Manson
(1844-1922)

Charles Louis Laveran

(1845-1922)

Ronald Ross
(1857-1932)

Giovanni Battista Grassi

(1854-1925)

In 1877-1879, Patrick Manson (1844-1922) demonstrated that the mosquito was the host of the filarial worm Wuchereria bancrofti, the etiologic agent of filariasis; he
suggested that the infectious agent that causes malaria is also spread by a mosquito.
In 1880, Charles Louis Alphonse Laveran (1845-1922), a French army doctor working in the military hospital in Constantine, Algeria described parasites inside red blood cells
of people suffering from malaria; he proposed that malaria was caused by this protozoan, the first time a protozoan was identified as causing disease.
In 1898, Ronald Ross (1857-1932), working in India, proved that avian malaria is transmitted by mosquitoes.
In 1898, Giovanni Battista Grassi (1854-1925) described the entire developmental cycle of the plasmodium in the body of Anopheles mosquitoes and proved that human
malaria is transmitted by Anopheles mosquitoes.
All of these discoveries may have been influential in guiding the work of Walter Reed and his colleagues in their discovery of the first human virus, yellow fever virus, and its
transmission cycle involving Aedes aegypti mosquitoes. However, after the Yellow Fever Commission had made its discoveries in 1900, Walter Reed cited only four influences
that had guided the research: (1) the work of Carlos Finlay on the yellow fever vector mosquito, Aedes aegypti, (2) the work of Henry Rose Carter providing evidence of a
mosquito extrinsic incubation period, (3) the work of Theobald Smith and his colleagues proving that the protozoan agent of Texas fever (Babesia bigemina) was transmitted
by an arthropod (the tick, Boophilus annulatus), and (4) the work of Friedrich Loeffler and Paul Frosch showing that the agent of foot-and-mouth disease was an ultrafilterable
virus. It has been said that the failure of the early malaria research to have influenced the work of the Yellow Fever Commission in Havana was due to the slowness of
communication of scientific findings at the time, the proof that human malaria is transmitted by Anopheles mosquitoes having only come in 1898.

1877> Patrick Manson, Charles Laveran, Ronald Ross, Giovanni Grassi discovery of the etiologic agent of malaria
page 50

Stanford Emerson Chaille, known as

the Father of Hygiene and Health
Education in America. Chaille gained
fame as the head of the U.S. Havana
Yellow Fever Commission of 1879,
organized to study the disease following
the 1878 epidemic in New Orleans.
A national figure, Chaille was
spokesman for the establishment of
community sewerage and drainage
systems, street paving, pure water
supplies and mosquito control. He was
also instrumental in the establishment
of the National Board of Health, the
forerunner of the U.S. Department of
Health and Human Services.

Stanford Emerson Chaille (1830-1911)

Yellow fever dragging down Florida, 1888
Artist Matt Morgans drawing in
Frank Leslies Illustrated Newspaper
There were more than 400 deaths in Jacksonville in 1888
leading to widespread panic

Thanksgiving Mass At Father Matthews Yellow Fever Camp,

near Memphis, during the epidemic of 1878

1879 Stanford Chaille father of hygiene and health education in the United States

page 51

Some of the Most Devastating Yellow Fever Epidemics

in the United States
YEAR AREA

1668
New York
1693
Boston
1699
Charleston & Philadelphia
1702
New York
1706
Charleston
1732
Charleston & New York
1734
Virginia
1741
Virginia
1762
Philadelphia
1793
Philadelphia (~5,000 deaths)
1794
Philadelphia, Baltimore
1798
Philadelphia, New York
1803
New York, Philadelphia
1839
Galveston (5% of population)
1841
Nationwide
1847
New Orleans
1850
Nationwide
1852
New Orleans (~8,000 deaths)
1855
Nationwide
1867
Galveston (5% of population)
1878
New Orleans, Mississippi Valley (~13,000 deaths)
1886
Jacksonville
1888 Florida
1905
New Orleans

During the yellow fever epidemic of 1878, fear and anxiety pervaded the country, in both the North and the South. Yellow fever evoked sympathy from many northerners,
and as they donated funds and prayed for southerners, the roots of post-Civil War national reconciliation were established. In the midst of their benevolence, northerners
consciously effaced memories of southerners as belligerents seeking to destroy the Union southerners now became stricken, honorable people in need. At the same time
southerners began to see northerners as fellow citizens. In effect, yellow fever helped open the way for southerners to accept national unity.
The 1878 epidemic began in New Orleans in May, likely with the arrival of a sailor infected in Havana. The disease spread in New Orleans during June, and the number of
victims began to multiply in frightening fashion from July onward. Cities were hardest hit: Memphis (5,000 of 40,000 residents died), New Orleans (>4,600 deaths) the disease
then struck large parts of Mississippi, Louisiana, Tennessee, Kentucky, Georgia, Ohio, and Missouri.
Local relief societies recognized that they could not handle the crisis on their own and pleaded for assistance. Southern requests for aid supplied northern newspapers and
magazines an opportunity not only to encourage the North to show compassion for the South, but also to describe southerners as full-fledged citizens of the nation. Leslies
Illustrated Newspaper maintained that sympathy must be made tangible with direct relief: These horrors should at least inspire a genuine sympathy with the suffering, and
induce on the part of all of us generous contributions . Donation boxes in public places served as physical proof of northern generosity to southerners as well as other
northerners. Leslies Illustrated Newspaper and Harpers Weekly regularly carried images of these donation boxes, allowing people to see themselves and others giving.

1668-1905 Yellow fever epidemics in the United States

page 52

Aedes aegypti

Carlos Juan Finlay (1833-1915)

Finlay hypothesized that the common house mosquito (then

Stegomyia fasciata, now Aedes aegypti) transmitted yellow
fever by directly injecting the blood from an infected person
into a susceptible person. He did not appreciate the need for
an extrinsic incubation period in the mosquito. In retrospect,
at most only 1 of his 104 experiments from 1881-1898
demonstrated mosquito transmission of yellow fever. Many
thought that Finlay had disproved his own hypothesis. However,
upon their arrival in Havana in 1900, Walter Reed and his
colleagues were greatly influenced by Carlos Finlays hypothesis
and he helped them initiate their studies, especially by providing
mosquitoes for the transmission experiments.

1881 Carlos Finlay initial experiments on the mode of transmission of yellow fever virus

page 53

Ilya Ilyich Mechnikov

(Elie Metchnikoff)
(1845-1916)

Paul Ehrlich
(1854-1915)

Jules Bordet
(1870-1961)

Emil Adolf von Behring

(1854-1917)

The founding of the science of immunology was celebrated by the Nobel Prize award to Elie Metchnikoff and Paul Ehrlich in 1908. Metchnikoff discovered phagocytosis by
macrophages and microphages as a critical host-defense mechanism and thus is considered the father of cellular innate immunity. Ehrlich described the side-chain theory of
antibody formation and the mechanisms of how antibodies neutralize toxins and induce bacterial lysis and thus is considered one of the fathers of humoral adaptive immunity.
Both Ehrlich and Metchnikoff provided a paradigm shift by developing concepts of how the host can deal with invading pathogens to overcome infectious diseases. Their ideas
evolved, however, on the shoulders of Pasteur and Koch, who had prompted another paradigm shift shortly before. Pasteur provided the ultimate disproof of spontaneous
generation by demonstrating absence of microbial growth after heat inactivation. Pasteurs conclusion that fermentation and putrefaction were the work of microorganisms
and that these processes occur not only in organic material but also in the living body laid the foundation for the germ theory of infectious disease. Koch clarified the
microbial etiology of anthrax, sepsis and tuberculosis. Bordets discovery that the bacteriolytic effect of acquired specific antibody is significantly dependent in vivo on the
presence of innate serum components which he termed alexine (which are now known as the component molecules of the complement system). This mechanism became
the basis for complement-fixation testing methods that enabled the development of serological tests for syphilis and many other diseases. von Behring developed the first
immunotherapeutic system, the highly effective serum therapy used to treat diphtheria. He also worked on the development of tetanus antitoxin with Emile Roux. The work of
these pioneers was quickly applied to the emerging science of virology in the first years of the 20th century.

1883> Elie Metchnikoff, Paul Ehrlich, others founding of immunology, hematology and chemotherapy
page 54

In 1881 and 1882, Louis Pasteur, Charles Chamberland,

mile Roux and Louis Thuillier began their research toward
developing a rabies vaccine. They modified Pierre-Victor
Galtiers technique by inoculating nervous tissue from a rabid
dog through a long series of dogs via subdural trephination.
After many passages, they obtained a virus of maximum
virulence and with a fixed incubation period of about 10
days. They estimated the degree of attenuation of brain
tissue recovered from each passage. They then passaged
the infectious agent serially in rabbits. This final attenuation
procedure consisted of suspending the spinal cord of a rabid
rabbit in a flask, in a warm dry atmosphere, to achieve slow
desiccation. They succeeded in producing attenuated viruses
of different strengths, that is a standardized range of viruses,
the weakest of which could be used to prepare the first dose
of a vaccine. Inoculating dogs with a sequence of spinal cords
of increasing virulence rendered the recipients resistant to
inoculation with fully virulent virus.

Louis Pasteur (1822-1895)

Pierre-Paul-mile Roux (1853-1933)

Painting byAlbert Edelfeldt, 1885

[from Institut Pasteur, used with permission]

1884> Louis Pasteur, mile Roux, Charles Chamberland, Louis Thuillier development of rabies vaccine

page 55

Pasteurs First Successful Postexposure Rabies Treatment: Joseph Meister, 6 July 1885
From Bazin H. Vaccination: a history. Montrouge-France: John Libbey Eurotext Ltd; 2011. Abridged; various translations.
Starting in 1879, Pierre-Victor Galtier (1846-1908) carried out extensive research on
the rabbit as an experimental rabies modelhe also transmitted rabies serially from
rabbit to rabbit. Galtiers work aroused the interest of Louis Pasteur (1822-1895) who
with Charles Chamberland (1851-1908), Pierre-Paul-Emile Roux (1853-1933), and
Louis Thuillier (1856-1883) wrote the first of their papers on rabies in 1881: It first
lodges and multiplies in the spinal cord and brain. Pasteur and his colleagues modified Galtiers technique by inoculating nervous system tissue from rabid dogs subdurally via trephination into further dogs. By successive passages in dogs, they obtained
a virus of maximum virulence and with a fixed incubation period of about 10 days.
They then passaged dog tissue serially in rabbits (again, subdural inoculation, via
trephination). With passage, they noted further progressive shortening of the incubation period down to 6 to 7 days; there was a coincident increase of the neurovirulence
of the agent (called fixed virus note: this was still in the era before the development of the concept of virus as we know it today). Spinal cords from rabbits infected
with fixed virus were progressively inactivated by dessication over dry potash for
periods of from 1 to 15 days. Pasteur: taking the greatest care possible to maintain
purity one removes from these cords sections a few centimeters in length, and then
suspends them in dry air, virulence slowly disappears until it finally disappears We
succeeded in producing attenuated viruses of different strengths, that is a standardized range of viruses, the weakest of which could be used to prepare the first dose of a
vaccine. Inoculating dogs with a sequence of spinal cords of increasing virulence rendered the recipients resistant to inoculation with fully virulent virus... These results
represent the scientific center of the method. Emulsified cord material was used as
vaccine in challenge trials in dogs. Pasteur: By the application of this method, I had
made fifty dogs of all ages and breeds refractory to rabies without a single failure.
These experiments had not yet been completed by 6 July 1885 when Joseph Meister, a
9-year old Alsatian boy, arrived to Pasteurs laboratory. He had been bitten 14 times
on his legs and thighs two days earlier by a clearly rabid dog. On the evening after
the boy was bitten, his parents requested the assistance of the local physician, who
cleaned the childs wounds with carbolic acid. Local people told the parents about
Pasteur and his work on rabies; they immediately decided to go to Paris to see Pasteur.
After examining the boy, Pasteur attended the weekly meeting of the Academy of Sciences where he saw Alfred Vulpian (1826-1887) and Jacques-Joseph Grancher (18431907), senior physicians at the faculty of medicine. Pasteur: they were good enough
to come to see immediately little Joseph Meister and to establish the condition and
number of his wounds. Their opinion was that by the intensity and the number of
bites, Joseph Meister was almost inevitably to come down with rabies. I then told
Vulpian and Grancher the new data that I had obtained. As the death of this child
appeared inevitable, [and with the support ot Vulpian and Grancher] I decided, not
without deep and severe unease, as one can well imagine, to try on Joseph Meister the
procedure which had consistently worked in dogs.

Pasteur: on July 6, at 8 in the evening, sixty hours after the bites of July 4, [the child
was injected with] one-half Pravaz syringe of spinal cord of a rabbit dead of rabies on
June 21 and conserved since then in a flask of dry air, that is to say for 15 days. On the
following days new inoculations were made always with the conditions which I give
here in the table The vaccine was administered by Jacques-Joseph Grancher MD,
Pasteur not being a physician and not legally able to treat patients.
Pasteur: In consideration of the methods of progressive attenuation of the fatal
virus, and the process by which one can determine it, and keeping in mind, on one
side, the influence of air in the attenuation, the first thought that comes to mind is
to understand the process: is it that the period of incubation of the rabid spinal cord
in contact with dry air progressively diminishes the intensity of virulence of the cord
up to the point of rendering it inactive? Pasteur argued with others for some time
about whether the drying effect on virulence
was qualitative [changing an intrinsic quality
of the virusas in the concept of inactivated
vaccines retaining native antigenicity, immunogenicity, as understood years later] or
quantitative [changing the amount of active
virus remaining after the dessication effect
later most importantly understood as residual
infectivity, a danger with inactivated vaccines
made from virulent viruses, as was the case
with inactivated polio vaccine (the Cutter
Incident)].
Pasteur next successfully treated a 15-yearold shepherd, Jean-Baptiste Jupille, who had
been bitten by a rabid dog 6 days earlier. A
year later, in 1886, Pasteur and his colleagues
reported the results of the treatment of 350
cases only one person developed rabies,
and this a child was treated six days after
exposure. Over the next decades hundreds of
thousands of people with potential rabies exposures were immunized with ever improving animal nervous system (brain and spinal
cord) vaccines, at the Institut Pasteur in Paris,
which was founded in 1888, and in other
locations throughout the world. Pasteurs
rabies post-exposure vaccination represented
the opening of the modern era
of viral vacinology.

1884> Louis Pasteur, mile Roux, Charles Chamberland, Louis Thuillier development of rabies vaccine
page 56

Apparatus for using Chamberland or other

filters: (a) glass mantle; (b) Chamberland filter;
(c) rubber stopper through which passes the
filter; (d) rubber tubing; (e) glass tubing;
(f ) perforated rubber stopper; (g) vacuum jar
with cotton-stoppered sterilized flask; (h) glass
stopcock; (i) vacuum gauge; (j) reservoir trap;
and (k) vacuum source. [it was common at the
time to use a running water vacuum gadget as
shown (Venturi effect).]
From a USDA laboratory manual
from the 1920s.

Charles Chamberland (1851-1908)

Theodor Albrecht Edwin Klebs (1834-1913) and Ernst Tiegel, in 1871, found that the causative agent of anthrax would not pass through a filter made of unfired clay it
was a non-filterable bacterium. As early as 1876, Louis Pasteur, in collaboration with the French physicist Jules Joubert, used a plaster-of-Paris filter connected to a vacuum
pump to confirm Krebs diacovery. The use of ultrafilters to retain and concentrate bacteria became standard practice after 1884, when Charles Chamberland and Emile
Roux, experimenting with a broken clay pipe purchased from Chamberlands tobacconist, developed unglazed porcelain ultrafilters (candles, bougies de Chamberland) that
retained bacteria. Chamberland filters were produced in quite a few porosities: L1 (most porous), L2, L3, L5, L7, L9, L11, L13 (least porous).
Chamberland C. Sur un filtre donnant de leau physiologiquement pure. C. R. Acad. Sci. Paris 1884;99:247-248.

1884> Charles Chamberland development of the porcelain ultrafilter, key to the discovery of the viruses

page 57

The Unique Role of Ultrafiltration1 in the Development of the Virus Concept

It is remarkable that one laboratory technology, ultrafiltration, lay at the center of
the development of the virus concept, yet disappeared from the scene a few decades
afterward. It might be thought that the technology was crude, simplistic, a one-off
way to identify viruses, but such is not the case: ultrafiltration was used by many
investigators in the first decades of the 20th century with rather elaborate equipment and thoughtful analyses of filters, filtration, controls and theory about the as
yet unknown nature of the viruses.

as the agent of bovine pleuropneumonia, could pass through filters with porosity
that would hold back most bacteria. Confusion occurred when only small amounts
of an agent passed through filters, whereas with agents like that causing foot-andmouth disease, very little was lost in filtration and conclusions were easier to make.
Although some studies of putative viruses involved use of filters with only one or
two porosities, the virus conceptthat of an infectious agent, very small but particulate in naturewas best advanced by use of a graduated set of filters, reaching the
point where the agent was held back while proteins passed through. Loeffler and
In the seminal book edited by Thomas Rivers, Filterable Viruses (1928, Baltimore;
Frosch used graduated Chamberland-Pasteur filters and then a minimally porous
Williams & Wilkins), Stuart Mudd wrote the chapter on Filters and Filtration; it is
full of details of the methodology of the day and has 189 references. Clearly, ultrafil- Kitasato filter to reach the limit point and conclude that foot-and-mouth disease is
caused by a very small but particulate moiety, a virus.
tration was importantbut the technology was also appraised most critically: Rivers wrote, Methods of filtration are crude and inaccurate and the most one can say There is still controversy whether early investigators presumed too much from early
regarding the viruses is that under given experimental conditions they either pass
ultrafiltration experiments. This bears on the question of attribution of the discovery
or do not pass through certain filters Filters, in spite of their faults, are useful in
of many of the viruses cited in this book. Was it enough that an infectious agent would
working with diseases of unknown etiology, and by means of them one is able at
not to grow on artificial media, that is required living cells for its replication, that it
times to determine quickly whether a given disease is produced by an agent smaller was not visible in the light microscope even when stained, that it was heat labile (for
than ordinary bacteria. Sometimes, however, small bacteria may still contaminate
exclusion of a toxin), and that it was ultrafilterable? Indeed this was the standard of
the filtrates or two viruses may be present in the filtered material Investigators
the day: interpretation of such data was usually published as discovery, and for many
should be extremely careful in working with filtrates not to be misled by their find- years attribution was so cited. In keeping with the historic record, this standard is used
ings and ascribe to an active agent an etiological role in a disease with which it has
throughout this book.
nothing more than accidental connection. Mudd wrote, The apparent simplicity of However, in some instances in subsequent years this standard was revisited by reviewfiltration has been a pitfall, leading usually to incomplete recording of essential data ers. This was as if to reset the standard of proof at the technologic level of the time of
and frequently to unwarranted conclusions. In view of the confusion of ideas exist- reviewignoring the historic context. This seems to have been most prominent when
ing in regard to filters, the authors will attempt to describe the common filters, the
the back-testing of ultrafiltrates had been done in human volunteers. Two examples
proper methods of using them, and the conclusions one is justified in making from stand out: yellow fever virus and influenza virus.
data obtained by their correct use.
In 1901, James Carroll, the microbiologist who was a member of Walter Reeds Yellow
Mudd had by 1928 written 10 papers on ultrafiltration methodology and pitfalls. In Fever Commission in Havana, using Berkefeld filters, showed that yellow fever virus is
his chapter in Rivers book he described all of the filters being used, reviewed their
ultrafilterable. He tested ultrafiltrates in three volunteers. His finding was confirmed
merits and failings, and cited the published data and opinions of others as to best
by several other investigators within just a few years. Yet, it is still common to attribute
quality. Three or four were considered most valuable, including:
the discovery of yellow fever virus to Adrian Stokes, Noel Hudson and Johannes
1. Chamberland-Pasteur unglazed porcelain filters (candles) made from kaolin (hydrated aluBauer, and to Constant Mathis, Andrew Sellards and Jean Laigret, who transmitted
minum silicate), then available in quite a few graduated porosities [L1 (most porous), L2, L3,
ultrafiltered virus to rhesus macaques in 1928.
L5, L7, L9, L11, L13 (least porous)].
2. Berkefeldt filters made from fired kieselguhr (diatomaceous earth), then available in three
porosities [V (viel, coarse, most porous), N (normal), W (wenig, small, least porous)].

The multiple producers of these filters were in the business of making filters for
water, drinking water and industrial water, and so production quality was rather
consistent. Use of the filters involved prefiltering to avoid clogging of the ultrafilters, control of filtration pressure, control of temperature and pH/ionicity of the
suspending fluid to avoid adsorption of the infectious agent to the filter, control of
sterility, proper use of bacterial controls, etc. Even at the time of the initial ultrafiltration experiments on foot-and-mouth disease virus by Friedrich Loeffler and Paul
Frosch in 1898, there was an understanding that small pleomorphic bacteria, such

In 1918-1919, during and following the great influenza pandemic, several investigators (Charles Nicolle, Charles Lebailly, Ren Dujarric de la Rivire, T. Yamanouchi,
and others) showed that ultrafiltered clinical samples (nasal washes, etc.) from
patients ill with influenza could cause respiratory disease in human volunteers. Yet,
it is still common to attribute discovery of influenza virus to Wilson Smith, Christopher Andrewes and Patrick Laidlaw, who transmitted ultrafiltered virus from
humans to ferrets and then from ferret to ferret in 1933. Seemingly, arguments like
these will be part of the world of virology for years to come.

1
The term ultrafiltration is used here to distinguish filtration of materials through bacteria-proof kieselguhr or porcelain filters, as opposed to filtration through coarser media.

1884> Charles Chamberland development of the porcelain ultrafilter, key to the discovery of the viruses
page 58

From 1860 onward, Louis Pasteur made known his germ theory, but
skeptics, especially Henri Charlton Bastian, argued against it because
in some cases germs (bacteria) seemed to appear in sealed vessels after
heating to 100C, Pasteurs standard inactivating temperature. Charles
Chamberland, working in Pasteurs laboratory, showed that some germs
(bacterial spores) could withstand a temperature of 110-120C. In the
course of this work, betweem 1879 and 1884 Chamberland invented the
autoclave (lautoclave Chamberland), expanding upon a precursor known
as the steam digester created by Denis Papin in 1679. A Parisian firm
marketed Chamberlands Autoclave in 1884 and by the turn of the century
steam-jacketed autoclaves from which steam could be evacuated at the end
of the heating cycle were in use widely.

mile Roux using one of Charles Chamberlands new autoclaves

[from Institut Pasteur, used with permission]

Pasteur L. Memoire sur les corpuscles organises qui existent dans latmosphere. Examen de la doctrine des generations spontanees.
Annales des Sciences Naturelles, 4th series. 1861;16:5-98.
Pasteur L, Joubert JF, Chamberland C. La thorie des germes et ses application la mdecine et la chirurgie. C R Acad Sci Hebd
Seances Acad Sci D 1878;86:1037-1043.
Chamberland C. Sur un filtre donnant de leau physiologiquement pure. C R Acad Sci Paris 1884;99:247-248.

1884 Charles Chamberland invention of the autoclave

page 59

Adolf Eduard Mayer (1843-1942)

Tobacco mosaic disease, Mayers work, 1866

Adolf Mayer was a German agricultural chemist and director of the Agricultural Experiment Station at Wageningen in the Netherlands. He is credited as the first person to
transmit tobacco mosaic virus by using juice extracts from diseased plants as the inoculum to infect other plants. Mayer published a paper in 1886 describing tobacco mosaic
disease in detail. He performed chemical analyses of healthy and diseased leaves to see if a difference in nutrition could explain the disease. He investigated temperature,
light, fertilization, and looked for fungi or animal parasites. He tried to follow Kochs Postulates and was able to culture organisms from his extracts, but none of these would
reproduce the disease. He was left with the conclusion that the infectious agent was likely some sort of microorganism. Although Mayer came to the wrong conclusion about
his finding, his work laid the groundwork for the experiments of Dmitry Ivanovsky and Martinus Beijerinck, who showed that the etiologic agent of tobacco mosaic disease
would pass through an ultrafilter, that is, it is an ultrafilterable virus.
Mayer, A. Concerning the mosaic disease of tobacco. Die Landwirtschaftliche Versuchsstationen 1886;32:451-467. Translation published in English as Phytopathological
Classics Number 7 (1942). American Phytopathological Society Press. St. Paul, MN.

1886 Adolf Mayer the concept of transmissibility and the earliest concept of an ultrafilterable virus
page 60

John Brown Buist (1846-1915) was a Scottish pathologist and medical practitioner who devoted much of his professional life to
vaccination. For a time he was also medical superintendent of the smallpox hospital at Barrow-in-Furness in Scotland. In the book
he published in 1887 (Vaccinia and Variola: a Study of their Life-History), Buist described and illustrated minute bodies which
he observed in films of lymph from vaccinia and smallpox vesicles after prolonged staining with the aniline dye gentian violet.
He regarded these bodies as the infectious agents. Although he believed the tiny bodies he saw were spores, he was nonetheless
the first person to see (and photograph) a virus. Although nearly all viruses are invisible under the light microscope, vaccinia and
variola virions are just big enough to be visible when heavily stained or observed by dark-field light microscopy. Vaccinia and
variola virions are brick-shaped, about 200x200x250nm in size.

Variola (smallpox) virus

negative contrast electron microscopy

1887-1922 John Buist, Edmund Cowdry discovery of elementary & inclusion bodies of vaccinia & variola viruses

page 61

Edmund Vincent Cowdry (1888-1975)

[from Rockefeller Archives, used with permission]

Edmund Cowdry was the founder of viral disease cytology and the person who initiated
our appreciation of inclusion bodies in cells infected by many viruses. But, his career at the
Rockefeller Institute and then Washington University School of Medicine was much more
diverse for example, here is a listing of the books he wrote over his long career: General
Cytology (1924), Special Cytology, Form and Functions of the Cell in Health and Diseases
(1928>) , Human Biology and Racial Welfare (1930), A Textbook of Histology (1934>),
Problems of Ageing (1939>), Microscopic Technique in Biology and Medicine (1943), Laboratory
Technique in Biology and Medicine (with Victor Emmel, 1964), The Care of the Geriatric
Patient (1968>), Etiology and Prevention of Cancer in Man (1968), and Cancer Cells (1955).

The variety of poxvirus inclusion bodies

Cowdry E. The supravital staining of vaccine bodies.
Journal of Experimental Medicine 1922;36:667-684.

1887-1922 John Buist, Edmund Cowdry discovery of elementary & inclusion bodies of vaccinia & variola viruses
page 62

Joseph J. Kinyoun (1860-1919)

The National Institutes of Health (NIH) was founded in 1887, within the Marine Hospital Service (MHS), the predecessor agency to the U.S. Public Health Service (PHS). The
MHS had been established in 1798 to provide for the medical care of merchant seamen. A clerk in the Treasury Department collected twenty cents per month from the wages
of each seaman to cover costs at a series of contract hospitals. In the 1880s, the MHS was charged by Congress with examining passengers on arriving ships for clinical signs
of infectious diseases, especially for cholera and yellow fever, in order to prevent epidemics. In the 1870s and 1880s scientists in Europe (Pasteur, Koch) presented compelling
evidence that microscopic organisms were the causes of several infectious diseases. In 1884, for example, Koch described a comma-shaped bacterium as the cause of cholera.
Officials of the MHS followed these developments with great interest. In 1887, they authorized Joseph J. Kinyoun, a young MHS physician trained in the new bacteriological
methods, to set up a one-room laboratory in the Marine Hospital on Staten Island, New York. Kinyoun called this facility a Laboratory of Hygiene in imitation of German
facilities and to indicate that the laboratorys purpose was to serve the publics health. Within a few months, Kinyoun had identified the cholera bacillus in suspicious cases
and used his microscope to demonstrate it to his colleagues as confirmation of their clinical diagnoses. In 1902, the Hygienic Laboratory was established in federal law as
the center for medical research within the federal government. The MHS was renamed the Public Health and Marine Hospital Service (PH-MHS), and the pathological and
bacteriological work as the Division of Pathology and Bacteriology was recognized by creating three new components that represented the most fruitful areas for research at
that time: the Divisions of Chemistry, Pharmacology, and Zoology. In 1912, another reorganization shortened the name of the PH-MHS to the Public Health Service (PHS or
USPHS). The National Institute of Allergy and Infectious Diseases (NIAID) was established at a unit of the NIH in 1955.

1887 Joseph Kinyoun founding of the National Institutes of Health

page 63

Sculpture of Jean-Baptiste Jupille

on the grounds of the Institut Pasteur

The Institut Pasteur was founded in 1887 by Louis Pasteur and his colleagues, whose experiments
led to the founding of the science of microbiology and virology. Pasteur discovered the principle of
food sterilization, which in the case of milk came to be known as pasteurization. His discoveries led
to the universal practice of surgical antisepsis. He also developed vaccination concepts and methods
to control several bacterial diseases and the viral disease rabies. As soon as the Institut was created,
Pasteur established five departments, each with a distinguished director: Emile Duclaux (general
microbiology research), Charles Chamberland (research applied to hygiene), Ilya Ilyich Mechnikov
(microbiology and immunology research), Jacques-Joseph Grancher (rabies research), and Emile Roux
(microbiology and virology research). One year after the inauguration of the Institut Pasteur, Roux set
up the first course of microbiology ever taught, then entitled Cours de Microbie Technique (course in
microbiological research techniques).

1887 Louis Pasteur and colleagues founding of the Institut Pasteur

page 64

Hermann Michael Biggs (1859-1923)

Hester Street, 1900, a time of cholera and smallpox outbreaks

Hermann Michael Biggs was the founder of public health laboratory sciences in the United States. He was born in the village of Trumansburg, N. Y. and attended Cornell
University and Bellevue Hospital Medical College. During his internship at Bellevue, he developed an interest in histopathology and the new science of bacteriology. In 1884
he visited Robert Kochs laboratory in Germany and in 1885 he visited Louis Pasteurs laboratory in France. When he returned from his first European trip, he became a senior
member of the newly established Carnegie Laboratory at Bellevue, devoted to bacteriology. This was the first bacteriological laboratory in the United States to apply this
science directly to public health. When a division of bacteriology was created within the New York City Department of Health in 1892 in response to the threat of cholera,
Biggs was appointed chief inspector and director of the laboratory. Biggs became the leading advocate for the use of the diagnostic laboratory; his leadership led to the
establishment of similar laboratories elsewhere: in Massachusetts in 1894, in Philadelphia in 1895, et al. In 1901 he was appointed general medical officer of New York City and
at the same time he was appointed to the board of directors of the newly formed Rockefeller Institute for Medical Research. In 1914 he became the commissioner of health for
the state of New York.

1892 Hermann Biggs founding of the New York City Department of Health microbiology laboratory

page 65

Mechanisms of Virus Neutralization by Antibody from: Occupancy and Mechanism in

Antibody-Mediated Neutralization of Animal Viruses. P. J. Klasse and Q. J. Sattentau.
Journal of General Virology, 2002.

George Miller Sternberg (1838-1915)

Sternberg GM. Wissenschaftliche untersuchungen ber das spezifische
infcktionsagens der blattern und die erzeugung knstlicher immunitt
gegen diese krankheit. Centralbl f Bakteriol Abt. I, 1896;19:857-869.
Klasse PJ, Sattentau QJ. Occupancy and mechanism in antibody-mediated
neutralization of animal viruses. J Gen Virol. 2002;83:2091-2108.
Klasse PJ, Sattentau QJ. Mechanisms of virus neutralization by antibody.
Curr Top Microbiol Immunol. 2001;260:87-108.

a. Ab binding to a proportion of the receptor-interactive structures on the virion may block virus attachment to
the surface of target cells. In the schematic example shown, the Abs are bound to protein spikes on an enveloped
virus, which is thereby prevented from making contact with either of the two cell-surface receptors that it uses for
attachment and entry.
b. Ab inhibition of the interactions between the viral envelope protein and the cell-surface receptors is shown to occur
after the virion has attached by binding via the receptors. For example, one receptor may serve as an attachment
point as well as a trigger of conformational changes that allow interactions with a co-receptor, which in turn
mediate later events such as membrane fusion. Ab interference with any of these necessary links in a chain of events
that lead to entry would constitute a neutralization mechanism.
c. In order to infect, some viruses require internalization by endocytosis and the lowering of the pH in the endosome
as a trigger of conformational changes in viral proteins. Abs that have not blocked virus attachment but allowed
internalization are shown. Their block of requisite interactions between viral and cell-membrane proteins would
delay or prevent the penetration of the viral core into the target-cell cytoplasm. The virion may thus ultimately be
destroyed through lysosomal degradation. Such effects of Ab would constitute a mechanism of virus neutralization.
In addition, Ab-mediated derouting of viruses that preferentially enter directly via the cell surface to a less
permissive endosomal compartment may abrogate infectivity.
d. The intercalation of Ab in the fusion interface between the cell membrane and the envelope of a virus may block
fusion at the cell surface, as illustrated, or in an endosome.
e. It has been conjectured that even a very low occupancy of antibody on the virion can cause global or internal
changes by transmitting a signal across the viral envelope or outer layer. These hypothetical changes would allow
the viral core to enter the cytoplasm but compromise further replicative steps.
g. The neutralization of naked viruses could potentially differ from that of enveloped viruses. Although naked viruses
have been shown in several instances to be neutralized by a block of viral attachment to target cells, as in (a),
conformational effects on the whole virion by Ab binding have also been registered as pI (isoelectric pH) shifts.
The possibility that the Ab-virion complex enters the cytoplasm and that the Ab blocks further replicative events is
shown.

1892 George Sternberg discovery of virus neutralization (vaccinia virus)

page 66

Theobald Smith (1859-1934)

Frederick Cooper Curtice (1856-1939)
Fred L. Kilborne (1858-1936)
Daniel E. Salmon (1850-1914)
The first proof of arthropod
transmission of a disease:
The disease: Texas fever.
The infectious agent: Babesia bigemina.
The tick vector: Boophilus annulatus.
As acknowledged by Walter Reed in
1901, this discovery was a key to the
discovery of the first human virus,
yellow fever virus, and its transmission
by mosquitoes.

Theobald Smith

Boophilus annulatus tick

Theobald Smith was born in Albany,

New York in 1859. He graduated from
Cornell University in 1881 and
received an MD from Albany Medical
College in 1883. From 1884-1895
he was director of the pathology
laboratory of the Bureau of Animal
Industry, USDA, where he investigated
infectious diseases of animals. From
1895 to 1915 he served as Professor
of Comparative Pathology, Harvard
Medical School. In 1915 he became
director of the Department of Animal
Pathology at the Rockefeller Institute
for Medical Research in Princeton.
He was president of the American
Associated of Pathologists and
Bacteriologists, the American Society
of Tropical Medicine, the Congress of
American Physicians and Surgeons,
and the International Society Against
Tuberculosis. He was a Fellow of the
AAAS. He received twelve honorary
degrees and eleven honorary medals,
among which was the Copley Medal
Babesia bigemina bovine blood film
of the Royal Society.
Smith T, Kilborne FL. Investigations into the nature, causation, and
prevention of Texas or Southern cattle fever. U. S. Bureau of Animal
Industry, Bulletin No. 1. U.S. Government Printing Office, 1893. 301
pages, 10 plates. Reprinted in Medical Classics, 1936-1937;1:372-597.

1893 Theobald Smith, Fred Kilborne, others first proof of arthropod transmission of a disease, Texas Fever

page 67

Frederick Cooper Curtice, collecting ticks from a cow dead of Texas fever, 1893

This photo served as the frontispiece through many editions of the book, Hagen and Bruners Infectious Diseases
of Domestic Animals, which was the authors textbook at the College of Veterinary Medicine, Cornell University.

There are infamous stories of how the authorship of the seminal monograph on the discovery of the etiology and mode
of transmission of Texas fever (Investigations into the Nature, Causation, and Prevention of Texas or Southern Cattle
Fever. BAI Bulletin no. 1, 1893) was manipulated, in the end frustrating all the investigators. According to Claude E.
Dolman, the monograph was written entirely by Theobald Smith, establishing his reputation for scrupulous logic,
detailed observation, abundant verification, and unrelenting patience. In Smiths draft he acknowledged the assistance
of Fred Kilborne and others, but not via co-authorship. The printed copies had Kilbornes name on the title page as
coauthorthis was the doing of the director of the BAI, Daniel Salmon. Smith considered this an injustice, which
along with similar concerns he has over credit for the discovery of Salmonella spp. and Mycobacterium bovis, led to his
resignation in 1895. Salmon, it has been stated, may have been reflecting the resentment of some BAI parasitologists
and veterinarians who saw in Smith, an MD, a usurping of their bailiwick. Dolman stated, Salmons vengeful act
possibly cost Smith a Nobel Prize. The Bulletin was later recognized as a classic, describing soundly based experiments
and observations, with all appropriate controls, and with clear evidence-based conclusionsfar ahead of its time.
Dolman CE. Theobald Smith, 1859-1934: A fiftieth anniversary tribute. ASM News 1984;50:577580.

From USDA/BAI Bulletin no.1, 1893: Experimental

fields from 1889. Scale: inch = 33 feet. a dwelling
house; b station laboratory; c house stable; d cow
stable; e breeding pens; f tool house; g shed in field.
Field Vfield of healthy Texas Longhorns heavily
infested with ticks. Field IVfield of healthy North
Carolina cattle without ticks. Experimental fields
I-III: On experimental field I, Texas longhorns
grazed together with North Carolina cattle. North
Carolina cattle became infested with ticks, and
subsequently they developed Texas cattle fever and
died. On experimental field II, Texas longhorns
without ticks (ticks were removed manually by
Smith and Kilbourne) grazed together with healthy
North Carolina cattle. North Carolina cattle
remained healthy. Next, healthy North Carolina
cattle were transferred to the heavily tick-infested
experimental field I where the cattle became
infested with ticks, developed Texas cattle fever,
and died. As a last step, Smith transferred grass and
ticks from experimental field I to experimental field
III. Subsequently, healthy North Carolina cattle
became tick-infested and died of Texas cattle fever.

1893 Theobald Smith, F. Cooper Curtice, others first proof of arthropod transmission of a disease, Texas Fever
page 68

Ivanovskys experiments involved passing sap from diseased tobacco plants through
Chamberland porcelain ultrafilters, which were considered to be the ultimate test for bacteria, as
bacteria would be retained in the filters but soluble toxins would not. He was surprised when the
infectious moiety passed through the filters and suspected that the filters was defective. Further
testing of the filters convinced him that they were not defective: According to the opinions
prevalent today, it seems to me that the latter is to be explained most simply by the assumption
of a toxin secreted by the bacteria present, which is dissolved in the filtered sap. Besides this there
is another equally acceptable explanation possible, namely, that the bacteria of the tobacco
plant penetrated through the pores of the Chamberland filter-candles, even though before every
experiment I checked the filter used in the usual manner and convinced myself of the absence of
fine leaks and openings. He presented his findings to the Academy of Science in St. Petersburg in
1892. Ivanovsky published a more detailed paper in 1903, which included a detailed description
of the disease, including microscopic observations on the two types of inclusions found in cells of
infected tissues and extensive unsuccessful efforts to culture the agent. In the end he still came to
the conclusion that the causal agent was an uncultivable bacterium.
Iwanowski D. Concerning the mosaic disease of the tobacco plant. St Petersburg Academy Imp
Sci Bul. 1892;35:67-70. Translation published in English as Phytopathological Classics Number 7.
American Phytopathological Society Press, St. Paul, Minnesota; 1942.
Iwanowski D. ber die mosaikkrankheit der tabakspflanze. Zeit Pflanzenkr Pflanzenpathol
Pflanzenschutz. 1903;13:1-41.

Dmitry Ivanovsky (1864-1920)

(or Dmitrii Iwanowski)

1892 Dmitry Ivanovsky discovery of tobacco mosaic virus

page 69

Beijerinck Laboratory, Delft

Martinus Willem Beijerinck (1851-1931)

Beijerinck repeated the ultrafiltration

experiments of his predecessors (although
he was apparently unaware of the work
of Ivanovsky) and concluded that what
passed through porcelain ultrafilters
remained infectious but did not contain
detectable microorganisms. He concluded
that it was a contagium vivum fluidum,
that is a contagious living fluid. He found
that from the sap of a diseased tobacco
plant ...an infinite number of healthy
plants may be inoculated and infected.
He concluded that, unlike a toxin, the
infectious agent reproduces in diseased
plants. He conducted diffusion experiments
in which he allowed the infectious agent to
penetrate agar he again concluded that
the infectious agent was soluble. He also
observed that the infectious agent in sap
extract remained viable after drying, but
was inactivated by heating to 90C.

Beijerinck MJ. Concerning a contagium vivum fluidum as cause of the spot disease of tobacco leaves. Verhandelingen der Koninkyke Akademie Wettenschapppen te
Amsterdam 1898;65:3-21. Translation published in English as Phytopathological Classics Number 7. American Phytopathological Society Press. St. Paul, Minnesota; 1942.

1898 Martinus Beijerinck discovery of tobacco mosaic virus

page 70

Friedrich A. J. Loeffler (1852-1915)

Paul Frosch (1860-1928)

Loeffler F, Frosch P. Berichte der kommission zur erforschung der maul-und klauenseuche bei dem institut fur
infektionskrankheiten. Part I. 1898;23:371-391. In Milestones in Microbiology: 1556 to 1940, translated and edited by
Thomas D. Brock, ASM Press; 1998.
Friedrich-Loeffler-Institute Bundesforschungsinstitut fr Tiergesundheit
(German Federal Research Institute for Animal Health)
on the Isle of Riems is named in Loefflers honor

1898 Friedrich Loeffler and Paul Frosch discovery of foot-and-mouth disease virus (the first virus: see text)

page 71

Who discovered the first virus?

There are many, many articles arguing the priority of the discovery of the first virus this is a place
for strongly held opinions
Dmitry K. Lvov: Centenary of Virology, in Concepts in Virology: From Ivanovsky to the Present
[Mahy BWJ (Ed.)]. Brian Taylor & Francis, Inc.; 1993. It was Ivanovsky
A. van Kammen: Beijerincks Contribution to the Virus Concept. In 100 Years of Virology The
Birth and Growth of a Discipline. [Calisher CH, Horzinek MC (Eds.)]. Archives of Virology, Suppl.
15; 1999. It was Beijerinck
Marc H. V. van Regenmortel: The Nature and Classification of Viruses, in Topley & Wilsons
Microbiology & Microbial Infections [Mahy BWJ, ter Meulen V (Eds.)]. Hodder Arnold; 2005.
It was Loeffler and Frosch...
An Opinion on the Priority of the Work of Ivanovsky, Beijerinck and Loeffler and Frosch by
Marc van Regenmortel, 2006 abridged:
...Although all historical accounts of the beginnings of virology refer to the work of Ivanovsky,
Beijerinck, and Loeffler and Frosch (working with Koch), there is disagreement among authors
about who should be credited with the discovery that viruses were a new type of infectious agent.
This debate concerns the question of what is a scientific discovery.
Although Ivanovsky was clearly the first one to show that the agent causing tobacco mosaic disease
passed through a bacteria-retaining filter, all his publications show that he did not grasp the
significance of his observation. He believed that the filter he used might have had fine cracks and
that small spores of a microbe might have passed through the filter.
Beijerinck on the other hand, realized he was dealing with something different from a microbe but
he thought that the virus was an infectious liquid and not a particle.
Only Loeffler and Frosch correctly concluded that the virus causing foot-and-mouth disease was a
small particle that passed through a Chamberland filter, but was stopped by a finer-grained Kitasato
filter.

Friedrich Loeffler
(1852-1915)

Robert Koch
(1843-1910)

The debate about who should be considered the founder of virology may be settled only if it is
accepted that, in order to make a discovery, it is not sufficient to make a novel observation (i.e.,
the filterability of an infectious agent), but that it is also necessary to interpret the observation
correctly.
Good science does not consist only in making new observations but it requires also unbiased,
imaginative thinking which enables the scientist to arrive at the correct interpretation of his
experimental findings.
Loeffler and Froschs interpretation of their filtration experiments came the closest to the modern
concept of a virus and so they should be acknowledged as the founders of virology.

1898 Friedrich Loeffler and Paul Frosch discovery of foot-and-mouth disease virus (the first virus: see text)
page 72

Friedrich A. J. Loeffler (1852-1915)

Paul Frosch (1860-1928)

In 1886, Friedrich Loeffler and Paul Frosch were commissioned by the German
government to investigate the cause of foot-and-mouth disease and find a way to
prevent it. They first reported on the task at hand, how to protect cattle, describing
the first successful use of a vaccine based on simultaneous inoculation of wild-type
infectious agent (ultrafiltered virus from vesicular lesions) and serum from recovered
animals (a similar approach was used at about the same time by Arnold Theiler and
his colleagues to prevent rinderpest). In 1898, Loeffler and Frosch wrote, However,
the filtration experiments have offered extremely important results in another area.
In these experiments the vesicular fluid was diluted with 39 parts of water, then
inoculated with an easily culturable and identifiable bacterial species-Bacillus
fluorescens-and then filtered two to three times through a sterilized Kieselguhr candle
[the equivalent of a Chamberland ultrafilter].... Filtrates tested in this manner were
always bacteria free. A series of calves were inoculated intravenously with measured
amounts of these filtrates corresponding to 1/10 to 1/40 cc. of pure vesicular fluid, in
order to ascertain if there was a dissolved substance which would aid in the production
of immunity. The results of these injections were quite surprising. The animals
inoculated with the filtrates died in the same time as had the control animals which
had received corresponding amounts of unfiltered fluid. We had the impression that
the activity of the vesicular fluid was not influenced by the filtration. On the basis of
meticulous calculations of the dilutions involved, they came to the conclusion that
no toxin could be sufficiently strong to have caused their results. They went on: In
order to confirm this important result, these experiments were repeated in a large
number of calves and hogs. Then they did the experiment that established the concept
of virus. The evidence that the virus is a corpuscular [particulate] and not a soluble
agent is provided by the repeated observation that the diluted vesicular fluid, filtered
several times through very fine Kitasato filters was no more able to infect susceptible
animals, even if an amount of filtrate corresponding to 4/50 ml of pure vesicular fluid
was used for injection. This study, apart from its purely scientific value, seems to offer
eminently practical possibilities. If it is confirmed by further studies of the commission
that the action of the filtrate, as it appears, is actually due to the presence of such
a minute living being, this brings up the thought that the causal agents of a large
number of other infectious diseases, such as smallpox, cowpox, scarlet fever, measles,
typhus, cattle plague, etc. which up to now have been sought in vain, may also belong
to this smallest group of organisms. If a bacteria free cowpox vesicular fluid could be
produced, this would ease the agitation against vaccination for smallpox. The bacteria
free filtrates of infectious material probably offer the most suitable material for the
acquisition of important new conclusions on the nature of the diseases named above.
All these speculations indicate that it would be highly desirable to continue the studies
on the action of the filtrate in a larger number of animals as soon as possible.
As argued by Marc van Regenmortel and Jean Witz, this work and its interpretation
came much closer to the modern concept of virus than the conclusions reached by
Dmitry Ivanovsky or Martinus Beijerinck in their earlier ultrafiltration experiments.

Loeffler and Froschs discovery should have been the basis for much excitement.
Hovever, as noted by Lise Wilkinson, the discovery was quickly confounded by
the discovery and ultrafiltration of the causative agent of bovine pleuropneumonia
at the Institute Pasteur by Edmond Nocard (1850-1903), Pierre Paul Emile Roux
(1853-1933), Amedee Borrel (1867-1936), Alexandre Salimbeni (!867-1942) and
Edouard Dujardin-Beaumetz (1868-1947). The causative agent was found to be
ultrafilterable, but it could be cultivated on enriched bacterial media and could be
see in the light microscope, barely, when stained with Giema stain. It is known now
to be a mycoplasma, Mycoplasma mycoides [ the agent so flexible that it fits through
the pores of many ultrafilters]. Wilkinson: The fact that it was at this early stage
recognized as a filterable agent probably to some extent delayed further development
of the virus concept in the first decades of the century. That is, because it was possible
to cultivate the causal organism and to see it under the light microscope, it gave
encouragement to those who believed that the failure to observe viruses with the
aid of the light microscope and to grow them in vitro represented only temporary
difficulties, to be resolved as techniques improved. By the turn of the century the
concept of the virus as a separate biological entity had emerged from its background of
general bacteriology, but still only in a tentative way. Only four agents had been shown
to pass through filter candles [tobacco mosaic virus, foot-and-mouth disease vkirus,
myxoma virus and the agent of bovine pleuropneumonia].
Nevertheless, as time would soon tell, the dam had burst and before any key new
technologies came on the scene, many more filterable agents were described. In
1903, Emile Roux published a review listing ten ultrafilterable agents and three years
later his colleague, Paul Remlinger, published a review listing nineteen.
Loeffler F, Frosch P. Berichte der kommission zur erforschung der maul-undklauenseuche bei dem Institut fr Infektionskrankheiten. Zbl Bakt I/Orig. 1898; 23:
371-391. In Milestones in Microbiology: 1556 to 1940, translated by Thomas Brock,
ASM Press; 1998.
Loeffler F. Bericht der komission zur erforschung der maul-und-klauenseuche bei
dem Institut fr Infektionskrankheiten in Berlin. Part IV. Zbl Bakt I/Orig. 1898;24:
569574.
Wilkinson L. The development of the virus concept as reflected in corpora of
studies on individual pathogens. I. Beginnings at the turn of the century. Med Hist.
1974;18:211-0221.
Witz J. A reappraisal of the contribution of Friedrich Loeffler to the development of
the modern concept of virus. Arch Virol. 1998;143:2261-2263.
van Regenmortel MHV. The nature and classification of viruses, in Topley & Wilsons
Microbiology & Microbial Infections [Mahy BWJ, ter Meulen V (Eds.)]. Hodder
Arnold; 2005.

1898 Friedrich Loeffler and Paul Frosch discovery of foot-and-mouth disease virus (the first virus: see text)

page 73

Timeline of the Discovery of the First Viruses

From the beginnings of virology in the last decade of the ninteenth century and its tentative immediate progress, there was in the first days of the twentieth century a
remarkable transformation, with ultrafilterable infectious agents reported by a diverse community of scientists in many different countries. Twenty of the first 30 viruses
discovered were animal pathogens; only nine were primarily human pathogens. This was certainly because of the technologies available at the time in this era before virus
cultivation in any experimental model system, the candidate specimen had to be obtained, usually from a diseased subject, ultrafiltered through Chamberland, Berkefeld,
Kitasato or similar candles (made from kieselghur, sintered diatomaceous earth), and then the filtrate back-titrated in susceptible subjects, whether animals or humans.
After the death of human volunteers in yellow fever studies, the latter continued only in particular circumstances. Looking back, it seems remarkable that early ultrafiltration
technology was so well done and so predictive that by 1927, when Thomas Rivers wrote his historic review, he was able to list 67 viruses from vertebrates, plants, arthropods
and bacteria (it must be noted that
Date
Discoverer(s)
Virus
the list did include several infectious
1
1892
D Ivanovsky, M Beijerinck
tobacco mosaic virus (the first virus?)
agents later found not to be viruses).
2

1898

F Loeffler, P Frosch

foot-and-mouth disease virus (the first virus?, the first vertebrate virus)

1898

G Sanarelli

myxoma virus (the first poxvirus)

1900

W Reed, J Carroll, A Agramonte, J Lazear

yellow fever virus (the first human virus, the first arbovirus, the first flavivirus)

1900

J MFadyean, T Edgington, A Theiler

African horse sickness virus (the first orbivirus)

1901

E Centanni, E Savonuzzi, A Lode, J Gruber

fowl plague virus (avian influenza virus, the first orthomyxovirus)

1902

A Aujeszky

pseudorabies virus (the first herpesvirus)

1902

A Borrel

sheeppox virus

1902

M Nicolle, M Adil Bey

rinderpest virus (the first morbillivirus)

10

1902

J Spruell, A Theiler

bluetongue viruses

11

1903

M Dorset, E De Schweinitz

classical swine fever virus (hog cholera virus, the first pestivirus)

12

1903

M Remlinger, Riffat Bey, A di Vestea

rabies virus (the first rhabdovirus)

13

1904

H Valle, H Carr

equine infectious anemia virus (the first retrovirus)

14

1905

H Carr, P Laidlaw, G Dunkin

canine distemper virus

15

1907

P Ashburn, C Craig

dengue viruses

16

1908

V Ellermann, O Bang

avian leukemia virus (the first leukemia virus)

17

1909

K Landsteiner, E Popper

poliovirus (the first enterovirus)

18

1909

R Doerr, K Franz, S Taussig

sandfly fever viruses (the first phleboviruses)

19

1911

J Goldberger, J Anderson

measles virus

20

1911

P Rous, J Beard

Rous sarcoma virus (the first solid tumor virus)

21

1914

A Hess, Y Hiro, S Tasaka

rubella virus (the first rubivirus)

22

1917

F Twort, F dHerelle

bacteriophages

23

1917

R Montgomery

Nairobi sheep disease virus (the first nairovirus, the first tick-borne virus)

24

1918

A Breinl, J Cleland, E French

Murray Valley encephalitis virus

25

1919

A Lwenstein

herpes simplex virus

26

1921

R Montgomery

African swine fever virus (the only asfarvirus)

27

1925

K Kundratitz, H Ruska, T Weller, M Stoddard

varicella-zoster viruschickenpox and zoster

28

1926

W Cotton, P Olitsky, J Traum, H Schoening

vesicular stomatitis viruses

29

1926

F Kraneveld, T Doyle

Newcastle disease virus (the first paramyxovirus)

30

1928

J Verge, N Christoforoni

feline panleukopenia virus (the first parvovirus)

1898-1928 Discovery of the first viruses

page 74

Roux, . 1903 Sur les microbes dits

invisibles [On the microbes known
as invisible]. Bull. Inst. Pasteur
1903;1:7-12, 49-56.
Remlinger P. Les microbes filtrants
[The filter-passing microbes]. Bull.
Inst. Pasteur 1906;4:337-345, 385392.
MFadyean J. The ultravisible viruses.
J comp path ther. 1908;21: 58-68, 168175, 232-242.
Rivers T. Filterable viruses
a critical review. J Bact. 1927;14:
217-258.

European rabbit (Oryctolagus cuniculus)

lesion of myxomatosis above eye

Myxoma virus

negative contrast
electron Microscopy

Giuseppe Sanarelli (1864-1940)

Myxoma virus was discovered quite serendipitously by Giuseppe Sanarelli of the University of Siena
when he was invited by the government of Uruguay to establish a Hygiene Institute in Montevideo.
In setting up laboratories, he procured domestic European rabbits (Oryctolagus cuniculus). Soon
after, his rabbit colony suffered an outbreak of a highly infectious, fatal disease. Sanarelli named the
disease myxomatosis and showed that it was caused by a ultrafilterable virus. In a series of classical
studies, Henrique de Beaurepaire Arago showed that Sylvilogus brasiliensis, which occurs widely in
South America, is the natural reservoir of myxoma virus and that the virus is transmitted by biting
mosquitoes. The systemic infection characteristic of myxomatosis does not occur in Sylvilogus
brasiliensis; instead the virus produces skin tumors which remain localized to the injection site and
eventually heal. These lesions serve as the source of the virus that is transmitted mechanically by
mosquitoes.
Sanarelli G. Das myxomatogene virus. Beitrag zum stadium der krankheitserrege ausser halb des
sichtbaren. Zentralbl Bakteriol Abt 1. 1898;23:865-873.
Arago HB. Myxoma dos coelhos. Mem Inst Oswaldo Cruz 1927;20:225-247.

1898 Giuseppe Sanarelli discovery of myxoma virus (the first poxvirus)

page 75

Henry Rose Carter (1852-1925)

Carter HR. A note on the spread of yellow fever in houses. Extrinsic

incubation. Medical Record 1901;59:933-937.

Henry Rose Carter, a physician in the Marine Hospital

Corps (forerunner of the U.S. Public Health Service)
spent 20 years in Louisiana where he observed the strange
but uniform timing of yellow fever outbreaks on ships
arriving from Central and South American ports. He
noted that within a day of leaving such ports, yellow fever
cases occurred, but then it was two weeks before the
next cases appeared. In 1898, he was called to two small
towns, Orwood and Taylor Mississippi, to investigate a
yellow fever outbreak. There he put his observations to the
test, meticulously documenting exposures and cases and
recording the timing of every case. He concluded that the
infectious agent leaving the patient must undergo some
change in the environment before it is capable of infecting
another man. He thought this period was about 2 weeks,
and he called it the period of extrinsic incubation.

Henry Rose Carter collecting mosquito

larvae in the field, North Carolina

1898 Henry Rose Carter discovery of the yellow fever virus mosquito extrinsic incubation period
page 76

Conquerors of
Yellow Fever

by Dean Cornwell

from the series Pioneers

of American Medicine
An allegorical depiction of
one of the great moments
in medical history. With
Walter Reed (in white
uniform) and Carlos
Finlay (with white hair)
looking on, Jesse Lazear,
who died a month later,
is shown applying an
infected mosquito to the
arm of James Carroll. The
painting includes Aristides
Agramonte (behind Lazear),
Leonard Wood (in brown
helmet), Jefferson Kean (in
white helmet) and several
of the volunteers who
subsequently were infected
in the same way. Carroll
became infected as a result
of this experimenthe
survived, and went on to
a distinguished career as a
microbiologist, but suffered
from chronic illness leading
to an early death, said to
be a consequence of yellow
fever infection.

1900 Walter Reed, colleagues discovery of yellow fever virus (the first human virus) and its transmission cycle

page 77

Walter Reed (1851-1902)

The history of the discovery of

yellow fever virus and its mosquito
transmission cycle is wonderfully
preserved in the Philip S.
Hench Walter Reed Yellow Fever
Collection at the University of
Virginia Health Sciences Library.

In 1900, the U.S. Yellow Fever Commission, led by Walter Reed, major U.S. Army Medical Corps, conducted experiments at stations just outside Havana, Cuba, that proved
that yellow fever is transmitted by mosquitoes, rather than by direct contact or fomites. He and the other members of the Commission, James Carroll, Aristides Agramonte,
and Jesse Lazear, proved that Aedes aegypti is the vector of yellow fever virus, and James Carroll conducted experiments that proved that the etiologic agent of yellow fever is
a virus the first human virus. The Commission operated as part of the American occupation force in Cuba after the Spanish-American War of 1898. During the war and the
following occupation yellow fever (and malaria, and dysentery) accounted for most deaths and disabilities. The Commissions discoveries focused efforts on the eradication
of the mosquito within six months yellow fever had been eliminated from Havana and within 10 years from Cuba. Although Reed received much of the credit for the
discoveries, he credited Carlos Finlay with the discovery of the yellow fever vector, the key to how the disease might be controlled. He also credited the insight provided by
Henry Rose Carters evidence of a mosquito extrinsic incubation period, by Theobald Smith and his colleagues proof that the agent of Texas fever was transmitted by an
arthropod, and Friedrich Loeffler and Paul Froschs proof that the agent of foot-and-mouth disease was a virus.

1900 Walter Reed, colleagues discovery of yellow fever virus (the first human virus) and its transmission cycle
page 78

American Association for the Advancement of Science Resolution:

Death of Dr. James Carroll from Yellow Fever Experimentation
Whereas, The late Major James Carroll, M.D., U.S. Army, was the first
to submit voluntarily to the bite of an infected stegomyia, and from
the bite of this mosquito, suffered a severe attack of yellow fever, the
effects of which led to his ultimate death, and
Whereas, This was the first experimentally produced case of yellow
fever leading to the present knowledge of this disease, which has
practically enabled its complete control, therefore, be it
Resolved, That the American Association for the Advancement of
Science now in session in Chicago, Illinois recommends to the
Senate and House of Representatives of the United States of America
the passage of a bill securing to Mrs. Jennie Carroll, widow of the
late Major James Carroll of the Yellow Fever Commission, of the
United States Army, a special pension for the support of herself and
her seven children.
[Adopted by the AAAS Council, December 30, 1907.]

James Carroll (1854-1907)

From: The Scientific Work and Discoveries of the late
Major Walter Reed, Surgeon in the Army of the United States.
Prepared by Major Jefferson Randolph Kean.
U.S. Senate Document No. 118, 57th Congress, 2nd Session.
Washington: Government Printing Office; 1903.

1900 Walter Reed, colleagues discovery of yellow fever virus (the first human virus) and its transmission cycle

page 79

Discovery of the First Human Virus

The original work of the Yellow Fever Commission was concluded in February 1901, but James Carroll was
allowed to return to Cuba in August of that year. Camp Lazear had been closed, so he worked at Las Animas
Hospital. To determine if the agent of yellow fever was ultrafilterable, he obtained some infected mosquitoes from
Juan Guitras and produced new cases. In August through October 1901, he produced six cases of yellow fever. A
Spaniard, Pablo Ruiz Castilo, was bitten by four mosquitoes on September 16 and became severely ill three days
later. Another Spaniard, Jacinto Mendez Alvarez, was bitten on October 9 by eight mosquitoes and developed a
mild illness.
On October 15, Paul Hamann, an American soldier, was injected with serum from Alvarez that had been passed
through a Berkefeld ultrafilter. Four days later, he developed yellow fever. Albert Wall Covington, another soldier,
was injected with the same ultrafiltered serum and became ill four days later. John R. Bullard, a civilian, was
given a similar injection. Four days later, he had a modest elevation in temperature and pulse, accompanied by a
headache and pain between his shoulders (not considered a certain case of yellow fever).
One week later, Bullard was injected again with ultrafiltered serum, this time from blood drawn from Paul
Hamann; he developed clinical signs of yellow fever just twenty-four hours later. Carroll was at a loss to explain
Bullards case: if he became infected from the first injection, the incubation period was nine days, three days
longer than any other case. If it was from the second, the incubation period was one day, less than half as long
as any other case. Carroll was inclined to believe that Bullards case was from the second injection, but he was
unsure.
It is certain that Carroll proved that the agent of yellow fever is an ultrafilterable agent and not a toxin in the
blood of yellow fever patients. In fact, James Carroll discovered the first human virus and demonstrated the
first experimental transmission of a viral disease from one human to another.
Shortly after this, Carroll received orders forbidding him from doing further human research this was partly
due to the public outcry over the death of the American nurse Clara Maass, and partly due to the fact that the
mosquito-control campaign had by this time practically eliminated the disease from Havana.
From: Pierce JR, Writer J. Yellow Jack,
How Yellow Fever Ravaged America
and Walter Reed Discovered its Deadly
Secrets. Hoboken, NJ: Wiley; 2005.

James Carroll (1854-1907)

Las Animas Hospital
Yellow fever ward
Havana, Cuba, 1901

1900 Walter Reed, colleagues discovery of yellow fever virus (the first human virus) and its transmission cycle
page 80

Jesse William Lazear (1866-1900)

The Washington Observer

September 29, 1900

Aristides Agramonte y Simoni

(1868-1931)

Aristides Agramonte y Simoni was a physician, pathologist and bacteriologist with expertise in tropical medicine. In 1898 he was appointed Acting Assistant Surgeon, Medical
Corps, U.S. Army and was sent to Cuba. Although he had no recollection of having had yellow fever as a child, he was thought to be immune because he had been born in
Cuba. While a member of the U.S. Army Yellow Fever Commission he autopsied numerous victims of yellow fever and helped eliminate the widely held notion that the disease
was caused by a common bacterium, Sanarellis Bacillus icteroides.
Jesse William Lazear was a physician and bacteriologist trained at Johns Hopkins, Columbia and the Institut Pasteur; in 1900 he reported for duty as an Assistant Surgeon,
Medical Corps, U.S. Army at Columbia Barracks (Quemados), Cuba, where he joined the Yellow Fever Commission. Three months later, without telling his colleagues, he
allowed himself to be bitten by yellow fever-infected mosquitoes and died. Although Lazear never admitted to experimenting on himself, when Walter Reed reviewed his
notebook he evidently found entries strongly suggesting Lazears infection was not accidental, as officially reported. Unfortunately, the notebook, so crucial to the preparation
of the Commissions initial paper, The Etiology of Yellow Fever A Preliminary Note, vanished from Reeds Washington office after his death in 1902.

1900 Walter Reed, colleagues discovery of yellow fever virus (the first human virus) and its transmission cycle

page 81

Camp Lazear, Quemados, Cuba, 1900

Left: Infected Mosquito Building

Center: Infected Clothing Building

The Yellow Fever Human Subjects Experimentation, Cuba, 1900-1901

The experimental work of the U.S. Army Yellow Fever Commission took place at a site at Quemados near
Havana, later named Camp Lazear. Here, Walter Reed designed two small wood-frame buildings, each 14
by 20 feet one conspicuously clean and well ventilated, the other filthy and fetid, all to test whether yellow
fever transmission involved contact and fomites or mosquitoes.
Three trials of twenty days each, involving seven men, were conducted in Building One, called the Infected
Clothing Building this was to determine whether contaminated bedding and clothing (bed linens, blankets,
nightshirts etc., recently worn and soiled by patients in the wards of Columbia Barracks Hospital and Las
Animas Hospital in Havana) could be involved in the transmission of yellow fever no cases occurred.
Building Two, called the Infected Mosquito Building, contained a principal room, divided into two sections
by a floor-to-ceiling wire mesh screen, so configured to keep infected mosquitoes inside one section only. A
separate room attached to the building held the laboratory where mosquitoes were raised and stored. Four
trials were conducted, each requiring two groups of volunteers, one to be exposed and another to serve as
controls. Loaded mosquitoes, that is mosquitoes obtained from rooms inhabited by known yellow fever
patients, were released into one screened section where volunteers remained for about twenty minutes,
enough time to receive several mosquito bites. These volunteers then exited to a quarantine tent outside.
The controls spent the night in the section of the room free of mosquitoes they returned to sleep in the
room for as many as eighteen more nights. As Reed stated, the absence of yellow fever in the controls
showed, that the essential factor in the infection of a building with yellow fever is the presence therein of
[infected] mosquitoes, and nothing more. Taken together, all the experiments led to 22 cases of yellow fever:
14 by mosquito bite, 6 by blood injection, and 2 by injection of ultrafiltered serum. There were no deaths in
the main series of experiments, but there were three in experiments carried out later by William Gorgas and
Juan Guitras one was the American nurse, Clara Maass.

1900 Walter Reed, colleagues discovery of yellow fever virus (the first human virus) and its transmission cycle
page 82

Hospital Corps Detachment, Camp Columbia, Havana, 1900

Most of the volunteers for the yellow fever experiments came from
this unit. Human subjects were needed to test the mosquito theory
because, at the time, no one knew of any animals susceptible to the
virus. The four members of the Army Yellow Fever Commission
agreed to experiment on themselves before requesting volunteers.
Agramonte was exempted since it was thought that he was immune,
having been infected in childhood. When Reed departed for
Washington to complete a typhoid report, only Carroll and Lazear
were left to share the risk with their volunteers. Several Spanish
immigrants also participated in the experiments as volunteers.

Clara Louise Maass (1876-1901)

The only woman volunteer.
After experimental mosquito exposure she
developed severe yellow fever and died.
Her death in 1901 led to a public outcry
that ended human experimentation.
Clara Maass was the first nurse honored
with an American stamp (issued in 1976).

1900 Walter Reed, colleagues discovery of yellow fever virus (the first human virus) and its transmission cycle

page 83

John McFadyean, Principal, Royal

Veterinary College, London (18921927). The teaching staff, 1895.
John McFadyean, center front row

John McFadyean (or MFadyean) (1853-1941)

MacFadyean J. African horse-sickness. J Comp Path Ther. 1900;13:1-30.
MacFadyean J. A further contribution to the pathology of African horsesickness. J Comp Path Ther. 1901;14:103-118.
MacFadyean J. The ultravisible viruses. J Comp Path Ther. 1908;21:58-68, 168175, 232-242.

John McFadyean, considered the father of veterinary research, was trained in Edinburgh
as a veterinarian and as a physician. Early on he became interested in bacteriology and
pathology. In 1888 he originated the Journal of Comparative Pathology and Therapeutics,
which he edited for many years. In 1892, he was appointed principal and professor of
bacteriology and pathology at the Royal Veterinary College, London, and for the next 35
years he had a profound effect on the development of veterinary science. In 1900, working
in London with blood from infected horses in Africa brought in sealed tubes by a returning
English veterinarian, McFadyean found that diluted blood passed through Berkefeld and
Chamberland F and B ultrafilters remained infectious he confirmed his results in many
experiments and proved the efficacy of the filters by mixing the blood to be tested with
known bacteria which the filters retained as usual. At the time he thought that the agent was
nothing more than a very small bacterium, but by 1908, in view of the inability to grow the
agent on artificial media, he now described the agent as ultravisible an obligatory parasite.
In 1901, McFadyeans ultrafiltration results were confirmed by Arnold Theiler in South Africa
and Edmund Nocard in France.

1900 John McFadyean discovery of African horse sickness viruses (the first orbiviruses)
page 84

Hugo Marie de Vries (1848-1935)

Oenothera lamarckiana (Evening Primrose)

drawn by Hugo de Vries

Hugo de Vries was a Dutch botanist who conceived the concept of genes, rediscovering the laws of heredity in the 1890s, while unaware of Gregor Mendels work thirty years
earlier. de Vries developed the laws of dominance and recessiveness, segregation, and independent assortment of inherited characters to explain the 3:1 ratio of phenotypes in
the second generation of his cross-breeding of evening primrose plants. In the late 1890s, de Vries became aware of Mendels obscure paper of 1866 and altered some of his
terminology to match. de Vries Mutation Theory (1900-1903) was one of the chief contenders to explain how evolution works, leading, for example, Thomas Hunt Morgan to
study mutations in the fruit fly this, in turn, stimulated Theodosius Dobzhansky and others to restate their definition of Darwinism in the modern evolutionary synthesis in
the 1930s and 1940s.

1900 Hugo de Vries founding of genetics, rediscovery of Mendels work

page 85

Rockefeller Institute for Medical Research, 1904

John D. Rockefeller Sr. (1839-1937)

and John D. Rockefeller Jr. (1874-1960)

The origins of the Rockefeller Institute for Medical Research lie, in part, in personal tragedy. After John D. Rockefeller Srs grandson died from scarlet fever in January 1901,
he formalized plans to establish the research center he had been discussing for three years with his adviser Frederick T. Gates and his son John D. Rockefeller Jr. At the time
of the institutes founding, infectious diseases such as tuberculosis, diphtheria, typhoid fever and yellow fever were considered the greatest threats to human health. New
research centers in Europe, such as the Institut Pasteur, were successfully applying laboratory science to increase understanding of these and other diseases. Following their
lead, the Rockefeller Institute became the first biomedical research center in the United States. From the beginning, Rockefeller researchers made important contributions to
understanding, preventing and treating disease, and within a few years their impact was felt globally, especially in the emerging discipline of virology.

1901 John D. Rockefeller Sr. founding of the Rockefeller Institute for Medical Research
page 86

Rockefeller Institute for Medical Research

First Board of Directors, Left to right:
Theobald Smith
Hermann Biggs
Simon Flexner (director)
William Welch (chair)
Theophil Prudden
Luther Holt
Christian Herter
Photo ~1909

Simon Flexner (1863-1946)

First director of the Rockefeller Institute

[from Rockefeller Archives, used with permission]

1901 John D. Rockefeller Sr. founding of the Rockefeller Institute for Medical Research

page 87

In 1901, three papers were published, all reporting

successful filtration experiments with the agent of fowl
plague. Two of the papers were by Italians, published
as reports to academies of medicine by Centanni and
Savonuzzi, and Maggiora and Valenti. The third paper
was by the Austrians Lode and Gruber. This almost
simultaneous appearance of three very detailed papers
reflects the rapidly growing interest in general in the
nature of the filterable viruses. Maggiora and Valenti
concluded that the agent behaved as a true virus, which
multiplied in the infected host, and not as a mere toxic
substance. Lode and Gruber considered three distinct
possibilities for the nature of the virus their notion
started out rather as a combination of the concepts of
Loeffler and Frosch and Beijerinck. Soon Lode concluded
from further ultrafiltration experiments using graded
Berkefeld filters that he was dealing with ultrasmall
bacteria, in keeping with the original concept of Loeffler
and Frosch.
Centannis comments were brief and objective: In view
of the incomplete methods currently at our disposal,
we must of course defer the question of whether the
reproduction of this virus involves living organisms or
complex chemical molecules, or even elements belonging
in some transitional area between the two.
During the next ten years, every attempt to isolate
and study unknown pathogens included attempts at
ultrafiltration. Review articles began to appear, recording
the rapidly growing number of known filterable agents.
When Emile Roux published the first review article in
1903 he included a total of nine known viruses. When
his colleague Paul Remlinger reviewed the situation three
years later the number had doubled to eighteen.

Eugenio Centanni (1863-1942)

Centanni E, Savonuzzi E. La peste aviaria I & II. Communicazione fatta
allAccademia delle Scienze mediche e naturali di ferrara, 1901.
Maggiora A, Valenti GL. Su una epizoozia di tifo essudativo dei gallinacei.
Accad Med di Modena, 20 June 1901.
Lode A, Gruber F. Bakteriologische studien fiber die aetiologie einer
epidemische erkrankung der hhner in tirol. Zentbl. Bakt Parasitkde Abt I.
1901;30:593-604.

More than fifty years later the virus of fowl plague was
shown to be an avian influenza A virus.
From: Wilkinson L. The development of the virus concept
as reflected in corpora of studies on individual pathogens.
1. Beginnings at the turn of the century. Medical History
1974;18:211-221.

In 1983 avian influenza virus

(H5N2) (fowl plague virus)
appeared in Pennsylvania, killing
17 million chickens, and costing
more than $65M. The same kind
of mutation to virulence occurred
in the commercial broiler industry
of Mexico in the 1990s and in Asia
from 1995 onward.

1901 Eugenio Centanni, others discovery of fowl plague virus (avian influenza virus, the first orthomyxovirus)
page 88

Aladr Aujeszky (1869-1933)

Aujeszky A. ber eine neue infektionskrankheit bei
haustieren. Zbl Bakt 1 Orig. 1902;32:353-357.
Pseudorabies (or Aujeszkys disease, and in cattle mad itch) is a disease of swine that is endemic in most
parts of the world. It is caused by pseudorabies virus (PRV), which is also called porcine herpesvirus 1
or suid herpesvirus 1. It is considered the most economically important viral disease of swine in areas
where classical swine fever has been eradicated. Aladr Aujeszky, a Hungarian physician and veterinarian,
recovered the virus from dogs, cats, and cattle as well as swine, and discovered that it was transmissible to
rabbits and guinea pigs. He proved it was a virus by ultrafiltration through graded Berkefeld ultrafilters.

1902 Aladr Aujeszky discovery of pseudorabies virus (the first herpesvirus)

page 89

Amde Borrel (1867-1936)

[from Institut Pasteur, used with permission]

Borrel A. Epitheliosee infecteuses et epitheliomas (infectious epithelioses and

epitheliomas). Annales de LInstitut Pasteur 1903;17:81-91.
Borrel A. tude exprimentale de las clavele. Annales de LInstitut Pasteur
1903;18:123-137.

1902 Amde Borrel discovery of sheeppox virus

page 90

A graphic explanation of the

Andy Warhol-like artistic
image of poxviruses: within the
typical complex brick-shaped
virion of all orthopoxviruses,
lies a dumbell-shaped core
containing the viral DNA.
Some idea of the complexity of
the poxvirus virion is indicated
by the fact that it contains more
than 200 proteins.
Sheeppox virus, along with
goatpox and lumpy skin disease
viruses, is responsible for
economically significant diseases
of domestic ruminants in many
areas of Africa and Asia.

Maurice Nicolle, brother of Nobel laureate Charles Nicolle, a staff member of the Institut Pasteur, Paris, went to
Constantinople (Istanbul) in 1893 at the request of the Sultan of the Ottoman Empire, Abdulhamid II. There, he
founded the Imperial Institute of Bacteriology (for human and animal diseases) and became its first director. This was
the third of the 31 Pasteurean institutes founded around the world.
Mustafa Adil Bey was a Turkish veterinarian, a graduate of the Ecole Nationale Vtrinaire dAlfort, who had been
trained by Edmond Isidore tienne Nocard. He was the founder of the Turkish Veterinary Bacteriology Institute.
Nicolle and Adil Bey worked on rinderpest from 1899 onward: they started mass production of bovine antiserum, they
showed varying breed resistance, they defined the incubation period of the infection, etc.
In 1902 they published their ultrafiltration studies, showing that the disease rinderpest was caused by a virus. Nicolle
and Adil Bey also demonstrated the ultrafilterability of vaccinia virus, five year before Negri and others, but their
publication was quite obscure.
Nicolle M, Adil Bey M. Etudes sur la peste bovine, (troisime mmoire). Experiences sur la filtration du virus. Ann Inst
Pasteur 1902;16:56-64.
Nicolle M, Adil Bey M. Sur la nature du virus vaccinal. Comptes Rendus de lAcademie des Sciences. Serie III, Sciences
de la Vie 1906;143:1196.

Maurice Nicolle (1862-1932)

Rinderpest, South Africa, ~1900

Mustafa Adil Bey

(1871-1904)
1902 Maurice Nicolle and Mustafa Adil Bey discovery of rinderpest virus (the first morbillivirus)

page 91

Arnold Theiler (1867-1936)

Bluetongue was first described in South Africa, where it had likely been endemic in wild
ruminants from antiquity. It was recognized when imported Merino sheep were found
to be highly susceptible to infection with fatal consequences. The first detailed studies
were done by James S. H. Spreull (1874-1948) in 1902-1905, when he was a beginning
staff member of the Veterinary Services of the Union of South Africa (founded in 1896;
Arnold Theiler, first Director), Grahamstown, South Africa. The separate Government
Veterinary Laboratory at Onderstepoort (now the ARC-Onderstepoort Veterinary
Institute), was commissioned in 1908; Spreull became an early staff member. and
Theiler its founding director. Spreull: Cause of the Disease [bluetongue]: Repeated
microscopic examinations have revealed no organism in this disease but as the blood
conveys the infection readily upon inoculation into a clean animal we can conclude that
the cause is ultra visible and this is confirmed by the fact that when virulent blood or its
serum only are passed through the Berkefeld filter the filtrate is found to be capable of
reproducing the disease (Robertson and Theiler) [William Robertson (1872-1918)]. Mr.
Dixon [Rowland W. Dixon (-1948)] and I performed this experiment in 1901 with
pipetted [ultrafiltered?] serum the results being exactly similar

Onderstepoort Veterinary Research Institute

founded in 1908, Arnold Theilers statue in front

Spreull J. Malarial catarrhal fever (bluetongue) of sheep in South Africa. J Comp Pathol Ther. 1905;18:321-337.
Theiler A. Bluetongue in sheep. Annual Report of the Director of Agriculture, Transvaal, 1904-1905, 110-121.

1902 James Spreull, Arnold Theiler discovery of bluetongue viruses

page 92

Bluetongue virus

Model: outer capsid layer (blue/red),

inner capsid (grey), one of the ten
segments of its double-stranded RNA
genome (aqua). Tom Goddard, UCSF

Classical swine fever virus

negative contrast electron microscopy

[Micrograph from Frank Weiland]

Emil Alexander de Schweinitz

(1864-1904)

Marion Dorset
(1872-1935)

De Schweinitz EA, Dorset M. New facts concerning the etiology of hog cholera [now called classical swine fever].
Bureau of Animal Industry, USDA, 20th Annual Report, 1903.
The first practical preventive measure used widely to prevent a livestock viral disease was anti-hog-cholera serum
injected together with virulent virus. This invention of Marion Dorset (with D. McBryde and W.B. Niles) was
successfully tested in 1907 and immediately commercialized.

Spleen from infected pig, showing diffuse, acute

necrosis of a lymphoid follicle surrounded by
red pulp hemorrhage, the typical lesion of
classical swine fever (hog cholera)

1903 Emil De Schweinitz, Marion Dorset discovery of classical swine fever virus (the first pestivirus)

page 93

Adelchi Negri (1876-1912)

Negri bodies, brainstem and Purkinje cell, human, Giemsa stain

In 1890, just after Pasteurs studies on rabies, the famous Camillo Golgi, professor of pathology at the University of Pavia, investigated the neuropathology of rabies, without
obtaining significant findings. Adelchi Negri, an assistant pathologist in Golgis laboratory, continued these studies. Using Mann stain, he observed, first in rabbits and then
in rabid dogs, characteristic inclusions in nerve cells of the spinal ganglia and brain. This discovery was presented in 1903 at a meeting of the Societ Medico-Chirurgica of
Pavia. Others immediately found the same inclusions in human rabies cases. Negri was convinced that the inclusions were specific features of rabies, and he believed he had
identified the etiologic agent responsible for the disease as a parasite. The report by Negri in 1903 immediately stimulated a scientific debate. Within a few months Alfonso
di Vestea in Naples and Paul Remlinger and Riffat Bey in Constantinople showed that the etiologic agent of rabies is an ultrafilterable virus. However, Negri tried until 1909
to demonstrate that the intraneuronal inclusions named after him corresponded to different steps of the developmental cycle of a protozoan. Despite his incorrect etiological
hypothesis, Negri had made a discovery of great practical importance his finding provided a breakthrough in the rapid diagnosis of rabies and the detection of Negri bodies
was immediately adopted by all the anti-rabies institutes of the world.

1903 Adelchi Negri discovery of the rabies inclusion bodythe Negri body
page 94

Rabies inclusions (Negri bodies);

remarkable camera lucida
drawings by Adelchi Negri
included in his original paper
(1903).
(a) Cerebral cortex of rabbit subdurally infected
with street virus: death occurred after 19 days.

(b) Ammons Horn, and

(c) Cerebellum, of dogs 19 days after experimental
subdural inoculation with street virus.
(d) Spinal ganglion of rabbit infected in the eye with
street virus: death occurred after 17 days.
Fixation in Zenkers solution. Manns staining.
Reproduced from Negris paper:
Negri A. Contributo allo studio delleziologia della
rabia. Bollettino della Societa Medico-Chirurgica,
Pavia 1903;2:88-115.

1903 Adelchi Negri discovery of the rabies inclusion bodythe Negri body

page 95

Paul Ambroise Remlinger (1871-1964)

[from Institut Pasteur, used with permission]

Remlinger PA, Riffat Bey.

Le virus rabique traverse
la bougie Berkefeld. CR
Soc Biol. 1903;55:730-731.

Rabies virus, negative contrast and thin section electron microscopy,

purified virus and brain of hamster infected with street virus (colorized)

Remlinger PA, Le passage

du virus rabique traverse
les filtres. Ann Inst
Pasteur 1903;17:834-849.

Rifat Bey (Riffat Bey)

The Sultan of the Ottoman State sent a mission to Paris in 1886 to learn how to prepare Pasteurs
rabies vaccine. Upon its return to Constantinople (now Istanbul), a Rabies Vaccine Laboratory (Rabies
Hospital) was established. In 1900, Paul Remlinger, a military physician, who had been at the Institut
Pasteur in Tunis, and had previously trained at the Institut Pasteur, Paris, was appointed director of
the Laboratory, where in succeeding years he carried out many remarkable studies on rabies. In 1902,
Remlinger, Rifat Bey, a Turkish veterinarian who had studied at the Institut Pasteur, Paris, and Hamdi
Efendi, a laboratory assistant, performed a key experiment: they mixed a fixed rabies virus brain
homogenate with fowl cholera bacteria (Pasteurella multocida), put the mixture through a Berkefeld
V ultrafilter, and inoculated the filtrate intracerebrally into rabbits. While the absence of Pasteurella in
the inoculated animals confirmed the success of the ultrafiltration, their later death from rabies made
clear that the etiologic agent of rabies was ultrafilterable. They repeated the experiment using both
fixed and street viruses, and graded Berkefeld and Chamberland filters. Remlinger later served for
many years as a senior staff member of the Institut Pasteur, Paris.

1903 Paul Remlinger, Rifat Bey, Alfonso di Vestea discovery of rabies virus (the first rhabdovirus)
page 96

Alfonso di Vestea (1854-1938)

Rabies virions at margin of a Negri body, hamster brain,
thin section electron microscopy
Remlinger P. Le passage du virus rabique a travers les filtres. Ann Inst Pasteur
1903;17:834-849.
Remlinger PA, Riffat Bey. Le virus rabique traverse la bougie Berkefeld. C R de
la Socit de Biologie. 1903;55:730-731.

Time series: development of Negri bodies

neuroblastoma cells, at 4, 8 and 24 hours post-infection

Di Vestea A. Sul trovato della filtrabilit del virus della rabbia Annali di igiene
sperimentale. 2005;15:147.

1903 Paul Remlinger, Rifat Bey, Alfonso di Vestea discovery of rabies virus (the first rhabdovirus)

page 97

Tiselius original liquid electrophoresis technology

Arne Wilhelm Kaurin Tiselius (1902-1971)

Preparative and analytical methods developed by separation scientists have played an important role
in the history of virology. This started in 1937 with the earliest liquid electrophoresis technique of
Tiselius; however, using liquid electrophoresis to separate molecules based on their charge without
stabilizing media has very poor resolution since molecules (or even virions) that are not stabilized
in a supporting medium, such as a gel, diffuse quickly after initially being separated. Early on, the
Rockefeller Foundations financial assistance brought Tiselius electrophoretic apparatus to selective
American research institutions this was seminal in the birth of molecular biology (and molecular
virology). Immediately, Tiselius understood the early shortcomings of liquid electrophoresis and
had ideas for their resolution: It seems practical to distinguish between boundary electrophoresis
as performed in the common moving boundary apparatus, and zone electrophoresis where the
migration of more or less completely separated zones is studied, usually by application of some
immobilizing medium to prevent convection. All the electrophoretic technologies that we know
today followed first paper electrophoresis, then starch block electrophoresis, but eventually many
gel electrophoresis methods.

1903 Arne Tiselius development of electrophoresis

page 98

Henri Valle (1870-1957)

Equine infectious anemia virus

thin section electron microscopy

Henri Carr (1868-1938)

In their 1904 publication, Henri Carr and

Henri Valle presented remarkable findings
from their experimental studies of equine
infectious anemia done at Alfort. They
showed that the disease was transmissible by
inoculation of blood, that the etiological agent
could be serially passaged in horses, and that
the singular disease in nature appeared in
three quite different forms, acute, subacute and
chronic. This was in an era when many experts
did not think that a single etiologic agent
could cause different clinical syndromes. Most
remarkable was their proof of the nature of the
etiologic agent (from a translation, abridged):
We have shown that the anemia virus in the
horse belongs to the category of microbes,
the so-called invisible microbes, those that
can pass through filters of diatomaceous earth
or porcelain, which hold back the microbes
large enough to be seen under the microscope.
New experiments have shown that regardless
of the time during the infection when blood
is taken, it contains the virus which does
not lose its infectivity even after the fifth
dilution [? five-fold dilution or 10-5 dilution]
before ultrafiltration, and the results are the
same whether using the diatomaceous earth
Berkefeld candles or porcelain Chamberland F
and B candles. These candles, however, retain
a fairly large amount of virus so the incubation
period of the disease produced by inoculation
of filtrates is always longer.... Finally, we have
shown, importantly, that horses that seem
absolutely cured of the chronic form of the
disease retain their infectivity. The inoculation
of blood from one of these horses into a horse
in excellent condition caused disease in its
acute form with death in 27 days. There is
reason to fear that the presence of silent virus
carriers in infected regions is a very big
obstacle to the extinction of the disease by any
prophylactic measures.

Ecole Nationale Vtrinaire DAlfort, ~1900 Carr H, Valle H. Sur lanmie infectieuse du
cheval. CR Acad Sci. 1904;139:331-334.

1904 Henri Valle, Henri Carr discovery of equine infectious anemia virus (the first retrovirus, the first lentivirus)

page 99

Yellow Fever Control / Elimination in Panama

1880: French company headed by Ferdinand de Lesseps breaks ground on

a sea-level canal in Panama.
1881: First construction crews arrive; first workers die of yellow fever.
1884: Work force of 19,000; it is estimated that 12,000 workers die during
the construction of the Panama Railway, and over 22,000 during the
French effort to build a canal.
1888: French abandon sea level canal and begin work on a lock canal.
1889: French canal company dissolved and work on canal stops; new
company formed and makes first excavations in Culebra Cut.

Gorgas in Panama, 1905

1898: New companys capital gone, offer to sell rights to the United States.
1898: At the end of the Spanish-American War, William Gorgas is
appointed Chief Sanitary Officer in Havana, and after the discoveries by
the Yellow Fever Commission he leads the campaign to eradicate yellow
fever from Havana, succeeding by 1902.
1902: President Theodore Roosevelt tries to buy rights to dig canal from
Colombia, but is refused.

Teddy Roosevelt at Pedro

Miguel Lock, 1906

1903: Roosevelt backs Panamanian uprising against Colombia by

stationing warships offshore. Panama declares independence and United
States signs a treaty with Panama allowing it to build canal. The treaty also
creates Canal Zone, a sovereign part of United States.
1904: United States buys French companys rights and properties, begins
construction; maximum workforce, 45,000 by 1911.
1904: Gorgas team arrives in Panama within a month all contract
malaria Gorgas urgent requests for action to eliminate mosquitoes are
ignored by John Findley Wallace, the Chief Engineer in charge of building
the canal.

William Crawford Gorgas (1854-1920)

1905 (March): Yellow fever outbreak causes panic in Panama, most

American canal workers flee and work is virtually halted.

Fumigation of buildings,
Panama, 1906

1905 (July): John Stevens arrives as new Chief Engineer and Gorgas public
health efforts are given top priority. As in Cuba, these efforts include the
draining of ponds and swamps, fumigation of buildings, mosquito netting of
beds, and public water systems to replace open standing water vessels.
1905 (December): Yellow fever eliminated from Panama.
1914: Official opening of Panama Canal S.S. Ancon makes first official
passage.

Draining swamp
Panama, 1911

1904 William Gorgas control of yellow fever, allowing construction of the Panama Canal
page 100

1904

1905

1904 William Gorgas control of yellow fever, allowing construction of the Panama Canal

page 101

Spraying
oil in
ditches,
Panama Canal
1906

Mosquito control gangs, Panama Canal, 1906

SS Ancon, first passage through the Panama Canal, 1914

1904 William Gorgas control of yellow fever, allowing construction of the Panama Canal
page 102

Le Docteur Henri Carr

Carr H. Sur la maladie des jeunes chiens. C R Acad Sci
(Paris) 1905;140:689-690 and 1489-1491.
Dunkin GW, Laidlaw PP. Studies in dog-distemper. I.
Dog-distemper in the ferret. II. Experimental
distemper in the dog. III. The nature of the virus. J Comp
Pathol. 1926;39:201-212, 213-221, 222-230.

Henri Carr (1868-1938)

Patrick Playfair Laidlaw

(1881-1940)

Phylogenetic studies have indicated that measles virus and rinderpest virus diverged during
the 11-12th centuries. The divergence of canine distemper virus seems to have occurred more
recently, with the first case described in 1905 by Henri Carr at Alfort, who then showed that
the infectious agent was ultrafilterable. It is now known that the virus affects all populations
of the domestic dog and importantly many species of wildlife. The virus contributed to the
near-extinction of the black-footed ferret and to the extinction of the Tasmanian tiger and
one species of African wild dog. The initial major canine distemper virus research program
was developed at the National Institute for Medical Research-Mill Hill (NIMR)(and later at
the Baker Institute for Animal Health at Cornell University). Rick Carver and John Skehel, in
their Mill Hill Essay, described that canine distemper research was central to the mission of
the NIMR from its founding in 1913. Contributions from dog owners through an appeal from
The Field magazine, allowed pathologist Patrick Laidlaw and veterinarian George Dunkin to
confirm Carrs work and by 1929 to develop the first protective vaccine. One key to the Mill
Hill success was the realization that dogs used in experiments had to be bred and maintained in
strict isolation to avoid introduced infection and that unique facilities were required to achieve
this Mill Hill was the site of the first high containment research animal facility. The NIMR
interest in canine distemper was also based on its similarity to human diseases such as measles
and influenza, just at the time of great measles outbreaks and the 1918 influenza pandemic.

[George W. Dunkin, MRCVS, was a staff member of the

National Institute of Medical Research, Mill Hill, and
later director of the Agricultural Research Council of
the UK and President of the Royal Society of Medicine,
Section on Comparative Medicine. No photo was found.]

Canine distemper virus

dog, trachea, intracytoplasmic inclusions

1905 Henri Carr, Patrick Laidlaw, George Dunkin discovery of canine distemper virus

page 103

Proof that Variola and Vaccinia are

Viruses:
Finding the initial proof of the viral nature of variola
and vaccinia, by passage through Chamberland
or Berkefeld filters, was difficult there are
contradictory, incomplete citations, many without
formal references, in the older literature. By ~1912,
many statements were being made that make it
seem as if the proof was common knowledge and
did not need referencing. Nothing was found in
newer literature to resolve this. In any case, quite a
few different investigators are mentioned by various
authors in the older literature:

Adelchi Negri
(1876-1912)

Negri A. Esperienze sulla filtrazione del virus

vaccinico. Nora prima. Gazzetta Medica Italiana,
Anno LVI 1905;13-46.

Paul Remlinger
(1871-1964)

Negri A. ber filtration des vaccinevirus. Zeitschrift

fr Hygiene und Infektionskrankheiten 1906;54:327346.
Remlinger P, Nouri-Osman. Le virus vaccinal
traverse la bougiede Berkefeld V. C R Societe de
Biologie, Paris, 1905, and, Sur le passage du
virus vaccinal travers la bougiede Berkefeld V. C R
Societe de Biologie, Paris, 1905.
von Prowazek S. Untersuchungen ber das wesen
des vaccineerregers. Deutsche Med Wochenschr S.
1905;19:752.
Siegel J. Untersuchungen ber die tiologie der
pocken und der maul-und-klauenseuche. Abhandl d
knigl preu. Akademie d Wissenschaften 1, 1905.
Rouget J. Contribution ltude du virus vaccinal. C
R Acad Sci Hebd Seances Acad Sci. Nr. 21, 1905.
Nicolle M, Adil Bey M. Sur la nature du virus
vaccinal. C R lAcad des Sci. 1906;143: 1196.

Maurice Nicolle
(1862-1932)

Stanislaus von Prowazek

(1875-1915)

And others, cited but without specific references:

Carini, Volpino, Paschen, Casagrandi, Galli, Valerio.

Variola virus

chorioallantoic membrane of embryonating

egg, thin section electron microscopy

1905 Adelchi Negri, Paul Remlinger, Stanislaus von Prowazek, others proof that variola and vaccinia are viruses
page 104

Ross Granville Harrison (1870-1959)

Diagram by P. R. White to illustrate the history of tissue

culture and relationships between workers in the field

From: Witkowski JA. Alexis Carrel and the mysticism of tissue culture.
Medical History 1979;23:279-296.
Using expert surgical and aseptic technique developed from successfully operating on tiny frog embryos, Ross Harrison explanted embryonic frog neural tube fragments into
a drop of fresh frog lymph on a sterile coverslip. Once the lymph clotted, he inverted the coverslip over the well in a glass depression slide creating a hanging drop culture, a
technique previously used by microbiologists for studying bacteria. He then watched the development of frog nerve fibers from the neurons in the explanted tissue. In doing
so, he solved several basic cell culture problems: medium, culture vessel, visualization and culture contamination (which he did encounter). Because they were relatively shortterm cultures he was not faced with the problems of feeding or subculturing. As he stated: The immediate object of the experiments was to obtain a method by which the end
of a growing nerve could be brought under direct observation while alive, in order that a correct conception might be had regarding what takes place as the nerve fiber extends
during embryonic development from the nerve center out to the periphery. In 2004, Haig Keshishian stated: Ross Harrisons [1910 paper] is likely the most important paper
ever published in the Journal of Experimental Zoology.... In a single stroke Harrison invented the method of tissue culture, and then used it to prove the neuron doctrine.
(Journal of Experimental Zoology 2004;301a:201-203.)
Harrison R. Observations on the living developing nerve fiber. Anat Rec. 1907;1:116-128. Read before the Society for Experimental Biology and Medicine, New York, 1907.
Harrison R. The outgrowth of the nerve fiber as a mode of protoplasmic movement. J Exp Zool. 1910;9:787-846.

1907 Ross Harrison development of the first cell cultures (nerve fibers from frog embryo tissues)

page 105

Percy Moreau Ashburn

(1872-1940)

Charles Franklin Craig

(1872-1950)

Susumu Hotta
(19182011)

Percy Ashburn and Charles Craig, working in Manila in 1906-1907, passed diluted blood from febrile dengue patients through Berkefeld ultrafilters and injected the filtrates
into healthy volunteers. They showed that the disease was caused by an ultrafilterable agent, a virus, the second human virus. In 1943, Ren Kimura and Susumu Hotta isolated
dengue virus 1 from patients during a dengue epidemic in Nagasaki, Japan. Hotta attributed his success to intracerebral inoculation of suckling mice, while others were
inoculating chick embryos or other animals. In 1944, Albert Sabin and Walter Schlesinger independently isolated dengue virus 1 from blood specimens from Hawaii and
Calcutta, India, and isolated dengue 2 virus from specimens from Papua New Guinea and Indonesia. Dengue viruses 3 and 4 were first isolated by William Hammon, Albert
Rudnick and Gladys Sather in the Philippines in 1960. Dengue 5 virus was
discovered in Sarawak (Malaysia) by Nikos Vasilakis and colleagues in 2013.
Ashburn PM, Craig CF. Experimental investigations regarding the etiology of
dengue fever. J Infect Dis. 1907;4:440-475.
Kimura R, Hotta S. Studies on dengue fever (VI). On the inoculation of dengue
virus into mice (in Japanese). Nippon Igaku. 1944;3e79:629; and Hotta S,
Kimura R, 1952. Experimental studies on dengue I. Isolation, identification and
modification of the virus. J Infect Dis 90: 19.
Sabin AB, Schlesinger RW, 1945. Production of immunity to dengue with virus
modified by propagation in mice. Science 101: 640642.
Dengue: geographic occurrence (green=consensus on dengue absence; red=dengue presence) Bhatt S, Gething PW, Brady
OJ, Messina JP, Farlow AW, Moyes CL, Drake JM, Brownstein JS, Hoen AG, Sankoh O, Myers MF, George DB, Jaenisch T, Wint
GR, Simmons CP, Scott TW, Farrar JJ, Hay SI. The global distribution and burden of dengue. Nature. 2013;496:504-507.

Hammon WM, Rudnick A, Sather GE, 1960. Viruses associated with epidemic
hemorrhagic fevers of the Philippines and Thailand. Science 131: 11021103.

1907> Percy Ashburn Charles Craig Susumu Hotta Albert Sabin William Hammon discovery of dengue viruses
page 106

Papillomavirus, negative contrast and thin section electron microscopy

Giuseppe Ciuffo, from the Fourth Italian Congress of Pathology, Paris, 1906: With a series of experiments...
I sought to clarify the problem of the etiology, pathogenicity and contagiousness of cutaneous or mucosal
papillomatous forms, such as the common wart and condyloma acuminatum, which are assuredly infectious. My
principal aim is to be able to find which is the specific microscopic germ or invisible virus that is responsible for
these lesions and what are its characteristics, its mode of transmission from man to man or to animals and finally
to determine whether a comparative clinical, anatomical and bacteriological examination could result in the
identification of the responsible agent. He proceeded to produce a wart on his own hand by the inoculation of a
wart extract which had been ultrafiltered through a Berkefeld N candle!
Ciuffo G. Innesto positivo con filtrado di verrucae volgare. Ital Mal Venereol. 1907;48:12-15.

1907 Giuseppe Ciuffo discovery of human papillomaviruses (the first papillomaviruses)

page 107

 IMMATURE

MATURE

Vilhelm Ellerman (1871-1924)

Oluf Bang (1881-1937)

Vilhelm Ellerman and Oluf Bang, in 1908, demonstrated that an extract ultrafiltered through a
Berkefeld filter could transmit leukemia among chickens. However, the scientific community largely
ignored this finding, as leukemia was not recognized as a neoplastic disease until after 1930. Peyton
Rous work two years later, although not ignored, was met with some indifference. The prevailing
attitude was that tumors that could only grow in chickens must bear little resemblance to human
cancers (which were thought to be noninfectious in origin) and were unlikely to provide any useful
information. Rous was eventually vindicated and his discovery earned him a Nobel Prize in 1966 (he
noted the work of Ellerman and Bang in his Nobel lecture).
Ellerman V, Bang O. Experimentelle leukemia im huhnem. Zentralbl Bakteriol Parasitenkd
Infektionskr Hyg. Abt. I (Orig.). 1908;46:595-609.

1908 Vilhelm Ellerman, Oluf Bang discovery of avian leukemia viruses (the first leukemia virus)
page 108

Karl Landsteiner (1868-1943)

Several investigators had, early

on, attempted to isolate an
etiologic bacterium from tissues
from poliomyelitis victims, but
all bacteriological findings were
negative; likewise, several attempts
to transmit the disease to the
usual laboratory animals, such
as rabbits, guinea pigs, and mice,
failed. In 1908, Karl Landsteiner
and Erwin Popper (1879-1955)
injected intraperitoneally a
suspension of spinal cord from a
9-year-old boy who had died of
poliomyelitis into two nonhuman
primates, a Hamadryas baboon
(Papio hamadryas) and a rhesus
macaque (Macaca mulatta). The
inoculated material, which was
bacteriologically sterile, yielded
negative results when injected into First electron micrograph of poliovirus , shadow-cast technique,
rabbits, guinea pigs, and mice. The 1953, RCA Laboratories, details unknown, from Sarnoff Library
two nonhuman primates, however,
exhibited lesions in the spinal cord,
medulla, pons, and brain stem that
were indistinguishable from those
in cases of human poliomyelitis.
The macaque developed complete
flaccid paralysis of both legs.
Landsteiner and Popper were
unable to passage the agent, but
this was achieved independently
in 1909 by others. In 1909,
Landsteiner and Constantin
Levaditi of the Institut Pasteur
successfully passed a suspension
of spinal cord material from a
poliomyelitis case through a
Berkefeld type V ultrafilter.

Landsteiner K, Popper E. Uebertragung der poliomyelitis acuta auf affen. Z Immunittsforsch.

1909;2:377-390.
Landsteiner K, Levaditi C. La transmission de la paralysie infantile aux singes. C R Soc Biol.
1909;67:592-594.
Poliovirus 1, negative contrast electron microscopy
of purified virus, Joseph Esposito, ~1975

1909 Karl Landsteiner, Erwin Popper discovery of polioviruses (the first enteroviruses)

page 109

Constantin Levaditi (1874-1953)

With Karl Landsteiner, Levaditi also discovered the presence of poliovirus

in tissues other than those of the nervous system. He isolated poliovirus in
tissue explants. He authored the first monograph dedicated to the disease, La
Poliomylite aigu pidmique (1913).

Karl Landsteiner (1868-1943)

In 1900 Landsteiner discovered that mixing the blood of two people
usually causes the erythrocytes to agglutinate, and in 1901 he found
that this effect was due to contact of the erythrocytes of one person
with the serum of the other. As a result he succeeded in identifying
the three major human blood groups A, B and O. Landsteiner
also found out that blood transfusion between persons with the
same blood group did not lead to agglutination and destruction of
erythrocytes, whereas this occurred between persons of different
blood groups. Based on his findings, in 1907 the first successful
blood transfusion was performed by Reuben Ottenberg at Mount
Sinai Hospital in New York. In 1930 Landsteiner was awarded
the Nobel Prize in Physiology or Medicine in recognition of these
achievements.
Two of many styles of computer modeling of the poliovirus virion surface

1909 Constantin Levaditi, Karl Landsteiner discovery of poliovirus in non-nervous system tissues
page 110

Global distribution of sandfly (phlebotomus) fever

Robert Doerr (1871-1952)

From the Transactions of the Society of Tropical Medicine and Hygiene, London, 1913 (abridged): In the year 1908, Doerr [Robert Doerr, K. Franz, S. Taussig] announced the
results of his experiments on the infectivity of the blood of the Dalmatian summer fever, and on the mode of the transmission of the virus by the phlebotomus [midge]. Since
then the closely related ailments in Malta [and Italy and Crete] have been investigated in a similar manner. It has been found that : (1) The subcutaneous injection of blood
or serum withdrawn during the first 24 hours of the patients illness caused fever 19 times. (2) Inoculation with the filtrate obtained by passing the diluted blood through a
porcelain candle, which retained the micrococcus melitensis, excited the disease 14 times. (3) Feeding experiments with infected sandflies were successful on 21 occasions.
This evidence is sufficient to show that the fever is specific, and that it is caused by a filter-passing virus, which circulates in the blood during the first day of the illness, and that
it is conveyed by the phlebotomus [midge]. During World War II, sandfly fever affected large numbers of U.S. Army personnel serving in Mediterranean operations. Study of
patients led to a clinical description, confirmation of the vector (Phlebotomus papatasii) and the distinction of the two viruses, namely sandfly fever Sicilian virus and sandfly
fever Naples virus.
Doerr R, Franz K, Taussig S. Das pappatacifieber. Leipzig und Wien: Franz Deuticke; 1909.

1909 Robert Doerr, K. Franz, S. Taussig discovery of sandfly (phlebotomus) fever viruses (the first phleboviruses)

page 111

The Fly Room, Columbia University

Thomas Hunt Morgan (1866-1945)

Nobel laureate Eric Kandel wrote of Morgan: Much as Darwins
insights into the evolution of animal species first gave coherence
to nineteenth century biology as a descriptive science, Morgans
findings about genes and their location on chromosomes helped
transform biology into an experimental science.

Thomas Hunt Morgan had become interested in species variation, and in 1911,
he established the Fly Room at Columbia University to determine how a species
changed over time. For the next 17 years, in this 16x23 ft. room, described by many as
cramped, dusty, smelly and cockroach ridden, Morgan and his students did groundbreaking genetic research using millions of fruit flies (Drosophila melanogaster),
and a combination of statistical-genetic methods (per Mendel) and microscopy
to characterize mutations. Though initially against the idea that the behavior of
chromosomes can explain inheritance, Morgan became the leading supporter of the
idea. Morgan and his students (Alfred Sturtevant, Calvin Bridges, Hermann Muller
and others), developed the ideas, and provided the proof for the chromosomal
theory of heredity, genetic linkage, chromosomal crossing over and non-disjunction
(the occasional failure of homologous chromosomes to segregate during and after meiosis). In 1915, they
published a seminal book, The Mechanism of Mendelian Heredity (Henry Holt and Company, New York,
1915). Its major tenets regarding the functions of the chromosomes as the bearers of heredity were:
1. Discrete pairs of factors located linearly on chromosomes like beads on a string bear hereditary
information. These factorsMorgan would soon call them genessegregate in germ cells and
recombine during reproduction, essentially as predicted by Mendelian laws. However:
2. Certain characteristics are sex-linkedthat is, occur together because they are located on the same
chromosome that determines gender. More generally:
3. Other characteristics are also sometimes associated because, as paired chromosomes separate during
germ cell division, genes proximate to one another tend to remain together. But sometimes, as a
mechanistic consequence of reproduction, this linkage between genes is broken, allowing for new
combinations of traits.
These rigorously proven tenants revolutionized our understanding of the processes of life, and of the cause
of many diseases. By demonstrating that genes are the driving forces for the evolution of all life forms,
Morgan established the correctness of Darwins hypothesis put forward in 1859.

1910> Thomas Hunt Morgan discovery of the nature and role of the chromosome (using Drosophila)
page 112

John F. Anderson joined the Marine Hospital Service in 1898, and

in 1909 was appointed director of the Hygienic Laboratory, the
predecessor of the National Institutes of Health. He was among the
early scientists who made this laboratory well-known in scientific
circles.
Joseph Goldberger joined the U.S. Public Health Service in 1899.
From 1902-1906, he held a variety of epidemiology postsin Mexico,
Puerto Rico, Mississippi, Louisiana, all in efforts to combat yellow
fever, typhus, dengue, and typhoid fever. He survived yellow fever and
dengue infections and almost died of typhus.
In 1911, Goldberger and Anderson transmitted measles to monkeys,
showed that it was caused by an ultrafilterable virus, and that infection
was transmitted from nasal and throat secretions. Their 1912 paper
was a landmark. Until then, there had been no experimental system
in which measles virus had been demonstrated to replicate or to
produce a clinical disease similar to that in humans. Indeed, it was
not until 1954, 43 years later, that measles virus was first propagated
successfully in the laboratory in cell culture by John Enders and his
colleagues. Goldberger and Anderson were successful in producing
a febrile response and varying degrees and extent of rash and they
successfully passed the virus to further monkeys. Their experimental
protocols and the descriptions of their work were remarkably
thorough, a model for future investigations.
Anderson JF, Goldberger J. Recent advances in our knowledge of
measles. Am J Dis Children 1912;4:20-26.

John F. Anderson (1873-1958) and Joseph Goldberger (1874-1929)

Multinucleated giant cells (syncytia), typical of
measles virus infection in cell culture

1911 John Anderson, Joseph Goldberger discovery of measles virus

page 113

Joseph Goldberger, middle row, fourth from left.

Joseph Goldberger (1874-1929)

In 1914, Joseph Goldberger was asked by US Surgeon General Rupert Blue to investigate pellagra,
an endemic disease in the Southern U.S. Goldbergers theory that pellagra was associated with diet
contradicted the then commonly-held medical opinion that pellagra was an infectious disease. After
multiple restricted-diet experiments with groups of volunteers spanning several years, Goldberger
was able to demonstrate that individuals who consumed heavily corn-based diets (to the virtual
exclusion of other foods) were at a greatly increased risk of contracting pellagra. Despite his careful
experiments, Goldbergers discovery proved socially and politically untenable and he made little
progress in securing support for treating pellagra. However, with further research, in 1926 Goldberger
was able to demonstrate that a vitamin B deficiency was the cause of pellagra. Conrad Elvehjem, in
1937, discovered the specific deficitpellagra is caused by a lack of the B vitamin niacin along with
insufficient levels of the essential amino acid tryptophan.
Goldberger was nominated five times for the Nobel Prize for his work on the etiology of pellagra.
Unfortunately, he died from cancer in January 1929, so his nomination was disqualified. Christiaan
Eijkman and Frederick G. Hopkins, who were selected for the Nobel Prize later that year for their work
on vitamins, stated that Goldberger would have been a most deserving companion.

1911 John Anderson, Joseph Goldberger discovery of measles virus

page 114

Francis Peyton Rous (1879-1970)

Joseph Willis Beard (1906-1983)

Joseph Beard and his wife Dorothy

with chickens used for research

Peyton Rous, Nobel Lecture, 1966: In 1910 I described a malignant chicken sarcoma which could be propagated by transplanting its
cells, these multiplying in their new hosts and forming new tumors of the same sort. In other ways the growth showed itself to be a
neoplasm of a classical sort, yet, as reported in 1911, its cells yielded a causative virus. Numerous workers had already tried by then
to get extraneous causes from transplanted mouse and rat tumors but the transferred cells had held their secret close. Hence the
findings with the sarcoma were met with down-right disbelief, though soon several other, morphologically different, spontaneous
chicken tumors were propagated by transplantation and from each a virus was got causing growths of its kind. Not until after some
15 years of disputation amongst oncologists were the findings with chickens deemed valid, and then they were relegated to a category
distinct from that of mammals because from them no viruses could be obtained. Only in 1925, through the efforts of a British worker,
W.E. Gye, was much attention given them by scientists. The virus causing the chicken sarcoma first studied, now generally termed
RSV, has been maintained for more than fifty-five years and is still studied in many countries.

1911 Francis Peyton Rous, Joseph Beard discovery of Rous sarcoma virus (first solid tumor virus)

page 115

Nobel Laureates Who Laid the Foundation

for the Use of Radioisotopes in Virology
1901
1903


1906
1908
1911
1918
1921
1921
1922
1924
1927

1934
1935

1935

Hevesy G. The absorption

and translocation of lead by
plants: a contribution to the
application of the method of
radioactive indicators in the
investigation of the change of
substance in plants. Biochem J.
1923;17: 439-445.

Wilhelm Rntgen
Henri Becquerel
Marie Curie
Pierre Curie
Joseph Thomson
Ernest Rutherford
Marie Curie
Max Planck
Frederick Soddy
Albert Einstein
Niels Bohr
William Einthoven
Arthur Compton
Charles Wilson
Harold Urey
Frederic Joliot
Irene Joliot-Curie
James Chadwick

1936
1938
1939
1943
1944
1946
1951

1956


1959
1961
1968
1977

Carl Anderson
Enrico Fermi
Ernest Lawrence
George de Hevesy
Otto Hahn
Hermann Muller
Glenn Seaborg
Edwin McMillan
John Bardeen
Walter Brattain
William Shockley
Emilio Segre
Robert Hofstadter
Luis Alvarez
Rosalyn Yalow
Roger Guillemin
Andrew Schally

Isotopes Commonly Used For Biological Radiolabelling

Isotope

Half Life

Emission Energy

Detection on Gel

Beta Emitters:
3

12.4 years

very low

Fluorography

14

5740 years

moderate

Autoradiography

35

87.4 days

moderate

Autoradiography

33

25 days

moderate

Autoradiography

32

14.3 days

high

Intensifying Screen

80 days

high

Intensifying Screen

C
S
P
P

Gamma Emitters:

George de Hevesy (1885-1966)

125

George de Hevesys introduction to practical radiochemistry came in Ernest Rutherfords laboratories at the University of Manchester. His work there, and in Vienna,
Budapest and Copenhagen, centered on the use of radium and lead isotopes. In 1923, in Copenhagen, he and Dirk Coster discovered the element hafnium (Latin Hafnia for
Copenhagen) in Mendeleevs periodic table a chemical element with 72 protons was missing and they concluded that there must be such an element a mineralogical
museum furnished the material for the research and characteristic X-ray spectra indicated that a new element was present this earned him the 1943 Nobel Prize in
Chemistry. [When Denmark was invaded in World War II, de Hevesy dissolved the gold Nobel Prizes of Max von Laue and James Franck with aqua regia (a mixture nitric
acid and hydrochloric acid) to prevent the Nazis from stealing them. He placed the resulting solution on a shelf in his laboratory at the Niels Bohr Institute. After the war, he
returned to find the solution undisturbed and precipitated the gold out of the acid. The Nobel Society then recast the Nobel Prizes using the original gold.] In 1923, De Hevesy
also started his pioneering work on the use of radioisotopic tracers to study metabolic processes of plants and animals he published the first study on the use of the naturally
radioactive 212Pb as a tracer to follow the absorption and translocation of atoms in the roots, stems and leaves of the broad bean plant. Much similar tracer biology followed.

1911> George de Hevesy, others development of radioisotopic labeling

page 116

Institut Pasteur, Kasauli, India. Patients waiting for post-exposure rabies

treatment, ~1910
Infected brain tissue placed on mosquito netting,
then suspended in bottle over ether fumes for drying

David Semple (1856-1937)

Emulsion of dried infected brain tissue being put through

a strainer, near final step in vaccine production
Institut Pasteur, Kasauli, ~1910. Wellcome Images

At the end of the 19th century, there was a massive epizootic of canine rabies in Europe; in 1878, there were 500 cases of canine rabies in Paris
alone. In the year following the vaccination of Joseph Meister, more than 600 people were treated with Pasteurs vaccine in Paris. Shortly thereafter, Pasteur Institutes were built in many cities around the world; many thousands of doses of vaccine were produced and many thousands of
people were treated. As this occurred, several changes were made in the vaccine and vaccination schedule, often without convincing evidence of
improved efficacy or safety. By the turn of the 20th century various inactivating agents were being added to the vaccine, mostly to overcome rising
concerns about safety, but again without much proof of merit. This changed with the work of David Semple, a British Army medical officer who
in 1900 founded the Pasteur Institute (later the Central Research Institute, CRI) at Kasauli in the Indian state of Himachal Pradesh. During 19031904 Semple treated 200 patients with antirabies serums and Pasteur vaccine. In 1911 he developed a rabies vaccine prepared from brain tissue
of virus-infected rabbits and later sheep inactivated with 0.5 percent carbolic acid. Over the years, the inactivating agent was changed to phenol,
then to formalin, and lastly to beta-propiolactone, but all the while it was the most commonly used rabies vaccine throughout the world. This
only changed at the beginning of the 21st century with the extension of cell culture vaccine production to most developing countries.
Semple noted three advantages of his vaccine: (1) It was safe in terms of vaccine-derived rabies disease, an isue of great concern in France (laboratory rabies, rage du laboratoire, much later shown to be an anti-myelin autoimmune disease): knowing that it is a dead vaccine we can dismiss
any doubts as to the possibility of its producing the disease which it is intended to prevent. (2) It could be sent to distant places in the Indian
empire without reducing its efficiencythis was considered a most important advance, given the tropical climate of most of the subcontinent. (3)
Vaccine production could be standardized. Vaccine could be produced at one central Pasteur Institute and then sent to the rest of India. Centralized production and decentralized delivery also had an impact on awareness about the disease and treatment. In turn, post-exposure rabies
vaccination became a key to rabies control programs based upon stray animal control, animal vaccination and public education. It also serves as a
clear example of how medical science can become ossified, in many places still based upon a 14-21 dose post-exposure regimen 75 years later.

1911 David Semple development of stable inactivated rabies vaccine

page 117

Two technicians at work in Alexis Carrels laboratory at the Rockefeller Institute, ~1936. They are dressed in the fulllength, black, hooded gowns adopted by Carrel. Between them is a rack of Carrel flasks that are being filled by the
technician on the right while the technician on the left is flaming rubber bungs prior to sealing the flasks. Carrels critics
continued: Tissue culture, although a delicate and exacting technique and one in which vigorous asepsis is absolutely
essential, has gained a spurious and unfortunate reputation for difficulty and almost for mysticism. From: Witkowski JA.
Alexis Carrel and the mysticism of tissue culture. Med Hist. 1979;23:279-296.
From Time magazine, 1938: A secluded labyrinth of black, dustless, germless laboratories
zigzags across the top floor of the main building of the Rockefeller Institute for Medical
Research in Manhattan. Black are the floors, black the furniture, dark grey the windowless
walls, shadowless the bleak illumination that comes through the skylights. Entrance to this
aseptic, dustless, reflectionless hideaway is by a spiral staircase from an anteroom on the
floor below. Only scientists particularly interested in fractioning life to its lowest common
denominators may mount that spiral. And all must wash their hands and faces, put on
gowns and hoods of black clothall except the master of this pure and dark domain, master
of its purified and black-clad servants. He, the most famed member of the Rockefeller
Institute, Dr. Alexis Carrel, distinguishes himself from the others with a little white
headpiece that looks like the hat of a U.S. bluejacket [U.S. Navy sailor].

1912> Alexis Carrel, Hugh and Mary Maitland, others development of many cell and tissue culture methods
page 118

Extending from his work

with cell/tissue culture,
Alexis Carrel worked with
Charles Lindbergh in the
mid-1930s to create a
perfusion pump, to allow
a diseased organ to be
removed, kept alive while
being repaired, and then
returned to the patient.
They thought that if this
process could be repeated
forever, it could potentially
render humans immortal.

Alexis Carrel (1873-1944)

In 1907, Ross Harrison of Yale University published a short note entitled Observations on the Living Developing Nerve Fibre, that aroused widespread interest, including by
Alexis Carrel and his colleagues at the Rockefeller Institute. Carrel became a pioneer in the development of tissue culture and also became its chief publicist. He made a
number of practical technical contributions, but more so greatly influenced his contemporaries. His tissue culture techniques were influenced by his surgical expertise and
they became increasingly complicated. Critics said that Carrels work, caused the method to be wrapped up from the beginning in a considerable cocoon of mumbo-jumbo,
derived from the practices that were prevalent at that time in the operating theatres of the world. Tissue culture, although a delicate and exacting technique and one in
which vigorous asepsis is absolutely essential, is gaining a spurious and unfortunate reputation for difficulty and almost for mysticism. Nevertheless, Carrel grew cells without
antibiotics for many years, which was in itself a considerable technical feat his tissue culture (explant culture) of a chicken heart embryo is said to have survived 34 years,
although the veracity of this claim is doubted by statements that chick heart cells were used in the constantly replaced nutritional medium. [Every year newspapers would run
a Happy Birthday column, celebrating the life of the culture.] Carrels development of more and more complexity in techniques dominated the field and led to a decline in
interest in tissue culture, which persisted for many years, until others developed simpler methods. The Nobel Prize in Physiology or Medicine of 1912 was awarded to Alexis
Carrel in recognition of his work on vascular sutures and the transplantation of blood vessels and organs, not for his later work with cell/tissue culture.
Abridged from: Witkowski JA. Alexis Carrel and the mysticism of tissue culture. Med Hist. 1979;23:279-296.

1912> Alexis Carrel, Hugh and Mary Maitland, others development of many cell and tissue culture methods

page 119

Hugh Bethune Maitland

(1895-1972)

Except for the work of Alexis Carrel and his colleagues at the Rockefeller Institute, cell culture had little impact on virology
research prior to the 1940s. However, key technical developments were made all along, most importantly, in 1928, by Hugh
Bethune Maitland and Mary Cowan Maitland, who devised a simplified method of growing vaccinia and a few other viruses
in suspension cultures of fresh minced adult chicken kidneys, in flasks, using a substrate of chicken serum and organic salts.
Thomas Rivers, Max Theiler and their colleagues introduced further refinements to the Maitlands technique and throughout
the 1930s demonstrated the cultivation of fowlpox, foot-and-mouth disease and yellow fever viruses, and the etiologic agent of
epidemic typhus, Rickettsia prowazekii. In 1936, George Gey developed roller tube culture methods, which became standard
for years. Albert Sabin and Peter Olitsky, in 1936, successfully cultivated poliovirus in human embryonic brain tissue and
there were other pioneering successes, but there was no general usage until the breakthroughs made in the laboratory of John
Enders. In 1948, Thomas Weller, Frederick Robbins and Enders successfully propagated mumps and influenza A viruses,
using a modification of the Maitlands method, before moving on to polio and measles viruses. One key to the successes
in the Enders laboratory was the addition of penicillin and streptomycin to culture media. Others rapidly followed these
initial, landmark successes. Enders coined the term cytopathic effect (CPE), that is, the visualization by low magnification
microscopy of cell death, a key to the practicality of titrating and serotyping viruses. Colorimetric indicators of pH change of
media caused by cell death were also introduced at this time, again making practical large surveys of the presence of viruses
and/or antibodies. Minimum Essential Medium (MEM), developed in 1959 by Harry Eagle, was a monumental breakthrough;
it still is one of the most widely used of all synthetic cell culture media. Early attempts to cultivate normal mammalian
fibroblasts and certain subtypes of HeLa cells revealed that they had specific nutritional requirements that could not be met
by Eagles initial Basal Medium (BME) additions to BME were made to aid growth of a wider variety of fastidious cells. MEM
incorporated the first of these modifications, included higher concentrations of amino acids so that the medium more closely
approximated the protein composition of mammalian cells. Since Eagles pioneering work, many, many different media have
been developed, most stemming from research into the nutritional requirements of particular cells, some for commercial
proprietary purposes, and some to meet the requirements of different culture systems (e.g., suspension culture systems).
Composition of a Medium Suitable for Cultivation of Mammalian Cells [1960s]
Amino acids
Arginine
Cystine
Glutamine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Trytophan
Tyrosine
Valine

Vitamins
biotin
choline
folate
nicotinamide
pantothenate
pyridoxal
thiamine
riboflavin

Salts
NaCl
KCl
NaH2PO4
NaHCO3
CaCl2
MgCl2

Other
glucose
penicillin
streptomycin
phenol red
whole serum

Proteins
(Serum-Free Media)
insulin
transferrin
specific growth factors

Maitland HB, Maitland MC. Cultivation of vaccinia virus without tissue culture.
Lancet 1928;2:596-597.
Eagle H. Nutritional needs of mammalian cells in culture. Science 1955;122:501-504.

Mary Logan Cowan Maitland

(~1896-1972)

Harry Eagle
(1905-1992)

Enders JF, Weller TH, Robbins FC. Cultivation of the Lansing strain of poliomyelitis
virus in cultures of various human embryonic tissues. Science 1949;109:85-87.

1912> Alexis Carrel, Hugh and Mary Maitland, others development of many cell and tissue culture methods
page 120

First cultivation of a virus in cell culture: vaccinia

virus (cellular outgrowths from explants of rabbit
and guinea pig cornea) and first demonstration
of virus neutralization (vaccinia virus, rabbit
corneal epithelium cell culture, and serum from
previously infected vs. uninfected rabbits)

Primary rabbit cornea cells

Edna Steinhardt Harde (1874-1941)

(photo from 1920 passport application)

Steinhardt E, Israeli C, Lambert RA. Studies on

the cultivation of the virus of vaccinia. J Infect
Dis. 1913;13:294-300.
Edna Steinhardt Harde is perhaps the most
accomplished virologist / microbiologist
to remain largely unrecognized. She led an
incredibly engaged, adventuresome and creative
life including being torpedoed while crossing
the English Channel during World War I on her
way to serve with the American Red Cross in
France and later with the Institut Pasteur she
deserves a full biography!

Robert A. Lambert (1883-1960)

[Rockefeller Archives, used with permission]

No photo of Clara Israeli was found.

1913 Edna Steinhardt Harde, Clara Israeli, Robert Lambert first cultivation of a virus in a cell culture (vaccinia)

page 121

1913 Edna Steinhardt Harde, Clara Israeli, Robert Lambert first cultivation of a virus in a cell culture (vaccinia)
page 122

Edna Steinhardt Harde (1874-1941)

Edna Steinhardt Harde is perhaps the best example of the many
accomplished virologists / microbiologists who made very important
contributions at crucial times in the development of the science of
virology, yet remain largely unrecognized today. Adding further details
about Edna Harde here is meant as a placeholder for the many others,
only a few of whom are particularized in this book.
Edna Harde, with Clara Israeli and Robert A. Lambert, in 1913-1914,
made two seminal discoveries while at the Research Laboratory, Board
of Health, and the Pathological Laboratory, College of Physicians and
Surgeons, New York City: (1) they were the first to grow a virus in cell
culture (vaccinia virus in rabbit and guinea pig corneal cells), and (2) they
were the first to demonstrate experimentally virus neutralization (vaccinia
virus, rabbit and guinea pig corneal cells and serum from infected/
recovered vs. uninfected rabbits).
But, beyond this, she led an incredibly engaged, adventuresome and
creative life. Unraveling this has been quite a difficult task, partly
because she changed her name in mid-career, not because of marriage,
but in concert with her father and the rest of her family. At the start
of World War I, in a wave of super-patriotism, German-Americans
often encountered persecution, with the result that many citizens
Americanized the spellings, forms and pronunciation of their names in
an attempt to disappear into mainstream America. In 1916, the name of
the entire Steinhardt family was changed to Harde. There is a note on
Edna Steinhardt Hardes 1916 passport application: This womans name
was not officially changed from Steinhardt to Harde until the issuance
of an order by the Supreme Court of New York, in New York City, Jan 4,
1916. However, her old passport #56371 of May 5, 1915 was issued under
Harde. At first, I had no idea why this name was chosen, but it is clear
enough: Steinhardt: stein=stone; hart=hardy, brave, strong; so hardy
becomes Harde.
She was born into an affluent New York City family and raised in hotelapartments along Fifth Avenue. She graduated as an MD from the
Womens Medical College (New York Medical College and Hospital for
Women) in 1890. She spent time at the Rockefeller Institute and the
University of Michigan before doing her best known work on vaccinia
virus. She traveled widely, visiting Europe several times as a young
woman. She had remarkably broad interests and a strong, determined
character the event that stands out most is her presence with her sister
on the British steamer Sussex when it was torpedoed and sunk by a
German U-boat in the English Channel in 1916, at a time when the United
States was still a non-belligerent nation. She behaved courageously, an
experienced physician helping fellow passengers.

1904

STEINHARDT E. The effect of filtration on bacteriolytic complement. J Med Res. 1904;12:479-489. (Research
Laboratory, Department of Health, New York City)

1905

STEINHARDT, EDNA, M.D. Some observations on bactericidal complement. (Research Laboratory,

Department of Health, New York City) Presented at the Fifth Annual Meeting of the American Association of
Pathologists and Bacteriologists, Chicago, 1905.

1907

STEINHARDT, EDNA and Alexandre Besredka. De lanaphylaxie et de lanti-anaphylaxie vis-a-vis du serum de

cheval. Ann de lInst Pasteur 1907;21:117-127.

1910

Banzhaf EJ, STEINHARDT E. Antianaphylactic vaccination. J Med Res. 1910;23:1-4. (Research Laboratory,
Department of Health, New York City)

1910

Banzhaf EJ, STEINHARDT E. Vaughans split products and unbroken proteins--a comparative study of their
effects. J Med Res. 1910;23:5-29. (Research Laboratory, Department of Health, New York City)

1910

STEINHARDT, EDNA. The value of collodion membranes as filters. (Hygienic Laboratory, University of
Michigan, Ann Arbor, MI)

1913
-1914

STEINHARDT, E, Israeli C, Lambert RA. Studies on the cultivation of the virus of vaccinia. J Infect Dis.
1913;13:294-300. STEINHARDT E, Lambert RA. Studies on the culture of the virus of vaccinia. II.
J Infect Dis. 1914;14:87-92. (Research Laboratory, Board of Health, and Pathological Laboratory,
College of Physicians and Surgeons, New York City)

1913

Poor DW and STEINHARDT, EDNA. A study of the virus of rabies, freed from the cells of the host and from
contaminating organisms. J Infect Dis. 1913;13:203-231 (Research Laboratory, New York Board of Health, New
York City)

1915

HARDE (EDNA S.) loccasion du procs-verbal; propos de la culture du vaccine. CR Soc Biol. Paris. 1915,
Ixxviii, 538-540. (Institut Pasteur)

1915

STEINHARDT, EDNA and Grund, Marie. Studies on the cultivation of the virus of vaccinia, III, with a note on
the glycerin resistance of various organisms. J Infect Dis. 1915;16:205-209.

1916

STEINHARDT, EDNA HARDE. Pasteur Institut, Paris, elected member, Society of Experimental Biology and
Medicine, 1916.

1916

Edna and Lillian Harde, on their way to Paris, were aboard the British steamer Sussex when it was torpedoed
and sunk by a German U-boat in the English Channel. The loss of American lives threatens to bring a grave
crisis in the relations between the United States and Germany. President Wilson has cabled to Ambassador
Page at London and Ambassador Sharp at Paris for full particulars...

1916

NY TIMES Dudley Harde, at 341 Central Park West, whose daughters, Dr. Edna S. Harde and Lillian S.
Harde were on board the Sussex on their way to join the American Red Cross in France. He said yesterday that
he had been notified that both Dr. and Miss Harde were safe in Paris.

1919

HARDE, EDNA. On the frequency of tetanus bacilli and of other anaerobic microorganisms upon the surface
of fragments of projectiles which have been extracted from wounds. Memoir by M. le Docteur E.S. Harde in
the Report of the Ambulance de lOcean, at La Panne, Tome II, Fasciculus I, p. 185. Ann Surg. 1919;69:68-71.
Edna Harde, staff member, Institut Pasteur, Paris.

1928

HARDE, EDNA. Greffes intracrbrales de tumeurs htrologues et homologes. CR Soc Biol. Paris
1928;98;1395-1403 and 99:261-269, and Ann Inst Pasteur 1928;42;1259-1266.

1935

DR. EDNA HARDE, American bacteriologist connected with the Pasteur Institute, awarded the Guy
Amerongen Prize for Cancer Research from La Ligue Francaise contre de Cancer in 1935. Special Libraries
Assn / DBIO

1936

HARDE, EDNA. Bullowa JGM, Rothstein AM, Ratish HD, Harde E. Cevitamic acid excretion in pneumonias
and some other pathological conditions. Proc Soc Exp Biol Med. 1936;34:1-7 (Bureau of Laboratories,
Department of Health, New York City)

1941

Death, Paris, septicemia

1913 Edna Steinhardt Harde, Clara Israeli, Robert Lambert first cultivation of a virus in a cell culture (vaccinia)

page 123

Rubella virus

cell culture, thin section electron microscopy

Alfred Fabian Hess (1875-1933)

Yoshibumi Hiro

(from Hokkaido University, Sapporo)

In 1914, Alfred Hess theorized that rubella was caused by a virus, based on his work with monkeys. Hess was a staff member of University & Bellevue Hospital Medical
College, New York University. He spent mornings at the Rockefeller Institute for Medical Research and afternoons at the Babies Hospital and Bellevue. He also made seminal
discoveries concerning rickets and scurvy. In 1938, Yoshibumi Hiro and S. Tasaka proved that rubella is caused by a virus by passing ultrafiltered nasal washings from acute
cases to susceptible children.
Hess A. German measles (rubella): an experimental study. Archives of Internal Medicine 1914;13:913-916.
Hiro Y, Tasaka S. Die rteln sind eine viruskrankheit. Monatsschr. Kinderheilk 1938;76:328-332 .

1914-1938 Yoshibumi Hiro, S Tasaka, Alfred Hess discovery of rubella virus (the first rubivirus)

page 124

Discovery and Rediscovery of Trypsinization for the Dispersal of Cells in Culture

1916: FP Rous and SF Jones - dispersal of cell clumps using trypsin
1953: W Scherer, JT Syverton and GO Gey - dispersal of HeLa cells using trypsin
1953: AW Frisch and V Jentoft - dispersal of monkey testicular tissue using trypsin
1954: JS Youngner - dispersal of monkey kidney cells using trypsin

(a key in the production of inactivated polio vaccine)
1916> Development of trypsinization for the dispersal of cells in culture

page 125

T4 bacteriophage
Twort FW. An investigation on the
nature of ultra-microscopic viruses.
Lancet 1915;2:1241-1243.
dHerelle F. Sur un microbe invisible
antagonistic des bacilles dysenterique.
C R Acad Sci Paris 1917;165:373-375.

Frederick William Twort (1877-1950)

Duckworth DH. Who discovered

bacteriophage? Bacteriol Rev.
1976;40:793-802.

Flix dHerelle (1873-1949)

In 1915, British bacteriologist Frederick Twort, working in London, discovered a glassy transformation of bacterial cultures. Among other ideas, he considered that the
transformation might be caused by a virus that grew in and destroyed the bacteria: (a) affected colonies would not grow on any medium; (b) examination of the glassy areas
revealed no bacteria; (c) if a pure culture of the bacteria was touched to one of the glassy colonies, the growth at the point touched started to become transparent and gradually
made the whole colony transparent; (d) after filtration of the glassy material through a Chamberland candle, it retained its ability to cause the same glassy transformation;
and (e) the glassy transformation could be conveyed to fresh cultures for an indefinite number of generations. Tworts work was interrupted by the onset of World War I and
a shortage of funding; seemingly he never got back to this work. Independently, French-Canadian microbiologist Flix dHrelle, working at the Pasteur Institute in Paris,
announced in 1917, that he had discovered an invisible, antagonistic microbe of the dysentery bacillus. In a flash I had understood: what caused my clear spots was in fact
an invisible microbe ... a virus parasitic on bacteria. DHrelle called the virus a bacteriophage but there was difficulty in defining this. Much work followed, leading to our
present understanding of the nature of bacteriophages, but there also developed a long-running public controversy between dHerelle and defenders of Twort over the priority
of discovery. This story has been wonderfully told by Donna Duckworth in her 1976 review, Who discovered bacteriophage?

1916-1919 Frederick Twort, Flix dHerelle discovery of bacteriophages

page 126

Montgomery RE. On a
tick-borne gastroenteritis
of sheep and goats
occurring in British East
Africa. J Comp Path.
1917;30:28-57.
Daubney R, Hudson JR.
Nairobi sheep disease.
Parasit. 1934;23:507-524.

Nairobi sheep disease virus, liver, mouse

thin section electron microscopy
In 1917, Robert Montgomery described a tick-borne gastro-enteritis of sheep and goats, which was eventually named Nairobi sheep disease. Working at the Kenya Agricultural
Research Institute at Kabete, near Nairobi, he showed that the causal agent was an ultrafilterable virus, transmitted by Rhipicephalus appendiculatus ticks. In succeeding years
his work was confirmed in detail. Nairobi sheep disease has been found across large areas of East and Central Africa, causing disease in sheep and goats with mortality rates
reaching 90%. Some human disease has been associated with the livestock disease, but its geographic range, prevalence rate and clinical disease data are incomplete. One
intriguing finding has been that Ganjam virus, first isolated in 1954 in India as the cause of sheep and goat disease, and also as the cause of widespread mild and severe human
disease, is serologically closely related to Nairobi sheep disease virus this has been confirmed by genomic sequencing: Ganjam virus is just an Asian variant of Nairobi sheep
disease virus. This finding, in turn, has pointed to failings in the classical USDA regulatory system used to proscribe the import of foreign animal disease agents although
work on Nairobi sheep disease virus has always been prohibited, virologists in several laboratories worked for many years on Ganjam virus, in many instances knowingly, while
federal regulatory officals remainded oblivious of the serologic and genomic data.

Robert Eustace Montgomery (1880-1932)

1917 Robert Montgomery discovery of Nairobi sheep disease virus (the first nairovirus, the first tick-borne virus)

page 127

Influenza epidemic, Boston, 1918

Camp Funston (now Fort Riley), Kansas, influenza epidemic, 1918

The only spike visible on the

20th century mortality graph
of the U.S. is the 1918-1919
influenza epidemic

People praying, influenza epidemic, 1918

1918-1919 Influenza pandemic, 40-100 million deaths worldwide, the most devastating disease episode in history
page 128

The 1918 Influenza Pandemic and the Causative Virus

Abstracted from the many outstanding papers of Jeffery Taubenberger and David Morens
Since the 1500s, there have been at least 14 influenza pandemics; in the era with
best data, pandemics have been recorded in 1889, 1918, 1957, 1968, 1977, and 2009.
Amid these, the 1918 pandemic stands out as the single most lethal disease episode
in human history, infecting ~500 million persons (~1/3 of the worlds population) and
killing ~50-100 million (500,000 to 700,000 in the U.S.). The case-fatality rate ranged
from 2.5 to 5% globally (compared to <0.1% in other pandemics), but in some remote
locations it was much, much higher. Influenza pandemics have always been thought
to develop in Asia and spread from there to the rest of the world; however, it has not
been possible to identify with certainty the geographic origin of the 1918 pandemic or
its causative virus, whether by use of historic and epidemiologic data or phylogenetic
analysis of the reconstructed genome of the causative virus. Nonetheless, some of the
origin theories set forth in recent years are intriguing: one theory places the origin in
Kansas at the U.S. Armys Camp Funston (now Fort Riley) in March of 1918. Another places the origin in China in the winter of 1917-1918. The first cases in Europe
appeared in April of 1918 among U.S. soldiers in a military camp close to Bordeaux,
France. In any case, the 1918 pandemic spread widely in three distinct waves over
about nine months; the first in the spring of 1918 with low mortality, followed in rapid
succession by much more lethal second and third waves in the fall and winter of 19181919. There is little evidence that the 1918 pandemic virus strain had been circulating in human populations much earliergiven how obvious local/regional outbreaks
were, it is difficult to imagine the disease going undetected for very long.
The curve of influenza deaths by age at death had historically been U-shaped, with mortality peaks in the
very young and the very old and comparatively fewer
deaths at all ages in between. In contrast, the agespecific death curve in the 1918 pandemic exhibited
a unique W-shape with an additional distinct peak
of deaths in young adults 15-35 years of age. Influenza and pneumonia death rates in this age group
were >20 times higher than in previous epidemics,
representing nearly half of all deaths. The basis of
this middle peak remains a fascinating mystery
there are several theories, no conclusion. Evidence
of a relatively lower-than expected mortality rate in
people older than age 65 in 1918 is consistent with
a residual protective effect from the circulation of
pandemic viruses in the 1830s and/or 1840s. Clinically, the 1918 pandemic presented the same clinical
signs and course of disease as influenza in other years and, pathologically, the disease
was similar in that severe complications were confined largely to the respiratory tract.
However, a substantially higher percentage of cases developed severe pneumonic complications. Nearly all victims had secondary bacterial bronchopneumonia.

In 1995, a team of scientists led by Jeffery Taubenberger identified lung tissues (paraffin tissue blocks taken at autopsy and tissues taken from corpses buried in arctic
permafrost) collected in the autumn of 1918 from influenza victims and began the
slow process of determining the genomic structure of the causative virus. This started
with RNA extraction, followed by reverse transcription and amplification using the
polymerase chain reaction (RT-PCR), then complete genome sequencing and reverse
genetics technology to reconstruct the virusputting together the complete genome
sequence of one virus and partial sequences of four others took a decade.
Viral sequence data and phylogenetic analyses suggested that the entire 1918 virus was
novel to humans, originating in, or shortly before, 1918; that is, it is unlikely to have
been produced from previously circulating human virus strains that acquired one or
more new gene segments by reassortment, as was the case in 1957 and 1968. The genomic sequence suggested that the genes encoding the hemagglutinin (HA) and neuraminidase (NA) surface proteins of the 1918 virus were derived from an otherwise
unknown avian-like virus. All of its 8 genome segments were found to be substantially
different from those of contemporary avian influenza viruses. Whether the adaptation
pathway between birds and humans was direct, or involved adaptation in an intermediate mammalian or other animal host, is unknown. All influenza A virus infections
since 1918 (excepting human infections caused by avian viruses such as H5N1 and
H7N7) have been caused by descendants of the 1918 virus.
Sequence analysis alone does not offer clues to the basis for the extreme virulence
of the 1918 virus. In fact, virulence determinants and transmissibility determinants
encoded in the RNA genome of influenza viruses are not the samefor example, the
H5N1 and H7N7 avian viruses found in recent years in Asia are quite virulent in humans but not very transmissible from human to human. Although full understanding
of the basis for the virulence (and the remarkable transmissibility) of the 1918 virus
remains obscure, one possible important step in its human host adaptation has been
found. Influenza virus infection requires binding of the viral HA protein (the protein
forming the major surface spikes on virions) to sialic acid receptors on the surface of
cells lining the respiratory tract. The HA receptor-binding site configuration is different for influenza viruses adapted to infect birds vs. those adapted to infect humans.
The former preferentially bind sialic acid receptors with (2-3) linked sugars, whereas
human-adapted viruses preferentially bind receptors with (2-6) linkages. The switch
in the HA protein from avian to human receptor configuration requires only one
amino acid changebased in the simplest kind of mutation. The gene encoding the
HA protein of all five of the 1918 viruses that have been sequenced have had this
change. Perhaps of even more interest are findings that the reconstituted 1918 virus is
lethal in mice and rhesus macaques, in the latter causing severe pulmonary lesions resembling lesions that were seen in rapidly progressing human cases in 1918. All these
findings certainly raise the unanswerable question of whether such a virus, and such a
pandemic, could ever occur again. As Nancy Cox, director of the Influenza Division of
CDC, says, We cannot predict, but we can prepare!

1918-1919 Influenza pandemic, 40-100 million deaths worldwide, the most devastating disease episode in history

page 129

The Discovery of Influenza Virus

The history of the discovery of influenza virus is more complex than that of most other viruses, intermixed with the great pandemic of 1918, confounded by many
contradictory claims, involving more than the usual number of players, and forced to overcome the insistence at the turn of the twentieth century by the prominent
bacteriologist, Richard Friedrich Johannes Pfeiffer (1858-1945), that the disease is caused by a bacterium, Pfeiffers bacillus (then Bacillus influenza, now Haemophilus
influenzae). By 1922, the notion that influenza was caused by an ultrafilterable virus had gained ground: the great microbiologist, Hans Zinsser (1878-1940) wrote in his
Textbook of Bacteriology (D. Appleton & Co.), All evidence must be considered in connection with recent experiments upon the possibility of causation by a filterable virus
He cited several studies from 1918-1919, studies done during the great pandemic, including: (1) Charles Nicolle (1866-1936) and Charles Lebailly, of the Institut Pasteur
Tunis, who ultrafiltered human blood and nasal secretions from uncomplicated grippe patients and instilled the filtrates into conjunctival sacs and nasal cavities of monkeys
(sic Macaccus Sinicus) and human volunteers, obtaining flu-like clinical signs in most. (2) Ren Dujarric de la Rivire (1885-1969), who ultrafiltered blood from four
influenza patients and injected the combined filtrate into himself, clearly obtaining a flu-like disease. (3) E. Leschke, who also produced flu-like clinical signs in monkeys with
ultrafiltered human nasal secretions. (4) H. Selter, who ultrafiltered nasal secretions from cases and sprayed the filtrate into his own throat and that of an assistant, producing
flu-like disease. (5) T. Yamanouchi, K. Sakami and S. Iwashima, of the Government Institute For Infectious Diseases, Tokyo Imperial University (now the Institute of Medical
Science, University of Tokyo), who carried out the most extensive experiments, processing nasophyrngeal secretions from 43 patients, ultrafiltering half, and instilling each
into the nose of 24 volunteers (six of those receiving the ultrafiltered material had just recovered from influenza) all of the 24, except those who had had influenza recently,
came down with a flu-like disease after an incubation period of two or three days. They did other experiments as well, confirming their initial impressive findings. (6) Peter
Kosciusko Olitsky (1886-1964) and Frederick L. Gates (1886-1933), who early in the pandemic instilled ultrafiltered material from patients into rabbits intratracheally, causing
fever, leucopenia, minute pulmonary hemorrhage, and pulmonary edema and emphysema. [This remains unexplained, since human influenza viruses usually do not cause
disease in rabbits.] Several other research groups published papers refuting the ultrafilterability of the etiologic agent. Thus, for years the viral etiology of human influenza
seemed only presumptively proven, denied by Pfeiffer and those he influenced. Then, 13 years later, a key breakthrough came when Richard E. Shope (1901-1966), of the
Rockefeller Institute, discovered the etiology of swine influenza. Shopes work stimulated American and British research groups to take up, once again, the search for the cause
of human influenza. In 1933, Alphonse Raymond Dochez (1882-1964) and colleagues, also of the Rockefeller Institute, produced apparent influenza via human nasopharyngeal
inoculation and succeeded in cultivating and serially passing a virus in primary chick embryo cultures, demonstrating that passage material still produced human disease. At
the same time, a British group that had been collaborating with Dochez, led by Christopher Howard Andrewes (1896-1988), Wilson Smith (1897-1965) and Patrick Playfair
Laidlaw (1881-1940), reported the isolation and serial propagation of human influenza virus in ferrets; their model system was used to confirm the etiology of the disease by
irrefutable modern methods. An explosion of research followed, which has continued unabated until the present time.
Overview: Taubenberger JK, Hultin JV, Morens DM. Discovery and characterization of the 1918 pandemic influenza virus in historical context. Antivir Ther. 2007;12: 581-591.
Nicolle C, Lebailly C. Quelques notions exprimentales sur le virus de la grippe [Certain experimental ideas about the infectious agent of influenza]. Comptes Rendus de
lAcadmie des Sciences 1918;167:607-610.
Dujarric de la Rivire M. La grippe est-elle une maladie virus filtrant? [Is influenza a disease of filter-passing microbes?]. Comptes Rendus de lAcadmie des Sciences
1918;167:606-607.
Leschke E. Untersuchungen zur aetiologie der grippe [Studies on the aetiology of influenza]. Berliner Klinische Wochenschrift 1919;56:11-12.
Selter H. Zur aetiologie der influenza [On the aetiology of influenza]. Deutsche Medizinische Wochenschrift 1918;44:932-933.
Yamanouchi T, Sakakami K, Iwashima S. The infecting agent in influenza. an experimental research. Lancet 1919;1:971.
Olitsky P, Gates F. Experimental study of the nasopharyngeal secretions from influenza patients. J Am Med Assoc. 1920;74:1497-1499.
Shope R. Swine influenza. I. Experimental transmission and pathology. J Exp Med. 1931;54:349-359; and Shope R. Swine influenza. III. Filtration experiments and etiology. J
Exp Med. 1931;54:373-385.
Smith W, Andrewes C, Laidlaw P. A virus obtained from influenza patients. Lancet 1933;2:66-68.
Dochez A, Mills K, Kneeland Y. Studies of the etiology of influenza. Proc Soc Exp Biol Med. 1934-1935;30:1017-1022.

1918-1919 Charles Nicolle, Charles Lebailly, Ren Dujarric de la Rivire, others discovery of influenza virus

page 130

Charles Nicolle (1866-1936)

T. Yamanouchi

Ren Dujarric de la Rivire

(1885-1969)

Peter Kosciusko Olitsky

(1886-1964)

Alphonse Raymond Dochez

(1882-1964)

1918-1919 Charles Nicolle, Charles Lebailly, Ren de la Rivire, T. Yamanouchi, others discovery of influenza virus

page 131

Anton Breinl (1880-1944)

John Burton Cleland (1878-1971)

Eric Lancelot French (1914-2004)

Murray Valley encephalitis virus was the first indigenous pathogenic virus isolated in Australia. In 1918 Anton
Breinl in Townsville infected monkeys intracerebrally with material from fatal human cases. In the same year,
John Burton Cleland, from Sydney, isolated the virus in the Murray River valley by passage of human brain
material in monkeys and sheep, but in both instances isolates were not kept. The virus was next isolated from
brain material from a fatal case by Eric French during the 1950-51 epidemic in the Murray-Darling basin. He
identified the virus as belonging to the Japanese encephalitis virus group, genus Flavivirus, family Flaviviridae.
Australian virologists established the courteous tradition of citing all of the earlier investigators when listing the
discoverers of the virus.
Breinl A. Clinical, pathological and experimental observations on mysterious disease, clinically aberrant form
of acute poliomyelitis. Med J Aust. 1918;1:209-213, 229-234.
Cleland JB, Campbell AW. Nature of recent Australian epidemic of acute encephalomyelitis. Med J Aust.
1919;1:234-236.
French EL. Murray Valley encephalitis - isolation and characterization of the aetiological agent. Med J Aust.
1952;1:100-103
S. Gray Anderson, Eric French and Leslie Frazer,
and their mobile laboratory used during the 1950-1951 outbreak of Murray Valley encephalitis

1918 Anton Breinl, John Cleland, Eric French discovery of Murray Valley encephalitis virus

page 132

Arnold Lwenstein (1882-1952)

Herpes simplex virus 1

budding from nuclear membrane, cell culture, thin section electron microscopy

In 1913, Wilhelm Grter (1882-1963), director of an ophthalmology clinic in Marburg, Germany initiated a series of studies in rabbits that demonstrated the infectious nature
of herpes simplex virus. He showed that the etiologic agent could be transmitted serially from rabbit to rabbit, and then in 1919 he transmitted the etiologic agent back to
the cornea of a blind man. [This led to the Grter test for herpes, used up until the 1940s, which involved swabbing vesicular fluid from a patients herpetic lesions onto the
cornea of a rabbit.] The following is from a 1932 review by Margaret Holden: Lwenstein showed that the infectious agent in the fluid of herpes vesicles is ultrafilterable, and
this was fully verified by later work of Luger & Lauda (1921), Blanc & Caminopetros (1921) and Levaditi, Harvier & Nicolau (1922). Many investigators had difficulty in filtering
herpes virus. Levaditi and Nicolau concluded that this was not necessarily due to the size of the virus particles, but to adsorption of the particles to the filter. According to
Rivers (1928), the electrical charge on the virus, the electrical charge on the filter, the adsorption of the virus by aggregates of protein or by cell detritus, the amount of protein
or other substances in the virus emulsion [etc.] . . . serve to influence the results of all filtration experiments. Recently Ward and Tang (1929) by emulsifying herpes virus in
broth instead of saline solution have been able to recover the virus consistently from the filtrate of a Berkefeld V candle.
Lwenstein A. Aetiologische untersuchungen ber der fieberhaften, herpes. Munch Med Wochenschr. 1919;66:769-770.
Holden M. Nature and properties of herpes virus. Journal of Infectious Diseases 1932;50:218-236.
McNair Scott TF. Historical aspects of herpes simplex infections. International Journal of Dermatology. 1986;25:63-70.

1919 Arnold Lwenstein discovery of herpes simplex virus 1

page 133

The term adjuvant is derived from the Latin word adjuvare, which means to aid or
to help it was coined by Gaston Ramon, who in 1924 developed an anti-tetanus
vaccine consisting of tetanus toxin treated with formaldehyde and heat. He proposed
that the efficacy of this vaccine was also enhanced by substances (adjuvants) such
as aluminium hydroxide. In 1926, A.T. Glenny confirmed the adjuvant activity of
aluminum compounds utilizing an alum-precipitated diphtheria toxoid. In the
mid-1930s, Jules Freund (1890-1960) developed a powerful adjuvant composed of
a water-in-mineral oil emulsion containing killed mycobacteria [Freunds complete
adjuvant (FCA)]. Although FCA is one of the most potent adjuvants known, it is
quite toxic, precluding its use in human vaccines, and now in most experimental
animal studies. Currently, only aluminum salt/gel-based (alum) adjuvants are
licensed for human use in the United States. Many of the newer vaccine candidates
are based on protective antigens, which are inherently less immunogenic than the
inactivated or live-attenuated virus vaccines of the past. Therefore, adjuvants have
become an increasingly important component of vaccines being developed today.
A number of novel adjuvants have been under development or are in preclinical
and clinical trials some have been approved in other countries. Adjuvants may
be divided into two classes: those with mechanisms affecting delivery systems and
those that are immunopotentiators:
Examples of Adjuvants, Divided by Their Class of Action
Antigen delivery systems

Insoluble aluminum compounds

Calcium phosphate
Liposomes
Virosomes
ISCOMS (structured complexes of
saponins and lipids)
Microparticles (e.g., PLG
- polylactide-co-glycolide
microparticles)
Emulsions [e.g., MF59,
microfluidized detergent stabilized
squalene oil-in-water emulsion),
Montanides [ISA-51 (stabilized
water-in-oil emulsion) and ISA-720
(stabilized water/squalene)]
Virus-like particles
& viral vectors

Gaston Ramon (1886-1963)

Gaston Ramon, a long time staff member of the Institut
Pasteur, also served as one of the first Directors General
of the Office International des Epizooties (OIE the
World Organization for Animal Health), the world
regulatory body for animal disease prevention and
control.
Ramon and many others advanced (1) the use of
formaldehyde to inactivate viruses for vaccines and
toxins for toxoids, (2) the means for measuring vaccine
potency, (3) the use of the first adjuvants, and many
other technological approaches.

Formaldehyde bottle from

Jonas Salks laboratory
1952

1920s> Gaston Ramon, others improvements in many vaccine technologies

page 134

Immunopotentiators

MPL (monophosphoryl lipid A) and

synthetic derivates
MDP (muramyl di-peptide) and
derivatives
Oligonucleotides (CpG, etc.)
Double-stranded RNA (dsRNA)
Alternative pathogen-associated
molecular patterns (PAMPs) (E.
coli heat labile enterotoxin (LT);
flagellin)
Saponins (Quils, QS-21)
Small-molecule immune potentiators
(SMIPs)
Cytokines & chemokines

African swine fever virus, thin section electron microscopy

Robert Eustace Montgomery (1880-1932)

Montgomery RE. On a form of
swine fever occurring in British
East Africa (Kenya Colony). J
Comp Pathol. 1921;34:159-191.

Many aspects of African swine fever virus are unusual, more than unusual enough
to warrant it being placed as the sole member of a genus, Asfivirus, and family,
Asfarviridae. The virus exhibits some similarities in genome structure and strategy of
replication with the poxviruses and phycodnaviruses, but it has a quite different virion
structure. It is transmitted by ticks (soft ticks, genus Ornithodoros), and is extremely
pathogenic in domestic pigs. Pigs respond to infection with such a minimal immune
response that it seems they cannot recognize the virus at all. It has been asked with
tongue-in-cheek, Is this a virus from outer space? Not really, but recent focus on the
nucleocytoplasmic large DNA viruses (NCLDV) will bring new attention to the virus.

1921 R. Eustace Montgomery discovery of African swine fever virus (the only asfarvirus)

page 135

Hans Gerhard Creutzfeldt (1885-1964)

Alfons Maria Jakob (1884-1931)

In 1921, a German neurologist/psychiatrist, Alfons Maria Jakob, described a clinicopathological disease in 6 patients
with spasticity and progressive dementia associated with neural degeneration (characterized pathologically by neuronal
loss, spongiform changes, and astroglial proliferation). Shortly thereafter, a German neuropathologist, Hans Gerhard
Creutzfeldt, independently published on a similar case. Jakob, a famous neurological scientist, gave credit to Creutzfeldt
for describing the syndrome first without realizing he had stumbled onto a new syndrome hence, the compound name.
Creutzfeldt-Jakob disease (CJD) is closer to a neuropathological syndrome than a single etiologic-nosological entity.
Some 84 forms, (types or variants) of CJD have been recorded on the basis of clinical and neuropathological criteria.
The question has been raised whether the cases that Creutzfeldt and Jakob described were instances of classical CJD
there are clinical, neurological and neuropathological reasons for doubting several of the cases, but most neurologists
who have studied the case records in detail believe that at least most of Jakobs cases, if not Creutzfeldts initial case, were
indeed classical CJD.

Creutzfeldt-Jakob disease, cerebrum, H&E

1921 Hans Creutzfeldt, Alfons Jakob initial description of Creutzfeldt-Jakob disease

page 136

The history of the development of the ultracentrifuge intersects with basic physics and
chemistry as they helped create the field of molecular biology. Simple centrifuges have
drawbacks that stem from the basic physics of a shaft-mounted rotor a spinning shaftmounted rotor running at speeds greater than a few hundred revolutions per second (rps) causes
a problem much like wheel imbalance in a car there is an inability to match the inertial axis
of the rotor exactly with the axis of its motor shaft resulting in uncontrollable shaking. Carl de
Laval, a Swedish engineer, overcame some of these limitations in 1883, when he developed a
centrifuge that featured a steam-powered rotor on a flexible shaft. Soon it was found that such
a centrifuge was too slow to separate the macromolecules that increasingly drew the attention
of scientists. In the 1920s, Theodor Svedberg at Uppsala University became interested in the
problems of centrifugation he was a colloid chemist working on proof of a theory by Albert
Einstein concerning Brownian motion of large molecules. It was through his studies of colloid
sedimentation that Svedberg came to invent the first ultracentrifuge, an instrument powerful
enough to separate macromolecules. By 1925, Svedberg and colleagues had constructed a
centrifuge with a maximum speed of 42,000 rpm. He later housed the rotor in a low-pressure
hydrogen atmosphere, where he was able to subject samples to forces up to a million times that
of gravity. The sample cells were constructed of rock crystal mounted on top of a vertical shaft,
all enclosed in a temperature controlled chamber. The housing had two windows for a beam of
UV light to shine through the sample cells during rotation and onto film in a special UV camera.
Using this ultracentrifuge, Svedberg calculated the molecular weights of hemoglobin and casein
as well as his colloids. The svedberg unit (symbol S, sedimentation coefficient), technically a
measure of time, defined as exactly 10-13 seconds (100 fentoseconds), is named after him.

Theodor H.E. Svedberg (1884-1971)

Th Svedberg with part of his massive oil-turbine driven

ultracentrifuge, Uppsala University, 1932. The rest of the
instrument was located in the basement below. By this time, his
ultracentrifuge was able to generate a force of 1,000,000g

1923 Theodor Svedberg development of the ultracentrifuge

page 137

Florence Rena Sabin (1871-1953)

Florence Rena Sabin was one of the first women to build a career as a medical research scientist in the
U.S. She was the first woman on the faculty of the Johns Hopkins University School of Medicine (1902),
and the first woman elected to the U.S. National Academy of Sciences (1925). Later, at the Rockefeller
Institute she applied her interests in the role of white blood cells, especially monocytes, to studies of
host defenses against infectious agents, including viruses.

Statuary Hall, United States Capitol

1925> Florence Sabin discovery of the role of monocytes and other white blood cells in viral/bacterial infections
page 138

In 1892, the Viennese physician Janos von Bkay

suggested a possible relationship between zoster
and varicella after observing that household
exposure to zoster cases could give rise to
varicella in susceptible children. Karl Kundratitz,
also in Vienna, confirmed this connection in
1925 by showing that vesicle fluid from patients
with either varicella or zoster could produce
varicella-like rash in unexposed children and
that these children could in turn transmit
chickenpox to non-inoculated contacts.
Nonetheless, it was not until the advent of
electron microscopy in the early 1940s that a
definite viral etiology was established when the
German physician Helmut Ruska first observed
the virus particles in vesicular fluid. The virus
was finally isolated in 1952 by Thomas Weller.
Using fluorescein-labeled antibodies, he later
resolved the remaining controversy that varicella
and zoster are caused by the same virus.

Helmut Ruska (1908-1973)

Thomas Huckle Weller (1915-2008)

Varicella-zoster virus capsids

negative contrast electron microscopy

Abstracted from: Nogueira RG, Traynor B. The

neurology of varicella-zoster virus. A historical
perspective. Arch Neurol. 2004;61:1974-1978.

Varicella-zoster virus infection, human skin

intranuclear inclusion bodies in infected cells

1925-1952 Helmut Ruska, Thomas Weller, others discovery of varicella-zoster virus

page 139

Henri Valle

Otto Waldmann

Herman Salomon Frenkel

(1874-1947)
(1885-1955)
(1891-1968)
Foot-and-mouth disease (FMD) vaccines were among the first to be developed, beginning at the end of the 19th century, by: Henri Valle (France), Otto Waldmann (Germany),
Herman Frenkel (Netherlands) and Paul Capstick (United Kingdom). FMD vaccines began to be produced on an industrial scale from 1950 onwards. The vaccine research was
done in secure government institutes: Insel Reims in Germany (founded in 1909), Pirbright Laboratory in the United Kingdom (1924), Lindholm Island in Denmark (1925),
State Veterinary Research Institute, the Netherlands (1929), Centro Panamericano de Fibre Aftsa (PanAftosa) in Brazil (1951), and Plum Island Animal Disease Center in the
United States (1953).
1925: Valle, the first vaccine, virus obtained from bovine tongue epithelial vesicles, inactivated with formalin.
1930s: Waldmann and Kbe , virus obtained by infecting cattle at an abattoir and collecting epithelium and vesicular fluid, vaccine produced by inactivation with formalin in

the presence of aluminum hydroxide gel.
1940s: Frenkel, large-scale in vitro culture system using freshly cut slices of normal bovine tongue epithelium obtained from an abattoir, the epithelium was infected in vitro

with virus, inactivated with formalin, clarified by centrifugation and filtered.
Today, the question of when and if to use foot-and-mouth disease (FMD) vaccine is incredibly complex and controversial, with policy varying by country. There are questions
whether vaccine supplies would be adequate in a large-scale, multi-country epidemic: for example, it has been stated that the total reserve held by EU-FMD member countries
is about 92 million doses. Further reserves are held in other vaccine banks (e.g., The North American Vaccine Bank). There are also questions whether additional amounts
of vaccine could be produced quickly enough to supplement present supplies, but FMD vaccine production can be ramped up very quickly (currently all FMD vaccines are
produced in BHK-21 cells grown in suspension in large bioreactors). One estimate is that the countries of the EU have the capacity to produce 155 million monovalent doses of
vaccine per year (13 million doses per month). Irrespective of whether FMD vaccine supply would be adequate in a multi-country emergency, because of its use in several parts
of the world where the disease is endemic, it is the most massive vaccine production economy in the world, except for poultry vaccines.

1925> Henri Valle Otto Waldmann Herman Frenkel, others development of foot-and-mouth disease vaccines
page 140

In researching the virologists cited in this book, the subject of personal inspiration and
the various kinds of influences upon career choices came up often. This page, reflecting
on one small aspect of this large subject, is a placeholder. Clearly, the subject is one of
the foundations of virology.
When virologists get together socially, sometime the question is asked, How did you
decide on your career? or What was your inspiration to become a virologist? Personal
experiences are often cited, but in the first golden era of virology the answer to such
questions often involved inspirational booksthis was an era before there were other
media. In the U.S. the book most often cited in biographies of research physicians and
scientists has been Arrowsmith, the novel by Sinclair Lewis, which was published in
1925, won the Pulitzer Prize in 1926 and contributed to his award of the Nobel Prize in
Literature in 1930. The book is arguably the earliest major novel to deal with the culture
of science. It reached several generations, significantly shaped public perceptions of science and medicine and inspired many career choices. It is still inspirational.
Paul de Kruif, after receiving a PhD in bacteriology from the University of Michigan in
1916, served in the U.S. Army during World War I and in 1920 became a staff member
of the Rockefeller Institute for Medical Research. He was encouraged to try his hand at
writing for a popular audience. His first article took medicine and medical research to
task for boastful claims of scientific authority while ignoring standards, such as use of
controls. This was interpreted as a veiled attack on the Institute and in 1922 De Kruif
was asked to resign. He became a prolific writer, in 1926 publishing his most famous
work, Microbe Hunters, which had great influence on aspiring medical scientists and is
still in print.

Sinclair Lewis (1885-1951)

Summers WC. Microbe Hunters

revisited. Internal Medicine
1998;1:65-68.

Paul Henry de Kruif (1890-1971)

Markel H. Reflections on
Sinclair Lewiss Arrowsmith: the
great American novel of public
health and medicine. Public
Health Rep. 2001; 116: 371-375.

In 1922, Lewis, already a celebrated author, and de Kruif began their collaboration on a
novel; De Kruif would provide the scientific material and character sketches based on
physicians and scientists he knew, and Lewis would supply the plot and dialogue. They
booked passage on a steamship to the West Indies, where they could work on the book
without distractions. The book, Arrowsmith, became a sensationit centers on the
title character, a modern scientific microbe hunter and his pursuit of the noble ideals of
medical research for the selfless benefit of mankind. The attractions of financial security,
recognition, even wealth and power are seen as distractions. In the course of the novel,
aspects of medical training, medical practice, scientific research, scientific fraud and
public health are described, themes that are still relevant today. The title character is
said to have been modeled on Flix dHerelle, the co-discoverer of bacteriophage (or on
de Kruif himself although he was not a physician), and the setting clearly was modeled
on the Rockefeller Institute. In the book, the climax deals with the discovery of a bacteriophage that destroys bubonic plague bacteria causing an epidemic on a Caribbean
island. De Kruif s contribution was so significant that he received 25% of the royalties.
In succeeding generations other books played the same role as Arrowsmith and Microbe
Hunters in inspiring career choicesin the 1990s Richard Prestons book, The Hot Zone
(1994), was read by many aspiring virologists. Since then, perhaps other kinds of media,
including films such as Contagion, Internet sites and social media may have become
dominant inspirational factors. Time will tell.

1925> Sinclair Lewis, Paul de Kruif, others Arrowsmith, Microbe Hunters, books inspiring careers in virology

page 141

Peter Kosciusko Olitsky (1886-1964)

Jacob Traum (1923-1995)

United States Department of Agriculture; Technical Bulletin TB0076; Report of the Foot-and-Mouth Disease Commission of the USDA, 1926: This extensive unsigned report
contains the results of a major research project on the nature of foot-and-mouth disease virus(es) and vesicular stomatitis virus(es). Proof that vesicular stomatitis is caused
by specific ultrafilterable agents (VSV-NJ and VSV-IND were already known as distinct entities) is presented in incredible detail. A section of the Bulletin entitled Filtration
Experiments 1925-26 states that J. R. Mohler and W. E. Cotton had earlier failed to show that VSV-NJ was ultrafilterable. In the many experiments reported in this Bulletin,
the VSV-NJ virus used was from fluid obtained from vesicular lesions on guinea pigs, horses or heifers and ultrafiltrates were tested by injecting guinea pigs which were then
evaluated for illness and lesions at 48 hours (using Bacillus prodigiosus as a filter control). In 13 of the first 18 filtration tests done in 1925 using Berkefeld type V candles the
virus passed through the filters. Then Berkefeld type N candles (finer than type V) were used: in 6 of 10 experiments the virus passed through the candles. Then Chamberland
bougies (candles) L3, L7 and L11 were used in a series of experiments also shown in tabular form: This experiment demonstrates that the virus can traverse the L3 and L7
types of Chamberland filters but not under ordinary conditions the L11.
Oltsky PK, Traum J, Schoening HW. Comparative studies on vesicular stomatitis and foot and mouth disease. J Am Vet Med Assoc. 1926;70:147-167. [Materials: Two strains
of vesicular stomatitis virus (Indiana, New Jersey), samples sent us through the kindness of Dr. W. E. Cotton, United States Bureau of Animal Industry, who had propagated the
strains in guinea pig foot pads for several years. William Edwin Cotton (1866-1951), Assistant Superintendent, Bureau of Animal Industry, USDA, Bethesda, Maryland)]
Cotton WE. Vesicular stomatitis and its relation to the diagnosis of foot-and-mouth disease, J Am Vet Med Assoc. 1926;69:313-332.

1926 Peter Olitsky, Jacob Traum, Harry Schoening, William Cotton discovery of vesicular stomatitis viruses
page 142

Frederik Cornelis Kraneveld

(1896-1957)

Thomas Michael Doyle

(1888-1981)

Highly pathogenic strains of Newcastle disease virus cause

severe, often fatal, disease in chickens, variably marked by
conjunctival reddening and swelling of the tissues of the head
The first recognized outbreaks of [velogenic (highly virulent), peracute] Newcastle disease in chickens occurred as if it were
a new disease, in 1926 in Java, Indonesia (by the work of Frederik Kraneveld) and in 1927 in Newcastle-Upon-Tyne, England
(independently, by the work of Thomas Doyle). This was a remarkable pair of discoveries in 1926-1927, considering the time
and places, the state of background knowledge of the viral diseases of poultry, and especially by the confounding presence
of fowl plague (avian influenza). There are three theories as to the explosive origin of the disease in poultry: (1) via a major
mutation of a low-virulence precursor virus; (2) emergence from a long standing presence of the virus in the village-level (rather
non-commercial) poultry environment of southeast Asia where the economic impact of the disease would not be appreciated;
and (3) emergence via transmission of virus from enzootically affected wild bird species, most likely shore birds (especially
cormorants) and/or psittacine species. Between 1926 and 1946, Newcastle disease appeared in all poultry-producing areas
of the world and became established as a major disease of chickens. Clearly, this was related to the global development of the
commercial poultry industry and its reliance on trade, including international trade. There have been three global pandemics of
velogenic Newcastle disease in the United States, these were in 1942-1946; 1971-1973 (in California alone in the eradication
campaign 12 million birds were destroyed at a cost of $56M); and 2002-2003 (in California and Arizona 3 million birds were
destroyed at a cost of $161M). In recent years, control has been impeded by the continuing presence of virus in unrecognized
backyard poultry flocks, legal and illegal pet bird import facilities and smuggling operations mainly centered on fighting cocks.
After the initial appearance of the virus in the 1920s, it became clear that other less severe diseases were caused by viruses
Newcastle disease virus
indistinguishable from Newcastle disease virus [mesogenic (intermediate virulence) and lentogenic (nonvirulent) virus strains].
negative contrast electron microscopy
Kraneveld FC. A poultry disease in the Dutch East Indies. Nederlands-Indische Bladen voor Diergeneeskunde 1926;38:448-450.
Doyle TM. A hitherto unrecorded disease of fowls due to a filter passing virus. J Comp Pathol Therap. 1927;40:144-169.

1926 Frederik Kraneveld, Thomas Doyle discovery of Newcastle disease virus (the first paramyxovirus)

page 143

The subject of virulence factors carried by bacteriophages is usually

lodged in the bacterial pathogenesis literature, especially in its historic
context. However, in that phages were central to the unraveling of
the principal mechanisms of viral replication and influenced early
viral pathogenesis concepts, the subject has a place here too. The
contribution of phages to the pathogenicity of their bacterial hosts
began to be uncovered as early as 1927, when Martin Frobisher Jr.
(1896-1984) and J. (James) Howard Brown (1884-1956) discovered that
non-toxigenic streptococci exposed to ultrafiltered supernatants of
toxigenic streptococcal cultures produced scarlatinal toxin. It took quite
a few years to show that these supernatants contained a bacteriophage
encoding the scarlatinal toxin and that the transfer mechanism was
transduction, i.e., the transfer of DNA between bacterial cells via phage
infection. The same long delay between discovery of the transmissibility
of toxins and the role of phage was the case with other bacterial
pathogens.

From Fortier LC, Sekulovic O. Importance of prophages to evolution and virulence of bacterial pathogens. Virulence 2013;4:354-365.

Bacterium

Phage

Gene Product

Phenotype

Escherichia coli

lambda phage

shigalike toxin

hemorrhagic diarrhea

Vibrio cholerae

Clostridium botulinum

Corynebacterium diphtheriae
Streptococcus pyogenes

CTX phage

clostridial phages

corynephage beta
T12

cholera toxin

botulinum toxin
diphtheria toxin

erythrogenic toxins

cholera

botulism (food poisoning)

diphtheria

scarlet fever

The CTX phage has received special attention because it was the first filamentous phage found to transfer
toxin genes to its host bacterium it provided the lesson that many different types of phage may carry
virulence factors. It provided another important lesson: some of the first vaccines against Vibrio cholera
had a deletion of the gene encoding cholera toxin. The attenuated bacterium was nonvirulent, and thus
considered a safe vaccine substrate. However, the vaccine strain readily acquired a functional copy of the
cholera toxin gene by infection with CTX phage, turning the vaccine organism into a fully virulent one.

Over time, many bacterial toxin genes were found to be phage encoded
and important in bacterial disease pathogenesis and in toxin gene
dissemination among bacterial populations. The most widely recognized
examples of phage-encoded exotoxins are those of Vibrio cholerae,
Corynebacterium diphtheriae, and Clostridium botulinum. It then
became increasingly clear that toxin genes are only a subset of the
diverse virulence factors encoded by phages; for example, in addition
to encoding bacterial exotoxins, phages can: (1) influence bacterial
adhesion, colonization, and invasion; (2) enhance bacterial resistance
to antibacterial serum factors and phagocytosis; (3) alter bacterial
susceptibility to antibiotics; and (4) enhance transmissibility of bacterial
pathogens. Diverse mechanisms are involved; for example: (1) some
phages encode regulatory factors that increase expression of virulence
genes not encoded by the phage; (2) some phages encode enzymes that
alter bacterial proteins involved in virulence and transmission; (3) some
phage structural components are directly pathogenic; and (4) some
phage-encoded genes undergo replication and transcriptional activation
following prophage induction, an effective amplification mechanism that
can be faster and more productive than processes linked to bacterial
replication per se. The latter, as early as 1960, was envisioned to be key to
the production of diphtheria toxin by Corynebacterium diphtheriae.
Frobisher M, Brown J. Transmissible toxicogenicity of streptococci. Bull
Johns Hopkins Hosp. 1927;41:167-173.
Fidelma Boyd E. Bacteriophage-encoded bacterial virulence factors and
phage-pathogenicity island interactions. Adv Virus Res. 2012; 82:91-118.
Fortier LC, Sekulovic O. Importance of prophages to evolution and
virulence of bacterial pathogens. Virulence 2013;4:354-365.

1927 Martin Frobisher Jr., J. Howard Brown role of bacteriophages in the pathogenicity of their bacterial hosts
page 144

Journal of Bacteriology 14: 217-258, 1927

Thomas Milton Rivers (1888-1962)

[Rockefeller Archives, used with permission]

Thomas Milton Rivers is considered the father of modern virology and the
father of American virology. He completed an MD at Johns Hopkins School
of Medicine in 1915. Entering the Army medical corps in 1918, he was assigned
to Fort Sam Houston, San Antonio, Texas, to a project headed by Rufus Cole
of the Rockefeller Institute for Medical Research, investigating outbreaks of
measles and pneumonia. The experience awakened Rivers interest in research,

and in 1922 Cole invited Rivers to join the Rockefeller Institute faculty and pursue research
in the still poorly understood field of virology. Between 1922 and 1955, Rivers molded the
Institute into the preeminent laboratory for research on viruses, and in over 100 papers
published during these years, Rivers addressed a range of topics relating to some of the most
devastating viral diseases, including smallpox, Rift Valley fever and the viral encephalitides.
He led the way toward delineating virology as a conceptually distinct discipline. In 1937,
Rivers became director of the Rockefeller Institute hospital, a position he held until 1953,
when he became vice president and medical director of the Institute. During World War
II, Rivers served as commander of the Naval Medical Research Unit in the South Pacific,
where he led programs to combat infectious diseases. He returned to the Rockefeller
Institute in 1946 and also served as medical director of the National Foundation for Infantile
Paralysis he played a major role in the testing of Jonas Salks polio vaccine. Rivers was
recognized widely: elected to the National Academy of Sciences (1932) and to the American
Philosophical Society (1942); served as president of the American Society for Clinical
Investigation (1932), the American Association of Immunologists (1934), and the Society of
American Bacteriologists (now the American Society of Microbiology)(1936).

1927 Thomas Rivers publication of the paper that established virology as a distinct field of science

page 145

Rivers T. Filterable virusesa critical review. Journal of Bacteriology 1927;14: 217-258.

1927 Thomas Rivers publication of the paper that established virology as a distinct field of science
page 146

Thomas Milton Rivers (1888-1962)

[Rockefeller Archives, used with permission]

Charles Edmund Simon

(1866-1927)

Charles E. Simon received an A.B. in 1888 from Johns Hopkins University and an M.D. in
1890 from the University of Maryland. From 1910 until 1920, he served on the faculty of
the University of Maryland. In 1920 he was appointed lecturer in medical zoology at the
Johns Hopkins University School of Hygiene and Public Health. There he started the first
known teaching program on filterable viruses in 1922 this became the first Department
of Filterable Viruses in the United States. He also compiled a large collection of virus
specimens, forming the first virus reference collection in the country. He was later
promoted to Professor of Filterable Viruses. From the inception of the American Journal
of Hygiene in 1921 to his death in 1927 Simon served as its managing editor. In 1928
Thomas Rivers dedicated his book, the first major virology reference work, to Simon.

1928 Thomas Rivers publication of the first major virology book, Filterable Viruses

page 147

Wilbur Augustus Sawyer

(1879-1951)

Thomas Milton Rivers

(1888-1962)

Frank Lappin Horsfall, Jr.

(1906-1971)

Max Theiler
(1899-1972)

Richard Shope
(1901-1966)

Wilbur Sawyer, mouse room,

Rockefeller Foundation
Virus Laboratory, 1934
[images from Rockefeller Archives, used with permission]

1928 founding of the Rockefeller Foundation Virus Laboratory (RFVL) and its international research programs
page 148

Fred Griffith (1881-1941)

Fred Griffith was killed in an air-raid in London in WWII

(this poor image is thought to be the best available)

In 1927, Fred Griffith conducted what is now known as Griffiths Experiment. Published in 1928 in the Journal of Hygiene, his paper carries the first widely accepted
demonstration of bacterial transformation, whereby a bacterium can distinctly change its form and phenotype (pathotype). In his experiments, Griffith typed pneumococcus
isolates searching for patterns to clarify the epidemiology of lobar pneumonia. He found that isolates occurred in two general formssmooth and rough. The smooth form
(S) has a polysaccharide capsule, which is associated with virulence (pneumonia in humans, death within days in mice). The rough form (R), lacking a capsule, was considered
avirulent. In Griffiths Experiment, as expected, when S bacteria were killed by heat and then injected into mice, they caused no illness despite being a virulent strain. When
a large amount of heat-killed S was injected along with live R, however, mice developed pneumonia and died. Recovering bacteria from dead mice, Griffith found that the R
bacteria had acquired a capsule to become S, and thereafter maintained this phenotype over many generations. The phenomenon was at the time attributed to an unidentified
transforming factor. Over the next 16 years Oswald Avery, Colin MacLeod, and Maclyn McCarty at the Rockefeller Institute for Medical Research demonstrated that DNA
was the transforming factor, a grand departure from the prevailing belief that the protein content of chromosomes probably made up genes, the stuff of inheritance. Avery and
his colleagues remain famous for this discovery, which was a key in the advance of molecular genetics.

1928 Fred Griffith discovery of transformation in bacteria, seminal to the development of molecular genetics

page 149

Principles / Methods of Viral Disease Diagnosis

Visual information leading to a presumptive diagnosis

Case history, clinical examination, chemistry, hematology

Pathology, histopathology, ultrastructural pathology
Detection of viruses by electron microscopy

Detection and identification of viral antigens

Enzyme immunoassay methods ( e.g., antigen-capture)

Immunochromatography, immunogold binding assays
Immunofluorescence
Immunohistochemistry (immunoperoxidase staining)
Immunoelectron microscopy
Radioimmunoassay
Latex particle agglutination
Immunodiffusion

Direct detection and identification of viral nucleic acids

Rebecca Lancefield
(1895-1981)

Edwin H. Lennette
(1908-2000)

Hybridization: in situ hybridization, Southern blot

hybridization and dot-blot filter hybridization
Polymerase chain reaction (PCR); isothermal amplification
Viral genomic sequencing and partial sequencing
Oligonucleotide fingerprinting and restriction
endonuclease mapping

Virus isolation and identification

Virus isolation in cultured cells

Virus isolation in animals and chick embryos
Virus quantitation, especially plaque assay

Detection / quantitation of anti-viral antibodies

(serologic diagnosis)

Nathalie J. Schmidt
(1928-1986)

Enzyme immunoassay (EIA)enzyme linked

immunosorbent assay (ELISA)
IgM class-specific antibody enzyme immunoassay
(EIA/ELISA)
Serum neutralization assay
Immunoblotting (western blotting)
Indirect immunofluorescence assay
Hemagglutination-inhibition assay
Complement-fixation assay
Immunodiffusion

1928> Rebecca Lancefield, Edwin Lennette, Nathalie Schmidt, others founding of viral disease diagnostics
page 150

Adrian Stokes (1887-1927)

Noel Paul Hudson (1895-1987)

Johannes Bauer (1890-1961) and Wilbur

Sawyer at Rockefeller Institute, 1933
After World War I, the Rockefeller Foundation expanded its
yellow fever activities to Africa. A West African Yellow Fever
Commission was formed in 1925 it was based near Lagos and
was to determine whether African yellow fever was the same
as the South American disease, to find the causative agent (as
if James Carroll had not done this in Havana in 1901) and to
study its epidemiology. Adrian Stokes, an Irish/British professor
of pathology, was a member. In 1927, blood from a 28-year-old
African named Asibi, suffering from a relatively mild illness,
was injected into a rhesus macaque (Macaca mulatta) imported
from India (African monkeys were resistant). The monkey
proved susceptible, establishing the infection for the first time
in a suitable laboratory host. This was the first time that Kochs
Postulates were proven for a virus. Tragically, very shortly
afterward Stokes died of yellow fever. Hideyo Noguchi arrived
in Lagos trying to verify his theory that leptospira caused yellow
fever he contracted yellow fever and died in 1928. A third
investigator, William Young, who had performed Noguchis
autopsy, also died of yellow fever.

Noel Paul Hudson served as field director of the International

Health Division of the Rockefeller Foundation, in which
capacity he worked with Adrian Stokes and Johannes Bauer
on yellow fever in Lagos. Later, he served as professor of
bacteriology and hygiene at the University of Chicago,
and then professor and chairman of the Department of
Bacteriology and dean of the Graduate School at the Ohio
State University.
Stokes A, Bauer JH, Hudson NP. The transmission of yellow
fever to Macaca rhesus [Macaca mulatta]. JAMA 1928;96:
253-254.

1928 Adrian Stokes, Noel Hudson, Johannes Bauer transmission of yellow fever virus to rhesus macaques

page 151

Constant Mathis (1871-1956)

Andrew Watson Sellards (1884-1942)

Jean Laigret (1893-1966)

At the same time that the Rockefeller Foundation West African Yellow Fever Commission was active, Andrew Watson Sellards, from the
department of tropical medicine at Harvard, was on his way to Dakar, Senegal, to study a yellow fever outbreak when he heard of the success
in infecting rhesus macaques (Macaca mulatta). Taking with him to Senegal some macaques and a strain of stegomyia (Aedes aegypti)
mosquitoes that came originally from Havana, he joined General Constant Mathis, director, and Jean Laigret of the Institut Pasteur Dakar, and
soon they reproduced the macaque infection. One day, Laigret noticed that the son of a woman with yellow fever did not appear for his daily
visit to the hospital. He visited the house of this man, Francois Mayali, and found that he had a fever. A sample of Mayalis blood produced
severe yellow fever in a rhesus macaque, though Mayali suffered only a relatively mild case. This strain of yellow fever virus became known
as the French strain. Sellards discovered that the virus survived freezing, allowing transport of infected macaque liver tissue (instead of
monkeys) to the United States for further study. As Sellards returned to the United States, Laigret serially passaged the French strain in mice
by intrcerebral inoculation; this led to neurotropic variants, which after hundreds of passages were used as candidate vaccines, first requiring
the use of immune serum along with vaccine to offset adverse effects due to under-attenuation. With more mouse passages (now up to 237)
and other manipulations they produced a vaccine strain they considered suitable for general use and started vaccinating large populations
by 1953, 56 million doses had been used. Some severe vaccine-associated central nervous system infections continued to occur, especially in
children, but considering the risk of yellow fever this was considered acceptable until 1982 when the vaccine was replaced by 17D vaccine.
Mathis C, Sellards AW, Laigret J. Sensibilit du Macaca rhesus au virus de la fivre jaune. C R de LAcademie des Sciences 1928;188:604-606.
Harvey AM. Applying the methods of science to tropical diseases-the story of Andrew Watson Sellards. Johns Hopkins Med J. 1979;144:45-55.
Monath TP. Yellow fever vaccines: the success of empiricism, pitfalls of application, and transition to molecular vaccinology. In: Plotkin S,
Fantini B, editors. Vaccinia, vaccination and vaccinology: Jenner, Pasteur and their successors. Paris: Elsevier; 1996.

Yellow fever virus

negative contrast electron

microscopy
Erskine Palmer, CDC

1928 Constant Mathis, Andrew Sellards, Jean Laigret transmission of yellow fever virus to rhesus macaques
page 152

Feline panleukopenia
in a kitten
Feline panleukopenia is a severe,
highly contagious, multisystemic
disease that is often fatal. Feline
panleukopenia virus is endemic
in cat populations worldwide, but
is rarely seen as a clinical entity
in developed countries today due
to the effectiveness of vaccines.
Young, unvaccinated kittens present
most commonly with this disease.
Unvaccinated feral cat colonies
and other wild felids also serve
as reservoirs of infection for the
domestic cat population.

Jean-Louis-Armand Verge (1892-1964),

with Jean-Marie Camille Gurin (1872-1961) and Gaston

Ramon (1886-1963), at Alfort, 1942
All three of these scientists were faculty members at Lcole Nationale
Vtrinaire DAlfort. Jean-Louis Verge was a distinguished professor, who
served for many years, making important discoveries pertaining to animal
viruses and veterinary vaccines. Camille Gurin worked with Albert Calmette,
discovering the tuberculosis vaccine, BCG (Bacille de Calmette et Gurin).
Gaston Ramon made seminal discoveries in vaccinology, such as the use
of formalin as an inactivating agent and the use of adjuvants that greatly
improved many vaccines for animals and humans.
Verge J, Christofferoni J. La gastro-enterite infectieuse des chats est-elle due a
un virus filgteerable? C R Soc Biol (Paris) 1928;99:312-314.

Feline panleukopenia virus

model built from X-ray
crystallographic data

1928 Jean-Louis Verge, J. Christofferoni discovery of feline panleukopenia virus (the first parvovirus)

page 153

Stefan S. Nicolau (1896-1967)

Stefan Nicolau started his career as an assistant Constantin Levaditi (1874-1953) at the Institut Pasteur.
Their collaboration was exceptionally fruitful, ranging from viral oncolytic therapy as a novel cancer
therapy, to the discovery, along with I. A. Galloway of the etiologic virus of Borna disease, a neurobehavioral disease, studied in sheep, cattle and horses. The name Borna refers to the city of Borna,
Germany, the site of an equine epidemic in 1895-1896 that crippled the Saxon cavalry in a war ongoing at
that time. In the past few years, similar viruses have been found in many animals, especially birds, but their
full natural history is just beginning to be unraveled. In 1990, Janice Clements and colleagues reported that
antibodies to a protein encoded by the Borna disease virus genome are found in the blood of patients with
behavioral disorders. Researchers in Germany, the U.S. and Japan studied 5,000 patients with psychiatric
disorders and 1,000 controls they found a significantly higher percentage of patients than controls were
positive for BDV antibodies. Subsequent studies have also shown an association between Borna disease
virus and human psychiatric disorders. However, as time has gone by, this association has become more
and more controversial.
Nicolau SS, Galloway I. Preliminary note on the experimental study of enzootic encephalo-myelitis (Borna
disease) Br J Exp Pathol. 1927;8:336-341.
Nicolau SS, Galloway I. Borna disease and enzootic encephalomyelitis of sheep and cattle. Spec Rep Ser
Med Res Council 1928;121:7-90.

Stefan S. Nicolau Institute of Virology

Bucharest, Romania

Ian A. Galloway
(1900-~1980)

1929 Stefan Nicolau, Ian Galloway discovery of Borna disease virus (the first bornavirus)
page 154

Borna disease virus

thin section electron microscopy

Robert Green was born in 1895 in Wadena,

Minnesota. He earned four degrees from the
University of Minnesota, including an MD
in 1922. He joined the staff at the University
of Minnesota in 1918 and became professor
of bacteriology and immunology in 1929.
Green was world renowned for his research
on diseases in animals caused by viruses. From
1932-1940, he directed the Minnesota wildlife
disease research program. During his tenure,
he developed a vaccine to prevent encephalitis
in foxes (infectious canine hepatitis virus) and
a vaccine against canine distemper (the first
canine distemper virus vaccine), thereby saving
Minnesotas fur industry.

Robert Gladding Green (1895-1947)

Green RG, Ziegler NR, Green BB, Dewey

ET. Epizootic fox encephalitis. I. General
description. Am J Hyg. 1930;12:109-129.

[from the University of Minnesota Archives, used with permission]

USDA Statement, 1928 Fox Distemper Being Investigated: Frequent requests from fox breeders for assistance in controlling infectious diseases have prompted the USDA
to investigate conditions on a number of fox farms. It was found that a thorough investigation would have to be made of so-called fox distemper to determine its cause. Dr.
Robert. G. Green, Medical School of the University of Minnesota, who with a group of associates, has investigated outbreaks on large farms near Minneapolis for the past three
years. Dr. Newell R. Ziegler, Ms. Beryl Green and Dr. Earle T. Dewey have been associated with Dr. Green since the time he began his investigations. Dr. Green has tentatively
called the disease epizootic fox encephalitis. [The infection in commercially reared foxes is much more neurotropic than in dogs; hence, the common name, fox distemper.
Later, Green and his colleagues found that the virus that they had discovered was indistinguishable from infectious canine hepatitis virus, the important virus of dogs.]

1930 Robert Green, Newell Ziegler, others discovery of infectious canine hepatitis virus (the first adenovirus)

page 155

Infectious canine hepatitis virus, MDCK cell

[stains: anti-NPCP (keratin, epithelial tight junctions) and anti-virus antibody]

Adenovirus, cell culture, thin section electron microscopy

1930 Robert Green, Newell Ziegler, others discovery of infectious canine hepatitis virus (the first adenovirus)
page 156

Leslie Tillotson Webster (1894-1943)

[from Leslie Webster III, used with permission]

The Genealogy of Swiss Mice (Swiss-Webster Mice)

Leslie Webster graduated from the School of Medicine at Johns Hopkins University and shortly afterward joined the Rockefeller Institute for Medical Research, where he
remained for the rest of his life. His work aimed to understand epidemiologic phenomena, especially the phenomenon that in a given population individuals differ greatly in
their susceptibility to infection, and that host factors play an important role in this regard. His work involved development of strains of laboratory mice having different levels
of resistance or susceptibility to particular infectious agents. He showed that such traits are inheritable and that strains of mice can be developed that are highly resistant
or exceedingly susceptible. He showed that there is no need for an infective agent of exceptionally high virulence to be present to start an epidemic; it is sufficient that the
number of susceptible hosts constitute a critical proportion of the population. In an epidemic, when the number of susceptibles reaches a certain low level, the infective agent
is not transmitted well enough to continue the infection chain. Individuals surviving an epidemic are resistant, not only through a process of active immunization but also
through their inherited resistance. In the course of his work, especially on rabies and the viral encephalitis viruses, Webster greatly advanced our understanding of viral disease
pathogenesis and at the same time advanced the use of laboratory bred mice (line-bred and inbred mice).

1930 Leslie Webster, Charles Armstrong, Max Theiler, others development of the mouse for virus research

page 157

Charles J. Armstrong (1886-1967)

Charles Armstrong graduated from the School of Medicine at Johns Hopkins University and became a
commissioned officer in the U.S. Public Health Service. In the fall of 1918 he served on a team investigating
pandemic influenza and soon became a life-long staff member of the Hygienic Laboratory as its name
changed to the National Institutes of Health. As chief of the Division (now Laboratory) of Infectious
Diseases he did seminal work on polio (transmitting the virus to cotton rats and then to mice), lymphocytic
choriomeningitis (discovery and characterization of the virus), Coxsackie viruses and psitticosis (nearly
dying of a laboratory-acquired infection). At the presentation of the Sedgwick Memorial Medal of the
American Public Health Association in 1941, Dr. T. E. Parran, the Surgeon General, said: He is unique in
that he has made a distinct contribution to our knowledge of every disease with which he has worked.

In 1947, at the Institute of Cancer Research (ICR) at Fox Chase

Center, 100 mice from a stock bred from 6 different sources,
including Websters lines of Swiss mice, were selected for
high production and high growth rate characteristics. They
achieved exceptional production parameters, with an average
15 pups per litter, >99% of pups weaned, <23 day interval
between litters. No sibling mating was used this line was
continued for 20 years, over 60 generations. Today, commonly
used outbred / linebred Swiss mouse stocks derive from these
mice, and are identified as to whether they descended through
ICR (ICR Swiss mice) or NIH (NIH Swiss mice) or Websters
colonies (Webster Swiss mice). Most of the common inbred
mice used today also derive from these mice.
From: Goios A, Pereira L, Bogue M, Macaulay V, Amorim A.
mtDNA phylogeny and evolution of laboratory mouse strains.
Genome Res. 2007;17:293-298.

1930 Leslie Webster, Charles Armstrong, Max Theiler, others development of the mouse for virus research
page 158

Richard Edwin Shope (1901-1966)

Paul Adin Lewis (1879-1929)

[from Rockefeller Archives, used with permission]

Swine influenza virus infection: mild bronchiolitis and alveolitis

with mononuclear inflammatory infiltration. H&E

Pig farmers in Iowa had reported two outbreaks, one in 1918 during the great influenza pandemic and another in 1929, of a highly contagious, influenza-like disease. In each
outbreak, thousands of pigs had become ill and many died. The disease bore such a remarkable resemblance to human influenza that it was named swine influenza. In 1930,
following a hunch, Richard Shope and his mentor Paul Lewis, working in the Rockefeller Institutes Department of Animal Pathology, Princeton, New Jersey, took mucus and
lung samples from infected pigs and attempted to isolate the etiologic agent. They isolated a bacterium that was quite similar to Hemophilus influenzae (then called Pfeiffers
bacillus), which many investigators still thought to be the cause of human influenza. They called the bacterium Hemophilus influenzae suis, but when they injected it into pigs,
it caused no disease. This must have been the last work done by Shope and Lewis before the latter left for a Rockefeller Foundation yellow fever investigation in Brazil; Lewis
died of yellow fever in Bahia, Brazil in 1929. Shope continued on, ultrafiltering (Berkefeld filters V and N) the samples and finding that the filtrates contained an infectious
agent. Shopes filtrate caused a highly contagious influenza-like disease in pigsalbeit a milder disease than seen in naturally infected animals. Mixing the filtrates with the
bacteria reproduced the severe disease. He concluded that the filterable agent caused the infection, which then facilitated secondary infection with the bacterium. Following
Shopes insight, Wilson Smith, Christopher Andrewes and Patrick Laidlaw, at the National Institute for Medical Research in London, in 1933 developed the ferret model of
human influenza and confirmed once-and-for-all the viral etiology of the human disease. Both Shope and the British trio later demonstrated that sera from humans that were
infected during the 1918 pandemic neutralized the swine virus, leading them to conclude that the swine virus was a surviving descendent of the 1918 human pandemic virus.
Shope RE. Swine influenza. I. Experimental transmission and pathology. J Exp Med. 1931;54:349-359.
Lewis PA, Shope RE. Swine influenza. II. A hemophilic bacillus from the respiratory tract of infected swine. J Exp Med. 1931;54:361-372.
Shope RE. Swine influenza. III. Filtration experiments and etiology. J Exp Med. 1931;54:373-385.

1930 Richard Shope, Paul Lewis discovery of swine influenza virus and the complex pathogenesis of the disease

page 159

Meyer KF, Haring CM,

Howitt B. The etiology of
epizootic encephalomyelitis
of horses in the San Joaquin
Valley, 1930. Science
1931;74:227-228.

Beatrice Howitt
(1891-1982)

Karl Friedrich Meyer (1884-1974)

The Structure of the Recombinant Alphavirus, Western Equine Encephalitis

Virus, Revealed by Cryoelectron Microscopy. Sherman MB, Weaver SC.
Journal of Virology, published online ahead of print 14 July 2010.
A 3D map of WEEV, with its 2-fold axis oriented perpendicular to screen.
Six trimer spikes are clearly visible. Green layers visible through the holes in
the outer glycoprotein layer represent the outer leaflet of the lipid membrane
separating the glycoprotein shell from the inner nucleocapsid.

Clarence Melvin Haring

(1878-1951)

[Dean, School of Veterinary Medicine

University of California, Davis]

1930 Karl Meyer, Beatrice Howitt, Clarence Haring discovery of western equine encephalitis virus (the first alphavirus)
page 160

Karl F. Meyer: Medical Research and Public Health An Oral History.

The Bancroft Library, University of California Berkeley Regional Oral History Office. Interview conducted by Edna Tartaul Daniel in 1961 and 1962.
ENCEPHALOMYELITIS (Abridged)
Reports and Suspicions

In July of 1930 a large number of horses were reported dying in the San Joaquin
Valley, particularly in the vicinity of Fresno, from botulism. Well, the moment that word is
mentioned I have to investigate, and I had my theoretical reservations because in summertime
you couldnt have botulism, there would not be an adequate amount of moisture in the feed
to permit botulinus to grow and produce its toxin. I sent Dr. Geiger down to a ranch outside
of Fresno where there were 670 horses. He came back and said, Yes, these horses are partly
paralyzed, they are unable to walk or if they walk they walk in circles.

So, I went out in the field. I took my moving picture camera with me and began
to photograph the horses, and when I projected them, after coming back, I realized from the
motions that this was really the result of inflammation of the brain and not any toxin action,
so botulism was out. Now, what was it? That was the question. These cases multiplied at a
terrific rate. The late Dr. Clarence H. Haring, who was in charge of the Division of Veterinary
Science, convinced us that this was an important thing because there were hundreds of deaths
of horses. Dr. Haring and I went to the valley together and standing on street corners just
counting the number of legs on trucks as they carted the horses to rendering plants, I roughly
estimated that there were between three and four thousand horses dead from this thing. At
any rate, I had carefully collected at least twelve horse brains; I did that all myself in the field,
and none took. By that time, it was the latter part of October and the number of cases became
less and less, and I was afraid this would begin to disappear with no solution. So, it was agreed
with Dr. Haring that we would set up a kind of listening post in the area of Merced where there
were still some cases. I thought that my failure to isolate this agent out of the brain was perhaps
attributable to the fact that I used only the brains from dead horses. I had already known from
the twenties that sometimes if there is a virus present in the brain, at the time of autopsy its
gone.
Procuring Fresh Tissue

I said that we must get a horse which has the first signs of it. Sure enough, one of the
stooges located a horse outside of Merced, and he went to the rancher and asked if he could buy
it and this fellow said, I wont sell the horse, and if you ever do anything to the horse I shoot
you. This was the news which came to me over the telephone, and I said, All right, let me try
my hand in this game. I went down and I had a $20 bill in my pocket. This was a depression
year and I was sure they would be glad to get rid of the horse for $20. When I walked into the
hotel in Merced there was a gloomy crowd sitting around. You shouldnt dare to do anything
out there. Youre going to get shot. I said, Im not going to talk to him. Im going to talk to his
wife. So I went out.

I talked to her and I said, look here, this horse is going to die anyhow, and when its
dead you havent anything. It just goes to the rendering plant and you get a couple of dollars.
On the other hand, you see, you could contribute to the knowledge of what this is and perhaps
to its prevention. Well, she said, My husband is just irate about this. I said, Yes, I can readily
understand, but look here, suppose I trust you, and I give you $20 and the next morning you
will find in the backyard the horse without a head? How are you going to do this? Look here,
about nine oclock at night when it is dark, Ill be over here behind some bushes on the corner
of the street, and I can see the window of your house. When your husband is sound asleep you
lift up the shade. I was at the corner of the street absolutely prepared for everything. I had a
syringe with strychnine, I had a good sharp knife, and I sat around there and smoked a pipe,

and sure enough about twenty minutes past nine the shade went up. Within about two minutes
I was over the fence and in another two minutes the strychnine was under the skin of the horse
and in another two or three minutes the horse was down, and in another five minutes the head
was off.

It was a heavy head, but I threw it over the fence and wrapped it in burlap and we
vanished as fast as we could to the most remote corner on the other side of the town of Merced
where Haring had located an old abandoned chicken coop, and there with the help of flashlights
I did a careful dissection of the brain and wrapped it up so that it was not contaminated, etc.
This was all done and we were about ready to go home by midnight. We drove back, and I tell
you, naturally, I was fantastically excited. Haring drove ahead of me, and he had the head of the
horse without the brain in the trunk of his car and the burlap began to turn loose and it was
flying just like a flag. Oh, it was a dramatic sight. We got back to the lab about six oclock in
the morning. I immediately got busy and made a suspension of the brain material. I was over
in Berkeley by about 9:30 and by ten oclock I had made two inoculations. I inoculated some
of the suspension directly into the eye of a horse and another part of the suspension I put into
the brain of the horse, which was a tricky techniquenobody ever had done that before, but I
had figured it out and I knew that if I could make a trephine hole just above the eye at a triangle
point I would get into the brain....

This was done in the canyon. Just behind the stadium [University of California,
Berkeley] was the veterinary science department and there we did this. We said Boo to nobody
because there could have been a lot of Meyer: criticism for bringing in an infection like this. The
rest of the brain was prepared by Miss Howitt, who was with me and who was very, very good,
and we had agreed that instead of merely using rabbits we would use mice, we would use guinea
pigs, and we would even use monkeys and we would put the material directly into the brain.
This gave us the virus. Both horses came down and both of them died from encephalitisthe
virus was in the brainone monkey came down with typical encephalitis manifestations but
recovered.... Here we were really in the midst of a most amazing type of observation. We had
a virus which was inoculable, passed from brain to brain. The next important thing was to try
to repeat it, but then the cases had disappeared. It had become cold. So it was most important,
during the winter months, to continue the passages, to keep it going. There were no difficulties
at all, it was there.

Then I began to plot on maps where the cases had occurred and what relationship
there was to the environment. In the course of doing this plotting I had received reports
that in Kern County Hospital there had been fantastic increases in cases of polio, all from
rural areas. I went down and saw some of these cases and said, This is not polio, this is
encephalitis. Then cases of human deaths occurred, and I had told every pathologist in valley
areas, If you do an autopsy, let me have the brain. Well, what these boobs did, instead of giving
the brain in the frozen state, they pickled it in formalin so naturally you couldnt do anything
but get a section and the sections again showed typical encephalitis. [Beatrice Howitt eventually
made an isolate from a fatal human case.]

Then, by looking at the map I began to see one crazy thing, that most of the cases
were in an irrigated area. The moment you went in the foothills, no cases. I said, Hmm, this
looks very much as if this might be transmitted by an insect; this might perhaps be mosquitoborne. But in 31, nobody had any techniques, nobody knew much how to do these things, but
it crystallized more and more that this was a mosquito-borne affair.

1930 Karl Meyer, Beatrice Howitt, Clarence Haring western equine encephalitis virus (the first alphavirus)

page 161

William Elfords Filtration System

William Joseph Elford

(1900-1952)

Christopher Howard Andrewes

(1896-1988)

Approximate Sizes of Certain Viruses as Determined by

Filtration Through Graded Collodion Membranes, 1934
(Table Prepared by Dr. J. H. Bauer of the International
Health Division of the Rockefeller Foundation)

The technology and equipment for producing, using and testing collodian membranes to estimate the size
of virus particles advanced very quickly, with several key papers published in the early 1930s that allowed
investigators in many laboratories to replicate Elfords work. One of the most remarkable things about the
early use of collodion membranes was its reproducability and its accuracy most of the virus particle size
estimates made using the method were remarkably close to measurements made many years later by more
sophisticated methods.
Elford WJ. A new series of graded collodion membranes suitable for general bacteriological use, especially in
filterable virus studies. J Pathol Bacteriol. 1931;34:505-521.
Elford WJ, Andrewes CH. Filtration of vaccinia virus through graded collodion membranes. Brit Jour Exp
Path. 1932;13:13-36.
Elford WJ, Andrewes CH. The sizes of different bacteriophages. Brit Jour Exp Path. 1932;13:446-456.
Bauer JH, Hughes TP. The preparation of the graded collodion membranes of Elford and their use in the study
of filterable viruses. Jour Gen Physiol. 1934;18:143-162.

1931 William Elford, Christopher Andrewes use of graded collodion membranes to determine virion size
page 162

In 1931, when Alice Miles Woodruff joined the

Department of Pathology at Vanderbilt University,
Ernest Goodpasture asked her to try to grow fowlpox
virus in tissue cultures made from chick kidney
cells this did not work. Well, lets try the chick
embryo itself, Goodpasture suggested, its made up
of living cells, and there are no bacteria within it.
Woodruff, using fertilized chicken eggs, cut a flap
in the shell with a tiny blade, injected fowlpox virus
into the embryo, sealed the window with a thin piece
of glass held in place with Vaseline, and replaced the
egg in an incubator. But this did not work because,
without exception, bacteria gained entrance and killed
the embryo. It was agreed that bacteria were being
transferred to the embryo together with the virus.
Henceforth all fowlpox samples were tested for an
absence of bacteria before transfers were made. Still,
the embryos died from bacterial infection. Every time
she and Goodpasture thought about abandoning the
experiments someone would say, Lets give it just one
more try. If we can only manage to get the virus to
grow in one embryo we can use that as a pure source
for all other experiments.
As luck would have it, Alice Woodruff discovered an
inoculated egg in which the embryo was thriving; it
also had a slightly swollen claw where the virus had
been placed. When the swollen tissue was transferred
to another embryo it produced more swollen tissues.
Soon she had a number of embryos that contained
pure virus. Under the microscope the swollen tissues
showed inclusion bodies. To make sure that this was
the same virus she started out with, she took some of
the swollen tissue from an embryo and injected it into
a grown chick; it developed typical fowlpox.

Alice Miles Woodruff (1900-1985)

and Ernest Goodpasture

[from Vanderbilt University Archives, used with permission]

[Alice Miles Woodruff s husband, C. Eugene Woodruff, also working with
Ernest Goodpasture at Vanderbilt, published the first evidence of the
relationhip between virions (elementary bodies) and viral inclusion
bodies, in 1928]

Ernest W. Goodpasture
(1886-1960)

Based on this work, Max Theiler of the Rockefeller

Institute, in 1937, grew yellow fever virus in eggs and
passed it through hundreds of mice and chicks until
he had a mutant that could be used in a vaccine
17D vaccine. Theilers work led to several other viral
vaccines as well.
From: Podolsky ML. Cures out of chaos. How
unexpected discoveries led to breakthroughs in
medicine and health. Chur, Switzerland, Harwood
Publishers; 1997.

Embryonating hens egg

from work of James Bumgardner Murphy
and Francis Peyton Rous, 1930s

1931 Alice Woodruff, Ernest Goodpasture development of embryonating hens eggs as host for viruses

page 163

Ernest W. Goodpasture (1886-1960)

and Frank Macfarlane Burnet (1899-1985)

Herald Rea Cox (1907-1986)

By 1932, Alice Miles Woodruff and Ernest Goodpasture used embryonating hens eggs for the isolation of herpes simplex virus
and shortly thereafter variola virus (grown on the choriallantoic membrane, CAM). By 1940, influenza A & B viruses were being
grown in the amniotic and allantoic cavities of embryonating eggs, and by the end of World War II when antibiotics (penicillin
and streptomycin) became available, embryonating eggs were a very important part of laboratory diagnostics and research. W. I.
B. Beveridge and F. M. Burnet published a book in 1946, The Cultivation of Viruses and Rickettsiae in the Chick Embryo [Medical
Research Council, Special Report 256, London], in which 37 viruses were described as cultivable in eggs, including the viruses
of smallpox, yellow fever, rabies, St. Louis and Japanese encephalitis, mumps and rinderpest. The usefulness of the method for
preparing vaccines was also emphasized. The embryonating egg reached its peak in the laboratory by the 1970s since then,
except for its use in influenza research and vaccine manufacture, it has
largely been replaced by cell culture methods.
Myxoma virus, chorioallantoic membrane,

embryonating hens egg

1931> Frank Macfarlane Burnet, Herald Cox development of embryonating hens eggs as host for viruses
page 164

Robert Daubney (1891-1977)

John Richard Hudson (1904-1990)

Percy Cyril Claude Garnham (1901-1994)

The first recorded outbreaks of Rift Valley fever occurred in Kenya in 1910 and 1913 in imported sheep; there were very large
numbers of abortions and deaths in newborn lambs and parturient dams. In 1930, Robert Daubney and John Richard Hudson
investigated a very large outbreak and carried out a classic study, isolating Rift Valley fever virus and describing the pathology
of the disease and much about its natural history. Subsequently, as imported animal numbers increased, further outbreaks
were reported in South Africa, sub-Saharan Central Africa and North Africa. The transmission cycle of Rift Valley fever virus
in eastern Africa involves virus survival in floodwater Aedes spp. mosquito eggs infected transovarially. These eggs survive
long periods of drought in dry water holes (called dambos) in fact, these eggs require a period of dehydration before they
will hatch. When dambos flood after heavy rainfall, the eggs hatch and the adults infect nearby domestic and wild animals. The
same flooding later leads to a population explosion of Culex spp. mosquitoes, which become infected by feeding on the viremic
animals these mosquitoes disperse widely, leading to extensive dissemination of virus and epidemics which often involve
humans as well as sheep and cattle. Since epidemics in East Africa are closely associated with the heavy rainfall that occurs
during the warm phase of the El Nio/Southern Oscillation, satellite-based mapping of excess surface water has been used
successfully to predict epidemics and guide intervention using livestock vaccine. In 1977, a large outbreak spread from Sudan
to Egypt along the Nile affecting 25%-50% of all sheep and cattle. The outbreak involved hundreds of thousands of human
infections with 18,000 officially confirmed clinical cases and 600 deaths (actual numbers were considered to be much higher).
There have been repeated epidemics in many different localities in Africa ever since. Following the opening of the Diama Dam
in 1987, an outbreak occurred in Mauritania. The relation between major irrigation projects and outbreaks clearly is due to an
increase in mosquito breeding habitat. Because of the importance of the livestock and human diseases, there is much ongoing
effort to develop / improve Rift Valley fever virus vaccines.

1931 Robert Daubney, J. Richard Hudson, Percy Garnham discovery of Rift Valley fever virus

page 165

Rift Valley fever virus, liver, mouse, thin section electron microscopy

[from an unpublished study by the author and Joel McKeith Dalrymple (1939-1992)]
Daubney R, Hudson JR, Garnham PC. Epizootic hepatitis or Rift Valley fever: an undescribed
virus disease of sheep, cattle and man from East Africa. J Pathol Bacteriol. 1931;34:545-579.
This classic paper influenced viral disease pathology / pathogenesis / natural history studies for
many years. For example, here is a snippet of text from the paper: Histologically the liver lesion
is in every way as diagnostic as the naked eye appearance of the organ. One might go further
and state that whenever the macroscopic changes are insufficiently pronounced to enable a
diagnosis to be made, which is not infrequently the case in adult sheep, histological examination
will enable one to establish the diagnosis beyond any possibility of error. Although the general
picture may vary with the susceptibility of the subject and with the degree of rapidity with
which a fatal termination or recovery is reached, the nature of the process remains essentially
Rift Valley fever, liver, human, from an epidemic in eastern Africa in
the same in all cases. It may be characterised briefly as a focal degeneration of liver cells
2006-2007 studied by Sherif Zaki of the Centers for Disease Control and
Prevention. Immunohistochemistry, paraffin section. The most distinctive closely followed by infiltration of the lesion with phagocytic cells, chiefly polymorphonuclear
leucocytes and histiocytes. The general sequence of these changes can most easily be followed
histopathologic change in liver tissue from fatal cases is extensive focal
hepatocellular necrosis without prominent inflammatory cell infiltration. in adult sheep, since in lambs the whole process is so rapid and the destruction of tissue is so
enormous as to obscure completely the actual nature of the lesion. In hyperacute cases in young
[from Sherif Zaki, used with permission]
lambs a section of the organ may be unrecognisable as liver.

1931 Robert Daubney, J. Richard Hudson, Percy Garnham discovery of Rift Valley fever virus
page 166

John Richard Hudson (1904-1990)

This page exemplifies another accomplished virologist/microbiologist/epidemiologist

who contributed importantly, yet remains largely unrecognized. J. Richard Hudson
played an important role in the discovery of one of the most important zoonotic
viruses, globally, Rift Valley fever virus. Upon initial searching, it became clear that
he had a long, distinguished career, serving in several institutions and in several
countries. He is an example also of the difficulty in learning more when except for a
name/address in research papers the trail has gone cold.

catarrhal fever (Alcelaphine herpes virus 1 and Ovine herpes virus 2; Rabies (rabies
virus); Nematodes (roundworms) of sheep and cattle; Cestodes (tapeworms) of sheep,
cattle, reptiles (a tapeworm of cobras!); Bovine hemorrhagic septicemia (Pasteurella
multocida); Fowl typhoid (Salmonella gallinarum); Redwater - Bovine babesiosis
(Babesia bovis and Babesia bigemina); Heartwater (Ehrlichia ruminantium);
Streptothricosis (Dermatophilus congolensis); Bovine trypanosomiasis - Nagana
(Trypanosoma brucei brucei and Trypanosoma vivax); Ticks; and Poisonous plants.

The reality is that the date and workplace in a key citation gives no clue about the
rest of the persons career. Hoping for an example of what Google might yield, I took
as my test case this single name and institutional address from the classic paper on
Rift Valley fever virus [Daubney R, Hudson JR, Garnham PC. Epizootic hepatitis or
Rift Valley fever: an undescribed virus disease of sheep, cattle and man from East
Africa. J Pathol Bacteriol. 1931;34:545-579.]. Immediately, it seemed clear that wildly
varying stories would emerge if this were done for many contributors to the virology
literature, some less, some more interesting, but all part of the warp and woof of the
science. I wondered if this Google search might make the case for future virologisthistorians to press on, using the ever expanding reach of the Internet to uncover other
pioneers. What follows is the very nice daisy chain that followed my test case.

Hudson was also a great naturalist and photographer, with a special interest in birds
and butterflies. He was editor of the Journal of the East Africa Natural History Society
and an officer of the society. After he moved to Australia, he became editor of the
Victorian Naturalist.

The seminal paper came from the Division of Veterinary Research, Kenya Colony.
Google found John Richard (Dick) Hudson on the alumni website of the King
Edward V Grammar School, Retford, UK. The site is maintained by John Palmer, who
in response to my email wrote: Hello Fred, What I know about John Richard Hudson
was sent me by his grandson Richard Hudson, who contacted me from Gillingham,
Dorset, UK. Wonderful emails from Richard followed: I am indeed the grandson of
John Richard Hudson. My grandfather was a very modest man and it was not until
after his death, when we were sorting out his study, that we became aware of his
achievements even my father, who was also a veterinary surgeon [John Richard
Hudson was at the midpoint of five successive generations of veterinary surgeons],
was unaware of the depth and extent of his research.
Hudson entered the Royal Veterinary College and qualified as MRCVS in 1925; he
was an outstanding student. He joined the Veterinary Service, Kenya Colony in 1925,
returning to England to marry Margaret Edith Davis in 1929. He progressed through
the East Africa Colonial Service ranks, early on with a research / laboratory bent; by
1946 he had been promoted to director of Veterinary Services for the colony.
He retired from the Colonial Service in 1947 and moved to the Central Veterinary
Laboratory, Weybridge, Surrey, UK, where he became the founding head of
the virology department. In 1956 he joined the UN/FAO bovine contagious
pleuropneumonia eradication program for Southeast Asia (Thailand), and from 1960
until 1967 he served as leader of the FAO/CSIRO pleuropneumonia eradication
program in Melbourne, Australia.
Hudsons infectious disease research was remarkably diverse; he published papers on:
Rift Valley fever (RVF virus); Nairobi sheep disease (NSD virus); Bovine malignant

An editorial in the Victorian Naturalist from 1968 states: Richard Hudson was a
visitor to Melbourne for a few years between 1960 and 1967. His life-long interest
in natural history, particularly birds and botany drew him to become a member of
the Field Naturalists Club of Victoria and the Bird Observers Club. He was quickly
recognized as a person of talent and ability and was elected editor in 1964 until just
before his retirement and return to Britain in 1966. Dick qualified as a veterinary
surgeon in London in 1925 and he did research, mainly on virus diseases, in Kenya
till 1947. After a short time in Britain he then worked for the Food and Agriculture
Organization of the UN in
various South-east Asian
countries before coming
to Melbourne in 1960 to
work on contagious bovine
pleuropneumonia. During
his stay in Kenya he edited
the Journal of the East Africa
and Uganda Natural History
Society. He is now enjoying his
retirement in Dorset, southern
England.
He capped off this diverse
record of accomplishment with
a paper in 1962: Veterinary
Education in Under-Developed
Countries (The Veterinary
Annual, Fourth Year). He died
in Dorset in 1990.
J. Richard Hudson, his wife
Margaret and their three
children, 1946

1931 Robert Daubney, J. Richard Hudson, Percy Garnham discovery of Rift Valley fever virus

page 167

Mammary adenocarcinoma, mouse mammary

tumor virus, prominent acinar architecture, H&E

Clarence Cook Little (1881-1971)

John Joseph Bittner (1904-1961)

Mouse mammary tumor virus (MMTV) is a milk transmitted retrovirus. It was formerly known as Bittner
virus, and before that as the milk factor, referring to the extra-chromosomal vertical transmission of
murine mammary cancer by adoptive nursing, as demonstrated in 1936 by John Bittner, while working at the
Jackson Laboratory in Bar Harbor, Maine. Bittner, a geneticist and cancer biologist, developed the idea that
a cancerous agent, a milk factor, a virus in milk, could be transmitted from mothers to their young. Several
mouse strains carry the virus endogenously, but in other mouse strains the virus is only transmitted via milk.
In the former, the virus is carried as a DNA provirus integrated in the DNA of lymphocytes in the mammary
glands. The virus is transported through the gastrointestinal tract of pups to Peyers patches where it infects
the new hosts macrophages, and then lymphocytes. In recent years, MMTV-like DNA sequences have been
found in human breast cancer tissue and most recently the virus has been shown to be able to productively
infect human cells, possibly suggesting that an MMTV like virus may play a role in human breast cancer
but this is quite controversial and largely unconfirmed.
Staff, Jackson Memorial Laboratory. The existence of non-chromosomal influence in the incidence of
mammary tumors in mice Science 1933;78:465-466.

Bittner JJ. Some possible effects of nursing on mammary gland tumor incidence in mice Science 1935;84:162.

1932 John Bittner, Clarence Little Jackson Memorial Laboratory discovery of mouse mammary tumor virus
page 168

Albert Bruce Sabin (1906-1993)

Frederick Parker Gay (1874-1939)

[from the U.S. National Academy of Sciences, used with permission]

The first documented case of human B virus infection occurred in 1932 when a researcher (patient W.B.) was bitten on the hand by an apparently healthy rhesus macaque
(Macaca mulatta) and died of progressive encephalomyelitis 15 days later. Two research groups obtained samples from patient W.B.: Frederick Gay and Margaret Holden
(University of California, later Columbia University) and Albert Sabin and Arthur Wright (Cincinnati Childrens Hospital). Both groups demonstrated a similar disease
progression in rabbits inoculated with nerve tissue from patient W.B. and characterized the agent as a herpesvirus. Neither group was able to produce disease in rhesus
macaques, presumably because the monkeys were already immune. Sabins group named the virus B virus (after patient W.B.). Formal and vernacular nomenclature have left
several synonymous names for this virus: Macacine herpesvirus 1, Cercopithecine herpesvirus 1, herpesvirus simiae, as well as B virus.
Gay FP, Holden M. The herpes encephalitis problem. J Infect Dis. 1933;53:287-303.
Sabin AB, Wright WM. Acute ascending myelitis following a monkey bite, with the isolation of a virus capable of reproducing the disease. J Exp Med. 1934;59:115-136.

1932 Frederick Gay, Albert Sabin, Margaret Holden, Arthur Wright discovery of B virus

page 169

Wilson Smith
(1897-1965)

Christopher Howard Andrewes

(1896-1988)

Patrick Playfair Laidlaw

(1881-1940)

Even though influenza was shown during the great pandemic of 1918-1919 to be caused by an ultrafilterable virus, by Charles Nicolle and quite a few others, and this was taken
as common knowledge by Thomas Rivers in his seminal book, Filterable Viruses, in 1928, there seemed to be lingering uncertainty. When in 1931, Richard Shope discovered
swine influenza virus and concluded that the disease in swine required the interaction of the virus and a bacterium, now called Haemophilus parasuis, the wish for further
proof of the etiology of the human disease became more vocal. It was Wilson Smith, Christopher Andrewes and Patrick Laidlaw at the National Institute of Medical Research
(NIMR) Mill Hill who settled the matter, irrefutably. Through the 1920s, Laidlaw had worked on canine distemper, culminating in discovery of the viral etiologic agent and
development of the first distemper vaccine. The key to this work was a ferret experimental animal model. When a severe influenza epidemic erupted in 1929, Laidlaw recruited
Smith and Andrewes and they infused ultrafiltered throat washings from eight influenza patients into rats, mice, guinea pigs, rabbits, monkeys, pigs, and horses. These efforts
failed. Curiously, ferrets, Laidlaws favorite model, were not among the first animals tested. However, soon after, following upon reports of an outbreak of a flu-like disease
among ferrets at Wellcome Laboratories, where the animals were being used to manufacture canine distemper vaccine, Smith instilled intranasally into two ferrets ultrafiltered
nasal washings taken from Andrewes, who had himself caught the flu. Within 48 hours the animals started sneezing and displaying signs of flu-like disease. Washings from
seven other patients also induced the disease. However, the etiologic agent was lost when canine distemper broke-out among the ferrets. By a twist of fate, shortly afterward
Smith developed influenza and Andrewes used his throat washing to infect a new batch of ferrets, now maintained in an isolation facility. The virus isolated from these ferrets
became the first influenza virus isolate (designated WS after Smith), the one used for validation of ultrafilterability, serial passage in ferrets, neutralization by convalescent
ferret serum, and transfer to mice. Most importantly, this work was seminal in shifting virological research from the perspective of its bacteriological roots to that of
experimental pathology the NIMR researchers identified themselves as experimental pathologists. This was the perspective of virology until the rise of molecular biology.
Smith W, Andrewes C, Laidlaw P. A virus obtained from influenza patients. Lancet 1933;2:66-68.
Bresalier M. Uses of a Pandemic: Forging the Identities of Influenza and Virus Research in Interwar Britain. Social Hist Med. 2011;25;400-424.

1933 Wilson Smith, Christopher Andrewes, Patrick Laidlaw ferret model confirming the viral etiology of influenza

page 170

Richard Edwin Shope

(1901-1966)

Francis Peyton Rous

(1879-1970)

Joseph Willis Beard

(1906-1983)

In 1933, Richard Shope learned from hunters in Kansas and Iowa of tumors around the head and shoulders of cottontail rabbits
(Sylvilagus spp.), appearing as large horny protruberances sometimes resembling antlers. The antlers were shown to be
epidermal papillomas, which because cornified cells continued to form but did not slough off, grew to appear as tough horns
[thought to be the origin of the mythical jackalope the jackrabbit with antelope horns or deer antlers]. Shope and E. Weston
Hurst showed that soluble extracts from the abnormal epdermal tissues from wild cottontail rabbits contained an ultrafilterable
(Berkefeld V filter) agent, the Shope papillomavirus (now cottontail rabbit papillomavirus), which can be transmitted and cause
disease in wild cottontail and domestic rabbits (Oryctolagus cuniculus). As covered later in this book, in 1935, Peyton Rous and
Joseph Beard showed the tumorigenic potential of the Shope virus, thereby recentering much of virus-cancer research for many
years. Meanwhile, other papillomaviruses were discovered and later became important research subjects: canine papillomavirus
was discovered by John McFadyean and Frederick Hobday in 1898; the first human papillomavirus was discovered by Giuseppe
Ciuffo in 1907; and, importantly because of its capacity to grow in cell culture, bovine papillomavirus was discovered by O.
Magelhaes in 1920.
Shope RE, Hurst EW. Infectious papillomatosis of rabbits: with a note on the histopathology. J Exp Med. 1933;58:607-624.
McFadyan J, Hobday F. Note on the experimental transmission of warts in the dog. J Comp Pathol Ther. 1898;11:341-344.
Magalhaes O. Verruga dos bovideos. Brasil-Medico 1920;34:430-431.

Horned hare by Benard. Summa

Dubiorum Circa Classes Quadrupedum
et Amphibiorum, 1743

1933 Richard Shope, Francis Peyton Rous, Joseph Beard discovery of rabbit (Shope) papillomavirus

page 171

Max Knoll (1897-1969), Ernst Ruska and their first electron microscope
One half of the 1986 Nobel Prize in Physics was awarded to
Ernst Ruska for his fundamental work in electron optics and
for the design of the first electron microscope, one of the most
important inventions of this century. Its development began at
the end of the 1920s with work carried out by Ruska as a young
student at the Berlin Technical University, under the supervison
of Max Knoll. Ruska found that a magnetic coil could act as a
lens to focus electrons, and that such electron lenses could be
used to obtain an image of an object irradiated with electrons. By
coupling two electron lenses he produced a primitive microscope.
Ernst Ruska (1906-1988)
He quickly improved on this and in 1933 built the first electron
microscope with a performance clearly superior to that of the
Ruska E, Knoll M. Die magnetische sammelspule fr schnelle
elektronenstrahlen. (The magnetic concentrating coil for fast electron light microscope. After completing his PhD in 1933, Ruska
joined Siemens, where in 1939 he led the development of the
beams.) Zeit Physik 1931;12:389-400 und 448.
first commercially-produced electron microscope. He also set up
Knoll M, Ruska E. Das elektronenmikroscop. Zeit Physik
a laboratory for visiting researchers, which was initially headed
1932;78:318-329.
by his brother Helmut, a physician who took the first electron
micrograph of a virus.

1933 Ernst Ruska, Max Knoll invention of the transmission electron microscope
page 172

The first transmission electron

microscope, Ernst Ruska and
Max Knoll (1933)
(replica)

The first commercially built transmission electron microscope

the Siemens bermikroskop M 100 (1939)

The first commercially produced electron microscope was produced by Siemens in 1939, as described by Ernst Ruska: As first collaborators [Max Knoll and I] started in
1937, and in 1938 we had completed two prototypes with condenser and polepieces for objective and projective lenses as well as airlocks for specimens and photoplates. The
maximum magnification was 30,000x. One of these instruments was immediately used for the first biological investigations by [my brother] Helmut Ruska and several medical
collaborators. By the end of 1939 the first serially produced Siemens instrument had been delivered to Hoechst [Hoechst AG, now Sanofi-Aventis]. By February 1945 more than
30 electron microscopes [bermikroskop] had been built in Berlin and delivered. By the late 1930s electron microscopes with theoretical resolving power of 10nm were being
produced. By 1946, resolving power had reached ~1.0nm, by 1970, 0.6nm, and by 1990, 0.1-0.2nm. [The theoretical resolving power of a an optical light microscope is 200 nm.]

1933 Ernst Ruska, Max Knoll invention of the transmission electron microscope

page 173

Ralph S. Muckenfuss (1899-1979)

St. Louis encephalitis virus, Culex pipiens salivary gland

The Metropolitan Health Council of St. Louis appointed Ralph Muckenfuss, Assistant
Professor of Medicine at Washington University School of Medicine, chairman of the
committee tasked with finding the cause of a major encephalitis epidemic still ongoing
in the area. [Muckenfuss later became the first director of The Public Health Research
Institute of the City of New York and director of the laboratories of the New York City
Department of Health.]

1933 Ralph Muckenfuss, Leslie Webster, Charles Armstrong, others discovery of St. Louis encephalitis virus

page 174

Leslie Tillotson Webster (1894-1943)

[from Leslie Webster III, used with permission]

Charles J. Armstrong (1886-1967)

Muckenfuss RS, Armstrong C, McCordock HA. Encephalitis: Studies on experimental transmission. Public Health Reports 1933;48:1341-1343. [virus isolation via intracerebral
inoculation of rhesus macaques]
Webster LT, Fite GL. A virus encountered in the study of material from cases of encephalitis in the St. Louis and Kansas City epidemics of 1933. Science 1933;78:463-465. [virus
isolation via intracerebral inoculation of Webster-Swiss mice]
In 1933, in five weeks in autumn an encephalitis epidemic of explosive proportions broke out in the vicinity of St. Louis, Missouri and the neighboring St. Louis County.
Over 1,300 cases were reported to the local health departments and the newly constituted National Institute of Health was appealed to for epidemiological and investigative
expertise. Time Magazine, 1937: The only scientific fact known about this disease is that it is caused by a specific virus. This was ascertained during the 1933 epidemic by one
of the most vigorous and concentrated attacks on a disease ever made by Medicine. Immediate discoverers of that virus were Dr. Howard McCordock, Washington University
School of Medicine, St. Louis; Dr. Charles Armstrong, virus expert of the U. S. Public Health Service; Dr. Leslie Webster of the Rockefeller Institute; and Dr. Ralph Muckenfuss,
then of Barnes Hospital, Washington University School of medicine, St. Louis, now director of NYCs Bureau of Laboratories.

1933 Ralph Muckenfuss, Leslie Webster, Charles Armstrong, others discovery of St. Louis encephalitis virus

page 175

Roberto Franco
(1874-1958)

Jerome Austin Kerr

(1925-1974

Studies of arboviruses, such as yellow fever

virus, were carried out for many years at the
Instituto Evandro Chagas, Belem, Brazil,
supported by the Rockefeller Foundation.
More than 50 different viruses were found,
many from tree platforms different
mosquito species harboring different
viruses were found at particular levels in
the canopy, Haemagogus spp. at the top of
the canopy.

Lus Patio Camargo

(1891-1978)

Jorge Boshell-Manrique
(1903-1976)

Fred L. Soper
(1893-1977)

The control of the urban mosquito vector of yellow fever, Aedes aegypti, in the major port cities of the western hemisphere in the
early decades of the twentieth century led to the notion that the disease could be eradicated. However, unexplained outbreaks in
inland rural forested areas continued after urban centers had been freed of Aedes aegypti. Roberto Franco (1874-1958) first studied
this in Colombia and coined the term yellow fever of the forests. In 1910-1911 he reported in an obscure journal that yellow fever
was transmitted by mosquitoes that bit during the day while men were working in the forest. From 1926 to 1932, Jerome Austin Kerr
(1925-1974)(Rockefeller Foundation staff member) and Lus Patio Camargo (1891-1978)(Rockefeller Foundation employee) found a
high level of yellow fever seropositivity in a forested region where there was no Aedes aegypti, but were unable to identify the vector.
Fred L. Soper (1893-1977), head of the Brazilian office of the International Health Division of the Rockefeller Foundation, prevented
the publication of the Colombian work, due to the dangerous nature of the findings, but then sent his own article to the American
Journal of Hygiene, intending to establish priority for the discovery of jungle yellow fever. For many years, Soper did indeed hold this
legacy, but now we understand the proper shared legacy. Soper went on to become the main architect of the Rockefeller Foundations
Victory Over Yellow Fever campaign in Brazil. He directed the program toward the nearly complete eradication of the urban
mosquito, Aedes aegypti, from Brazil and other Latin American countries. Soper later also led the Rockefeller Foundations yellow
fever vaccination campaign in the region. In 1936, Jorge Boshell Manrique (1903-1976)(also a Rockefeller Foundation employee)
confirmed the role of arboreal Haemagogus spp. mosquitoes and the endemic forest canopy / monkey
cycle of the virus. Haemagogus spp. mosquitoes live at the top of the jungle canopy, but are brought to
ground level when trees are felled, thereby transmitting virus to forest workers. In Africa, several species
of Aedes mosquitoes were later shown to maintain a cycle of jungle yellow fever and carry virus to the
edges of urban habitats, where again Aedes aegypti (and other urban Aedes species) cause epidemics.
Quevedo E, Manosalva C, Tafur M, Bedoya J, Matiz G, Morales E. Knowledge and power: the
asymmetry of interests of Colombian and Rockefeller doctors in the construction of the concept of
jungle yellow fever, 1907-1938. Can Bull Med Hist. 2008;25:71-109. (History of Medicine Center,
Universidad Nacional de Colombia)
Soper FL, Penna HA, Cardoso E, Serafim J, Frobisher, M, Pinheiro J. Yellow fever without Aedes aegypti:
study of rural epidemic in Valle do Chanaan, Espirito Santo, Brazil. Am J Hyg. 1932;18:555-587.

Hemagogus janthinomys

1910-1933 R Franco, JA Kerr, L Patio Camargo, F Soper, J Boshell Manrique discovery of jungle yellow fever cycle
page 176

William W. Dimock
(1880-1953)

Elvis Roger Doll

(1912-1967)
There are now 9 equine/equid herpesviruses:
Equine herpesvirus 1 exists in horse populations
worldwide and is associated with acute respiratory
disease. It is also responsible for abortion in mares and
neurological disease, though the latter is rare.
Equine herpesvirus 2 is of unclear clinical significance,
but it is consistently found in the respiratory mucosa,
conjunctiva and leukocytes of clinically normal horses
and has been associated with keratoconjunctivitis, upper
respiratory tract disease, generalized malaise and fever,
pharyngitis and lymphadenitis.
Equine herpesvirus 3, also called equine coital
exanthema virus, is the cause of a sexually-transmitted
genital vesicular disease of some importance, especially in
breeding mares and stallions.
Equine herpesvirus 4, like EHV-1 causes acute
respiratory disease in horses worldwide and is rarely the
cause of neurological sequelae in infected horses.

Michael J. Studdert

Equine fetus, lung, equine herpesvirus 1. (top) Necrotizing

bronchiolitis with intranuclear eosinophilic inclusions and
chromatin margination (H&E) (bottom) Same, with virus antigen
detected using a monoclonal antibody recognizing a specific
viral structural glycoprotein. (Immunohistochemistry
and hematoxylin)

The other viruses mostly infect other equid species.

1933> William Dimmock, Elvis Doll equine abortion virus and equine rhinopneumonitis virus (herpesviruses)

page 177

The Etiology of Mumps.

C.D. Johnson and E.W. Goodpasture, 1934:
We have recently reported the production of a nonsuppurative parotitis in the rhesus monkey (Macaca mulatta)
by the inoculation of the glands, through Stensons duct, with
saliva collected from early cases of epidemic parotitis. The
etiological agent of the experimental parotitis was found to be
a filterable, cytotropic virus which is resistant to freezing,
drying and glycerination. This virus was not found in normal
saliva nor did it correspond to any known virus with which we
were familiar.
The difficulty of proving the etiological relationship of a virus
to a particular disease is apparent when one realizes that the
two means of establishing microbial causation, heretofore so
successfully applied to infectious diseases, namely cultivation
and microscopic morphology, are completely lacking in the
study of viruses. Therefore, the only means one has to rely
on are the specific inhibition of a particular virus by immune
serum from an animal or man recently recovered from an
attack of the specific infection, and the reproduction of the
specific disease in the natural host by inoculation with the
virus under investigation.

Mumps N protein (turquoise) in

the cytoplasm of a cultured cell.
Immunofluorescence

Ernest W. Goodpasture (1886-1960)

Johnson CD, Goodpasture EW. An investigation of the etiology of
mumps. J Exp Med. 1934;59:1-19.
Johnson CD, Goodpasture EW. Experimental immunity to the virus
of mumps in monkeys. Am J Hyg. 1936;23:329-339.
Johnson CD, Goodpasture EW. The histopathology of experimental
mumps in Macacus rhesus. Am J Pathol. 1936;12:495-510.

1934 Claud Johnson, Ernest Goodpasture discovery of mumps virus

page 178

Mumps virus, with prominent

nucleocapsids in budding virions
thin section electron microscopy

Claud D. Johnson (1910-1976)

Vanderbilt University School of Medicine,
Class of 1932
[from Vanderbilt Archives, with permission]

Beckman Acidimeter (1934)

with lemons

Arnold O. Beckman (1900-2004)

Model G pH Meter (1935)

The California citrus industry needed an accurate gauge of the level of acidity of fruit and derived products. At the time, early in the 1930s, the most common method for
determining acidity was litmus paper (litmus is a soluble powder derived from lichens). When the paper turned color, color charts helped the examiner determine the acidity
of the solution. Chemists also used electrochemical methods to measure the concentration of hydrogen ions, but these did not work well on citrus products. When approached
about the problem, Arnold Beckman recommended using a vacuum tube voltmeter to amplify weak electrical signals from a glass electrode submerged in the sample to be
tested. In 1934, Beckman decided he would build the instrument himself it worked so well that he was asked to make another one just like it. He soon concluded that all
the parts should be put together in an integrated instrument, initially called an acidimeter. Beckman not only figured out how to measure pH accurately, he revolutionized
instrumentation by building the first chemical instrument that consisted of one compact unit that was portable. This simplified research as the user no longer had to assemble
various components an instrument could just be purchased. [The pH (hydrogen potential) scale, ranging from 0 to 14, was developed in 1909 by Sren Srenson, a Danish
biochemist.]

1934 Arnold Beckman invention of the electronic pH meter

page 179

Malcolm H. Merrill (1903-1987)

Charles Lacaillade, Jr. (1904-1978)

Carl Ten Broeck (1885-1966)

Eastern equine encephalitis virus, Aedes triseriatis mosquito salivary gland, thin section electron microscopy

[Ten Broeck photo from Rockefeller Archives; Lacaillade photo from St. Johns University Archives, used with permission]

1934 Malcolm Merrill, Charles Lacaillade, Carl Ten Broeck discovery of eastern equine encephalitis virus
page 180

Japanese encephalitis (JE) is the leading cause of viral encephalitis in

Asia, with an estimated annual incidence of 45,000 human cases and
10,000 deaths. However, JE cases are under-reported the true annual
incidence of encephalitis cases is estimated to be closer to 175,000.
Case-fatality rates (hospitalized) range from ~25-40%, with serious
neurologic sequelae occurring in ~45-70% of survivors depending
on the population and age of infected individuals. Outbreaks of JE in
northern India and Nepal between 2005 and 2007 have resulted in at
least 11,000 cases and over 2,000 deaths, highlighting the continued
burden of disease in developing countries and the continuing westward
spread of the virus.
Seminal experiments conducted near Tokyo in the 1950s elucidated
the transmission cycle of JE virus, with pigs and wild birds identified
as amplifying hosts and Culex tritaeniorhynchus incriminated as the
primary vector mosquito species. In Japan, it has been shown that pigs
are necessary for pre-epizootic amplification of the virus, although
some epidemics do occur in the absence of large pig populations (as is
certainly the case in India and Pakistan). Humans and horses develop
fatal encephalitis, but they are only incidentally infected and are deadend hosts of the virus.

Michitomo Hayashi (1885-1973)

Shiro Kasahara

[from Kitasato Institute, used with permission]

Hayashi M. Ubertragung des virus von encephalitis epidemica auf affen. Folia Psych et Neurol
Japonica 1935;1:419-465.
Kasahara S, Ueda M, Okamoto Y, Yoshida S, Hamano R, Yamada R. Experimental studies on
epidemic encephalitis. I. Transmission tests of the Japanese encephalitis in 1935 and some
characteristics of the infectious agent. Kitasato Arch Exp Med. 1936;13;48-65.[origin of prototype
Nakayama strain of virus]
Kasahara S, Yamada R, Hamano R. Experimental studies on epidemic encephalitis. 4.
Immunological comparison of viruses of the Japanese (B type) and the American (St. Louis) types.
Kitasato Arch Exp Med. 1937;14:229-233.
Hashimoto H, Kudo M, Uraguchi K. Experiences in the summer epidemics of acute encephalitis in
Tokyo. JAMA. 1936;106:1266.

1934 Michitomo Hayashi, Shiro Kasahara, others discovery of Japanese encephalitis virus

page 181

Top: TMV, purified, negative contrast electron microscopy, minimum exposure study, Robley
Williams,
1970s. Bottom: TMV in paracrystalline array in the cytoplasm of a tobacco plant cell,
Wendell Meredith Stanley (1904-1971)
thin section electron microscopy.
In 1935, Wendell Stanley of the Rockefeller Institute for Medical Research at Princeton emerged as a scientific celebrity. The media hailed his work as revolutionary, providing
the key to the riddle of life. By crystallizing tobacco mosaic virus (TMV) he seemed to have demonstrated that viruses are protein molecules, a special class of enzymes, and to
have proved the chemical nature of life. This was the symbolic beginning of molecular biology. Yet Stanleys work was flawed by technical errors and misconceptions. His virus
sample contained water and impurities and thus was not a true crystal. It also contained a significant fraction of nonprotein material: about 6% ribonucleic acid (RNA), which
Stanley had missed entirely, and which turned out to be the crucial component, the stuff of heredity. Furthermore, the property of self-replication in viruses was not based on
any enzymatic activity and crystal growth, as Stanley claimed, but was a direct consequence of the structure of the nucleic acid. Nevertheless, the wonder of crystalline life
and living molecules captured the imagination of many scientists, especially the Rockefeller Institute and the University of Uppsala, where the crystallization of TMV was
seen as a validation of the chemical nature of life and validation of the merit of research based in new technologies.
Stanley WM. Isolation of a crystalline protein possessing the properties of tobacco-mosaic virus. Science 1935;81:644-645.

1935 Wendell Stanley purification and crystallization of tobacco mosaic virus

page 182

Patient Asibi
Ghana, 1935

Max Theiler
(1899-1972)

Hugh Hollingsworth Smith

(1902-1995)

In June 1927, a blood sample was obtained by Johannes Bauer and Alexander Mahaffy from a 28-year-old man named Asibi, a resident of the village of Kpeve, Gold Coast
(now Ghana), who had a mild case of yellow fever. It was inoculated into a rhesus macaque at the Rockefeller Foundation field laboratory in Accra; the animal was moribund 4
days later. Blood from this animal was inoculated into a second macaque, which was transported during the incubation period to the Rockefeller Foundation main laboratory
at Yaba, near Lagos, Nigeria, where it developed clinical yellow fever the day after arrival. Adrian Stokes, Johannes Bauer and Noel Hudson established this, the Asibi strain,
by continuous direct passage in rhesus macaques. Starting in 1932, this virus strain was adapted to grow in mouse embryonic tissue by Max Theiler and Hugh Smith. After 17
mouse tissue passages the virus was passaged 58 times in whole chicken embryonic tissue and thereafter, until passage 114, in denervated chicken embryonic tissue. At this
stage, Theiler and Smith injected the virus intracerebrally into rhesus macaques the virus showed a marked reduction in its viscero- and neuro-tropism as compared to wild
type. This virus was further passaged, until passages 227 and 229, and then used to immunize 8 human volunteers. Results were satisfactory there were no adverse reactions
and all 8 recipients seroconverted within 2 weeks.
Theiler M, Smith HH. The effect of prolonged cultivation in vitro upon the pathogenicity of yellow fever virus. J Exp Med. 1937;65:767-786.
Monath TP. Yellow fever vaccines: the success of empiricism, pitfalls of application, and transition to molecular vaccinology. In: Plotkin SA, editor. History of vaccine
development. New York: Springer; 2011. p109-135.

1935 Max Theiler, Hugh Smith development of yellow fever vaccine

page 183

Max Knoll
(1897-1969)

Manfred von Ardenne

(1907-1997)

The theory of the scanning electron microscope (SEM) was

developed by Max Knoll in 1935 he then built one, but did
not patent it. Two years later Manfred von Ardenne built an
electron microscope with a probe for scanning transmission
electron microscopy (STEM) and also tried it as an SEM. Soon
afterwards, Vladimir Zworykin and colleagues built a dedicated
SEM. The general use of the SEM began in 1965 when the
Cambridge Instrument Company marketed its Stereoscan 1,
which was developed by Charles Oatley, Gary Stewart and
Dennis McMullan at Cambridge University (this was followed
about 6 months later by an instrument built by JEOL in Japan).
By 1952 the Stereoscan had achieved a resolution of 50 nm
and produced the first micrographs showing the striking
three-dimensional imaging characteristics of the modern-day
SEM. [Vladimir Zworykin also invented the iconoscope,
kinemascope, and storage principle that became the basis of
TV as we know it, and Manfred von Ardenne achieved the first
transmission of a television picture.]

Vladimir Zworykin
(1888-1982)

Charles Oatley
(1904-1996)

Ebola virus, scanning electron microscopy,

from Cynthia Goldsmith, Centers for
Disease Control and Prevention

Stereoscan 1
Cambridge Instrument Company
1965, the first commercially produced
scanning electron microscope

1935 Max Knoll, Manfred v Ardenne, Vladimir Zworykin, Charles Oatley the scanning electron microscope
page 184

Frederick Charles Bawden (1908-1972)

Norman Wingate Pirie (1907-1997)

Wendell Stanley was nominated for a Nobel Prize in chemistry for the first time in 1938 and 13 times after that until he was awarded it in 1946 it was the first Nobel Prize
in virology. The basis for the award was his crystallization of tobacco mosaic virus (TMV). Stanley concluded that the crystals contained protein, and he likened TMV to
an autocatalytic protein which, for the present, may be assumed to require the presence of living cells for multiplication. Frederick Bawden and Norman Pirie also purified
TMV: their results in 1936 were dramatic. Pirie later stated, In a few weeks using the methods that had been standard in protein chemistry for half a century, we got liquidcrystalline preparations of an infective nucleoprotein. They showed that TMV particles contain about 95% protein and 5% RNA, that the virus particles are rod-shaped,
can form liquid crystals, and have a regular substructure detectable by X-ray diffraction. Stanley had not detected nucleic acid in his preparations and at first disputed the
evidence for its presence later he admitted that he was wrong and agreed with Bawden and Pirie. However, this admission was not much aired in public venues. Later, some
knowledgeable people suggested that the Nobel Prize should have gone to Bawden and Pirie.
Bawden FC, Pirie NW, Bernal JD, Fankuchen I. Liquid crystalline substances from virus infected plants. Nature 1936;138:1051-1052.
Bawden FC, Pirie, NW. The isolation and some properties of liquid crystalline substances from solanaceous plants infected with three strains of tobacco mosaic virus.
Proc R Soc Lond. 1937;B123:274-320.

1936 Frederick Bawden, Norman Pirie tobacco mosaic virus is comprised of nucleoprotein, the nucleic acid is RNA

page 185

Jean Cuill (1872-1950)

Paul-Louis Chelle (1902-1943)

By 1755, the industrial revolution was in full swing and wool was in short supply. The British
parliament asked about the economic effects of a spreading disease in sheep. Thus began the recorded
history of scrapie. By 1850, C. Besnoit and his colleagues, at the Ecole Vtrinaire de Toulouse,
recognized the characteristic neuronal vacuolation in scrapie; they attempted to transmit the disease
via brain and blood from affected sheep. They kept animals for only a few months and the results
were negative. Jean Cuill and Paul-Louis Chelle, taking note of epidemiological studies indicating
incubation periods of >18 months, succeeded in 1936 in transmitting scrapie to two healthy sheep by
intraocular inoculation of brain tissue. They then transmitted the disease via intracerebral, epidural and
subcutaneous routes, and by passing brain tissue through an ultrafilter. The incubation period varied
from one to two years. This was the first step in the long path from thinking that the etiologic agent of
scrapie, and later the agents of other spongiform encephalopathies, was a virus, to the work of Stanley
Prusiner and his colleagues and their conclusion that the agent is a prion, an infectious protein.
Cuill J, Chelle P-L. La maladie dite tremblante du mouton est-elle inoculable? C R Acad Sci.
1936;203:1552-1554. [Ecole Vtrinaire de Toulouse]

Scrapie, sheep brain (geniculate nucleus)

(top) H&E; (bottom) H&E, counterstained with
antibody to glial fibrillary acidic protein (GFAP)

[micrographs courtesy of Robert Higgins, University of California,Davis]

1936 Jean Cuill, Paul-Louis Chelle transmission of scrapie prion in sheep using ultrafiltered CNS tissue
page 186

Charles J. Armstrong (1886-1967)

Thomas Milton Rivers (1888-1962)

[from Rockefeller Archives, used with permission]

Erich Traub (1906-1985)

In the 1930s, lymphocytic choriomeningitis (LCM) virus was discovered independently in three laboratories.
Charles Armstrong and Ralph Lillie, at the National Institutes of Health, isolated the virus along with St. Louis
encephalitis virus during the 1933 encephalitis epidemic in St. Louis. They showed that LCM virus was quite
distinct based on unusual pathological changes in the brains of experimentally infected monkeys and mice
(especially a massive lymphocytic infiltration of the choroid plexus and meninges). Armstrong went on to do
extensive virologic and pathogenetic research on LCM. Meanwhile, Erich Traub at the National Naval Research
Laboratory found the same virus in laboratory mice and went on to do the first epidemiological research on
the virus in mouse colonies. Thomas Rivers and T.F. McNair Scott at the Rockefeller Institute isolated the virus
from two cases of aseptic meningitis and went on to do the first prevalence studies of the infection in humans.
Armstrong C, Lillie RD. Experimental lymphocytic choriomeningitis of monkeys and mice produced by a virus
encountered in studies of the 1933 St. Louis encephalitis epidemic. Pub Health Rep. 1934;49:1619-1627.
Traub EA. Filterable virus recovered from white mice. Science 1935;81:298.
Rivers TM, Scott TFMcN. Meningitis in man caused by a filterable virus. Science 1935;81:439.

1936 Charles Armstrong, Thomas Rivers, Erich Traub lymphocytic choriomeningitis virus (the first arenavirus)

page 187

During the years 1911-1914, Peyton Rous worked at

disproving objections to generalizations made from
his discovery of virus-induced tumors in chickens. He
wrote, The findings with the chicken tumors largely
demolish the theoretical basis in which objections to an
extrinsic cause for cancer have been built up (1912).
He and Joseph Beard showed that tumors induced by
different viruses were capable of invading tissues and
of metastasizing to distant organs; thus they were true
cancers. Moreover, each independently isolated virus
caused a tumor of a different kind. In order to find
more generally acceptable evidence, Rous and Beard
attempted to extend their observations especially
through carefully devised experiments with the tumors
of other species of animals (1911). Evidently, for the
viral etiology of cancers to be accepted, similar findings
were needed in mammals. The strategy of their work
was determined at that time, but the extension of their
ideas to other species came only after Richard Shopes
discovery of rabbit papillomavirus in 1933. Shope
reported the first experimental studies on cottontail
rabbit papillomavirus (CRPV) infection and its life
cycle. From then on, the CRPV rabbit model was used
extensively to study virological aspects of papillomavirus
infections in vivo, virus-induced carcinogenesis,
immunological features of virus infection leading to
host immune-mediated tumor regression, interactions
between papillomas and co-carcinogens, viral gene
function, etc.

Francis Peyton Rous (1879-1970)

Joseph Willis Beard (1906-1983)

Rous FP, Beard JW. The neoplastic traits of a mammalian growth due to a filterable virusthe
Shope rabbit papilloma. Science 1934;79:437-438.
Rous FP, Beard JW. A virus-induced mammalian growth with the characters of a tumor (the
Shope rabbit papilloma). I. The growth on implantation within favorable hosts. J Exp Med.
1934;60:701-722.

Papillomaviruses infect epithelial tissues of cutaneous

and mucosal origin and are ubiquitous throughout the
animal kingdom from reptiles to primates. In humans,
more than 100 papillomavirus (HPV) types have been
defined genetically, with approximately 15-20 types
associated with various cutaneous and mucosal cancers,
most notably cervical cancer.

Rous FP, Beard JW, Kidd JG. Observations on the relation of the virus causing rabbit
papillomas to the cancers deriving therefrom. II. The evidence provided by the tumors; general
considerations. J Exp Med. 1936;64:401-424.
Rous FP. The virus tumors and the tumor problem. Am J Cancer 1936;28:233-271.

1936 Francis Peyton Rous, Joseph Beard progression of rabbit papillomavirus lesions to carcinomas
page 188

Vesicular exanthema virus of swine

negative contrast electron microscopy

Jacob Traum (1923-1995)

Traum J. Vesicular exanthema of swine. JAVMA 1936;88:316-327.

Vesicular exanthema of swine first occurred in 1932 and was thought to be foot-andmouth disease because of the clinical similarities. In 1935, Jacob Traum of the University of
California Davis first isolated the etiologic agent, vesicular exanthema virus of swine (VESV).
The outbreaks were associated with the feeding of uncooked garbage to pigs. Following
legislation requiring the cooking of garbage fed to domestic animals, by 1956 the disease
was eliminated and the virus was declared extinct. Epidemiologic evidence had prompted
Stuart Madin of the Naval Biological Laboratory at the University of California Berkeley to
postulate that the virus had its origin in some natural reservoir and was only incidentally
transmitted by feeding uncooked garbage to swine. The later identification of San Miguel sea
lion viruses (SMSV) from aborted sea lion fetuses on the island of San Miguel in California
confirmed his belief the viruses appeared to be nearly identical to VESV. It was found
that sea lion carcasses were fed to pigs causing the initial outbreaks the virus was then
spread widely via uncooked pork scraps from railroad dining cars being fed to pigs at distant
locations. There are 13 genotypes of VESV, and presently 17 genotypes of SMSV recent
phylogenetic comparison of these viruses has shown that they are, indeed, very closely
related, so closely that they should be classified as a single genotype, not just separate viruses
within a genus of the family Caliciviridae.

1936 Jacob Traum discovery of vesicular exanthema virus of swine (the first calicivirus)

page 189

Nothing in Biology Makes Sense

Except in the Light of Evolution
Theodosius Dobzhansky was a prominent geneticist and evolutionary biologist, and in his day the
central figure in developing the unifying modern evolutionary synthesis. The following is a summary
of the modern synthesis, which bridged the gap between experimental geneticists and naturalists, and
between both and paleontologists. It states that:
1. All evolutionary phenomena can be explained
in a way consistent with known genetic
mechanisms and the observational evidence of
naturalists.
2. Evolution is gradual: small genetic changes,
recombination ordered by natural selection.
Discontinuities amongst species (or other taxa)
are explained as originating gradually through
geographical separation and extinction (not
saltation).
3. Natural selection is by far the main mechanism
of change; even slight advantages are important
when continued. The object of selection is the
phenotype in its surrounding environment.
4. The role of genetic drift is equivocal. Though
strongly supported initially by Dobzhansky,
it was downgraded later as results from
ecological genetics were obtained. [The role of
genetic drift in the evolution of viruses, such as
influenza viruses, came much later.]

Theodosius Dobzhansky (1900-1975)

Dobzhansky T. Genetics and the origin of species. New York:
Columbia University Press; 1937.

5. Thinking in terms of populations, rather than

individuals, is primary: the genetic diversity
existing in natural populations is a key factor
in evolution. The strength of natural selection
in the wild is greater than previously expected;
the effect of ecological factors such as niche
occupation and the significance of barriers to
gene flow are all important.
6. In paleontology, the ability to explain
historical observations by extrapolation from
microevolution to macroevolution is proposed.
Historical contingency means explanations at
different levels may exist. Gradualism does not
mean a constant rate of change.

1936 Theodosius Dobzhansky publication of Genetics and the Origin of Species, seminal in evolutionary synthesis
page 190

Fred Robert Beaudette (1897-1957)

Avian infectious bronchitis virus

thin section and negative contrast electron microscopy

Fred Beaudette started his career in avian pathology in 1919 after receiving a DVM from Kansas State College. He left Kansas in 1923 for Rutgers University, where he rose
through the ranks becoming professor, a position he held for the rest of his life. Dr. Beaudette was an interesting character. He was a brilliant man, spoke fluent Russian and
Portuguese. The original poultry pathology books were all written in Portuguese so he had learned the language. Avian infectious bronchitis costs the U.S. poultry industry
millions of dollars annually and it remains one of the top research priorities for the commercial poultry industry worldwide. It is highly contagious and is extremely difficult
to control because immunological variants emerge often due to mutations and recombination events. In addition to genetic drift, genetic shift (recombination via template
switching) can lead to dramatic phenotypic (pathotypic) changes and can result in unique variant viruses requiring new vaccine formulations.
Beaudette FR, Hudson CB. Cultivation of the virus of infectious bronchitis. JAVMA. 1937;90:51-58.

1937 Fred Beaudette, Charles Hudson discovery of avian infectious bronchitis virus (the first coronavirus)

page 191

X-ray crystallographic diffraction

pattern by Keiichi Namba, 1982,
of a preparation of TMV made by
Ken Holmes working in
Don Caspars laboratory

John Desmond Bernal

(1901-1971)

Dorothy Crowfoot Hodgkin

(1910-1994)

Rosalind Elise Franklin

(1920-1958)

Aaron Klug

John Bernal, at Cambridge in the early 1930s, was a pioneer in x-ray crystallography and one of the first to recognize its application to biology. Along with Dorothy Hodgkin and
others, he developed or refined many of the techniques and analytical methods that would become crucial to the advance of knowledge of virus structure and assembly. In the
mid-1930s Bernal and Isidore Fankuchen (1904-1964) took the first ever x-ray diffraction images of tobacco mosaic virus (TMV); it was a landmark study. From 1937 onward, many
prominent structural researchers (including Rosalind Franklin, Aaron Klug and Donald Caspar) joined Bernal in his move to Birbeck College, University of London. To study the
complex structure of TMV, they used both X-ray diffraction and electron microscopy. Bernal and Fankuchen found that TMV was composed of identical protein subunits; James
Watson, during a short stay in 1952, established that the protein subunits were arranged in a spiral. Franklins group (Aaron Klug, Kenneth Holmes, John Finch and Donald Caspar)
in 1954 found that TMV particles were of a uniform length, and that the protein subunits were of uniform size. Donald
Caspar, in 1955, found that TMV had a hole down the center of its cylindrical form. Then, a new crystallographic technique made it possible to discern the central hole and a separate groove that held the RNA. By the end of 1955, Franklin
and her group constructed a model of TMV, which was exhibited at the Brussels Worlds Fair in 1956.
TMV is composed of one strand of RNA wrapped in a protein coat that is composed of 2,130 copies of a small protein
(capsomeres), which stack like bricks in a cylindrical chimney. One of the many surprises found during the study of
TMV was its mode of assembly. A two-step process was found, comprising nucleation and elongation: (a, in graphic)
it begins with the insertion of a hairpin loop formed by the initiation region of the viral RNA into the central hole in a
two-layer disk where it binds; (b) the loop intercalates around the first turn of the disk, opening up the base-paired stem
as it does so; (c) this interaction causes the disk to dislocate into the helical lock-washer formthis structural transformation closes the jaws made by the rings of subunits, trapping the viral RNA inside. Then for elongation, the longer
RNA tail is doubled back through the central hole of the growing helix, forming a traveling loop at the growing end of
the virion; (d) the loop inserts itself into the center of a nascent disk and binds within open jaws of the rings, this interaction converting the new disk into a helical lock washer; this continues until the full length of the RNA is covered.
Bernal JD, Fankuchen I. Structure types of protein crystals from virus-infected plants. Nature 1937;139:923.
Franklin RE. Structure of tobacco mosaic virus. Nature 1955;175: 379-381.
Butler PJG, Klug A. The assembly of a virus. Scientific American 1978;239:62-69.

1937> John Bernal, Dorothy Hodgkin, Rosalind Franklin X-ray crystallographic structure of tobacco mosaic virus

page 192

Viruses and Kochs Postulates

From Thomas Rivers Presidential address delivered before the Society of American
Bacteriologists (now the American Society for Microbiology) at its Thirty-eighth
Annual Meeting, Indianapolis, Indiana, December 29, 1936:
...very early in the bacteriological era a few discerning individuals appreciated the fact
that there was no reason, except analogy, for assuming that all infectious agents must
be living autonomous organisms. Through the activities of these investigators a group
of disease-producing agents, known as viruses, has gradually become recognized. The
exact nature of these agents is not known; some may be the midgets of the microbial
universe, others may represent forms of life unfamiliar to us, while still others may
be inanimate incitants of disease. Regardless of lack of complete knowledge of their
nature, it is decidedly incorrect to say that these agents are unknown. The incitants
of smallpox, vaccinia, poliomyelitis, yellow fever, fowl plague and tobacco mosaic are
known Thus, to the initiate the term virus used in connection with an infectious
agent has lost its old indefinite meaning and has acquired a new significance similar in
exactness to that borne by the words bacterium and spirochete. Such a statement does
not imply that all viruses are alike in nature and that a subdivision of the viral group is
not essential. The proper time for this subdivision, however, has not yet arrived.
...At the time when they were formulated Kochs postulates were essential for the
progress of knowledge of infectious diseases; but progress having left behind old rules
requires new ones which some day without doubt will also be declared obsolete. Thus,
in regard to certain diseases, particularly those caused by viruses, the blind adherence
to Kochs postulates may act as a hindrance instead of an aid. It is obvious that Kochs
postulates have not been satisfied in viral diseases. Moreover, it is equally evident
that proof of the etiological significance of viruses has been obtained without their
satisfaction. Such a statement, however, does not imply that certain conditions do not
have to be met before the specific relation of a virus to a disease is established. The
conditions are [as listed in the separate box]
...To summarize, Kochs postulates as proposed by him do not have to be fulfilled in
order to prove that a virus is the cause of a disease. However, the spirit of his rules of
proof still holds in that a worker must demonstrate that a virus is not only associated
with a disease but that it is actually the cause. The methods of doing this are different
from the ones used by Koch but are equally efficient.

Thomas Milton Rivers (1888-1962)

Rivers Criteria for Proof of Viral Disease Causation

A specific virus must be found associated with a disease with a degree of regularity.
The virus must be shown to occur in the sick individual not as an incidental or accidental finding but as the cause of the disease under investigation.
Information concerning the presence of antibodies against the agent and the time of their appearance in the serum of patients is equally important as evidence
of etiological significance of the virus.

1937 Thomas Rivers criteria for proof of viral disease causation: the Henle-Koch postulates revisited

page 193

Zilber LA, Levkovich EN,

Shubladze AK, Chumakov MP,
Soloviev VD, Shboldaeva AD,
Safonova TI. Etiology of springsummer encephalitis. Arch Biol Sci.
1938;52:162-183. (in Russian)
The discovery of tick-borne
encephalitis in the far east of
the USSR in 1937 was marked
by courage and tragedy there
were infections in members of the
expedition and deaths when the
first vaccine was tested in 1938. Lev
Zilber and two others were arrested
on charges of subversive activities:
spreading Japanese encephalitis in
the guise of discovering a new virus.

Lev Alexandrovich Zilber

(1894-1966)

Elizabeth N. Levkovich
(1900-1982)

Mikhail Petrovich Chumakov

(1909-1993)

Tick-borne encephalitis virus, mouse brain, thin section electron microscopy

1937 Lev Zilber, Mikhail Chumakov, Elizabeth Levkovich discovery of tick-borne encephalitis virus
page 194

Plaque honoring Elisabeth Wollman and Eugne Wollman

at the Institut Pasteur, Paris

August H. Doermann (1918-1991)

From Esther Zimmer Lederberg: ...regarding the famous geneticist, lie Leo Wollman, long time staff member and
deputy director of the Institut Pasteur: His parents, Elisabeth Wollman and Eugne Wollman, were researchers at
the Institut Pasteur. It was during World War II. The Gestapo arrested these Jewish scientists (they were sent to
Auschwitz and never seen again). Their son, lie, was walking towards the Institut to meet his father. Friends of the
parents quickly grabbed the young boy, and hid him from the Gestapo officers who were waiting for him, with his
father. Later, these friends hid the young boy in the catacombs of the Institut Pasteur, and took care of him until
the end of the war. He survived the Holocaust. Andr Lwoff had known the Wollmans in the thirties as scrupulous
and dogged experimentalists: they did seminal work leading to the development of the concept of the eclipse phase
in virus (bacteriophage) replication. lie Wollman later fought with the maquis in the south of France, and in 1945
joined Lwoff s unit at the Pasteur, just a few weeks before Jacque Monod. From: Judson, HF. The eighth day of
creation: the makers of the revolution in biology. New York: Simon and Schuster; 1979.
Doermann AH. The eclipse in the bacteriophage life cycle. In: Cairns J, Stent GS, Watson JD, editors. Phage and the
origins of molecular biology. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1992. p. 79-87.

1937-1943 Elizabeth Wollman, Eugne Wollman, August Doermann discovery of the eclipse period

page 195

In quantitating a virus by serial dilution assay, the measured endpoint is usually taken as
the dilution of virus in which half of treated animals or cells show the desired reaction
(death LD50, infection ID50, cytopathology TCID50, per volume of specimen, etc.),
and the other half do not. That is, the dilution endpoint is taken as the highest dilution that
will produce the desired reaction in 50% of the animals or cells used. Accurate endpoint
dilution measurements are very difficult and expensive to perform, so reasonable numbers
of replicate samples at dilutions around the predicted endpoint are tested in animals or cells
and the results are interpolated by classic methods: the methods of Reed and Muench, from
1938, or the method of Spearman-Krber, from 1931. More modern methods are available,
including several based in complex computer programs, but in most virology laboratories
the old classic methods are still used. Most virologists never think about the statistical bases
for the Reed-Muench and Spearman-Krber methods the methods lend themselves to
plugging data-points (dilutions vs. effects) into the formulae as shown in the two boxes.
These are taken from the book, Diagnostic Procedures for Viral and Rickettsial Diseases, by
Edwin H. Lennette, Nathalie J. Schmidt, several editions of which were published over the
years by the American Public Health Association.
Krber G. Beitrag zr kollektiven behandlung pharmakologischer reihenversuche. Arch Exp
Path Pharmak. 1931;162: 480-487.
Reed LJ, Muench H. A simple method of estimating fifty percent endpoints. American
Journal of Hygiene 1938;27: 493-497.

Lowell J. Reed (1886-1966)

Hugo Muench (1894-1972)

1938 G. Krber, Lowell Reed, Hugo Muench development of methods for estimating fifty percent endpoints
page 196

Vladimir Kubes
(1904-1982)

Francisco A. Rios

Ralph Walter Wyckoff

(1897-1994)

Journal of Virology
cover art from Scott Weaver
from Scott Weaver, used with permission

1938 Vladimir Kubes, Francisco Rios discovery of Venezuelan equine encephalitis virus

page 197

Handbuch der Virusforschung. Edited by Prof. Dr. R. Doerr

and Prof. Dr. C. Hallauer. With Contributions by F.M.
Burnet, R. Doerr, W.J. Elford, G.M. Findlay, M. Haitinger,
C. Hallauer, M. Kaiser, and W.M. Stanley. Erste Hlfe.
Illustrated, pp. I-XII, 1-546. Wien:Julius Springer; 1938

Robert Doerr (1871-1952)

Curt Hallauer (1900-1994)

From an unsigned review, Journal of the American Medical Association, 1939: This is a
collective work by a most competent group of specialists in a field of remarkable activity, of
astounding results and of greatest scientific significance. The viruses lie at the lowest margin
of the living world or beyond it, depending on the concept of the limits of living substance.
They appear to be protein substances capable of self propagation within living cells and to
be composed of units mostly but not wholly beyond the limits of visibility or photographic
detection even with the ultramicroscope. They can be centrifuged, filtered through graded
collodion membranes, purified by filtration and high speed centrifugation from organic
contaminants, and inactivated and reactivated by various chemical procedures. Their
dimensions can be determined within limits and their activity retained between a series of
crystallizations. They differ among themselves not only in their pathologic consequences
but in dimensions and in physical and chemical properties. They can be cultivated in tissue
cultures and grown on chick allantoic membranes Perhaps this is a realistic view of the
virus world in 1939 from outside the citadel even today, the viruses are mysterious to many
people who know quite a bit about biology in general.

1938 Robert Doerr, Curt Hallauer publication of the major virology book, Handbuch der Virusforschung

page 198

The First Virus Visualized by Electron Microscopy

There is a large, confused literature on this, with many claims of primacy, but
the actual record is quite clear Bodo von Borries, Ernst Ruska and Helmut
Ruska published the first paper, which clearly showed the virions claimed.
Helmut Ruska went on to study several viruses by the same methods:
1938: von Borries B, Ruska E, Ruska H. Bakterien und virus in
bermikroskopischer aufnahme. Klin Wochenschr. 1938;17:921-925. [images
of ectromelia virus and vaccinia virus]
1939: Kausche GA, Pfankuch E, Ruska H. Die sichtbarmachung von
pflanzlichem virus [TMV] im bermikroskop. Naturwissenschaften
1939;27:292-299.
1940: Ruska H, von Borries B, Ruska E. Die bedeutung der bermikroskopie
fr die virusforschung (The significance of electron microscopy for virus
research). Arch Ges Virusforsch. 1940;1:155-169.
1940: Ruska H. Die sichtbarmachung der bakteriophagen lyse im
bermikroskop. Naturwissenschaften 1940;28:45-46.

Bodo von Borries (1905-1956)

Ernst Ruska (1906-1988)

1942: Ruska H. ber das virus der varicellen und des zoster. Klin Wochenschr.
1942;22:703-704.

Ectromelia (mousepox) virus

T2 bacteriophage

Helmut Ruska (1908-1973)

1938 Bodo von Borries, Ernst Ruska, Helmut Ruska first electron micrographs of viruses (ectromelia, vaccinia)

page 199

Archiv fr die Gesamte Virusforschung / Archives of

Virology, 1939, Volume 1, Number 1 Table of Contents
P. Jordan. Die Stellung der Quantenphysik zu den aktuellen
Problemen der Biologie. Archives of Virology, 1939, Volume
1, Number 1, Pages 1-20.
A. Gratia and P. Manil. Recherches sur les virus des plantes.
Archives of Virology, 1939, Volume 1, Number 1, Pages 2145.
Erich Khler. ber das Auftreten abweichender Varianten bei
den Cs-Stmmen des Kartoffel-X-Virus. Archives of Virology,
1939, Volume 1, Number 1, Pages 46-69.
C. Hallauer. Studien ber die Variabilitt des Hhnerpestvirus
im Gewebsexplantat. Archives of Virology, 1939, Volume 1,
Number 1, Pages 70-84.
M. Kaiser. Die Trockenkonservierung von Virusarten. Archives
of Virology, 1939, Volume 1, Number 1, Pages 85-90.
Helga Jahn. Experimentelle Untersuchungen ber das Virus
der Ektromelia infectiosa. Archives of Virology, 1939,
Volume 1, Number 1, Pages 91-103.
W. Gavrilov and A. Fester. Quelques proprits du virus
de sarcome Rous. Archives of Virology, 1939, Volume 1,
Number 1, Pages 104-113.

Robert Doerr (1871-1952)

In the first volume of Archiv fr
die gesamte Virusforschung, there
was an article on the significance of
ultramicroscopy in the evaluation of the
nature of viruses by Helmut Ruska (19081973). The paper was written with his
brother, Ernst Ruska (1906-1988), and his
brother-in-law Bodo von Borries (19051956).

Curt Hallauer (1900-1994)

1984

R. W. Fairbrother and A. E. Martin. Further observations

on the value of heated elementary body suspensions in
immunization against experimental influenza. Archives of
Virology, 1939, Volume 1, Number 1, Pages 114-119.
A. Paillot and A. Gratia. Essai disolement du virus de la
grasserie des vers soie par lultracentrifugation. Archives of
Virology, 1939, Volume 1, Number 1, Pages 120-129.
A. Gratia and A. Paillot. tude srologique du virus de la
grasserie des vers soie isol par ultracentrifugation.
Archives of Virology, 1939, Volume 1, Number 1, Pages 130139.
K. Burckhardt. Die Durchlssigkeit der Blut-Liquor-Schranke
und der Placenta fr Bakteriophagen. Archives of Virology,
1939, Volume 1, Number 1, Pages 140-154.
Helmut Ruska, Bodo v. Borries and Ernst Ruska. Die
Bedeutung der bermikroskopie fr die Virusforschung.
Archives of Virology, 1939, Volume 1, Number 1, Pages 155169.

1939 First international virology journal, Archiv fr die gesamte Virusforschung (now Archives of Virology)
page 200

Charles J. Armstrong (1886-1967)

While still doing research work on lymphocytic
choriomeningitis (LCM) virus in 1939, Charles Armstrong
adapted a human strain of poliovirus (Lansing type 2 virus)
from monkeys to small rodents, first to cotton rats and then
to mice. This accomplishment was considered revolutionary.
His bust is included in the Polio Hall of Fame at Warm
Springs, Georgia.
Armstrong C. The experimental transmission of
poliomyelitis to the Eastern cotton rat, Sigmodon hispidus
hispidus. Pub Hlth Rep. 1939;54:1719-1721.

Scientists who played leading roles in poliomyelitis research met beneath bronze busts at the dedication
of the Polio Hall of Fame in Warm Springs, Georgia. Left to right: Dr. Thomas M. Rivers, Dr. Charles
Armstrong, Dr. John R. Paul, Dr. Thomas Francis, Jr., Dr. Albert B. Sabin, Dr. Joseph L. Melnick, Dr. Isabel
Morgan, Dr. Howard A. Howe, Dr. David Bodian, Dr. Jonas E. Salk, Mrs. Eleanor Roosevelt, and Mr. Basil
OConnor. February, 1958.

1939 Charles Armstrong adaptation of poliovirus to cotton rats and mice

page 201

The Polio Hall of Fame, Warm Springs, Georgia

(left to right)
1. Jakob Heine (1800-1879) Described infantile paralysis in 1840.
2. Karl Oskar Medin (1847-1927) Described polio as an acute infectious disease (1890).
3. Ivar Wickman (1872-1914) Described epidemic polio and non-paralytic polio (1907).
4. Karl Landsteiner (1868-1943) Discovered poliovirus and transmitted the virus to monkeys.
5. Thomas M. Rivers (1888-1962) Planned and ran the successful 1954 inactivated vaccine field trials.
6. Charles Armstrong (1886-1967) Transmitted poliovirus to cotton rats and mice.
7. John R. Paul (1893-1972) Described polio natural history, spread and epidemiology.
8. Albert Sabin (1906-1993) Developed the attenuated live-virus polio vaccine that bears his name.
9. Thomas Francis, Jr. (1900-1969) Conducted the field trials of the Salk inactivated vaccine.
10. Joseph L. Melnick (1914-2001) Developed assays of polio immunity in populations.
11. Isabel Morgan (1911-1996) Tested an experimental inactivated-virus vaccine in monkeys.
12. Howard A. Howe (1901-1976) Showed that chimpanzees can acquire polio infection by mouth.
13. David Bodian (1910-1992) Showed initial viremia before central nervous system invasion.
14. John F. Enders (1897-1985) Developed cell culture methods for growing poliovirus in volume.
15. Jonas E. Salk (1914-1995) Developed the inactivated-virus vaccine that bears his name.
16. Franklin D. Roosevelt (1882-1945) Founded the National Foundation for Infantile Paralysis.
17. Basil OConnor (1892-1972) The architect of the first national polio vaccination campaign.

Charles J. Armstrong (1886-1967)

1939 Charles Armstrong adaptation of poliovirus to cotton rats and mice
page 202

Paul Hermann Mller

(1899-1965)

p,p-dichloro-diphenyltrichloroethane
[DDT]

DDT, dichloro-diphenyl trichloroethane, was first synthesized

in 1874, but it gathered dust until 1939 when Paul Mller, of JR
Geigy AG of Basle, Switzerland, discovered its amazing insecticidal
qualities. DDT had a major role in improving the health of military
personnel in World War II . A 1945 military document states, At
present the armed services are using all the DDT available and it
is not expected that any will be available for civilian use soon
Already the army has checked a typhus epidemic in Naples and
is preventing malaria and dengue epidemics in the Pacific. After
the war, more than 4 billion pounds of DDT were used throughout
the world (1.35 billion pounds in the U.S.), until 1973 when the
Environmental Protection Agency stopped all use in the U.S. and
most other countries rapidly followed suit. The largest amount of
this usage was in agriculture, where as the illustrations here show,
very high concentrations were used regularly. The ecologically
damaging effect of this usage in turn affected the scientific-political
debate whether usage at very low concentration to repel mosquitoes
is safe when DDT is applied to the inside wall of homes it is
very effective as a mosquito repellent; there is little environmental
effect and little development of DDT resistance of mosquitoes that
carry malaria, dengue and other pathogens. Nevertheless, very few
jurisdictions are willing to allow its use. The mode of action of DDT
has never been clearly established, but in some complex manner
it opens sodium and potassium ion channels in neurons of insects
and prevents normal nerve impulse transmission.
Mller P. Triclorethane insecticidal composition and methods. US
patent 2329074, issued 1943, assigned to JR Geigy AG.

1939 Paul Mller discovery of the insecticidal qualities and use of DDT for the control of vector-borne diseases

page 203

Max Ludwig Henning Delbrck

(1906-1981)
[from Caltech Archives, used with permission]

Emory Leon Ellis (1906-1993)

[from Caltech Archives, used with permission]

In 1937, Max Delbrcks initial introduction to Caltech was quite difficult. One day he inadvertently failed to attend a seminar on bacteriophages by Emory Ellis, and went
to him later to find out what he had missed: I had sort of the vaguest notions that viruses might be an interesting experimental object for a study of reproduction at a basic
level. Ellis showed him the simple materials and techniques needed for experiments, and Delbrck saw for the first time plaques made in a lawn of bacteria where single
bacteriophage had initiated infection. Ellis also demonstrated one step-growth curves, revealing the kinetics of the cycle of bacteriophage multiplication. According to
Ellis, Delbrcks first comment was, I dont believe it, but Delbrcks recollection was, This seemed to me just beyond my wildest dreams of doing simple experiments on
something like atoms in biology and I asked him whether I could join him in his work, and he was very kind and invited me to do so. Ellis and Delbrck invented and greatly
refined the one-step growth curve (the single-burst experiment), which permitted study of bacteriophage multiplication in individual cells, a key in later research.
Ellis EL, Delbrck M. The growth of bacteriophage. J Gen Physiol. 1939;22:365-384.

1939 Emory Ellis, Max Delbrck the one-step growth experiment (now called the single burst experiment)
page 204

Kenneth C. Smithburn
(1904-1973)

West Nile virus (WNV) was first isolated from a feverish adult
woman in the West Nile District of Uganda in 1937 during research
on yellow fever. Serosurveys in 1939 found antibody ranging from
1.4% (Congo) to 46.4% (White Nile region, Sudan). The virus was
subsequently identified in Kenya (1942) and India (1953); a 1950
serosurvey in Egypt found 90% of those over 40 years of age had
WNV antibodies. The ecology of the virus was characterized in
1953 in studies in Egypt and Israel. The virus became recognized
as a cause of severe human meningoencephalitis in elderly patients
during an outbreak in Israel in 1957. In the 1960s the disease was
found to be widespread in southern Europe and southwest Asia.
Smithburn KC, Hughes TP, Burke AW, Paul JH. A neurotropic virus
isolated from the blood of a native of Uganda. Am J Trop Med Hyg.
1940;20:471-492.

Alexander W. Burke (1886-1944)

John Rodman Paul

(1893-1971)

Thomas P. Hughes (1900-1995)

with Wilbur Sawyer at Rockefeller Institute, 1930s

West Nile Virus, Global Distribution

1940 Kenneth Smithburn, Thomas Hughes, Alexander Burke, John Paul discovery of West Nile virus

page 205

Ernst Mayr was the leading evolutionary biologist of

his day. He was also a renowned taxonomist, tropical
explorer, ornithologist, historian of science and naturalist.
His work contributed to the conceptual revolution that
became the modern evolutionary synthesis, unifying
concepts from (a) Mendelian genetics, (b) systematics, (c)
paleontology, and (d) the original Darwinian principles.
From Mayrs book, Systematics and the Origin of Species,
Columbia University Press, New York, 1942, and from
What Evolution Is, Basic Books, New York, 2001: ... I
was much struck how entirely vague and arbitrary is the
distinction between species and varieties. Darwin,
1859.

Ernst Walter Mayr (1904-2005)

In many of his writings, Mayr rejected reductionism in

evolutionary biology, arguing that evolutionary pressures
act on the whole organism, not on single genes, and that
genes can have different effects depending on the other
genes present. He advocated a study of the whole genome
rather than of isolated genes only. After articulating
the biological species concept in 1942 (acknowledging
Theodosius Dobzhanskys earlier work in this area), Mayr
played a central role in the problem of defining species.
He staunchly defended the biological species concept
against the many definitions that others proposed. Mayr
insisted throughout his career that the gene as the target
of selection cannot and should not be considered a valid
idea in modern evolutionary thought: The idea that
a few people have about the gene being the target of
selection is completely impractical; a gene is never visible
to natural selection, and in the genotype, it is always
in the context with other genes, and the interaction
with those other genes make a particular gene either
more favorable or less favorable. Therefore people
like Dawkins in England who still think the gene is the
target of selection are evidently wrong. In the 30s and
40s, it was widely accepted that genes were the target of
selection, because that was the only way they could be
made accessible to mathematics, but now we know that
it is really the whole genotype of the individual, not the
gene. Except for that slight revision, the basic Darwinian
theory hasnt changed in the last 50 years.

1940 Ernst Mayr, others development of the conceptual basis of the modern evolutionary synthesis
page 206

Barbara McClintock (1902-1992)

Barbara McClintock began her studies at the College of Agriculture, Cornell University, in 1919. Her interests became focused when she took her first genetics course taught by
C. B. Hutchison in 1921. He was impressed by her interest, and telephoned to invite her to participate in his graduate genetics course; later, she said that Hutchinsons invitation
was the reason she continued in genetics: Obviously, this telephone call cast the die for my future. I remained with genetics thereafter. In 1948, working as always with maize,
she discovered the first transposons she noticed insertions, deletions, and translocations, caused by these transposons. She hypothesized that gene regulation via transposons
(sequences of DNA that can move to different positions within the genome of a cell) could explain how complex multicellular organisms made of cells with identical genomes
can have cells with vastly different functions. These changes in the genome could, for example, lead to changes in the color of corn kernels. About 50% of the total genome of
maize consists of transposons. [The most common transposon in humans is the Alu sequence, which is ~300 bases long and can be found between 300,000 and a million times
in the human genome.] The importance of McClintocks contributions came to light in the 1960s, when Franois Jacob and Jacques Monod described the molecular basis for
genetic regulation of the bacterial lac operon, a parallel system to that which she studied years earlier. Their work also explained the mechanism of transposition
McClintock B. The origin and behavior of mutable loci in maize. Proc Natl Acad Sci USA. 1950;36:344-355.

1940> Barbara McClintock development of the concept of transposable elements, transposons

page 207

Katherine K. Sanford (1915- )

Wilton R. Earle (1902-1964)

The fibroblast-like L cell line was established in 1940 by Wilton Earle from normal C3H/An mouse subcutaneous areolar
and adipose tissue; it was one of the first cell lines to be established in continuous culture. Clone L929 was the first cloned
cell line to be established; this was done by Kay Sanford, Wilton Earle, and G.D. Likely in 1948, using Sanfords capillary
technique for single cell isolation. The cloned cell line was given the designation ATCC CCL-1 in the catalog of the newly
founded American Type Culture Collection. Both the cell line and its cloned derivative became widely used in virology
because of their broad spectrum of virus susceptibility: pseudorabies virus, vesicular stomatitis viruses, herpes simplex
virus, B virus, vaccinia virus, and many others replicate well in these cells.

1940-1948 Katherine Sanford, Wilton Earle development of the L cell line and L929 cloned cell line
page 208

Keith Roberts Porter (1912-1997)

Keith Porter is considered the father of cell biology. In the 1940s, using thin-section
electron microscopy, he initiated studies of the fine structure of cells and contributed
to an appreciation of the importance of understanding the relationship between cell
structure and cell function. He was the driving force behind the founding of the Tissue
Culture Society, the American Society for Cell Biology, the Electron Microscopy
Society of America (now the Microscopy Society of America), the Journal of Cell
Biology, and the International Federation for Cell Biology. In 1977, Porter was awarded
the U.S. National Medal of Science, presented by President Jimmy Carter.

1940s> Keith Porter, others description of the fine structure and biochemistry of cellular organelles

page 209

U.S. National Medal of Science

(Recipients Cited in this Book)

U.S. Presidential Medal of Freedom

(Recipients Cited in this Book)

Carl Ten Broeck

James E. Darnell
William McDowall Hammon
John R. Paul
John F. Enders
Thomas Francis Jr.
James D. Watson
Jonas E. Salk
Albert Bruce Sabin
D.A. Henderson
Anthony Fauci
William Foege

David Baltimore
Paul Berg
J. Michael Bishop
Herbert W. Boyer
Mario R. Capecchi
Stanley N. Cohen
Theodor O. Diener
Harry Eagle
Donald A. Henderson
Maurice R. Hilleman
Leroy Hood
Robert J. Huebner
Har Gobind Khorana
Arthur Kornberg
Joshua Lederberg
Ernst Mayr
Barbara McClintock
Daniel Nathans
Severo Ochoa
George Emil Palade
Keith Roberts Porter
Stanley Prusiner
Albert B. Sabin
Phillip A. Sharp
Maxine F. Singer
Howard M. Temin
Harold Varmus
James D. Watson
Carl R. Woese
Rosalyn S. Yalow

Copley Medal of the Royal Society

(Recipients Cited in this Book)

1753: Benjamin Franklin

1845: Theodor Schwann
1864: Charles Darwin
1874: Louis Pasteur
1892: Rudolf Virchow
1902: Lord Lister
1906: Elias Metchnikoff
1917: Emile Roux
1933: Theobald Smith
1939: Thomas Hunt Morgan
1945: Oswald Theodore Avery
1949: George Charles De Hevesy
1959: Frank Macfarlane Burnet
1969: Peter Medawar
1975: Francis Crick
1977: Frederick Sanger
1979: Max Perutz
1983: Rodney Porter
1985: Aaron Klug
1989: Cesar Milstein
1991: Sydney Brenner
1993: James D. Watson
1995: Frank Fenner
2001: Jacques Miller
2009: Martin Evans

Recipients of U.S. Presidential Medal of Freedom, National Medal of Science and Copley Medal of the Royal Society
page 210

Albert Claude (1899-1983)

Christian de Duve (1917-2013)

George Palade (1912-2008)

The Nobel Prize in Physiology or Medicine of 1974 was awarded jointly to Albert Claude, Christian de Duve and George Palade for their discoveries concerning the structural
and functional organization of the cell. The cell had been studied since the middle of the 19th century, but its structure, composition and functions were quite limited until
two different approaches, both introduced at The Rockefeller Institute, changed everything. One was the application of electron microscopy, the other was biochemical
analysis of cellular components (organelles) that were being seen in the electron microscope. Tissues or cells were homogenized and components separated by differential
ultracentrifugation. Fractions were re-examined by electron microscopy and then subjected to many biochemical analyses. The two technologies taken together gave rise
to the discipline of cell biology. Albert Claude, from Belgium, working at the Rockefeller Institute, was the first to apply the two technologies for the study of animal cells.
He published the first electron micrographs of cells and cell components that provided new and relevant biological information in 1945; shortly thereafter he published the
methodology of differential ultracentrifugation. His approach was taken up by George Palade, from Romania, also working at the Rockefeller Institute, in 1947. He added
important methodological improvements both to differential centrifugation and electron microscopy. His early work, done in collaboration with Keith Porter, was mainly
descriptive of the cytoplasmic organelles the endoplasmic reticulum (originally discovered by Claude and Porter), ribosomes (showing with others that they are the site
of protein synthesis), secretory granules and secretory processes, and the Golgi complex. Christian de Duve, from Belgium by way of the United Kingdom, also working at
the Rockefeller Institute, studied the distribution of different enzymes among the four major differential ultracentrifugation fractions [nuclei, mitochondria, microsomes
(fragmented endoplasmic reticulum) and the soluble fraction]. He found that particular enzymes sedimented with particular fractions, and discovered lysosomes and their
complex, compartmentalized proteolytic degradative functions.

1940s> Albert Claude, Christian de Duve, George Palade fine structure and biochemistry of cellular organelles

page 211

Many viruses can agglutinate erythrocytes, usually under specific conditions

(species of mammal or bird providing the erythrocytes, temperature, pH, etc.).
Examples of viruses that hemagglutinate include influenza viruses, parainfluenza
viruses, adenoviruses, rubella virus, alphaviruses, bunyaviruses, flaviviruses and
some picornaviruses. Influenza viruses have an envelope protein complex, the
hemagglutinin (HA), which binds to sialic acid receptors on erythrocytes (and the
same receptors on airway epithelial cells). Other virion surface protein complexes
comprise the hemagglutinin of other viruses. Virions bind to erythrocytes in a
cross-linking fashion, causing the formation of a compact mass of erythrocytes
that settles to the bottom of the well or tube as a pellet or button. This is the
basis of a classical rapid assay to determine levels of virus present in a sample.
To conduct the assay, two-fold serial dilutions of a sample containing virus are
prepared, mixed with a specific amount of erythrocytes, and added to tubes or the
wells of a plastic tray. Where the erythrocytes are cross-linked by virions a pellet
or button is seen at the bottom of the tube or well. Where the erythrocytes are
not cross-linked by virions they settle out smoothly covering the whole bottom of
the tube or well. The assay can be performed within 30 minutes, and is therefore a
quick indicator of the relative quantity of virus present in a sample.

George K. Hirst (1910-1994)

Hirst GK. The agglutination of red cells by allantoic fluid of chick
embryos infected with influenza virus. Science 1941;94:22-23.
Hirst GK. The nature of hemagglutination by viruses. Harvey Lect
Series 1948;44:84-98.

The hemagglutination assay can be easily modified to determine the level of

antibodies present in a serum sample this is called the hemagglutinationinhibition (HAI) assay. In HAI assays the specificity is usually rather narrow:
with influenza it is strain specific, with flaviviruses it is somewhat broadly crossreactive. A fixed amount of virus is added to each tube or well; then, two-fold
dilutions of each serum to be tested are added, and finally erythrocytes are added
and left at a given temperature for 30 minutes. Antibodies present in the serum
sample inhibit the formation of a cross-linked pellet of erythrocytes the highest
dilution of serum that prevents hemagglutination is called the HI titer of the
serum. While less accurate than a plaque assay or plaque-inhibition assay, HA and
HAI assays are cheaper and quicker and for years were widely used.

1941 George Hirst discovery of influenza virus hemagglutination, hemagglutination-inhibition, neuraminidase

page 212

Norman McAllister Gregg (1892-1966)

Gregg NM. Congenital cataract following German measles in the mother.
Trans Ophthalmol Soc Aust. 1941;3:35-46.

The notion that rubella was only a mild illness of children was dispelled in 1941, when Norman
Gregg, an Australian ophthalmic surgeon, reported the devastating teratogenic effects of the
virus. In 1940, Australia had experienced an exceptional epidemic of rubella, the magnitude
of which was probably enhanced by wartime mobilization. Gregg observed an unusually large
number of cases of congenital cataracts in newborn children, and the cataracts were often
associated with other deformities. Today we know that the congenital rubella syndrome includes
deafness, eye abnormalities (especially cataract, microphthalmia), congenital heart disease
(especially patent ductus arteriosus), mental retardation, and other less common defects. Gregg
obtained careful histories from the mothers of the affected babies in an attempt to assign a cause
to the episode. He had 13 such cases in his own practice, and with colleagues he managed to
collate a total of 78 cases of children with cataracts; of these, 68 mothers gave a history of rubella
infection early in pregnancy. Soon after, he unraveled the same relationship between congenital
deafness and maternal rubella infection. Thus, the idea that viruses could be teratogenic agents
was introduced. Greggs report initially drew little attention. It was supported by his Australian
colleagues, who further reported other defects associated with maternal rubella, but it was
not until the syndrome was confirmed in Europe and the U.S. that the significance of Greggs
observations was accepted.

1941 Norman Gregg discovery of congenital abnormalities caused by rubella virus infection during pregnancy

page 213

Culex tarsalis

Hammon WM, Reeves WC,

Brookman B, Izumi EM,
Gjullin CM. Isolation of the
viruses of Western equine and
St. Louis encephalitis from
Culex tarsalis mosquitoes.
Science 1941;94:328-330.

William McDowall Hammon

(1904-1989)

William Carlisle Reeves

(1917-2004)

News Item: Mosquitoes and Encephalitis in the Yakima Valley (JAMA. 1942;119:1430-1431.)
In a series of recent studies by Hammon, Reeves and their colleagues much new information has been made
available on the transmission of encephalitis in the Yakima Valley of Washington state. In a four month period
during the summer of 1941 over 15,000 arthropods were collected, frozen and inoculated into laboratory animals
for the purpose of isolating the encephalitis virus. From Culex tarsalis Coquillett three strains of St. Louis
encephalitis virus and five strains of western equine encephalomyelitis virus were isolated. Virus was not isolated
from other species of mosquitoes. For the isolation intracerebral inoculation of five Swiss mice of a suitable strain
was proved by comparative tests to be more satisfactory than intracerebral inoculation of one 200 Gm. guinea pig
or two 8 to 12 day chick embryos. By means of precipitin tests it was demonstrated that C. tarsalis feeds in nature
on cows, horses, man, pigs, dogs, chickens (other birds?) and sheep. Since C. tarsalis has been shown to be an
efficient vector of western equine and St. Louis viruses, this widespread feeding makes it possible for the species
to spread the infection among many animals and birds. In the course of these investigations an efficient trap for
collecting live mosquitoes was developed. Finally the investigators summarize the evidence against Culex tarsalis
and conclude that it is the most important vector of western equine and St. Louis encephalitis viruses in the
Yakima Valley. Its possible role elsewhere is wisely left to be judged on the basis of local observations.

Bill Reeves and Bill Hammon, Yakima Valley

1941 William Hammon, William Reeves quantitative study of natural history of arboviruses in vector arthropods
page 214

The Beckman Model DU Ultraviolet Spectrophotometer

Arnold O. Beckman (1900-2004)

Beginning in 1940, the first in what became a series of Beckman spectrophotometers was
developed at the National Technical Laboratories Company headed by Arnold Beckman.
This later became the Beckman Instrument Company. Initially, the sole product of the
company was the worlds first pH meter, which Beckman had invented. Next, in 1941,
came the Beckman Model DU ultraviolet spectrophotometer, which was considered the
Model T of laboratory instruments. It was referred to by Nobel laureate Bruce Merrifield
as probably the most important instrument ever developed towards the advancement of
bioscience. The Model DU was an immediate success and it had a commercial lifetime
of 35 years, with more than 30,000 sold. Among other uses, the Model DU was used in
quantifying protein and DNA concentrations in all sorts of samples. Several amino acids
found in most proteins, including tryptophan, absorb light in the 280nm range and DNA
absorbs light in the 260nm range. Reasonable estimates of protein or DNA concentration
were made by simple measurements of absorbance of light at these wavelengths relative
to standards. Additionally, the ratio of 260/280nm absorbance was found to be a good
general indicator of the relative purity of a solution in terms of protein and DNA.
Eventually, every virology laboratory in the world had, or had access to, a Beckman
pH meter, a Beckman DU spectrophotometer, a Beckman analytical ultracentrifuge, a
Beckman preparative ultracentrifuge, and other Beckman instruments.

1941 Arnold Beckman development of the ultraviolet spectrophotometer (the Beckman DU)

page 215

Thomas Foxen Anderson (1911-1991)

The earliest electron micrographs of many
bacteriophage particles showed them to be tadpoleshaped structures with round or oval heads to which
tails of various thicknesses and lengths were attached
this was realized from the early work of Helmut Ruska
in 1941, but was made much clearer by the work of
Thomas Anderson. The secret of his success, before the
invention of negative contrast electron microscopy, was
his development of the critical point drying method
and Robley Williams development of a freeze-drying
technique, both reducing distortions in specimens.
Anderson stated in 1953 that, It turns out that the
heads of all the phage particles so far prepared by these
methods are polyhedral in shape. He studied T1, T2, T5
and P1 bacteriophages.

These images illustrate negative contrast electron microscopy of various bacteriophages. This method was invented
in 1959 by Sydney Brenner and Robert Horne it was not available when Thomas Anderson did his work in
the 1940s. His images were not as revealing, but he made the right interpretation of the images he produced by
the methods he invented, especially his critical point drying technique. Clockwise: Escherichia coli T1 phage,
Staphylococcus aureus 3A phage, Listeria A118 phage, Salmonella phage.

1941 Thomas Anderson discovery of the amazing variety of bacteriophage morphologies

page 216

John F. Enders
The Cultivation of the Poliomyelitis Viruses in Tissue Culture
Nobel Lecture, December 11, 1954
These indices of viral multiplication in suspended cell cultures, useful as they
were in our earlier investigations, proved inconvenient... Accordingly, other
means were sought by which viral cytopathogenicity might be conveniently and
rapidly demonstrated. At first we explored the method of explanation which
had been used in the past by other workers, in particular by C.H. Huang, to
test the effect of viruses on cell viability. This consists in placing the fragment
taken from a suspended cell culture in a drop of clotted chicken plasma and
observing whether or not cell outgrowth occurs upon subsequent incubation.
Results of experiments with poliomyelitis virus carried out in this manner were
unequivocal...

Chen-Hsiang Huang (~1914-1988),

Frederick A. Murphy,
Robert E. Shope (1929-2004)
WHO Regional Office-New Delhi, 1980

To quantitate virus, this approach was adapted to serial dilutions of virus

inoculated into cell culture tubes, with the endpoint determined by color change
using a pH indicator. C.H. Huang first did this using Western equine encephalitis
virus and primary chick embryo cells, while at the Rockefeller Institute in 1942,
where he worked with Richard Shope.
Huang CH. Further studies on the titration and neutralization of the Western
strain of equine encephalomyelitis virus in tissue culture. J Exp Med.
1943;78:111-126.

1942 Chen-Hsiang Huang development of the quantitative virus neutralization assay

page 217

CDC, Savannah, Georgia, 1946

Joseph W. Mountin (1891-1952)

The U.S. Centers for Disease Control and Prevention
originated as the Office of National Defense Malaria Control
Activities (1942); it then became the Office of Malaria Control
in War Areas (1942); then later, the Communicable Disease
Center (1946); then the National Center for Disease Control
(1967); then the Center for Disease Control (1970); then the
Centers for Disease Control (1980); and finally the Centers for
Disease Control and Prevention (1992) but, since 1946 it has
retained its initials, CDC.

CDC, Virus & Rickettsia Section

Montgomery, Alabama, 1950s

Left to Right: Alan Eschenbreriner, Alan Bernstein,

Matt Bucca, Roy Chamberlain, ?, ?, ?, ?, Brownie
Yarbrough, James Paine, Rachel Gorrie, ?, Robert
Kissling, Morris Schaeffer, Don Nelson, Dorothy
Reese, Gerald Taylor, Lila Pope, Sy Kalter,
W. Daniel Sudia.

CDC, Downtown Atlanta, Georgia, 1952

Etheridge EW. Sentinel for health: a history of the Centers for Disease Control. Berkeley:
University of California Press; 1992.
Historical perspectives: history of CDC. MMWR Morb Mortal Wkly Rep. 1996;45;526-530.
60 Years of public health science at CDC. Morb Mortal Wkly Rep. 2006;55(Sup2);1-24.

1942 Joseph Mountin, others founding of the U.S. Centers for Disease Control and Prevention
page 218

CDC, 1960, view from the front of the campus

CDC, 1965, view from the back of the campus

Virology building (Bldg 7) at back-right

Virology building (Bldg 7) at left, Alex Langmuir at front

CDC, 1988, Virology building (Bldg 15) at top-left

CDC, 1988, Virology building (Bldg 15)

1942 Joseph Mountin, others founding of the U.S. Centers for Disease Control and Prevention

page 219

(left) Mumps virus, rhesus macaque parotid gland, frozen section immunofluorescence,
Coons & colleagues, 1949 (colorized); (right) Cell labeled with five fluorophores, 2006,
from The fluorescent toolbox for assessing protein location and function. Giepmans BN,
Adams SR, Ellisman MH, Tsien RY. Science 2006;312:217.
Coons AH, Creech HJ, Jones RN, Berliner G. The demonstration of pneumococcal antigen in
tissues by the use of fluorescent antibody. J Immunology 1942;45:159-170.

Albert Hewett Coons (1912-1978)

Coons AH, Kaplan MH. Localization of antigen in tissue cells. II. Improvements in a method
for the detection of antigen by means of fluorescent antibody. J Exp Med. 1950;91:1-13.

Albert Coons initiated a major revolution in immunology cell biology and virology that continues to this day he developed the immunofluorescent technique for labeling specific
antibodies with fluorescent dyes, thus permitting the detection of antibodies and antigens in cells and tissues. His methods are also the basis for all immunohistochemical methods
now in such wide use. The development of immunofluorescence in the early 1940s was a technical tour de force, taking several years to accomplish after he had the casual idea that
putting a visible label on antibody molecules would provide a valuable research tool. With the idea in mind, Coons received encouragement from John Enders and Hans Zinsser,
among others, and expert assistance from a number of researchers including Hugh Creech and Norman Jones, who were working on the conjugation of isocyanates to proteins.
After false starts with other molecules, Coons obtained from Louis Fieser and Ernst Berliner fluorescein isocyanate, which fluoresces with a brilliant apple green color not seen in
normal tissues. Coons had further good luck Allan Grafflin was just assembling a fluorescence microscope, not a common instrument at the time. Coons labeled antibody to a
pneumococcal polysaccaride and in phagocytic cells of mice injected intravenously with the same pneumococcus he detected large numbers of fluorescing bacteria. He published
two papers, but then the work was interrupted by World War II. After the war, he had to synthesize the fluorescent compounds himself and solve many other technical problems,
the most troublesome being background tissue fluorescence in the frozen sections of tissues from animals infected with various bacteria and viruses (solved using acetone-dried
tissue extracts as an absorbing agent). The papers reporting this work had great impact, most notably on virologists at a time when pathogenesis research was booming.

1942 Albert Coons development of immunofluorescence labeling methods

page 220

A one-million liter sphere at Fort

Detrick, called the Eight-Ball, is the
largest aerobiology chamber ever built.
The sphere is on the National Register
of Historic Places it has not been used
since 1969.
Camp Detrick, later Fort Detrick, the U.S. Army installation in Frederick, Maryland, was from 1943 to 1969 the home of the U.S. Army Biological Warfare Laboratories.
During World War II, it became the site of intensive biological warfare research using various pathogens, including many viruses. The work was cloaked in the deepest
wartime secrecy, matched only by the Manhattan Project. For example, 5,000 bombs containing anthrax spores were produced there during the war. In 1969, the United
States ratified the 1925 Geneva Protocol prohibiting the use of chemical and biological weapons, and President Richard Nixon issued an executive order outlawing offensive
biological warfare research. At the same time the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) was formed to carry out a defensive mission. From
then on work has focused on diagnostics, prevention and therapeutics against infectious agents used in biowarfare and bioterrorism.

1943 U.S. Army, Camp Detrick development of military high-containment virology laboratories

page 221

Kenneth C. Smithburn
(1904-1973)

Alexander John Haddow

(1912-1978)

Smithburn KC, Haddow AJ. Semliki Forest virus. I. Isolation and pathogenic
properties. Journal of Immunology 1944;49:141-157. [From the Yellow Fever
Research Institute, Entebbe, Uganda]
Kenneth Smithburn (abridged): Between 1937 and 1948 a number of viruses
were encountered by staff members of the International Health Division of The
Rockefeller Foundation and their colleagues during long-term investigations of
yellow fever in West Africa... No systematic studies were possible until 1949,
when a comprehensive study of them was undertaken by means of a complete
series of cross-neutralization tests. The results of the study indicate that 11 of the
agents are hitherto unknown viral entities [this includes Semliki Forest virus]. All
of the agents were discovered, so to speak, accidentally by virtue of the fact that
the methods employed in the isolation of yellow fever virus are effective also for
other viral entities which are neurotropic for Swiss mice [this is] without intent
to imply that they are necessarily neurotropic in their natural hosts, whatever
these may be.

Semliki Forest virus, purified virus, thin section of ultracentrifuge pellet, by Carl von Bonsdorff

1944 Kenneth Smithburn, Alexander Haddow discovery of Semliki Forest virus

page 222

Colin Munro MacLeod

(1909-1972)

Oswald Theodore Avery (1877-1955)

Maclyn McCarty
(1911-2005)

Rough avirulent type II pneumococcal

colonies (left) and Smooth virulent type III
colonies (right).
Avery OT, MacLeod CM, McCarty M. Studies
on the chemical nature of the substance inducing
transformation of pneumococcal types. Induction
of transformation by a desoxyribonucleic acid
fraction isolated from pneumococcus type III.
J Exp Med. 1944;79:137-158.

Fred Griffiths 1928 experiment, cited earlier in this volume, described the discovery of bacterial transformation, whereby a bacterium (Streptococcus pneumoniae) was made
to change its form and pathogenicity. The phenomenon was attributed to an unidentified transforming factor. After years of work, in 1944, Oswald Avery, Colin MacLeod,
and Maclyn McCarty of the Rockefeller Institute identified the factor as DNA. They found that they could induce transformation not only with killed bacteria, but also with
extracts of bacterial cells. Treating the extract with enzymes to destroy polysaccharides (including the polysaccharide of the bacterial capsule) had no effect; neither did a lipase
to destroy bacterial lipids, proteases to destroy bacterial proteins, and RNase to destroy bacterial RNA. But, treating the extracts with DNase to destroy bacterial DNA did
abolish the transforming activity. Exhaustive studies confirmed that only DNA was capable of transforming bacteria from one type (type II rough avirulent) to another (type
III smooth virulent). The paper was met with skepticism it was a departure from the prevailing belief that the protein content of chromosomes probably made up genes. It
took another decade, and the discovery by Watson and Crick, for DNA to be widely accepted as the stuff of inheritance.

1944 Oswald Avery, Colin MacLeod, Maclyn McCarty identification of DNA as the material of inheritance

page 223

Erwin Schrdinger (1887-1961)

Erwin Schrdinger was an Austrian theoretical

physicist who was one of the fathers of
quantum mechanics he was awarded the
Nobel Prize in physics in 1933. In 1939, he
was dismissed from his university position in
Germany for political unreliability. He ended
up in Dublin, where in addition to continuing
his career in physics he wrote the book, What
is Life? This was in 1944, at a time when DNA
was not yet accepted as the stuff of heredity,
but a time when the concept of complex
molecules containing the genetic code for
living organisms was being discussed in
scholarly circles. Schrdinger assumed that the
gene is a one-dimensional crystal, which lacks
a periodic repeat, i.e., an aperiodic crystal.
Not being aware of Avery, MacLeod and
McCartys proof that the gene is composed of
DNA (the book was based upon lectures given
in 1943), Schrdinger believed that the gene
was composed of protein. He concluded, We
are faced with a mechanism entirely different
from the probabilistic one of physics, one
that cannot be reduced to the ordinary laws
of physics... Living matter, while not eluding
the laws of physics ... is likely to involve other
laws of physics hitherto unknown... The book
received many uncomplimentary reviews,
but nevertheless became quite popular and
influential. Eventually, the book was translated
into seven languages, and total sales exceeded
well over 100,000. The molecular virologist,
Gunther Stent, later stated that the book
probably had little influence on professional
biologists, but it had a great impact on
physical scientists, who were only too happy
to focus their intellect on a new and refreshing
problem in the post-war years when physics
was seen only as contributing to the specter
of nuclear war. Many of the first molecular
virologists came to biology from backgrounds
in physics, influenced by Schrdingers book
Max Delbrck, James Watson, Francis Crick
and others cited the book in their memoirs as
inspirational and career changing.

Schrdinger E. What is life? Cambridge:

Cambridge University Press; 1944.

1944 Erwin Schrdinger publication of book, What is Life? motivating many scientists toward molecular biology

page 224

Saul Krugman, a nurse and visitors, including the

NYC mayors wife, at Willowbrook State School
Much of the early work in the United States on viral hepatitis
(hepatitis A and hepatitis B) was done at the Willowbrook State
School, a notoriously overcrowded institution for mentally
disabled children in Staten Island, New York. At a time before
much was known about these infections, Saul Krugman and his
colleagues discovered that nearly all patients and staff became
infected shortly after arrival at the institution unless they had
Frederick O. MacCallum
Saul Krugman
been previously infected. Most infections had gone unrecognized.
( ~1912-1994)
(1911-1995)
They found that hepatitis A and B co-existed and many cases
of relapse were actually infections with two different viruses.
In the 1940s, F. O. MacCallum realized that a number of soldiers who received yellow fever vaccine
Krugman and his colleagues showed that injecting patients
developed hepatitis a few months later. The yellow fever vaccine contained human serum as a stabilizing
upon admission with immunoglobulin provided protection from
agent, and MacCallum was aware of other hepatitis cases reported in the medical literature that followed
illness. This was an important advance, but in a contentious set
inoculation with vaccines containing human serum. He also knew of cases that followed the reuse of
of experiments, viruses were injected and fed as well Krugman
unsterilized syringes, needles and instruments that could be contaminated by human blood. MacCallum
argued that the development of a vaccine would outweigh any
came to suspect that a virus carried in human blood caused hepatitis.
minor harm to the children. He also argued that the children were
A series of observations of volunteers by MacCallum and others during and shortly after the war
bound to be infected anyway, and only children whose parents
strengthened this hypothesis and made it clear that hepatitis could also be spread by other means than
through blood. MacCallum coined the term hepatitis A for the form of the disease that is spread primarily who gave consent were included. The Willowbrook experience
contributed importantly to the public debate over research
through food and water contaminated with fecal material and the term hepatitis B for the form that is
ethics and gave impetus to regulation. In the 1970s, the ethical
transmitted mainly by exposure to contaminated blood.
controversy reached medical journals and newspapers, and led
MacCallum FO, Bauer DJ. Homologous serum jaundice transmission experiments with human volunteers. to Congressional hearings. Many regulations of institutions
The Lancet 1944;1:13:622-627. [Virus Reference Laboratory, Public Health Laboratory Service, Colindale, like Willowbrook were instituted at state and national levels in
London, UK]
succeeding years.

1944-1958 Frederick MacCallum, Saul Krugman, others separation of two forms of hepatitis, hepatitis A and B

page 225

Max Ludwig Henning Delbrck

(1906-1981)

Franois Jacob, Max Delbrck, Matthew Meselson,

Ronald Rolfe,Gunther Stent and Sydney Brenner, 1960

Salvador Edward Luria

(1912-1991)

Salvado Luria
and Max Delbrck

Alfred Hershey
and Seymour Benzer

Gunther Stent
(1924-2008)

In 1945, Max Delbrck initiated the first course

at the Cold Spring Harbor Laboratory the
Phage Course, which covered the fundamentals of
bacteriophage biology and methodology. It instilled
the Phage Groups distinctive math- and physicsoriented approach to biology and played a seminal
role in the global development of molecular biology
and molecular genetics. Faculty and participants
in the first years included Salvador Luria, Alfred
Hershey, Seymour Benzer, Gunther Stent, James
Watson, Frank Stahl, Renato Dulbecco, and other
young scientists who went on to build the science
we know today. The course has continued for more
than 60 years, now under the title, the Molecular
Genetics of Bacteria & Phages.

1945> Max Delbrck, Salvador Luria, others founding of the Phage Course at Cold Spring Harbor Laboratory
page 226

Thomas Francis, Jr.

(1900-1969)

George K. Hirst
(1910-1994)

Frederick Davenport
(1914-1982)

Gordon Meiklejohn
(1911-1997)

Edwin Kilbourne
(1921-2011)

The first influenza vaccine was developed by Thomas Francis, Jr., working at the Rockefeller Institute in 1933, just as Smith, Andrewes and Laidlaw developed the ferret
model of influenza Francis was the first American to isolate the virus (by instillation of ultrafiltered respiratory secretions from patients intranasally in ferrets). In 1937, he
first attempted to vaccinate humans using a virus grown in minced chick embryo cultures. Francis was appointed director of the Commission on Influenza of the U.S. Army
Epidemiological Board in 1941, from where he conducted what are now called phase one clinical trials, involving volunteers including conscientious objectors. The trials,
which would be considered unethical today, proceeded to a successful double-blinded field trial in soldiers (12,000 subjects). By 1943, the method of vaccine production
evolved into that which is still used today: vaccine virus is inoculated into the allantoic cavity of embryonating eggs, incubated, harvested, inactivated using formalin or
b-propriolactone, and purified. By 1945, all 7 million U.S. military personnel were immunized and vaccine was made available to the public. The vaccine of the 1940s was
monovalent; in the 1960-70s vaccines were bivalent; since 1978 they have been trivalent, containing one strain of influenza B and two of influenza A virus.

1945> Thomas Francis, George Hirst, Fred Davenport, others development of influenza vaccines

page 227

Crimean-Congo hemorrhagic fever, human liver,

massive focal necrosis and hemorrhage. H&E

Mikhail Petrovich Chumakov

(1909-1993)

Ghislain F. G. J. Courtois
(1912-1971)

Crimean hemorrhagic fever was first observed by Russian scientists in 1944-1945 when about 200 Soviet
military personnel were infected during an epidemic in the Crimea. At that time it was established by
Mikhail Chumakov in studies in human volunteers that the etiological agent was ultrafilterable and
transmitted by the tick Hyalomma marginatum. The virus was not maintained in the laboratory. Congo
virus was first isolated in 1956 in Zaire by Ghislain Courtois and his colleagues from the blood of a febrile
patient. The virus was isolated using newborn mice and with difficulty was serially passaged. Jordi Casals
later showed that the viruses isolated in the Crimea and in Zaire were antigenically indistinguishable
hence, the hyphenated name. Since then, the virus has been shown to have an extremely wide geographic
distribution and is a significant cause of severe disease and substantial mortality.
Chumakov MP, Butenko AM, Shalunova NV, Martianova LI, Smirnova SE, Bashkirtsev IuN, Zavodova TI,
Rubin SG, Tkachenko EA, Karmysheva VIa, Reingold VN, Popov GV, Savinov AP, New data on the viral
agent of Crimean hemorrhagic fever. Vopr Virusol. 1968;13: 377 (in Russian).
Woodall JP, Williams MC, Simpson DI. Congo virus: a hitherto undescribed virus occurring in Africa. II.
Identification studies. East Afr Med J. 1967;44:93-98.

1945> Mikhail Chumakov, Ghislain Courtois, colleagues discovery of Crimean-Congo hemorrhagic fever virus
page 228

Joshua Lederberg (1925-2008) in his lab, 1958

Edward Lawrie Tatum (1909-1975)

1958 Nobel Prize

in Physiology or
Medicine
Plaque at Yale University
1946 Joshua Lederberg, Edward Tatum discovery of genetic recombination in bacteria

page 229

In 1946, at Yale University Joshua Lederberg and Edward Tatum carried out a series
of experiments demonstrating genetic recombination in bacteria for this in 1958
they shared the Nobel Prize in Physiology or Medicine. Joshua Lederberg was a genius
(at 33 he was the second youngest Nobel laureate ever), but he also was the most
wonderful writer; the following are quotes clipped from his autobiographical pieces:
The important biological discovery of 1944 was the identification by Avery,
MacLeod & McCarty, at the Rockefeller Institute, of the substance responsible for
pneumococcal transformation. This phenomenon, which Fred Griffith had stumbled
on in 1928, appeared to be the transmission of a gene from one bacterial cell to
another... Sleepwalking, we were all groping to discover just what was important
about the chemical basis of this biological specificity. My immediate private response
to reading the 1944 paper was that the research was unlimited in its implications
What could be done to incorporate this dramatic finding into the mainstream
of biological research; how could one further advance these new hints about the
chemistry of the gene?... It is difficult to find a clear instance of a scientific revolution
in the history of biology, in the strict sense of a paradigm shift barely coupled to
experimental evidence The Darwinian revolution comes very close, especially in
its application to microbiology My work of 1946-1947 became my dissertation I
spent the summer of 1947 at Woods Hole (and the magnificent library of the Marine
Biological Laboratory), completing my dissertation. The stacks gave me a wonderful
opportunity to explore the history of microbiology: how its pioneers had sought to
cope with the perplexities of bacterial variability, totally isolated from the intellectual
apparatus of modem genetics.
My first important discovery came about through a rather unusual process. More
than almost everything else that Ive ever done, it was theory driven rather than data
driven. It was theory driven in the sense that a postulate had arisen out of the course
of examination of the contemporary scene, namely Avery et al., had shown transfer
of heritable characteristics in a bacterium. One was deeply motivated to want to
know more about whether there were things like genes in bacteria. The approach
that speculatively arose and how to respond to it is: could one find out that there is a
possibility of genetic recombination, of crossing between bacterial cells?... One was
able to put together a thought experiment that said, If bacteria can be crossed, and
if you start out with two different nutritional mutants, and if they exchange with one
another, they will form prototrophic [wild type, able to grow on unsupplemented
minimal medium] genotypes, you will be able to select for them, and define their
occurrence even if they happen very, very rarely [this could be done] by a rather
simple procedure plating the mixtures on minimal agar. So here was a case where
the experimental design was worked out [in theory] it was a theoretical postulate:
does genetic recombination occur? Does it not occur? And that was all worked out in
advance of ever doing the experiment.
Well somewhat to my surprise it worked very, very promptly. I was rather fearful
when the first positive results came in. I was worried deeply that this was going to
be an artifact, that my hopes would be dashed. I didnt want to get too excited about

it. I was scared. I knew it was a very important finding, but I didnt want to be out
on a limb until I could be absolutely certain and I didnt want to commit myself
emotionally to the consequence until then. So it was a matter of going back to the
grindstone and repeating the experiment many times, doing it different ways and
being sure it was a totally reliable and reproducible result. Put in every control that I
could think of to be sure it had controlled for possible artifacts. Happily, with bacteria
one can do these experiments, you can run two or one or two cycles a day in this kind
of experimentation so within a few weeks it was possible to get total validation.
The experiment was as follows: two strains of Escherichia coli with different
nutritional requirements were used. Strain A would grow on a minimal medium only
if the medium was supplemented with methionine and biotin; strain B would grow on
a minimal medium only if it was supplemented with threonine, leucine, and thiamine.
Each strain of bacteria was plated into dishes containing only unsupplemented
minimal medium. Some of the dishes were plated only with strain A bacteria, some
only with strain B bacteria, and some with a mixture of strain A and strain B bacteria
that had been incubated together for several hours in a liquid medium containing
all the supplements. No colonies arose on plates containing either strain A or strain
B alone, showing that back mutations cannot restore prototrophy. However, the
plates that received the mixture of the two strains produced colonies at a frequency
of 1 in every 10,000,000 bacteria plated (i.e., 1x107). This observation showed that
recombination of genes had taken place between the genomes of the two strains to
produce prototrophs.
Between 1946 and 1952, Lederberg and his lab group, then at the University of
Wisconsin, significantly reshaped the field of bacterial genetics. By showing that
certain strains of bacteria reproduce by mating that is, by recombining their
genetic material, he overturned prevailing assumptions that bacteria were primitive
organisms not suitable for genetic analysis. Rather, he demonstrated that bacteria
could serve as a powerful experimental system, with widespread application in genetic
research. He also entered the world of virology, making seminal discoveries such as
transduction, using bacteriophage.
In 1950, Bernard Davis (1916-1994) showed that the donor and recipient bacteria
involved in genetic recombination must be in contact, and in 1952 William Hayes
(1918-1994) showed that the mechanism of transfer of genes between bacteria is quite
complex, unidirectional and employs a unique filamentous mating structure.
Lederberg J, Tatum EL. Novel genotypes in mixed cultures of biochemical mutants of
bacteria. Cold Spring Harbor Symp Quant Biol. 1946;11:113-114.
Lederberg J, Tatum EL. Gene recombination in Escherichia coli. Nature 1946;158:558.
Lederberg J. Genetic recombination in bacteria: a discovery account. Annu Rev
Genet. 1987;21:23-46.
Lederberg J. Forty years of genetic recombination in bacteria. A fortieth anniversary
reminiscence. Nature 1986;324:627-628.

1946 Joshua Lederberg, Edward Tatum discovery of genetic recombination in bacteria

page 230

Max Ludwig Henning Delbrck

(1906-1981)

Alfred Day Hershey

(1908-1997)

[with the same model fraction collector used by the

author in his thesis research at the University of
California Davis]

In 1946, Alfred Hershey demonstrated the independence of different mutation types occurring in the same bacteriophage: this was the first indication that a virus may contain
more than one gene. In the same year, Max Delbrck discovered an unexpected genetic interaction between viruses infecting the same cell. He and Hershey independently
advanced this work and demonstrated that the phenomenon was due to genetic recombination and that it could be used to construct the genetic map of the bacteriophage.
This finding led, about 10 years later, to the complete mapping of the T2 bacteriophage genome by Seymour Benzer. Salvador Luria was able to support Delbrck and Hersheys
interpretation of their findings as showing genetic recombination with experiments in bacteriophages in which genomic damage caused by radiation could be repaired by gene
exchange following infection of the same host bacterium with several damaged bacteriophage particles. These findings opened tremendous possibilities for analysis of the
structure of the genetic material, DNA.
Delbrck M, Bailey WT Jr. Induced mutations in bacterial viruses. Cold Spring Harb Symp Quant Biol. 1946;11:33-37.
Hershey AD, Rotman R. Genetic recombination between host range and plaque-type mutants of bacteria in single bacterial cells. Genetics 1949;34:44-71.

1946 Max Delbrck, Alfred Hershey discovery of genetic recombination in viruses (bacteriophage)

page 231

Smithburn KC, Haddow AJ, Mahaffy AF. A neurotropic virus

isolated from Aedes mosquitoes caught in the Semliki forest. Am J
Trop Med. 1946;26:189-208.
The virus was isolated in September 1943 from a large lot of Aedes
mosquitoes caught in an area in the Semliki Forest [Uganda], known
as Bunyamwera, hence the name provisionally adopted, Bunyamwera
virus. In recent years a considerable number of previously
unknown filterable viruses have been discovered, many of them
being neurotropic in action. These include the viruses of eastern,
western and Venezuelan equine encephalomyelitis; lymphocytic
choriomeningitis; St. Louis, Japanese B and Russian spring-summer
encephalitis; and louping ill. Also, in this laboratory, and quite
incidental to our main problem (yellow fever), we have encountered
four hitherto unknown viruses and have found humoral immunity
to each to occur in man. Some of these agents have been isolated
from man or animals, others from insects and a few from both.
Some have been discovered during the course of investigations of
specific diseases in man or animals, while others were encountered
accidentally, so to speak. Some are related antigenically, others are
not known to be. The significance of at least three of the agents in
human and/or veterinary medicine is not yet known. All of these
facts indicate that a concerted attack on the problem of virus etiology
of disease (including pyrexias of unknown origin) might not only
increase our knowledge of the known viruses and their properties
but also uncover further viruses at present unknown as the causative
agents of disease.

Kenneth C. Smithburn
(1904-1973)

Alexander John Haddow

(1912-1978)
Uganda Virus Research Institute
(UVRI), Entebbe, Uganda
The UVRI was established in 1936 as
the Yellow Fever Research Institute by
the Rockefeller Foundation. In 1977,
it became a Ugandan government
public health research institution.
The Institute has a grand record in
virus discovery; more than 20 new
arboviruses were discovered by its
staff members, including West Nile
virus, Semliki Forest virus, and here,
Bunyamwera virus.

Alexander Francis Mahaffy (1891-1952)

Yellow fever vaccination campaign, Uganda, 1944

1946 Kenneth Smithburn, Alexander Haddow, Alexander Mahaffy discovery of Bunyamwera virus

page 232

Lloyd Florio (1911-1976)

Florio L, Stewart MO, Mugrage ER. The etiology of Colorado tick fever.
J Exp Med. 1946;83:1-10. [Department of Public Health and Laboratory
Diagnosis, University of Colorado School of Medicine]
Florio and his colleagues reported the transmission of the etiologic
agent of Colorado tick fever to Syrian hamsters and human volunteers
after ultrafiltration through a series of Gradocol (collodion)
membranes, thereby showing that the agent was a virus. However, even
though the experiments were repeated and controls were satisfactory,
the findings were not cut-and-dried and their interpretation of their
findings did not predict the virion size later determined by others.

Colorado tick fever virus: (top) BHK-21 cell culture, thin section electron
microscopy; (bottom) negative contrast electron microscopy.

1946 Lloyd Florio, Mabel Miller, Edward Mugrage discovery of Colorado tick fever virus (the first coltivirus)

page 233

Frank Macfarlane Burnet (1899-1985)

Alfred Gottschalk (1894-1973)

Ernst Klenk (1896-1971)

The interaction of viruses with cellular receptors initiates a chain of dynamic events that enables entry of the virus into the cell. This is a multistep process, usually
with multiple attachment receptors used sequentially with or without co-receptors. As more and more research has been done on this subject, it has gotten more and
more complex, with less and less merit in generalizations. Extrapolation from cell culture studies, to in vivo studies, to natural host studies, has further confounded our
understanding. Nevertheless, it is virus+receptor interactions that provide keys to our understanding of virus tissue tropism, host range, disease pathogenesis and the antiviral
immune response (protective or immunopathogenic). Since the discovery of the first cellular receptors for viruses, so many investigators have made so many discoveries that
this subject cannot be covered properly here. Schneider-Schaulies J. Cellular receptors for viruses: links to tropism and pathogenesis. J Gen Virol. 2000;81:1413-1429.

1947> Frank Macfarlane Burnet, Alfred Gottschalk, Ernst Klenk discovery of viral receptors on target cells

page 234

Comparison of readouts from (a) Schlieren, (b) interference, (c)

absorbance, and (d) photoelectric optical systems. Schachman H.
Ultracentrifugation in Biochemistry. Academic Press, New York, 1959

Spinco Model E Analytical Ultracentrifuge 1947, Spinco (now Beckman Coulter)

This was the first commercial analytical ultracentrifuge

Jerome Vinograd (1913-1976),

a pioneer in the use of analytical
ultracentrifugation in virology, with
his Model E, Caltech, at the time of the
Meselson-Stahl experiments, 1956
Analytic ultracentrifugation
of foot-and-mouth disease
virus, and as controls, X174
bacteriophage and southern
bean mosaic virus,
Schlieren optics.

1947 Arnold Beckman development of the Spinco Model E analytical ultracentrifuge

page 235

Mettler Analytical Balance

Model H5, Mettler Instruments AG

Only someone who had prepared cell

culture media from scratch, weighing
out the many ingredients on a two-pan
beam balance, could fully appreciate the
advance in laboratory virology brought
about by the Mettler single-pan balance.

1947 Mettler Instruments AG development of the first single-pan precision analytical balance

page 236

Frank John Fenner (1914-2010)

John Curtin School of Medical Research, Canberra

Left: Growth curves of ectromelia virus in foot, spleen, blood,
and skin of mice inoculated in the foot with a small dose
of Moscow strain of ectromelia virus. Development and
disappearance of primary lesion and rash are shown, as is the
occurrence of inclusion bodies in skin in sections stained with
Manns stain.
Right: The original classic figure from Fenners 1948 paper in The Lancet
the first quantitative virus pathogenesis study, the model for many
that followed the figure has been copied and redrawn many times,
appearing in most virology and experimental pathology textbooks.

1948 Frank Fenner founding of viral disease pathogenesis research (ectromelia virus infection in mice)

page 237

Co-Evolution of Virus and Host: Myxoma Virus and Rabbit Myxomatosis

Myxoma virus occurs naturally as a mild infection of rabbits in South America
and California (Sylvilagus spp.), in which it produces a benign fibroma from which
virus is transmitted mechanically by biting insects. However, in European rabbits
(Oryctolagus cuniculus, the species domesticated and used in laboratories), the virus
causes a lethal infection, a finding that led to its use for biological control of wild
European rabbits in Australia. The European rabbit was introduced into Australia
in 1859 for sporting purposes; by 1930 it had spread over the entire continent, its
numbers had reached an estimated three billion, and it had decimated pasturage
and become the most important impediment to domestic livestock agriculture and
pastoral industries. Several trials were conducted in Australia in the 1930s to evaluate
the effectiveness of myxoma virus for biocontrol. However, the virus failed to become
established, possibly because there were insufficient arthropod vectors to spread the
virus beyond the sites of introduction. Further trials were conducted after the Second
World War, and in the summer of 1950-1951, the virus spread explosively from an
experimental site in the Murray Valley in southeastern Australia. The virus was spread
mechanically by mosquitoes and it was a wet year and there were extraordinary
numbers of mosquitoes. Within less than a decade, the feral rabbit population was
reduced by ~95%.
When originally introduced the virus produced case-fatality rates of over 99%. This
highly virulent virus was readily transmitted by mosquitoes. Farmers undertook
inoculation campaigns to introduce virulent myxoma virus into wild rabbit
populations. It might have been predicted that the diseaseand with it the virus
would disappear at the end of each summer, owing to the greatly diminished numbers
of susceptible rabbits and the greatly lowered opportunity for transmission by
mosquitoes during the winter. This must often have occurred in localized areas, but it
did not happen everywhere. The capacity of the virus to survive the winter conferred
a great selective advantage on viral mutants of reduced lethality, since during this
period, when mosquito numbers were low, rabbits infected by such mutants survived
in an infectious condition for weeks instead of a few days and thereby had more
opportunity to be perpetuated. Within three years such attenuated mutants became
the dominant strains throughout Australia. Thus the original highly lethal virus was
progressively replaced by a heterogeneous collection of strains of lower virulence, but
most of them still virulent enough to kill 70-90% of genetically unselected rabbits.
Rabbits that recover from myxomatosis are immune to reinfection. However, since
most wild rabbits have a life-span of less than one year, herd immunity is not so
critically important in the natural history of myxomatosis as it is in infections of
longer lived species. The early appearance of viral strains of lower virulence, which
allowed 10-30% of genetically unselected rabbits to recover, allowed selection for
genetically more resistant animals to occur. In areas where repeated outbreaks
occurred, the genetic resistance of the rabbits increased steadily such that the casefatality rate (measured in laboratory experiments) fell from 90% to 50%. Subsequently,
in areas where there were frequent outbreaks of myxomatosis, somewhat more

virulent strains of myxoma virus became dominant, largely because they produced
the kind of disease that was best transmitted in populations of genetically resistant
rabbits. Thus, the ultimate balance struck between myxoma virus and Australian
rabbits involved adaptations of both the virus and host populations, reaching
a dynamic equilibrium that finds rabbits greatly reduced compared with their
pre-myxomatosis numbers, but still too numerous for the wishes of farmers and
conservationists. The co-evolution of rabbits and pathogens used for biological
control in Australia remains dynamic: rabbit hemorrhagic disease virus (a calicivirus)
has been introduced with great success, and the introduction of rabbit flea species
from other countries that mechanically transmit myxoma virus in arid regions has
also been helpful.
Fenner F, Woodroofe GM. The pathogenesis of infectious myxomatosis: The
mechanism of infection and the immunological response in the European rabbit
(Oryctolagus cuniculus). Br J Exp Pathol. 1953;34:400-410.
Fenner F, Marshall ID. A comparison of the virulence for European rabbits
(Oryctolagus cuniculus) of strains of myxoma virus recovered in the field in Australia,
Europe and America. J Hyg. 1957;55:149-151.
Fenner F, Ratcliffe FN. Myxomatosis. Cambridge University Press, Cambridge. 1965.
Spiesschaert B, McFadden G, Hermans K, Nauwynck H, Van de Walle GR. The
current status and future directions of myxoma virus, a master in immune evasion.
Vet Res. 2011;42:76.

Rabbits,_Myxomatosis_Trial,_Wardang Island, Australia,1938

1948> Frank Fenner, Francis Ratcliffe, Ian Marshall, Gwen Woodruff co-evolution of myxoma virus and rabbits
page 238

Gilbert Dalldorf (1900-1979)

Grace Mary Sickles (1898-1959)

Gilbert Dalldorf graduated from New York

University and Bellevue Hospital Medical
School in 1924, and trained as a pathologist
in Freiburg and at the New York Hospital.
He contracted tuberculosis, probably in the
morgue, and poliomyelitis, probably from
his children. These infections influenced his
research in following years. In the summer of
1947, there were outbreaks of poliomyelitis
in upstate New York. Dalldorf, then director
of the New York State Department of Health
Wadsworth Laboratory in Albany, and his
associate, Grace Mary Sickles, investigated
these outbreaks, in particular seeking
polioviruses that would replicate in mice. This
was motivated by having heard that suckling
mice could be infected with Theilers virus (a
parvovirus). Dalldorf and Sickles made fecal
suspensions from two children suspected of
having poliomyelitis, and inoculated these
into suckling mice the mice developed
paralysis caused by widespread necrosis of
skeletal muscles, not by the central nervous
system lesions seen with polioviruses. This
was the first instance of isolation of new
viruses using suckling mice. The viruses were
named Coxsackieviruses, after the New York
village of the same name. In subsequent years
many different Coxsackieviruses, divided
into groups A or B depending upon their
pathology in suckling mice, were isolated and
associated with a variety of clinical syndromes:
In general, group A Coxsackieviruses infect
the skin and mucous membranes, causing
herpangina, acute hemorrhagic conjunctivitis,
and hand-foot-and-mouth disease. Group
B viruses infect the heart, pleura, pancreas,
and liver, causing pleurodynia, myocarditis,
pericarditis, and hepatitis. Both group A and
group B Coxsackieviruses cause nonspecific
febrile illnesses, rashes, upper respiratory tract
disease, and aseptic meningitis.
[Wadsworth Center, New York State Department of
Health, used with permission]

1948 Gilbert Dalldorf, Grace Sickles discovery of Coxsackieviruses

Coxsackievirus A4

mouse hind limb muscle, thin section electron microscopy

Coxsackievirus
structural model

Dalldorf G, Sickles GM. An unidentified, filterable agent

isolated from the feces of children with paralysis. Science
1948;108:61-62.
page 239

Thomas Milton Rivers

(1888-1962)

Frank Lappin Horsfall Jr.

(1906-1971)

Igor Tamm
(1922-1995)

Viral and Rickettsial Infections of Man

First Edition: Thomas M. Rivers, Editor. 1948.

Second Edition: Thomas M. Rivers, Editor. 1953.
Third Edition: Thomas M. Rivers and Frank L. Horsfall, Editors. 1959.
Fourth Edition: Frank L. Horsfall and Igor Tamm, Editors. 1965.
First edition: Twenty-seven chapters/contributors, including General Aspects of Viral and Rickettsial
Infections, by Thomas M. Rivers; Physical and Chemical Procedures, by W. M. Stanley and Max A.
Lauffer; Serologic Reactions in Viral and Rickettsial Infections, by Joseph E. Smadel; Chick Embryo
Techniques, by E. W. Goodpasture and G. John Buddingh; Propagation of Viruses and Rickettsiae
in Tissue Cultures, by John F. Enders; Epidemiology, by Kenneth F. Maxey; and Bacterial Viruses
(Bacteriophages), by A. D. Hershey and J. Bronfenbrenner.

1948-1965 Thomas Rivers, Frank Horsfall, Igor Tamm publication of book, Viral and Rickettsial Infections of Man

page 240

Gueh-djen (Edith) Hsiung

Frances Whitman Doane

Pekka Halonen

Phillip Gardner

Kenneth McIntosh

Caroline Breese Hall

(1918-2006)

(1925-1994)

(1928-2001)

(1927-2001)

(1930-2012)

1948> Gueh-djen Hsiung, Phillip Gardner, Pekka Halonen, others broad application of viral disease diagnostics

page 241

Spinco Model L Ultracentrifuge

The first commercial preparative ultracentrifuge
Maximum speed: 40,000rpm, ~275,000g

The vacuum ultracentrifuge was invented by Edward Greydon Pickels. His was
the contribution of the vacuum system, which allowed a reduction in friction
on the rotor generated at high speeds. The vacuum system also enabled the
maintenance of constant temperature. In 1940, interest in the isolation of viruses
brought Pickels and Johannes Bauer of the International Health Division of the
Rockefeller Institute together to build the first high-speed air-driven vacuum
centrifuge suitable for the study of filterable viruses the first virus they worked
with was yellow fever virus. Later, Pickels went on to develop the more convenient,
electrically driven ultracentrifuge.
In 1946, Pickels co-founded Spinco (Specialized Instruments Corp.) and marketed
an air-driven ultracentrifuge based on his design. Pickels, however, considered his
design to be too complicated and developed a more foolproof version. But even
with the enhanced design, sales of his centrifuge remained low, and Spinco almost
went bankrupt. In 1949, Spinco introduced the Model L, the first preparative
ultracentrifuge to reach a speed of 40,000 rpm. In 1954, Beckman Instruments
(now Beckman Coulter) purchased the company, forming the basis of its Spinco
centrifuge division. Arnold Beckman immediately went to work making major
improvements in the instrument it became an incredibly rugged workhorse,
such that by the 1960s every virology laboratory had at least one Model L
running night and day.
Pickels EG, Bauer JH. Ultracentrifugation studies of yellow fever virus. J Exp Med.
1940;71:703-717. [From the Laboratories of the International Health Division of
The Rockefeller Foundation]

1949 Edward Pickels, Johannes Bauer development of the Spinco Model L preparative ultracentrifuge
page 242

John Franklin Enders

(1897-1985)

Thomas Huckle Weller

(1915-2008)

Frederick Chapman Robbins

(1916-2003)

Nobel Prize Award Ceremony, Presentation Speech, by Sven Gard, member of the Staff of Professors of the Royal Caroline Institute
(abstracted):
in 1949 there appeared from a Boston research team a paper, modest in size and wording but with a sensational content. John
Enders, director of the Childrens Hospitals Research Laboratory and his associates Thomas Weller and Frederick Robbins reported
the successful cultivation of the poliomyelitis virus in test-tube cultures of human tissues. A new epoch in the history of virus
research had started. In their first experiments they used human embryonic tissue. To the great surprise of everybody except
perhaps the experimenters themselves they registered a hit in their first attempt. The virus grew not only in brain tissue but equally
well in cells derived from skin, muscle, and intestines Typical changes appeared in the cellular structure, finally leading to complete
destruction, easily recognizable under the microscope a convenient method of reading the results applicable also in immunity
tests. The virologists finally had a tool in the same class as the culture technique of the bacteriologists These discoveries incited a
restless activity in the virus laboratories the world over. The tissue-culture technique was rapidly made one of the standard methods
of medical virus research, among which it now holds an undisputed first place Weller has succeeded in cultivating the agents
causing varicella and herpes zoster, Enders that of measles, viruses previously almost inaccessible for study. The method has also been
successfully applied to several problems in the field of veterinary medicine Much effort and a considerable time will be required for
equivalent achievements in the fight against the virus diseases as has been the case with the bacterial diseases. However, thanks to
Enders, Weller and Robbins discovery we may look with confidence to the future.

Thomas Weller, Frederick Robbins and

John Enders at the Nobel Prize ceremony, 1954

1949 John Enders, Thomas Weller, Frederick Robbins development of cell culture methodology for polio, measles

page 243

Andr Lwoff (1902-1994)

In 1949, Andr Lwoff started to work on lysogeny. He had fixed his choice on a lysogenic Bacillus
megaterium, a particularly large bacterium, because he wanted to study single bacteria. With a
microscope and a micromanipulator he inoculated bacteria into individual microdrops and let
them grow. Why this methodological choice? Because, as he said, he: disliked mathematics, and
wanted to avoid formulas, statistical analysis and, more generally, calculations as much as possible.
By following the kinetics of phage production, he realized that only a relatively small fraction of
the bacteria lysed and produced bacteriophage. Why did some lyse, while others did not? With
Louis Siminovitch and Niels Kjeldgaard he tried, day after day, to obtain lysis of the entire bacterial
population in a culture vessel. After several unsuccessful months, he decided to irradiate the
bacteria with ultraviolet light. A UV lamp, used by Jacques Monod to mutagenize Escherichia coli,
was available. A bacterial culture was irradiated for a few seconds, and after an hour the culture was
entirely lysed. Lwoff wrote: As far as I can remember, this was the greatest thrill of my scientific
careerfor the first time in my life, I had the feeling of having discovered something. This was a
far-ranging discovery. First of all, a clear picture of lysogeny emerged: with the bacteriophage in a
lysogenic state, the bacterium perpetuates the genome of the bacteriophage (named by Lwoff the
prophage); induction of the prophage leads to its vegetative multiplication and lysis of the host
bacterium. The subsequent discovery of induction by Franois Jacob and Elie Wollman, and the
uncovering of the mechanism of genetic repression by Jacob and Jacques Monod extended this line
of research to the eventual award of Nobel Prizes to Lwoff, Jacob and Monod.
Lwoff A. The specific effectors of viral development (The First Keilin Memorial Lecture). Biochem J.
1965;96:289-301.

1949 Andr Lwoff, Louis Siminovitch, Niels Kjeldgaard discovery of lysogeny and induction (bacteriophage)

page 244

In 1950, Erwin Chargaff discovered two rules (Chargaff s Rules) that helped lead to the discovery of the
double helix structure of DNA: (1) That in natural DNA the number of guanine molecules equals the
number of cytosine molecules and the number of adenine molecules equals the number of thymine
molecules. This strongly hinted of the base-pairing structure of DNA, although Chargaff did not make
this connection himself. This work is credited with disproving the tetranucleotide hypothesis that
was widely accepted at the time. Chargaff met Crick and Watson at Cambridge in 1952 and explained
his findings to them a seminal meeting, indeed. (2) That the composition of DNA varies from
one species to another, in particular in the relative amounts of A, G, T, and C bases. Such evidence
of molecular diversity, which had been presumed absent from DNA, made DNA a more credible
candidate for the genetic material than protein.

Erwin Chargaff (1905-2002)

Chargaff E. Chemical specificity of nucleic acids and mechanism of their enzymic degradation.
Experientia 1950;6:201-209.
Chargaff E, Lipshitz R, Green C, Hodes ME. The composition of the deoxyribonucleic acid of salmon
sperm. J Biol Chem. 1951;192:223-230.

1950 Erwin Chargaff discovery that in DNA the number of molecules of A+T equals that of G+C

page 245

From his holistic view to the

study of viral pathogenesis in
experimental animals, Cedric
Mims taught the merit of a holistic
view of the infectious diseases
per se. From the Preface of his
book: It is my conviction that the
centrally significant aspects of the
subject of infectious diseases are
the mechanisms of infection and
pathogenicity, and that the principles
are the same, whatever the infectious
agent. When we consider the entry
of pathogens into body, their spread
through tissues, the damage they
cause, their exit and spread, the role
of the immune responses, etc., the
general features are the same...

Cedric Mims
Mims CA. Aspects of the pathogenesis of virus
diseases. Bacteriological Reviews. 1964;28:30-71.
Mims CA, Nash A, Stephen J. Mims Pathogenesis of
Infectious Disease (Fifth Edition). London: Elsevier;
2001.

Cedric Mims illustration of the power of the frozen-section

immunofluorescence technique (colorized). A, trachea 24 hours after the
intranasal infection of a mouse with Sendai virus. B, Section of thymus and
nearby muscle of a newborn LCM virus carrier mouse, showing infected
thymus and neighboring intercostal muscle. C, Leg muscle of a mouse showing
infected muscle cells 96 hours after infection with Ross River virus. D. Uterus
of a pregnant LCM virus carrier mouse, showing infected endometrium and
glands.

1950-1970s Cedric Mims development of immunofluorescence in viral pathogenesis research

page 246

HeLa cells

George Otto Gey (1899-1970)

Gey GO, Coffman WD and Kubicek MT. Tissue culture studies of the
proliferative capacity of cervical carcinoma and normal epithelium.
Cancer Res. 1952;12:264.

The HeLa cell line was derived from a biopsy of a cervical carcinoma suffered by Henrietta Lacks,
who died from this carcinoma in 1951. The cells were propagated by George Otto Gey, who with his
wife, Margaret, had started the Tissue Culture Laboratory at Johns Hopkins University in the 1950s.
The cells were obtained without Mrs. Lacks knowledge or permission at that time permission was
not required. The issue of ownership was later brought up in a Supreme Court of California case,
Moore v. Regents of the University of California. The court ruled that a persons discarded tissue and
cells are not their property and can be commercialized without permission. HeLa cells were used
by Jonas Salk in his polio vaccine studies in the 1950s and since then in many other kinds of studies.
They are very widely used in virology.
In 2010, a book, The Immortal Life of Henrietta Lacks, by Rebecca Skloot (Crown Publishing Co.),
brought the travails of the Lacks family to public attention, becoming a best-seller and the basis for
reopening the question of legal ownership of materials like biopsies and cells and molecular products
derived from such materials.

1951 George Gey establishment of HeLa cell line (human cervical adenocarcinoma; named for Henrietta Lacks)

page 247

Ludwik Gross
(1904-1999)

Frank J. Rauscher Jr.

(1932-1993)

Charlotte Friend
(1921-1987)

Herbert T. Abelson

John B. Maloney
(1924-2007)
From Wallace P. Rowe, 1973: The history
of tumor virology is one of the more
stormy and interesting chapters in the
history of 20th century science. From
the very beginning of tumor virology,
the pioneers in this field had to go
through successive cycles of optimism
and disillusion as each technical and
conceptual breakthrough failed to
achieve any degree of success in dealing
with the problem of human cancer. In
addition to struggling with themselves,
these pioneers had to deal with intensive
opposition from other parts of the
scientific community. The virus-cancer
question has been such a highly polarized
issue that the question Do you believe
that viruses cause cancer? has always
been as much a test of faith as Do you
believe in God?; and if the answer was
Yes, it was as likely to be greeted by a
patronizing half-smile half-sneer.

More than four decades passed between the

discoveries of the first avian retroviruses by
Ellermann and Bang and Rous to the isolation
of murine leukemia virus by Ludwik Gross
(1951). Gross found that cell-free filtrates
prepared from the spontaneous leukemias in
AKR strain mice could transmit the disease to
the low leukemia C3H strain. Gross success
where everyone else had failed had three main
bases: his serendipitous use of newborn mice as
recipients; his fortuitous choice of C3H mice as
recipients (the only low leukemia strain available
that was susceptible to the virus carried by AKR
mice); and Gross dogged persistence when no
one expected positive results. The scientific
community was skeptical, unbelieving until
Jacob Furth took pains to repeat the experiments
under original conditions. The virus isolated
became Gross murine leukemia virus. During
the next two decades, many such viruses that
cause neoplastic disease in mice were identified
and became important model systems, actively
studied at the cellular and molecular levels
to this day. Friend (1957), Maloney (1960),
Rauscher (1962), Abelson (1970) and other
murine leukemia and sarcoma viruses provided
models for the study of many aspects of
oncogenesis.
Gross L. Spontaneous leukemia developing in
C3H mice following inoculation in infancy, with
AK-leukemic extracts, or AK-embryos. Proc Soc
Exp Biol Med. 1951;76:27-32.
Gross L. Mouse leukemia. Ann N Y Acad Sci.
1952;54:1184-1196.
Rowe WP. Genetic factors in the natural history
of murine leukemia virus infection. Cancer
Research 1973;33:3061-3068.
Coffin JM, Hughes SH, Varmus HE.
Retroviruses. Cold Spring Harbor: Cold Spring
Harbor Laboratory Press; 1997.
Javier, RT, Butel JS. The history of tumor
virology. Cancer Research 2008;68:7693-7706.

1951> Ludwik Gross, Charlotte Friend, others discovery of murine leukemia and lymphoma viruses
page 248

Esther Miriam Zimmer Lederberg (1922-2006)

The bacteriophage (lambda) has been the most widely used bacteriophage in molecular biological
research. Esther Lederberg was the first to isolate this DNA virus, from Escherichia coli K-12, in 1951.
The genome consists of a double-stranded DNA molecule with 5 twelve-base-pair sticky ends, which
permit circularization of the DNA molecule. It shows a lytic cycle and a lysogenic cycle. Studies on the
control of these alternative cycles have been very important for our understanding of the regulation of
gene transcription. Study of over the past 50 years has also provided valuable insights into virus life
cycles, the regulation and expression of DNA, and the mechanism of integration and excision of viral
genes and genomes into host chromosomes. The mechanism of integration of DNA into bacterial
DNA was first worked out by Lederbergs colleague, Allan Campbell, in 1962. has also been used as a
vector for the cloning of recombinant DNA.

Bacteriophage

negative contrast electron microscopy

Lederberg EM. Lysogenicity in Escherichia coli strain K-12. Microb Genetics Bull. 1950;1:5-8.
Lederberg EM, Lederberg J. Genetic studies of lysogenicity in Escherichia coli. Genetics 1953;38:51-64.

1951 Esther Lederberg discovery of the bacteriophage

page 249

Linus Carl Pauling (1901-1994)

John Cowdery Kendrew (1917-1997)

Max Ferdinand Perutz (1914-2002)

Studies leading
to resolution of
the structure of
hemoglobin:
Max Perutz with
his diffractometer,
the crystal
diffraction pattern
of hemoglobin 1c,
and a molecular
ribbon model of
hemoglobin 1c

1951> Linus Pauling, John Kendrew, Max Perutz discovery of the structure of proteins using X-ray crystallography
page 250

Over the years, there had been quite a few

references to a mysterious viral interference
phenomenon, but starting in 1951 the mystery was
slowly resolved, starting with the work of Preben
von Magnus and his wife and colleague, Herdis,
at the Statens Seruminstitut in Copenhagen. In
their work with influenza viruses, they concluded
that the interfering agent was an incomplete form
of the virus, lacking ability to cause infection on
its own. They observed that repeated passage of
an influenza virus, using as inoculum undiluted
supernatant of the previous passage, caused
the virus yield to decrease with each successive
passage. This became known as the von Magnus
effect. With influenza virus the interference was
first attributed to the production of particles with
a lower ratio of nucleoprotein antigen to surface
protein antigens than normal infectious particles.
Later, it was found that defective influenza
particles usually had a missing or truncated
genome segment.

Preben & Herdis von Magnus

(1912-1973) (1912-1992)

Others entered the field, most notably Alice

Huang, at first working with Robert Wagner and
later with David Baltimore. She used vesicular
stomatitis virus to demonstrate high multiplicityof-infection interference. She found that defective interfering (DI)
virus particles were shorter-than-normal length particles (called
truncated = T particles)(normal-length infectious particles are called
bullet-shaped = B particles). Molecular details have been studied ever
since, and quite a few other viruses have been shown to exhibit the
von Magnus effect: Rift Valley fever virus, mumps virus, measles
virus, Sendai virus, fowl plague (avian influenza) virus, Sindbis virus,
LCM virus, polioviruses, reoviruses, et al.

Alice Huang
(photo from 1970s)

von Magnus P. Propagation of the PR8 strain of influenza A virus

in chick embryos. II. The formation of incomplete virus following
inoculation of large doses of seed virus. Acta Pathol Microbiol Scand.
1951;28:278-293.

Defective interference (DI) phenomenon exemplified by vesicular stomatitis virus

The truncated DI virus particles (T particles) cannot replicate by themselvesthey need the presence
of parental wild-type virus (standard virus, B particles); at the same time they can interfere with and
decrease the yield of the wild-type virus. These DI particles, which are actually deletion mutants, are
easily demonstrated in cell culture, but their role in in vivo infections and disease is not clear.

Huang AS, Wagner RR. Defective T particles of vesicular stomatitis

virus. II. Biologic role in homologous interference. Virology
1966;30:173-181.
Huang AS, Baltimore D. Defective viral particles and viral disease
processes. Nature. 1970;226:325-327.

1951> Preben & Herdis von Magnus, Alice Huang, David Baltimore discovery of defective interfering (DI) viruses

page 251

Myron Brakke, who spent many years as a U.S. Department of Agriculture staff member at the
University of Nebraska-Lincoln, invented sucrose density-gradient (rate-zonal) ultracentrifugation
for the purification and characterization of viruses, nucleic acids, proteins, cellular organelles and
other macromolecules. It was one of the keys to the development of modern virology and molecular
biology. Rate zonal centrifugation separates particles based on their size, shape, and density
larger, denser and more compact macromolecules migrate faster through the gradient during
centrifugation. At first rather slow centrifugation was used, but in 1953 in a collaboration with
Josef Blum, the head instrument maker at the Rockefeller Institute for Medical Research, Brakke
designed the first high-speed horizontal (swinging-bucket) rotor, which essentially eliminated the
streaming effects (wall effects) of fixed angle-head rotors, optimally stabilizing sedimenting viruses
and macromolecules in the protective density/viscosity gradient. The methodology was first used
to purify potato yellow-dwarf virus. With colleagues he also developed equipment to facilitate
the building of density gradients, the collection of fractions after centrifugation and the direct
densometric scanning of fractions collected from gradients. Through Brakkes work: (1) virus bands
could be visualized by light scattering, (2) the sedimentation value (S) of viruses could be calculated,
and (3) the buoyant density of viruses could be determined. His work led to the development of
modern preparative ultracentrifuge methods for isolation of the large amounts of viruses needed for
crystallography, vaccinology and other technologies. Brakke also made seminal contributions to the
development of plant tissue culture.

Myron Kendall Brakke (1921-2007)

Brakke MK. Density gradient centrifugation: A new separation
technique. J Amer Chem Soc. 1951;73:1847-1848.
Brakke MK. Zonal separations by density-gradient centrifugation. Arch
Biochem Biophys. 1953;45:275-290.
Brakke MK. Density gradient centrifugation and its application to plant
viruses. Adv Vir Res. 1960;7:193-224. Brakke MK.

1951 Myron Brakke development of rate zonal density gradient centrifugation

page 252

In 1944, the Avery-MacLeod-McCarty experiments demonstrated that DNA rather

than proteins is the carrier of genetic information. Though the work was well done, it
met with resistance from much of the scientific community and for the next decade
Avery was forced to repel attacks. Finally, in 1952, Alfred Hershey, at Cold Spring
Harbor Laboratory, set out to settle the issue Hershey believed that proteins, with
their complicated structures, were more likely to be the carriers of genetic information
than was the simple DNA molecule. Hershey and his technician, Martha Chase, decided
to track the transfer of proteins and DNA between a virus and its host. They chose the
bacteriophage T2 as the vehicle for delivering the genetic material to the host Escherichia
coli the phage would inject its genetic material into the bacterium, leaving its protein
shell attached to the outside of the bacterial cell wall. It was thought that certain proteins
were transferred from the virus to the bacterium upon attachment; if the genetic
information was in fact carried by these proteins, it was necessary to demonstrate that at
least a portion of it was transferred to the interior of the bacterium.

Martha Cowles Chase (1927-2003)

Alfred Day Hershey (1908-1997)
[Karl Maramorosch took this famous photo]

In their first experiment, Hershey and Chase tagged the phage DNA with radioactive
phosphorus-32 (32P). Because phosphorous occurs in large quantities in DNA, but in
only trace amounts in protein, the location of the phage DNA could be determined.
They then allowed the labeled phage to begin infecting samples of E. coli. After the
phage was mixed with the bacteria, they used a Waring blender to violently disturb the
infected bacteria, causing the phage protein shells to detach from their hosts. Then,
using a centrifuge, they separated the bacteria from the phage and protein. Once the
separation was complete, they measured the 32P in the bacteria and the separated
phage protein shells. The 32P appeared in large quantities only in the bacterial sample,
demonstrating that DNA was transferred from the phage to the bacteria. Further, despite
the protein shells being detached while infection of the bacteria was still ongoing, the
phage replicated in the bacteria as usual. This, in turn, suggested that the protein shell
was not necessary for the replication process following the initial insertion of the genetic
material.
Shocked by their findings, Hershey and Chase decided to perform the experiment again,
this time using a different tracer sulfur-35 (35S), because sulfur is present in proteins, but
not in DNA. After labeling the proteins, infecting the bacteria, and separating the phage
shells from the bacteria, they tested for the presence of 35S. It could only be found in the
protein shells, not in the bacteria. And again, phage replication went on normally.
Sufficiently impressed by the significance of their findings, they examined the progeny
of the 32P-labeled phage and found that they also had 32P-labled DNA, but their protein
lacked any trace of radioactivity.
At first, Hershey and Chase were inclined to believe that their experiments were flawed,
but no faults were found. The results were no mistake and the importance of their work
was clear: they had produced direct, irrefutable, biological evidence that DNA, not
protein, is the stuff of inheritance. Later that year, they reported their findings in a short
paper. As they say, The rest is history.
Hershey AD, Chase M. Independent functions of viral protein and nucleic acid in growth
of bacteriophage. J Gen Physiol. 1952;36: 39-56.

1952 Alfred Hershey, Martha Chase Waring blender experiment proof of DNA as the material of inheritance

page 253

The Virus Laboratory, University of California Berkeley, 1952

Wendell Meredith Stanley (1904-1971)

In 1948, Wendell Stanley went to the University of California Berkeley from the Rockefeller Institute to be director of a new research organization, the Virus Laboratory (always
known as the virus lab). Stanley assumed the additional task of recruiting staff for a new Department of Biochemistry to be attached to the College of Letters and Science. In
1952, this department, as well as the Virus Laboratory, moved from temporary quarters into the new Virus Laboratory building near the East Gate. The Department of Virology
was established in 1958, in recognition of the prominent role that graduate and post-doctoral training had assumed in the Virus Laboratory. The staff, under the chairmanship
of Stanley, organized a course of study and research leading to MA and PhD degrees in virology. The department emphasized in its teaching and research the biochemical,
biophysical, and biological aspects of animal, plant, and bacterial viruses. It was the second Department of Virology founded in a major university, the first being at Johns
Hopkins University. In 1962, a broader vision of the role of virology culminated in the creation of the Department of Molecular Biology. Its initial staff numbered 16: among its
members were three Nobel Prize winners and five members of the National Academy of Sciences. In 1964, the Department of Virology was subsumed into the Department of
Molecular Biology. In 2005, the Virus Laboratory building was demolished to make room for a new biosciences and bioengineering facility.
[In 1962, the author (FAM), while a graduate student at the University of California Davis, enrolled in the basic virology course at Berkeley it was taught by Gunther Stent
and Harry Rubin and was held in the lecture room in the virus lab. It was the best course ever incredible in its scope, depth and context, demanding to an extreme, and
of great influence upon everyone who ever took it. Part of the aura was that nearby in the building were leaders in all the booming facets of virology research, including
bacteriophage research (it was said that Gunther Stent and his colleagues, along with colleagues at Caltech, represented branches of the Phage School at Cold Spring Harbor
Laboratory). Of course students taking that great basic virology course did not know all this at the time!]

1952 Wendell Stanley establishment of the Virus Laboratory at the University of California Berkeley
page 254

Between 1947 and 1955, Joshua Lederberg and his colleagues at the
University of Wisconsin described several important experimental
techniques and results which transformed the science of bacterial genetics
and helped define the classical era of molecular biology. Their most
important discoveries were that of transduction the transfer of genetic
fragments from one cell to another by a virus and of extra-chromosomal
genetic elements called plasmids. Lederberg later said: We were exploring
a completely new territory that we only dimly understood. We werent
looking for transduction we bumped into it. We werent looking for
plasmids we bumped into them. Every time we turned around we found
something unexpected.

Joshua Lederberg (1925-2008)

Norton Zinder (1928-2012)

Lederberg and the other members of his small laboratory, in particular

his wife, Esther Zimmer, and graduate students Norton Zinder and Larry
Morse, conducted a systematic search for genetic recombination in bacteria.
Streptomycin-resistant mutants of Salmonella proved especially important.
In 1951, Lederberg and Zinder discovered the third mechanism of genetic
transfer in bacteria, (the first being transformation, discovered by Oswald
Avery, the second being mating, discovered earlier by Lederberg). Lederberg
named the new phenomenon transduction. They used a glass tube, bent
into a U-shape and fitted with an extremely fine glass filter (an ultrafilter)
at its crook, and filled it with broth containing none of the nutrients their
Salmonella mutants needed to grow. They then added one mutant parent
strain to each arm, and pumped the broth back and forth through the filter.
They expected to find no recombinants, because the bacteria in Lederbergs
original crossing experiments had to be in direct contact with one another
for conjugation to take place. To their surprise, several bacteria nonetheless
appeared and multiplied, an indication that recombination had occurred
and had enabled each mutant strain to compensate for the nutritional
deficiencies of the other. Obviously, a biological agent small enough to
pass through the filter was at work. When they purified the material, they
found that it did not consist of pure DNA or RNA; instead, it proved to be
a bacteriophage. The Salmonella Lederberg and Zinder had used turned
out to be lysogenic: it carried phage DNA integrated into its chromosome,
but occasionally this was excised and the phage replicated. Prior to lysis,
the phage particles picked up random snippets of bacterial DNA, which
they then carried through the filter The phage acted as vectors for bacterial
genes, genes that complemented the mutual genetic (nutritional) defects of
the parent bacteria.
Zinder ND, Lederberg J. Genetic exchange in Salmonella. J Bact.
1952;64:679-699.
Morse ML, Lederberg EM, Lederberg J. Transduction in Escherichia coli
K-12. Genetics 1956;41:142-156.

1952 Joshua Lederberg, Norton Zinder discovery of transduction: DNA transfer between bacteria by bacteriophage

page 255

Karl Maramorosch

Tobacco necrosis virus A, negative contrast electron microscopy

Karl Maramorosch wrote in 1953: There exists a group of viruses which constitutes a link between animal and plant viruses. These viruses were shown to multiply both in
the plants they infect and in their insect vectors. The best studied of these versatile viruses is the one that causes aster-yellows disease. The aster-yellows virus requires for
its survival in nature two alternating hosts: a susceptible plant and a susceptible insect. The host range in the plant kingdom is very wide, as species in more than 50 families
have been found susceptible. However, only one species of cicadellid leafhopper, Macrosteles fascifrons Stal, the common aster leafhopper, has been found susceptible to the
virus in the Eastern United States. At the same time, arthropod transmission of plant viruses was also discovered in Japan by Teikichi Fukuushi. This pattern of rather broad
plant host specificity and narrow arthropod host specificity tuned out to be quite common. Over many years, Karl Maramorosch led studies of the natural history of many
arthropod-borne plant viruses, many of which cause economically important crop and ornamental flower diseases. Many, as well, exhibit complex, intriguing mechanisms by
which they survive and propagate in nature, including: mechanical transmission (flying pin), biological transmission (requiring virus replication in the arthropod and delivery
of virus to the feeding organs), vertical transmission (requiring infection or carriage in the egg or embryo of the arthropod), and fomite transmission (often via carriage on farm
equipment and implements). [In recent years, some plant diseases thought to be caused by viruses have been attributed to mycoplasmas; this includes aster yellows.]
Maramorosch K. Multiplication of aster-yellows virus in its vector. Nature 1952;169:194-195.
Maramorosch K. Do developmental stages occur in the reproductive cycle of aster-yellows virus? Cold Spring Harbor Symposia in Quantitative Biology 1953;18: 51-54.

1952 Karl Maramorosch discovery that some viruses can replicate in both plants and insects
page 256

Western equine encephalitis virus,

plaques in Vero cells
Dulbecco R. Production of plaques in monolayer tissue
cultures by single particles of an animal virus. Proc Natl
Acad Sci USA. 1952;38:747-752.
Dulbecco R, Vogt M. Plaque formation and isolation
of pure lines with poliomyelitis viruses. J Exp Med.
1954;99:167-182.

Renato Dulbecco (1914-2012)

Marguerite Vogt (1913-2007)

Interview with Renato Dulbecco by Shirley K. Cohen, 1998. [Caltech Oral History] (Unpublished) http://resolver.caltech.edu (section abridged): You know, accidents are
wonderful in science, provided you can take advantage of them. [In ~1950 Dulbecco, then a fellow at Caltech, was called into the office by Max Delbrck regarding some new
funding for virus research.] at the time, phage work didnt appeal to me so much. I needed something new, so I said yes Immediately I thought that to do anything in the
field, you needed tissue cultures. And I already had a background in that. So I said yes [After trips to several labs, to] learn about what people were doing, I thought of how
to develop a good system for assay of animal viruses [I told Max,] You know, the virus I need is the western equine encephalitis virus, which was known to be pathogenic.
They were very worried about that, so they decided to send me to the second basement at the end of the corridor, where there was a room by itself Immediately I was
thinking that my goal was to develop a plaque system like that of phage and for that, you need a uniform layer of cells. [Dulbecco passed minced mouse embryo tissue
through a fine screen so dispersed cells could be spread on Petri plates and infected with highly diluted virus.] For a week or two, nothing seemed to happen. And then one day
I put one of the cultures in a tangent light, and saw that it was full of plaques that were not visible in transmitted light [Someone, name forgotten, suggested using a vital stain
one that stained only living cells clear holes were seen in a red background.] The paper was published in 1952 in the Proceedings of the National Academy of Sciences
Then people from the National Science Foundation came and said that I should try to develop a system for polio [important since vaccine development was then ongoing the
NSF people also told Dulbecco about the use of trypsin to disperse cells]. Of course, people here at Caltech were very, very worried about work in polio. [So, a lab was rented
in a nearby hospital. At that time, Marguerite Vogt joined Dulbecco as a research fellow. Using monkey kidney cells, they developed a plaque system which worked very well.]
We succeeded in demonstrating two things: One is that if you want to have a pure strain, you have to pick one plaque, and second, that by picking different plaque types or
size, you can get mutants. I think this was essential for Sabin vaccine development.

1952 Renato Dulbecco, Marguerite Vogt development of virus quantification by plaque assay (WEE, polio viruses)

page 257

5-hydroxymethyl cytosine

cytosine

Seymour Cohens work on bacterial viruses, begun in 1945, was the first systematic exploration
of the biochemistry of virus-infected cells and of how viruses multiply. In 1952, he and Gerard
R. Wyatt independently discovered that one of the T-even viruses that infect E. coli contained a
new pyrimidine, 5-hydroxymethylcytosine, in its DNA, instead of the normal cytosine. Later, in
1957, he and Joel G. Flaks found that the enzyme that induced this pyrimidine was not found in
uninfected bacteria. They reasoned that the virus caused the E. coli cells to produce the enzyme
that would then induce the formation of 5-hydroxymethyl cytosine. This discovery made it
possible to develop drugs that would inhibit the enzymes induced by the virus without harming
healthy cells. The discovery that virus infection creates new metabolic pathways and affects flux
through existing pathways was of importance because it suggested that virus-specified enzymes
could be used as biochemical targets for antiviral agents.

Seymour Stanley Cohen

Separately, when it was discovered in 1952 that T2, T4 and T6 bacteriophages lacked cytosine,
it was realized that this would make the double-helix model impossible. The demonstration that
these bacteriophages contained the modified form 5-hydroxymethylcytosine and that this was in
equal quantities to guanine removed this obstacle to the double-helix model.
In recent years, 5-hydroxymethylcytosine has been found in large amount in mouse, bovine,
rabbit and human zygotes and in human stem cells. It is eventually enzymatically converted to
cytosine, but research is ongoing questioning the ancient phylogenetic basis for this seeming
anomaly in a key building block of the stuff of inheritance.
Wyatt GR, Cohen SS. The bases of the nucleic acids of some bacterial and animal viruses: the
occurrence of 5-hydroxymethylcytosine. Biochem J. 1953;55:774-782.

1952 Seymour Cohen discovery that the DNA of some bacteriophages contains 5-hydroxymethylcytosine

page 258

Wallace Rowe (1926-1983)

Human adenovirus 5, cell culture

thin section electron microscopy

From a memoir by Harry Ginsberg: Robert Huebner of the National Institutes of Health visited Western Reserve to present a seminar. Afterward he came to my laboratory to
hear about my research, but before I could begin, he told the following exciting news: Dr. Wallace Rowe, a postdoctoral fellow in his laboratory, had made a thrilling discovery.
In an attempt to isolate the virus of the common cold, he had used an explant of an adenoid in cell culture, into which he inoculated secretions from a patient with a cold.
Dr. Rowe being a very smart young scientist also used control uninoculated cultures. After only a few days he noted that the cells in the control uninoculated cultures, as well
as the cells in those inoculated with patient secretions, became rounded and formed grape-like clusters, but the cells did not lyse. If he disrupted the cells, he could obtain
and pass on the cytopathic agent to other cells. Hence, the adenoids were obviously infected with a latent virus I asked Dr. Huebner if he would send me the virus after
publication of this exciting discovery. He immediately responded that prior publication was not necessary. He then called Dr. Rowe and told him to send me samples from his
adenoid cultures. (Can you imagine that occurring today?). On receipt of the sample, I demonstrated that it could replicate and be passaged in HeLa cells. Shortly, thereafter
Dr. Maurice Hilleman, who was then in the U.S. Army, visited Western Reserve... [Hilleman was working on acute respiratory diseases of recruits and had isolated a virus
from recruits who were thought to have influenza, but this was not influenza virus Ginsberg showed that Hillemans virus was related to Rowes virus by complement fixation
assays but distinct by neutralization assay. Since Rowe first isolated an adenovirus from human adenoid cells, more than 100 different viruses have been identified from humans
and various mammals, all related to each other by various methods, all distinct by neutralization assay.]
Rowe WP, Huebner RJ, Gilmore LK, Parrott RH, Ward, TG. Isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture.
Proc Soc Exp Biol. 1953;84:570-573.
Ginsberg HS. The life and times of adenoviruses. Adv Vir Res. 1999;54:1-13.

1953 Wallace Rowe, Robert Huebner, others discovery of human adenoviruses

page 259

James Dewey Watson

Watson J, Crick F. Molecular structure of nucleic

acids. A structure for deoxyribose nucleic acid.
Nature 1953;171:737-738.

Francis Crick (1916-2004)

Maurice Hugh Wilkins (1916-2004)

The lives of Francis Crick and James Watson changed when they met at Cambridge in 1951; neither was supposed to
be working on DNA, but they at once fell into often loud discussions on how the problem of its structure might be
approached. Jim and I hit it off immediately, Crick wrote, partly because our interests were astonishingly similar
and partly, I suspect, because a certain youthful arrogance, a ruthlessness and an impatience with sloppy thinking
came naturally to both of us. Their approach was to build an exact scale model of a DNA molecule that was driven
by the limited information available from X-ray crystallography. They constructed the backbone of a DNA molecule
in the form of a double anti-parallel spiral, or helix, with the two helices held together in the middle by ionic forces
the key change to this model came when Watson played with cardboard cutouts of the four bases, A, T, G, C,
and noticed that an A-T pair was identical in shape with a G-C pair. He immediately perceived how the bases could
point inward, holding the spiral together, provided that adenine always paired with thymine, guanine with cytosine
(Chargaff s rule). The pairing rule at once explained how one DNA chain could serve as the template for building
another, the essential requirement for any molecule that encoded hereditary information. Building the final model
took only a few days after this. It has not escaped our notice, they wrote in their paper in Nature, that the specific
pairing we have postulated immediately suggests a possible copying mechanism for the genetic material. The
personal/professional intrigues surrounding the work, involving Crick, Watson, Maurice Wilkins, Rosaland Franklin,
et al, which have been written about ever since, are not cited further here.

1953 James Watson, Francis Crick, Maurice Wilkins, Rosalind Franklin discovery of the structure of DNA
page 260

Rosalind Elise Franklin (1920-1958)

James Watson and Francis Crick with their structural model of DNA

Rosalind Franklins photo 51X-ray diffraction image of sodium salt of DNA, B form

This image provided critical evidence in identifying the structure of DNA. The photo was taken by Rosalind
Franklin while working at Kings College, London. James Watson was shown the photo by Maurice Wilkins.
Watson said, The instant I saw the picture my mouth fell open and my pulse began to race. Whether or not
Wilkins had Franklins approval to share the image has been a matter of endless controversy. The Nobel prize
is not awarded posthumously; Franklin, whose breakthrough data was used to formulate the
DNA structure, had died in 1958, and thus was not eligible for nomination.

1953 James Watson, Francis Crick, Maurice Wilkins, Rosalind Franklin discovery of the structure of DNA

page 261

Morphology of Viruses!
by Frederik B. Bang

The Johns Hopkins University

[Annual Reviews of Microbiology, 1955]
The Microbe is so very small
You cannot make him out at all,
But many sanguine people hope
To see him through a microscope.
His jointed tongue that lies beneath
A hundred curious rows of teeth;
His seven tufted tails with lots
Of lovely pink and purple spots,
On each of which a pattern stands.
Composed of forty separate bands;
His eyebrows of a tender green;
All these have never yet been seen. But,
scientists, who ought to know.
Assure us that they must be so . . . .
Oh! let us never, never doubt
What nobody is sure about.
(CAUTIONARY VERSES
by Hilaire Belloc)

James Bumgardner Murphy (1921-2007)

Frederik B. Bang (1916-1981)

Murphy JS, Bang FB. Observations

with the electron microscope on cells
of the chick chorio-allantoic membrane
infected with influenza virus. J Exp Med.
1952;95:259-268.

Influenza virus, budding from

the plasma membrane of an
infected cell in culture. Thin
section electron microscopy.
from: Noda T, Sagara H, Yen A,
Kawaoka Y, et al. Architecture
of ribonucleoprotein complexes
in influenza A virus particles.
Nature 2006;439:490-492.

1953 James Murphy, Frederik Bang discovery of virus release by budding at the cell surface

page 262

Sendai virus infection

adult mouse, 24 hours

after intranasal instillation
of virus, small bronchus
showing infection of nearly all
epithelial cells, frozen section
immunofluorescense

Nakao Ishida (1923-2009)

Sendai virus murine parainfluenza virus 1

negative contrast electron microscopy

In 1952, there was an outbreak of pnemonitis in newborn babies in the university hospital in Sendai, Japan. A virus was isolated from lung tissues from five fatal cases
by intranasal instillation of mice. All of the mice died between 5 to 7 days and showed a consolidation of the lungs, resembling that of influenza. The histopathological
findings in the mice were similar to that seen in the newborn babies. The virus was cultivated in embryonating eggs, and its hemagglutinin was compared with that of other
hemagglutinating viruses known at the time the hemagglutinin associated with the virus was unique. However, all this was smoke further characterization established the
virus as murine parainfluenza virus type 1, an important pathogen of laboratory mice but not of humans. Only later when methods improved and human parainfluenza virus
types 1, 2, 3, 4a/4b were discovered by Robert Chanock and his colleagues and shown to be important pathogens of humans did it become clear that the presence of the murine
virus, Sendai virus, was confounding the study of coincident human parainfluenza viruses. For years this served as an object lesson in the need for verification of virus identity.
Kuroya MK, Ishida N, Shiratori T. Newborn virus pneumonitis (type Sendai). 2. The isolation of a new virus possessing haemagglutinin activity. Yokohama Medical Bulletin
1953;4:217.
Ishida N, Homma M. Sendai virus. Adv Virus Res. 1978;23:349-383.

1953 Nakao Ishida, colleagues discovery of Sendai virus (murine parainfluenza virus 1)

page 263

Rupert E. Billingham (1921-2002)

Peter Brian Medawar (1915-1987)

Leslie Baruch Brent

In 1945, Ray Owen, who was interested in bovine blood groups, discovered that most dizygotic-twin cattle fetuses had placental anastomoses and shared their blood supply
in utero. These cattle maintained a stable mixture of each others red cells throughout their lives. Unlike the situation with non-twin cattle, even when each had a different
blood group, transfusions between the twins did not cause any transfusion reaction. This mosaicism provided the first clue of immune tolerance. In 1949, Rupert Billingham
and Peter Medawar came across Owens work in a monograph by Frank Macfarlane Burnet and Frank Fenner, entitled The Function of Antibodies. This publication helped
them make sense of their work. The authors postulated that the age of the animal at the time of its first encounter with a foreign body was the critical factor in determining its
responsiveness and, hence, its recognition of nonself-antigens.
In a paper in Nature in 1953, Billingham, Leslie Brent, and Medawar reported the successful transplantation in mice of cells from normal mouse bone marrow, spleen, and
lymph nodes. The donor cells were obtained from adult mice and infused intravenously into normal newborn mouse recipients whose immune system was not yet sufficiently
developed to reject the cells. When the recipient mice grew up, they could freely accept skin and other tissue grafts from the original cell donor mouse strain, but from no
other donor. These first studies of acquired transplantation tolerance led to Medawars award (with F. Macfarlane Burnet) of the 1960 Nobel Prize. Medawar shared his
portion of the cash award with Billingham and Brent. [The mechanism of the phenomenon discovered by Billingham, Medawar and Brent is T-cell tolerance it is based on
the deletion of autoreactive T cells in the thymus so that the animal is rendered tolerant to self. T-cell tolerance has a very important role in advancing our understanding of
the pathogenesis, the immunopathogenesis of viral infections.]
Billingham RE, Brent L, Medawar PB. Activity acquired tolerance of foreign cells. Nature 1953;172:603-606.

1953 Rupert Billingham, Peter Medawar, Leslie Brent discovery of immunological tolerance
page 264

Wilhelm Bernhard (1920-1978)

Albert J. Dalton (1905-1992)

Between 1953 and 1966, Wilhelm Bernhard, Albert Dalton, Franois

Haguenau, Etienne de Harven, Odile Croissant, Ludwik Gross and others
demonstrated the structure and intracellular development, first of Rous
sarcoma virus, then of other retroviruses including mouse mammary tumor
virus, avian endothelioma virus, avian erythroblastosis and myeloblastosis
viruses, murine leukemia viruses, and others. It was Bernhard who proposed
the classification of types A, B, and C (and later D) retroviruses, now
embedded in the definitions and names of the subfamilies and genera of
the family Retroviridae. This book uses vernacular virus nomenclature and
taxonomy for formal taxonomy and nomenclature, the reader is referred
to the current editions of the report of the International Committee on
Taxonomy of Viruses (ICTV) [http://talk.ictvonline.org/]. Nevertheless, for
clarity in this instance, it seems necessary to cite the formal family, subfamily
and genus taxonomy and nomenclature:

Thin section electron microscopy of retroviruses. (A) Intracisternal

type A particles. (B) Type B particles, mouse mammary tumor virus.
(C) Type C particles, murine leukemia virus. (D) Type C particles,
avian leukosis virus. (E) Type D particles, Mason-Pfizer monkey
virus. (F) Deltavirus, bovine leukemia virus. (G) Lentivirus, bovine
immunodeficiency virus. (H) Spumavirus, bovine syncytial virus.

Family Retroviridae, Subfamily Orthoretrovirinae

Genus Betaretrovirus Mouse mammary tumor virus (the former type B particles)
Genus Gammaretrovirus Murine leukemia virus (some of the former type C particles)
Genus Alpharetrovirus Avian leukosis virus (some of the former type C particles)
Genus Deltaretrovirus Bovine leukemia virus (some of the former type C particles)
Genus Epsilonretrovirus Walleye dermal sarcoma virus
Genus Lentivirus Human immunodeficiency virus 1
Family Retroviridae, Subfamily Spumaretrovirinae
Genus Spumavirus Simian foamy virus
Bernhard W. Electron microscopy of tumor cells and tumor viruses: A review. Cancer Res.
1958;18:491-509.

1953-1966 Wilhelm Bernhard, Albert Dalton, others morphology/morphogenesis/classification of retroviruses

page 265

Albert Bruce Sabin (1906-1993)

Mammalian reovirus 3 (strain Dearing)

In 1959, Albert Sabin coined the acronym reovirus as a descriptive term for a new group of viruses. The acronym refers to the fact that although reoviruses can be isolated
from the respiratory and enteric tracts, there is no association of carriage with clinical disease, hence, they are orphan viruses (r-e-o). There are three common reovirus types,
based on their hemagglutination-inhibition activity. Prototypical laboratory strains of each were isolated from the respiratory and enteric tracts of children: Type 1 Lang, Type
2 Jones, Type 3 Dearing. All three virus types are found ubiquitously in the environment, including in such sources as water and sewage. This, combined with the fact that the
viruses are environmentally stable, explains why as many as 50% of adults carry antibodies to the viruses.
Sabin AB. Reoviruses. A new group of respiratory and enteric viruses formerly classified as ECHO type 10 is described. Science. 1959;130:1387-1389.

1953 Albert Sabin discovery of orthoreoviruses (the first reoviruses)

page 266

Dulbecco and Vogts growth curve of WEE virus in

a monolayer of chicken embryo cells

Renato Dulbecco (1914-2012)

Marguerite Vogt (1913-2007)

Actually, the first one-step growth experiment was done in 1939 by Emory L. Ellis and Max Delbrck,
using bacteriophage proving that lysis of the host bacteria and release of the daughter burst of phage
indeed occur strictly on the dot [i.e. at a precise time interval.] But, it was considered an important advance
when Renato Dulbecco and Marguerite Vogt did this with a vertebrate virus (Western equine encephalitis
virus) in cell culture, since at that time few believed that discoveries made using bacteriophage pertained
directly to the viruses of vertebrates.
Dulbecco R, Vogt, M. One-step growth curve of western equine encephalomyelitis virus on chicken embryo
cells grown in vitro and analysis of virus yields from single cells. J Exp Med. 1954;99:183-199.

1954 Renato Dulbecco, Marguerite Vogt concept of the latent period, eclipse period, single cell burst size

page 267

Jonas Salk (1914-1995) and Julius Youngner

Children lining up for polio vaccine, 1955

In 1949, John Enders, Thomas Weller, and Frederick Robbins grew poliovirus in monkey kidney cell culture. That same year, the virus
was separated into three immunological types. The development of an inactivated poliovirus vaccine moved forward quickly from this
point, under the leadership of Jonas Salk and Julius Youngner at the University of Pittsburgh and Pierre Lpine at the Institut Pasteur
in Paris. Salk used formalin to inactivate virus, Lpine used formalin followed by -propiolactone (the latter prevented the persistence
of residual virus that led to the Cutter Incident 200 cases of polio and 11 deaths in vacinees, all very soon after initial commercial
vaccine manufacture). In 1952, Salk vaccinated a group of children who had already had polio and recovered after vaccination,
their antibody titer increased. Next were volunteers who had not had polio, including Salk himself, his wife, and their children all
produced antibodies, and none developed polio. Next, in 1954, came field trials run by Thomas Francis of the University of Michigan
on behalf of the National Foundation for Infantile Paralysis; these trials were unprecedented in scope and size, involving 1.8 million
children in the U.S., Canada and Finland. These trials were among the first to use the
double-blind-placebo-controlled system that has since become standard. Scale up of
trivalent vaccine production involved six U.S. pharmaceutical companies, eventually
led by Connaught Laboratories in Montreal, where by 1955 most of the vaccine used in
North America was produced. Over 4 million doses had been given by August of 1955:
by 1957 polio cases in the U.S. had dropped by 85-90%.

Pierre Lpine
(1901-1989)
1954 Jonas Salk, Julius Youngner, Pierre Lpine, Thomas Francis development of inactivated polio vaccine
page 268

Margaret Gladys Smith (1896-1970)

Owls Eye intranuclear inclusions of cytomegalovirus

Top: from Jesionek & Kiolemenoglov, 1904, then thought to be a
protozoan agent. Bottom: recent image.

Cytomegalovirus infections are characterized by the enlargement of infected cells (a process called cytomegaly by Ernest Goodpasture in 1921) and the presence of intranuclear
inclusions. The first proof of the viral nature of an agent responsible for cytomegaly was by Rufus Cole and Anne Kuttner in 1926. They ultrafiltered homogenates of
submaxillary gland tissue from infected adult pigs through a Berkfeld N filter and inoculated the filtrate into inclusion-free guinea pigs. Subsequently, inclusions were observed
in the inoculated animals guinea pig cytomegalovirus. Murine cytomegalovirus was first isolated in cell culture in 1954 by Margaret Smith from salivary gland tissues of
infected laboratory mice. This followed demonstration in 1934-36 by Anne Kuttner and Shao-Hsun Wang that the etiological agent of intranuclear inclusion bodies in murine
tissues could be transmitted by the inoculation of (unfiltered) tissue homogenates into healthy mice.
Smith MG. Propagation of salivary gland virus of mouse in tissue cultures. Proc Soc Exp Biol Med. 1954;86:435-440.
Jesionek A, Kiolemenoglou B. Uber einen befund von protozoenartigen gebilden in den organen eines hereditar-luetischen foetus. Muenchner Med Wochenschr.
1904;51:1905-1907.

1954 Margaret Smith discovery of murine cytomegalovirus

page 269

Sigurdssons Institute for Experimental Pathology, Iceland

Bjrn Sigurdsson (1913-1959)

During the 1930s a flock of Karakul sheep was imported into Iceland from Germany. The intention was to start a lamb fleece industry. This flock, although apparently healthy
while kept in quarantine for several months, brought several diseases to the previously isolated Icelandic native breed of sheep. These included diseases caused by maedivisna virus and the scrapie prion. Maedi is a slowly progressive interstitial pneumonia (adenomatosis) of adult sheep, whereas visna is a slowly progressive encephalomyelitis
caused by the same lentivirus. Owing to Icelands sheep farming practices, using vast common summer pastures, these diseases spread widely causing enormous losses.
Annual mortality often reached 20-30%. In the face of these diseases, the Government, with financial aid from the Rockefeller Foundation, built the Institute for Experimental
Pathology, at Keldur, and made it a unit of the medical faculty of the University of Iceland. The director was Bjrn Sigurdsson, who had been a fellow at the Rockefeller
Institute, Princeton, New Jersey from 1941 until 1943. Sigurdsson isolated maedi-visna virus and worked on other diseases with long incubation periods and protracted
courses of illness, affecting largely the central nervous system and/or the lymphoreticular system, usually culminating in death. In 1954, Sigurdsson coined the term slow virus
infections to bring attention to this poorly defined group of diseases, which were eventually found to be caused by several quite different viruses and prions.
Sigurdsson B. Rida, a chronic encephalitis of sheep: with general remarks on infections which develop slowly and some of their special characteristics.
Br Vet J. 1954;110:341-354.

1954 Bjrn Sigurdsson development of the concept of slow viruses (e.g., maedi-visna virus, scrapie prion)
page 270

The first microtiter system was made in Budapest in 1951 by Gyula Taktsy; it consisted of (1) a plate with small wells drilled
into it, (2) micro-droppers, and (3) coiled wire loops for making dilutions. He machined by hand 6 rows of 12 drilled wells,
each ~5mm in diameter and 10mm deep, in a Lucite block he soon changed this to 8 x 12 wells the ubiquitous 96-well
plate. His original loop for making dilutions looked like the platinum loop used everywhere in bacteriology labs, but the
end of the wire was wound into a loose ball so that capillary action would draw up a constant volume into the interstices
of the coiled wire. Calibrated loops and droppers enabled him to perform dilution series in the wells of the block. Serial
dilutions were made by swizzling the loops in successive wells of the plates. Holding eight or twelve of the loops in one
hand, he was able to complete large numbers of serial dilutions at one time. The wire loops were quickly replaced by thin
steel knitting needles with a cut-out cup on the end that could be flame-sterilized. The system took years to make it to the
west, not because of patent protection, but because Taktsys publications went unnoticed. It wasnt until John Sever, at
the NIH, found Taktsys 1955 paper that the system was introduced internationally. Sever wanted to do large-scale viral
disease serologic surveys, but the method of transferring reagents and making dilutions, using small Cornwall syringes,
was very slow. He acquired a Taktsy device through a dealer in Budapest and Frank Cooke (Cooke Engineering, now
Dynex Technologies) improved it to serve Severs needs. They developed commercially improved loops and droppers and
injection-molded 96-well (polystyrene) plates. A tape dispenser was made to seal the plates, an especially valuable addition
for micro-neutralization tests. The plates were read from the bottom, using a concave shaving mirror mounted in a frame.
The microtiter system was a great success in virology and serology, for example for hemaglutination-inhibition assays,
complement-fixation assays, cell culture neutralization assays, etc., where very large numbers of tests were run as part of
large serosurveys. The first automated liquid handling device for automating serial dilutions, the Autotiter, was developed
in 1967 by Astec Inc. (now Tomtec Inc.). The first microplate readers evolved into the Multiskan photometer and later by
various spectrophotometric readers. By 1990 there were more than 15 companies producing a wide range of microplate
systems with different features. Today, it is estimated that 125 million microplates are used annually.

Gyula Taktsy (1914-1980)

Taktsy G. The use of spiral loops in serological and virological micromethods. Acta Microbiologica et Immunologica
Hungarica 1955;3:191-202. [State Institute of Public Health, Budapest, Hungary]
Sever J. Major technological advances affecting clinical and diagnostic immunology. Clin Diag Lab Immunol. 1997;4:1-3.

Taktsys second dilution loop

Cookes modification of dilution loop
96-well plate reader
Holding 8 dilution loops in each hand!
Virus titration in 96-well plate

1955 Gyula Taktsy invention of the microtiter system for virologic and serologic assays

page 271

1967

George K. Hirst (1910-1994)

1955

Peter Wildy (1920-1987)

1967

Lloyd Kozloff (1924-2012), Robert Wagner (1923-2001), and Norman Salzman (1926-1997)

1955> George Hirst, Peter Wildy, Lloyd Kozloff, Robert Wagner, Norman Salzman three major virology journals
page 272

Richard Moreland Taylor (1887-1981)

Richard Taylor bleeding Dinka

people, Sudan, 1955

Telford H. Work (1921-1995) and Herbert

Hurlbut (1908-2001), bleeding a hooded
crow (Corvus corone), Egypt, 1955

Richard Moreland Taylor and young scientists Herbert

Hurlbutt and Telford H. Work, working under the auspices
of the Rockefeller Foundation at NAMRU-3, Cairo, Egypt
in 1954-55, were evaluating yellow fever endemicity
following a severe outbreak which struck secluded Nubian
populations in preceding years. In the course of this work
they discovered Sindbis virus.
Taylor RM, Hurlbut HS, Work TH, Kingston JR,
Frothingham TE. Sindbis virus: a newly recognized
arthropod-transmitted virus. Am J Trop Med Hyg.
1955;4:844-862. [Departments of Virology and Entomology,
U. S. Naval Medical Research Unit No. 3, Cairo, Egypt.]

Richard Moreland Taylor (1887-1981)

1955 Richard Taylor, Herbert Hurlbut, Telford Work, others discovery of Sindbis virus

page 273

Heinz Fraenkel-Conrat (1910-1999)

Robley Cook Williams (1908-1995)

Fraenkel-Conrat H, Williams RC. Reconstitution of active tobacco mosaic virus from its inactive protein and nucleic acid
components. Proc Natl Acad Sci USA. 1955;41:690-698. [from the Virus Laboratory, University of California Berkeley.]
Summary. The preparation from TMV of protein and RNA fractions, which tend to recombine to form a nucleoprotein
carrying virus activity, is described. An electron microscopic search revealed no TMV rods in either of the two component
solutions at a concentration level thirty fold to three hundred fold greater than those at which the reconstituted virus was
assayed. In the reconstituted virus, on the other hand, up to about one-third of the material consisted of rods of the typical
diameter and length of TMV, many, if not all, containing a nucleic acid core.The evidence thus seems reasonably complete
that, under the conditions described, TMV nucleic acid enters into combination with TMV protein subunits and favors
aggregation to rods, some of which are of sufficient length and structural integration to carry infectivity.

Tobacco mosaic virus

from minimum-exposure study

by Robley Williams, 1974
negative contrast electron microscopy

1955 Heinz Fraenkel-Conrat, Robley Williams reconstitution of tobacco mosaic virus from its protein & nucleic acid

page 274

Seymour Benzer (1921-2007)

In 1954, Seymour Benzer began mapping the fine structure of a region of the genome of
bacteriophage T4; by the time he finished his work in 1961 he had developed the neoclassical
concept of the gene i.e., a discrete chromosomal region which (1) is responsible for a specific
cellular product, and (2) consists of a linear collection of potentially mutable units, each of which
can exist in several alternative forms and between which crossing over can occur. Benzer called
this unit a cistron, basing his definition on a criterion known as the cis-trans test for a functional
unit of the genome. In the diagram of the cis-trans complementation test, m1 and m2 are
fictional alleles representing simple recessive mutations for years this was the way one told if
two recessive genes were part of the same gene or on two different genes. The test originated
with Edward B, Lewis who at Caltech in 1942 presented the concept as his PhD thesis. [Lewis
was awarded a Nobel Prize in 1995 for discovering the group of nearly identical master control
genes that orchestrate the development of body parts in all animals.]
Today, many different definitions of the gene are in use, all deriving from our evolving
understanding of molecular genetics. Maxine Singer and Paul Berg suggested the following
popular definition: A eukaryotic gene is a combination of DNA segments that together constitute
an expressible unit. Expression leads to the formation of one or more specific functional gene
products that may be either RNA molecules or polypeptides. Each gene includes one or more
DNA segments that regulate the transcription of the gene and thus its expression. Thus, a gene
includes: [1] a transcription unit, which includes the coding sequences, introns, leader and
trailer flanking sequences, and [2] regulatory sequences, which flank the transcription unit and
which are necessary for its specific function. As usual, virologists have been left to extend such
definitions to the RNA virus world.
Benzer S. The elementary units of heredity. In: McElroy WD, Glass B, editors. The chemical
basis of heredity. Baltimore: John Hopkins University Press; 1957.
Singer M, Berg P. Genes and genomes: a changing perspective. Mill Valley: University Science
Books; 1991.

Paul Berg and Maxine Singer

1955> Seymour Benzer, Paul Berg, Maxine Singer definition of a gene (cis-trans test, molecular genetic definition)

page 275

Zvonimir Dinter
(1914-1990)

Werner Schfer (1912-2000)

Clinical signs of fowl plague include:

sudden death, labored breathing and
coughing, nasal and ocular discharge,
swollen face, cyanosis/necrosis of
comb and wattles, paralysis, paresis.

A number of orthomyxoviruses morphologically indistinguishable from influenza virus and possessing the
ribonucleoprotein antigen common to influenza A viruses were isolated early on from different species of birds. The first
virus to fall into this category was classical fowl plague virus, discovered in 1909 but not identified as an influenza A virus
until 1955, by Werner Schfer. Another virus, related to but not identical to, fowl plague virus was described by Zvonimir
Dinter in 1949 and called virus N it was identified as an influenza A virus by Rudolph Rott and Werner Schfer in 1960.
Schfer W. Vergleichende sero-immunologische untersuchungen ber die viren der influenza und klassischen geflgelpest.
Z Naturforsch. 1955;10b:81-91.
Dinter Z. Eine variante des virus der geflgelpest in Bayern. Tierrztl Umschau 1949;4:185-186.
Rott R, Schfer W. Physikalisch-chemische und biologische eigenschaften des virus N und seine beziehungen zur
influenza-a-untergruppe der myxoviren. Zbl Vet-Med. 1960;B7:237-248.

1955> Werner Schfer, Svonimir Dinter discovery that fowl plague virus is an influenza virus, likely zoonotic
page 276

Respiratory syncytial virus infection in cell culture

showing typical multinucleated syncytium formation
multiple fluorescent probes, confocal microscopy

Respiratory syncytial virus was first recovered in 1956 by J. Anthony Morris and colleagues
from chimpanzees during an outbreak of coryza in a colony at the Walter Reed Army
Institute of Research. In 1956, Robert Chanock and colleagues recovered the same virus
from an infant with pneumonia and from another infant with croup. Subsequently, the
virus, respiratory syncytial virus, has been found to be responsible for a considerable
proportion of the severe respiratory illness afflicting infants and small children. Chanock
wrote in 1961, It is clear that an RS virus vaccine, whether live attenuated or inactivated,
should receive a very high priority in any future plans for immunoprophylaxis of pediatric
respiratory disease.

Robert Merritt Chanock (1924-2010)

Blount RE Jr, Morris JA, Savage RE. Recovery of cytopathogenic agent from chimpanzees
with coryza. Proc Soc Exp Biol Med. 1956;92:544-549.
Chanock R, Roizman B, Myers R. Recovery from infants with respiratory illness of a virus
related to chimpanzee coryza agent (CCA). I. Isolation, properties and characterization.
Am J Hyg. 1957;66:281-290.
Johnson KM, Chanock RM, Cook MK, Huebner RJ. Studies of a new human hemadsorption
virus. I. Isolation, properties and characterization. Am J Hyg. 1960;71:81-92.

Bovine respiratory syncytial virus, calf, 4 days postinfection,

infection of bronchial epithelium, immunohistochemistry

1956 J. Anthony Morris, Robert Chanock, others discovery of respiratory syncytial virus (the first pneumovirus)

page 277

William J. Mogabgab (1921-2001)

The first isolations of rhinoviruses were reported

in 1956 by William Pelon, William Mogabgab and
Winston Price, who had noted the growth in monkey
kidney cells of ultrafilterable agents associated
with mild respiratory disease. Their viruses had
many similarities, but they were antigenically
distinct. Serological evidence indicated widespread
human infection, and a causal relationship with
respiratory disease. Early studies were hampered by
the insensitivity and variability of the tissue culture
systems used, but by 1958 the use of roller drums,
rather than stationary racks of cell culture tubes, and
of second generation monkey kidney cells had added
significantly to the sensitivity of the isolation system.
Sensitivity was soon improved further by the use of
WI-38 diploid human embryonic lung cells. By 1965,
an international workshop was held to organize the
classification and nomenclature of the very many
similar but distinct viruses being isolated around the
world, and in 1967 the numerical nomenclature system
was published in Nature. The signatories of this paper
were: A.Z. Kapikian (Chairman), R.M. Conant, V.V.
Hamparian, R.M. Chanock, P.J. Chapple, E.C. Dick,
J.D. Fenters, J.M. Gwaltney, Jr., D. Hamre, J.C. Holper,
W.S. Jordan, Jr., E.H. Lennette, J.L. Melnick, W.J.
Mogabgab, M.A. Mufson, C.A. Phillips, J.H. Schieble,
and D.A.J. Tyrrell.

Phylogenetic
tree
depicting the
relationships
among 101
human
rhinovirus
serotypes
(P1-P2 region
of 5NCR)

Price WH. The isolation of

a new virus associated with
respiratory clinical disease
in humans. Proc Natl Acad
Sci USA. 1956;42:892-896.
Mogabgab WJ, Pelon W.
Problems in characterizing
and identifying an
apparently new virus
found in association with
mild respiratory disease in
recruits. Ann NY Acad Sci.
1957;67:403-412.
Consortium. Rhinoviruses:
a numbering system. Nature
1967;213:761-763.

Winston H. Price (1924-1981)

William S. Pelon (1926-2005)

and Albert Sabin

Model of human rhinovirus 3,

from H. Adam Steinberg and
Jean-Yves Sgro, University of
Wisconsin-Madison

1956 Winston Price, William Mogabgab, William Pelon discovery of human rhinoviruses, numerical nomenclature
page 278

Alfred Gierer

Gerhard Schramm
(1910-1969)

Heinz Fraenkel-Conrat
(1910-1999)

Beatrice A. Singer
(1923-2005)

Until about 1948, most attention was focused on the proteins in viruses as the likely stuff of inheritance. Although it was
known that enzymes were proteins, the function of the nucleic acids was not known. Then, in 1949, Roy Markham and
Kenneth Smith showed that a plant virus, turnip yellow mosaic virus, contained two classes of particles, one infectious and
containing a nucleoprotein, the other non-infectious and containing an identical protein but no nucleic acid (in this case
RNA). This suggested that the viral RNA was essential for infectivity. This pioneering work was done just after Oswald Avery,
Colin MacLeod, and Maclyn McCarty demonstrated that DNA was the bacterial transforming factor (1944) and just before
the Hershey-Chase experiment (1952). However, doubts as to whether the RNA in viruses was really the genetic material
persisted. Heinz Fraenkel-Conrat and Robley Williams showed in 1955 that upon incubating a solution containing a mixture
of a non-infectious disassembled tobacco mosaic virus (TMV) protein and purified, apparently non-infectious, viral RNA,
normal-looking infectious TMV particles were formed. This experiment had a big impact because it appeared that a living
molecule had been produced in vitro from non-living constituents. The next year, 1956, the pioneering experiments of
Alfred Gierer and Gerhard Schramm showed that TMV RNA alone was capable of initiating virus replication; the same result
was obtained at the same time by Heinz Fraenkel-Conrat and his wife Beatrice Singer. Thus it became clear that the infectivity
observed a year before in Fraenkel-Conrats TMV reconstitution experiment was a result of the infectivity of TMV RNA.
Fraenkel-Conrat and Singer then did reconstitution experiments using different host range variants to provide protein and
RNA: reconstituted viruses had the host range specificity characteristic of the virus from which the RNA was obtained, not
the protein.

Gerhard Schramm (center)

observing sedimentation of a protein,

Schlieren optics camera, air-driven
ultracentrifuge

Gierer A, Schramm G. Infectivity of ribonucleic acid from tobacco mosaic virus. Nature 1956;177:702-703.
Fraenkel-Conrat H, Singer B. Virus reconstitution and the proof of the existence of genomic RNA. Phil Trans R Soc Lond B
1999;354:583-586. (historical review)

1956 Alfred Gierer, Heinz Fraenkel-Conrat, others discovery of the infectivity of viral RNA (tobacco mosaic virus)

page 279

Stewart Harvey Madin

(1918-2002)

Charles J. York
(1920-2000)

Delbert Grant McKercher

(1914-1988)

The first report on infectious bovine rhinotracheitis

appeared in 1954; it was described as an apparently new
upper respiratory disease of dairy cattle in California. It
appeared suddenly and was characterized by high fever
and agalactia in addition to respiratory signs. The following
year, a similar disease was described in Colorado feedlots
and dairies. Delbert McKercher showed by comparative
serology that the respiratory disease found in California
dairy and beef cattle and in Colorado beef cattle were the
same. Stewart Madin, Charles York and Delbert McKercher
isolated the causative agent from nasal washing from
clinically affected cattle: infectious bovine rhinotracheitis
virus, now also called bovine herpesvirus 1 (BHV-1). In
1961, J.A. Armstrong, Helio Pereira and Christopher
Andrewes showed that the virus is a herpesvirus.

School of Veterinary Medicine, University of California Davis, 1961

Madin SH, York CJ, McKercher DG. Isolation of

the infectious bovine rhinotracheitis virus. Science
1956;124:721-722.

1956 Stewart Madin, Charles York, Delbert McKercher discovery of infectious bovine rhinotracheitis virus
page 280

Chikungunya disease was first described by Marion Robinson and William Hepburn Russell
Lumsden in 1955, following an outbreak in 1952 on the Makonde Plateau, along the border between
Mozambique and Tanganyika (today Tanzania). According to Marion Robinsons initial 1955 report
on the epidemiology of the disease, the term chikungunya is derived from the Makonde root verb
kungunyala, meaning to dry up or become contorted, in reference to the stooped posture developed
as a result of the arthritic clinical manifestations of the disease. Subsequent authors apparently
overlooked the references to the Makonde language and assumed that the term derived from
Swahili, the lingua franca of the region the result has been many erroneous interpretations of the
name.
From Lumsden: The epidemic to be described here was first recognized in 1952 by Dr. Marion
C. Robinson of the Lulindi and Newala Hospitals of the Universities Mission to Central Africa.
The occurrence of the epidemic was notified to the Virus Research Institute [Entebbe] by the
Director of Medical Services, Tanganyika, in February, 1953, and a team was sent by air to attempt
virus isolation and to carry out general epidemiological studies. Dr. R. W. Ross and I arrived in
Newala on 21 February, 1953, and began immediately the collection of sera from acute cases of the
disease and the catching of biting insects from which to attempt virus isolation The description
of the epidemic falls naturally into three parts, the clinical features of the disease (Dr. Marion C.
Robinson), the general description of the environment and the epidemiology (Dr. W.H.R. Lumsden),
and the characteristics of the viruses concerned (R. W. Ross) and it seems convenient to record the
results in this form.
Robinson MC. An epidemic of virus disease in Southern Province, Tanganyika territory, in 19521953. I. Clinical features. Transactions of the Royal Society of Tropical Medicine and Hygiene
1955;49:28-32.
Lumsden WHR. An epidemic of virus disease in Southern Province, Tanganyika territory, in
1952-1953. II. General description and epidemiology. Transactions of the Royal Society of Tropical
Medicine and Hygiene 1955;49:33-57.
Ross RW. The Newala epidemic. III. The
virus: isolation, pathogenic properties
and relationship to the epidemic. J Hyg
(Lond). 1956;54:177-191.
[R. William Ross, at the time a staff
member of the Rockefeller Foundation
sponsored Virus Research Institute at
Entebbe, Uganda, later spent many years
as a faculty member of the Department
of Pathology, University of Cambridge.
Despite much searching, no photo of him
has been found.]

William Hepburn Russell

Lumsden (1914-2002)
1956 R.William Ross, Marion Robinson, W.H.R. (William) Lumsden discovery of chikungunya virus

page 281

Phylogenetic tree of the family Paramyxoviridae,

showing the human parainfluenza viruses (HPIVs)

Robert Merritt Chanock (1924-2010)

The human parainfluenza viruses (HPIVs) were discovered by Robert Chanock and his colleagues between
1956 and 1960. HPIV1, 2 and 3 were first isolated from infants and children with lower respiratory tract
disease and HPIV4a and b from children and young adults with minor upper respiratory tract infections.
Soon afterward, the viruses were shown to be major causes of croup (HPIV1, 2,3) and pneumonia and
bronchiolitis (HPIV3). As a group these viruses are second only to respiratory syncytial virus as the cause of
severe, sometimes fatal respiratory tract disease in infants and children.
Chanock RM, Parrott RH, Cook K, Andrews BE, Bell JA, Reichelderfer T, Kapikian AZ, Mastrota FM,
Huebner RJ. Newly recognized myxoviruses from children with respiratory disease. N Engl J Med.
1958;258:207-213.
Chanock RM, Johnson KM. Infectious disease: respiratory viruses. Annu Rev Med. 1961;12:1-18.

1956 Robert Chanock, others discovery of human parainfluenza viruses

page 282

Parainfluenza virus 4a

Thin section and negative contrast electron microscopy

Margaret Gladys Smith

(1896-1970)

Thomas Huckle Weller

(1915-2008)

Wallace P. Rowe
(1926-1983)

Human CMV, kidney, H&E

Human cytomegalovirus (CMV) was isolated in cell culture only after the development of the technology to culture human cells by
John Enders, Thomas Weller and Frederick Robbins. In 1955, Margaret Smith isolated human CMV from the salivary gland of a patient
at autopsy; the virus grew in human but not in mouse cell culture. Her paper was rejected because she was also working with murine
CMV and the editor thought she might have had a contaminant. It was only in 1956 when she reisolated the virus and isolated the same
virus from the kidney of another patient that her paper was accepted. In 1957, Weller, working on toxoplasma in human embryonic cell
culture, isolated a virus from an infant suspected of having congenital toxoplasmosis, and Wallace Rowe, working on respiratory viruses,
isolated a virus from adenoid tissue of a patient suspected of having varicella. Rowe took his material to Wellers lab for confirmation,
where it was found to be similar to the virus Weller had isolated. The three independently isolated viruses were exchanged among the
three groups, and their similarity was established in advance of publication indeed, a commendable example of cooperation. Weller
named the virus cytomegalovirus. [Ho M. The history of cytomegalovirus and its diseases. Med Microbiol Immunol. 2008;197:65-73.]
Smith MG. Propagation in tissue cultures of a cytopathogenic virus from human salivary gland virus (SVG) disease. Proc Soc Exp Biol
Med. 1956;92:424-430.
Rowe WP, Hartley JW, Waterman S, Turner HC, Huebner RJ. Cytopathogenic agent resembling human salivary gland virus recovered
from tissue cultures of human adenoids. Pro Soc Exp Biol Med. 1956;92:418-424.

Wellers image of human CMV

inclusions in cell culture, 1956

Weller TH, Macauley JC, Craig JM, Wirth P. Isolation of intranuclear inclusion producing agents from infants with illnesses resembling
cytomegalic inclusion disease. Proc Soc Exp Biol Med. 1957;94:4-12.

1956 Margaret Smith, Thomas Weller, Wallace Rowe discovery of human cytomegalovirus

page 283

Aleutian disease: lung, mink, multisystemic immunologically

mediated lymphoplasmacytic perivascular and diffuse lesions

John R. Gorham (1922-2011)

James B. Henson

Aleutian disease virus of mink was first recognized in 1956 by John Gorham and
G. R. Hartsough in ranch-raised mink (Mustela vison) carrying the Aleutian coat
color gene, a gene which produces a highly desired gun-metal grey pelt. In the
1960s, it was common practice for mink ranchers to make their own vaccine against
canine distemper virus, an important pathogen of mink, by emulsifying tissue from
distemper-infected mink and formalizing it. This practice led to severe outbreaks
of Aleutian disease and extensive virus spread (Aleutian disease virus, a parvovirus,
is more resistant to formalin than canine distemper virus). In adult mink, Aleutian
disease is a persistent, slowly progressive lethal infection, marked by high levels
of antiviral antibodies and hypergammaglobulinemia. However, the virus is not
neutralized and severe immune complex glomerulonephritis and vasculitis develop.
Massive accumulations of macrophages and plasma cells occur in many organs and
tissues. Antibody-coated virions infect macrophages leading to antibody-dependent
enhanced disease. Because of this exceptional immunopathology, leaders in viral
disease pathogenesis research did extensive work on the disease throughout the 1960s
and 1970s. Included in this were research groups at the Rockefeller Institute led by
Robert Leader (1919-1996) and the Scripps Research Institute led by Frank Dixon
(1921-2008). At the same time, research continued at Washington State University led
by John Gorham and James Henson.
Hartsough GR, Gorham JR. Aleutian disease in mink. Nat Fur News 1956;28:10-11.

Frank Dixon (1921-2008)

Robert Leader (1919-1996)

Henson JB, Gorham JR, Leader, RW, Wagner, BM. Experimental

hypergammaglobulinemia in mink. J Exp Med. 1962; 116:357-364.

1956 John Gorham, James Henson, Robert Leader, Frank Dixon discovery of Aleutian disease of mink virus

page 284

Niels Kaj Jerne (1911-1994)

Frank Macfarlane Burnet (1899-1985)

Early theories of immunity explained the large repertoire of

antibody specificities with the notion that antigen somehow acts
as a template to transfer information to the globulin-producing
machinery in certain cells. This was termed an instruction theory;
another theory, termed a selective theory, postulated that antigen
exposure induces the elaboration of a sample of antibodies from a
range of pre-existing specificities. In 1955, Niels Jerne suggested
that the information for all antibody specificities is inherent in the
host, and that the sole function of antigen is to select the matching
antibody and transport it to the appropriate cells where, somehow,
they would be stimulated to produce more of the same. Jernes
idea attracted little support at the time; its surviving claim-tofame lies in the fact that David Talmage and Frank Macfarlane
Burnet independently saw in its selectionist approach the seed of
an interesting idea. In 1957, Talmage suggested, it is tempting to
consider that one of the multiplying units in the antibody response
is the cell itself only those cells are selected for multiplication
whose synthesized product has affinity for the antigen injected.
Burnet, having also read Jernes paper, and pondering heavily on
it, asked why it was so attractive, though obviously wrong. By
replacing molecules with cell clones and their membrane receptors,
Burnet said the whole picture fell into shape. In his 1957 paper,
Burnet assumed that a large repertoire of lymphoid cell clones were
precommitted to producing a similarly large repertoire of antibody
specificities. As he put it, ... antibody production is a function not
only of the cells originally stimulated, but of their descendants.
When an antigen enters the body, a number of cell clones that
happen to produce antibodies against that antigen are selected for
and multiply into much larger clones of cells, producing antibody
molecules specific against the intruding antigen. Although it
encountered some skepticism in the beginning, the clonal selection
theory rapidly gathered support, and within less than a decade the
immunological community switched from believing in templates
to believing in selection. Since then, clonal selection has been the
explicit theoretical foundation of immunology.
Jerne NK. The natural-selection theory of antibody formation. Proc
Nat Acad Sci USA. 1955;41:849-857.
Talmage DW. Allergy and immunology. Annu Rev Med.
1957;8:239-256.
Burnet FM. A modification of Jernes theory of antibody production
using the concept of clonal selection. Aust J Sci. 1957;20:67-69.

David W. Talmage (1919-2014)

Burnets clonal selection diagram

1957 Niels Jerne, David Talmage, Frank Macfarlane Burnet clonal selection as the central mechanism in immunity

page 285

Confocal microscopy offers advantages over conventional optical microscopy, including the ability to control
depth of field, to eliminate background information away from the focal plane, and to collect serial optical sections
(tomographic sections) from thick specimens. It uses filtering techniques to eliminate out-of-focus light or glare
in specimens whose thickness exceeds the immediate plane of focus. Current instruments are highly evolved from
the earliest versions, but the principle of confocal imaging advanced by Marvin Minsky, and patented in 1957, is
employed in all modern confocal microscopes. Today, image formation in a confocal microscope is achieved by
scanning one or more focused beams of light, usually from a laser, across the specimen, which is usually labeled
with one or more fluorescent probes. The pinhole point of illumination is brought to focus in the specimen by the
objective lens and a sequence of points of light from the specimen are built up by scanning the specimen and sending
the light signal to a photomultiplier tube and from there to a computer.

Marvin Lee Minsky

Marvin Minsky, from the Massachusetts Institute of Technology, was a leader in the development of artificial
intelligence concepts and technologies, and only worked on confocal microscopy on the side: he only published his
invention as a patent. Yet he not only built a prototype microscope and made it work, but he also tried to see that it
was applied however, as he said, I demonstrated the confocal microscope to many visitors, but they never seemed
very much impressed with what they saw on that radar screen [he used a surplus radar screen as the photomultiplier
tube]. Only later did I realize that it is not enough for an instrument merely to have a high resolving power; one must
also make the image look sharp. Perhaps the human brain requires a certain degree of foveal compression in order to
engage its foremost visual abilities. In any case, I should have used film - or at least have installed a smaller screen! It
took many years for confocal microscopy to catch on, but when it did there was an explosion in its use, due in part to
the relative ease with which extremely high-quality images can be obtained from specimens usually prepared in the
same way as they would be for conventional fluorescence microscopy. In fact, confocal technology is proving to be
one of the most important advances ever achieved in optical microscopy.

Sagittal section of rat cerebellum, confocal microscopy, multiple fluorescent probes

1957 Marvin Minsky invention of the confocal microscope

page 286

Alick Isaacs (1921-1967)

Jean Lindenmann

In 1956, Alick Isaacs was investigating influenza

viruses at the National Institute for Medical Research
(NIMR), Mill Hill, when he had a cup of tea with Jean
Lindenmann, who had just arrived as a postdoc from
Switzerland. They began investigating the mechanism
of viral interference, that is, the capacity of cells
infected with one virus to resist infection by another.
To study the matter they set up a simple system:
pieces of chick embyro chorio-allantoic membrane
were placed in culture medium in test tubes, treated
with heated virus (later ultraviolet light inactivated
virus) and rotated in a roller drum for 24 hours. The
membranes were then removed, washed, and put
into tubes and challenged with fresh virus. The
fluids in these tubes were tested for the presence
of viral hemagglutinin as proof whether virus was
replicating or not. True to form, the first virus
seemed to be thwarting the growth of the second.
They wrote, It was surprising to find that after 24
hours considerable interfering activity remained in
the surrounding fluid after the treated membranes
were removed and new membranes added. So the
possibility was considered that fresh interfering
activity was produced by the membranes. The active
substance was christened interferon (a hybrid of
interference and the suffix on, which was in vogue
at the time), and it was concluded that establishment
of interference was accompanied by the liberation
of interferon. The initial discovery was recorded in
Isaacs lab notebook under the entry: In search of an
interferon. Lindenmann took it all in stride, saying,
I thought it quite natural that when you did research
you discovered things. As stated by Christopher
Andrewes, who was director of the NIMR at the time,
The paper, in the Proceedings of the Royal Society
reporting this discovery must be accounted one of the
outstanding landmarks in biological science The
term breakthrough is one of those most abused and
overworked especially in popular accounts of science,
but it can justly be used concerning the discovery of
interferon.
Isaacs A, Lindenmann J. Virus interference. I. The
interferon. Proc Roy Soc London Set B 1957;147:258273.

1957 Alick Isaacs, Jean Lindenmann discovery of interferons

page 287

Telford Hindley Work (1921-1995)

Telford Work and Pushpa

Kyasanur Forest disease virus, the etiologic agent of a hemorrhagic disease in

humans (mortality rate ~10%), was first isolated in the Shimoga district of Karnataka
state (then Mysore) of India in 1957. The virus commonly infects the black faced
langur (Presbytis entellus, from which the first virus isolate was made) and red faced
bonnet monkey (Macaca radiata); it is transmitted between monkeys by the bite
of the tick, Haemaphysalis spinigera, especially in its nymphal stage. The virus also
circulates in rodents, shrews and birds. The cause of the sudden emergence of the
virus in India in the late 1950s and its subsequent localization to the Karnataka state
alone are not clear. It has been argued that the virus and other hemorrhagic and
encephalitic tick-borne viruses almost certainly exist in other parts of India and in
the region in general, but their presence has not yet been recognized. Phylogenetic
analyses of the virus and other tick-borne flaviviruses suggest that they evolve
gradually, whereas mosquito borne viruses evolve more rapidly and become more
genetically and ecologically divergent in the course of their evolution. The original
paper on the virus and disease by Telford Work, Pravin Bhatt, F.R. Roderiguez and
their colleagues reminds us of the classic terse style of ecologic and epidemiologic
description of the era:
Work TH, Roderiguez FR, Bhatt PN. Virological epidemiology of the 1958 epidemic
of Kyasanur Forest disease. Am J Public Health Nations Health 1959;49:869-874.

Pravin Bhatt, bleeding a subject, 1959

1957 Telford Work, F.R. Rodriguez, Pravin Bhatt discovery of Kyasanur Forest disease virus
page 288

Equine viral arteritis is a contagious disease of horses caused by equine arteritis

virus (EAV). The disease was first described about 200 years ago. Horsemen also
recognized long ago that otherwise healthy stallions could transmit the disease to
susceptible mares at breeding, and that these carrier stallions could be a source of
infection for many years. The virus was fist isolated by Elvis Doll, John Bryans, William
McCollum and M.E.W. Crow during an outbreak of respiratory disease and abortion
on a breeding farm in Bucyrus, Ohio in 1953. The infection of equine species (horses,
donkeys, and mules) occurs throughout the world, although its incidence varies
markedly between countries and among horses of different breeds. The vast majority
of infections are inapparent or subclinical, but outbreaks occur that are characterized
by any combination of influenza-like illness in adult horses, abortion in pregnant
mares, and fatal interstitial pneumonia in young foals. The global dissemination of
the virus and the rising incidence of the disease likely reflect the incredible amount of
international movement of horses for competition and breeding,

Elvis Roger Doll

(1912-1967)

John T. Bryans
(1924-2004)

Doll ER, Bryans JT, McCollum WH, Crowe MEW. Isolation of a filterable agent
causing arteritis of horses and abortion by mares. Its differentiation from the equine
abortion (influenza) virus. Cornell Vet. 1957;47:3-41.

1957 Elvis Doll, John Bryans, William McCollum discovery of equine arteritis virus (the first arterivirus)

page 289

James R. Gillespie
Alan O. Betts (1927-2005)
Kyu M. Lee
Cell culture clean room
Veterinary Virus
Research Institute
(now Baker Institute
for Animal Health)
Cornell University
1956

James R. Gillespie (1918-2011)

The complex pathogenesis of bovine

viral diarrhea virus infection and
mucosal disease

Bovine viral diarrhea is a very important disease of cattle, worldwide. When it was first identified and studied,
however, by James Gillespie and Kyu M. Lee at Cornell University, A.B. Hoerlein and his colleagues at Colorado State
University, and Albert L. Fernelius at what is now the National Animal Disease Center in Ames Iowa, only the most
severe forms of the disease were recognized most dramatically, fatal mucosal disease. As is now known, bovine
viral diarrhea (BVD) virus infection may be inapparent or may cause severe disease, with abortion, infertility, and
immunosuppression that underlies calf respiratory and enteric diseases. BVD virus causes two patterns of infection: (1)
infection of a seronegative animal usually causes no clinical illness the virus is eliminated by the humoral and cellular
immune response and the animal is protected against further infection. (2) In contrast, infection of the dam between
the second and fourth month of gestation often leads to persistent infection of the fetus the fetus is immunotolerant
to the infecting BVD virus strain and at birth is antibody negative. The calf sheds large amounts of virus during
its entire lifetime [Infection later in gestation may lead to abortion, malformation, or to the birth of a normal,
immunocompetent calf ]. If the persisting virus mutates into a cytopathogenic virus biotype or if the persistently
infected animal is superinfected with an antigenically related biotype, it will succumb to lethal mucosal disease. The
virus is maintained in nature by the small population of animals that become immunotolerant and persistently infected;
these animals often die prematurely but some live a relatively normal life for several years, all the time shedding large
amounts of virus and serving as a very efficient virus reservoir.
Lee KM, Gillespie JH. Propagation of virus diarrhea virus of cattle in tissue culture. Am J Vet Res. 1957;18:952-953.
Underdahl NR, Grace OD, Hoerlein AB. Cultivation in tissue culture of cytopathogenic agent from bovine mucosal
disease. Proc Soc Biol Med. 1957;94:795.

1957 James Gillespie, Kyu M. Lee, Norman Underdahl, others discovery of bovine viral diarrhea virus
page 290

Matthew Meselson

Frank W. Stahl

Jerome Vinograd (1913-1976)

In 1957, Matthew Meselson and Frank Stahl were seeking a means to implement their idea for a bold experiment to verify the hypothesis that
DNA replication involved the separation of the two parental strands, one going into each of the two daughter DNA molecules see following
pages for this, what has been called the most beautiful experiment in history. They initially wanted to make the parental strands heavier than
normal by incorporation of 5-bromouracil and sought Jerome Vinograds advice as to whether 5-bromouracil-containing DNA could be separated
from normal DNA by velocity ultracentrifugation. Vinograd indicated that this seemed unlikely unless the difference could be magnified by
approximately matching the density of the DNA to the density of the salt solution in which the DNA was suspended. From this germinated
the concept of equilibrium sedimentation of macromolecules in density gradients. In equilibrium sedimentation methods the macromolecule
(e.g., DNA) is dissolved in a salt solution (e.g., CsCl) of the appropriate density and centrifuged to equilibrium (approximately 24 hours). At
equilibrium, the CsCl builds its own stable concentration (density) gradient, increasing in density with the radius. At equilibrium the suspended
DNA molecules form a narrow band at the CsCl density that is equivalent to their own density. Vinograds notion worked beautifully. In several
later papers they refined the theory and extended the application of their density gradient technique to other macromolecules: hemoglobin,
MS2 bacteriophage RNA, T7 bacteriophage DNA, X174 bacteriophage, and southern bean mosaic virus. They also found that the addition of
dimethylsulfoxide enhanced the ability to band RNA in CsCl without aggregation.
Meselson M, Stahl FW, Vinograd J. Equilibrium sedimentation of macromolecules in density gradients. Proc Natl Acad Sci USA. 1957;43:581-588.

1957 Matthew Meselson, Frank Stahl, Jerome Vinograd invention of isopycnic density gradient ultracentrifugation

page 291

Meselson M, Stahl FW. The replication of DNA

in Escherichia coli. Proc Natl Acad Sci USA.
1958;44:671-682. [original figure 4 legend below and
analytic ultracentrifuge Schlieren optics photographs
at right]
Ultraviolet absorption photographs showing DNA
bands resulting from density gradient centrifugation
of lysates of bacteria sampled at various times after
the addition of an excess of 14N substrates to a
growing 15N-labeled culture. Each photograph was
taken after 20 hours of centrifugation at 44,770 rpm.
The density of the CsCl solution increases to the
right. The degree of labeling of a species of DNA
corresponds to the relative position of its band
fully labeled and unlabeled DNA are shown in the
lowermost frame, which serves as a density reference.
The DNA in the band of intermediate density is just
half-labeled proof is provided by the frame showing
the mixture of generations 0 and 1.9 the peak of
intermediate density is centered at 50 (2) percent of
the distance between the 14N and 15N peaks.

For those who learned their molecular biology after the

Meselson-Stahl experiment (the vast majority of scientists living
today), it will be surprising to learn of the grave doubts about
the semiconservative mechanism for the replication of DNA
implied by the Watson-Crick paper of 1953. Even Max Delbrck
worried about the means of unwinding the strands of DNA
during replication. The result of the Meselson-Stahl experiment
more or less resolved the doubts about all this, leading to broad
support from other scientists for the Watson-Crick structure
and the semiconservative mechanism of the replication of DNA.

1958 Matthew Meselson, Frank Stahl discovery of the semi-conservative mode of replication of DNA
page 292

Howard Martin Temin

(1934-1994)

Harry Rubin

Despite the discoveries of viruses causing avian leukosis and sarcoma by Vilhelm
Ellerman, Oluf Bang and Peyton Rous in the first decade of the 20th century,
even by the middle 1950s the viral causation of cancer was not considered to
have any connection with cancer in general, and tumor viruses were studied
in only a few laboratories. Rous sarcoma virus (RSV), the first virus accepted
as a cause of cancer, was first assayed by its ability to form tumors in chickens
and then by its ability to form pocks on the chorioallantoic membrane of the
developing chicken embryo. All this changed in 1957 when Howard Temin,
a graduate student at Caltech, met Harry Rubin, a postdoctoral fellow. Rubin
told Temin about a paper he was refereeing for the journal Virology by Robert
Manaker and Vincent Group on morphological alterations in cells infected
with RSV. Rubin suggested that Temin try to make a quantitative tissue culture
assay based on these observations. Soon thereafter Temin switched from
embryology to virology and moved into the laboratory of Renato Dulbecco, who
with Marguerite Vogt had just developed the plaque assay for quantification
of cytolytic viruses. Temin immediately set out to develop a quantitative cell
culture assay for RSV under Rubins direction. The breakthrough came when
Temin decided not to plate chicken embryo cells at a density that would form a
monolayer, as was done for cell culture assays of cytolytic viruses, but to dilute
them to form a sparse cell layer. When RSV was applied to such sparse cell
layers and the cells were subsequently allowed to divide, foci of morphologically
transformed cells appeared in numbers proportional to the concentration of
the virus applied. In uninfected cell monolayers there is usually a phenomenon
call contact inhibitionwhen cells touch each other they stop dividing. When
cells are infected with RSV they lose their characteristic contact inhibition and
continue to proliferate, forming foci of massed rounded cells. This was the first in
vitro quantitative assay for neoplastic transformation. It established that RSV by
itself could transform cells. It was the birth of modern tumor virology.
One problem, however, was that the focus assay often did not work. Production
of foci seemed a hit-or-miss occurrence, without obvious cause. Eventually the
problem was linked to particular batches of chicken embryo cells; Harry Rubin
discovered that some embryos were infected by a virus that caused resistance
to RSV. This virus was called Rous-interfering virusor more commonly, RIF,
for resistance-inducing factor. RIF is an avian leukosis virus that is vertically
transmitted from the hen through the egg yolk to the chick. Testing embryos for
infection by RIF beforehand eliminated the problem.

RSV focus assay in chick embryo fibroblast monolayers as described by Temin and
Rubin in1958, showing a 1:100 and 1:1,000 dilution of the virus stock. Each stained
spot represents a focus of transformed cells

This assay was followed by similar cell culture assays for transformation by
polyoma virus and other DNA tumor viruses and then for transformation by
chemicals and irradiation. Such assays opened the way to the research that
defined reverse transcription, oncogenes, and proto-oncogenes and has shown
the fundamental connections between retroviral carcinogenesis and all other
carcinogenesis.
Temin HM, Rubin H. Characteristics of an assay for Rous sarcoma virus and
Rous sarcoma cells in tissue culture. Virology 1958;6:669-688. Citation Classic.

1958 Howard Temin, Harry Rubin focus assay for Rous sarcoma virus, the first assay for neoplastic transformation

page 293

Throughout the 1940s and 1950s everyone

dismissed the notion that viruses could cause cancer
that is, except for Sarah Stewart. She began her
career at NIH in 1936. When Bernice Eddy arrived
the following year the two became friends and
developed a long-running collaboration.
In 1944, Stewart asked for support for research
on the possible link between animal tumors and
viruses, but she was told she was not qualified. So,
she enrolled in Georgetown University School of
Medicine, graduating in 1949 (as the first woman to
receive an MD from that institution).
After an internship, she returned to the NIH, to the
National Cancer Institute, just as Ludwik Gross at
the Rockefeller Institute reported that some mouse
leukemias and parotid gland tumors were caused by
viruses.

Bernice Eddy (1903-1989)

Sarah Elizabeth Stewart (1906-1976)

In 1956, in Bernice Eddys laboratory, Stewart

injected tumor extracts from mice into other
laboratory animals and into cell cultures. Mouse
tumor material passaged in rhesus monkey cell
cultures and then inoculated into newborn mice and
mouse embryos caused 20 distinct types of tumors.
Tumors were also produced in hamsters, rabbits,
guinea pigs and rats. The virus, at Eddys suggestion,
was called polyoma virus.
The findings caused an international sensation
there was even a Time magazine cover story the
director of the NCI, John Heller, said, Right now,
the hottest thing in cancer research is research on
viruses as possible causes. Thus, the field of viral
oncology was born.
Stewart SE. Eddy BE, Borgese N. Neoplasms in
mice inoculated with a tumor agent carried in tissue
culture. J Nat Cancer Inst. 1958;20:1223-1243.

Murine polyoma virus

model built from X-ray
crystallographic data

Eddy BE, Stewart SE, Young R, Mider GB.

Neoplasms in hamsters induced by mouse tumor
agent passed in tissue culture. J Nat Cancer Inst.
1958;20:747-760.

1958 Sarah Stewart, Bernice Eddy discovery of murine polyoma virus (the first polyomavirus)
page 294

After Albert Coons (1912-1978) first developed

immunofluorescence technology in 1942, its first practical
application was in 1956 by William Cherry, who used it to
identify several bacterial pathogens. Then in 1958, Robert
Kissling and a guest in his lab at CDC, Robert Goldwasser,
developed an immunofluorescence (fluorescent antibody)
test for rabies. Impression smears of brain tissue of
animals suspected of having exposed humans were fixed
and overlayed with fluorescin isothiocyanate-conjugated
hyperimmune rabies globulin. Positives clearly showed
apple-green stippling and large aggregates of rabies viral
antigen (Negri bodies) when examined in a microscope
equipped with an appropriate light source and UV
filters. Thousands of diagnostic specimens were tested
to compare the new technique with other diagnostic
methods of the day, especially intracerebral inoculation of
mice (taking 21 days), as first developed by Leslie Webster
in the 1930s. The massive comparative testing was done in
several labs, including those of James McQueen (Florida),
Donald Dean and Melvin Abelseth (New York) and the
Fourth Army Medical Lab, Ft. Sam Houston, Texas, where
the author was a fledgling veterinary corps officer, and
where many of the specimens submitted to him from
the area were rabies positive. The comparative testing
proved that the fluorescent antibody test was at least as
good as mouse inoculation and results could be reported
in hours of specimen receipt. The test was as good from
the beginning as today conjugates made according
to the CDC recipe were excellent and microscopes
and filters were good enough (but were improved over
the years). For the first time, laboratory results greatly
influenced post-exposure human treatment (vaccine, later
supplemented with human immune globulin). In recent
years, despite success with several other rapid diagnostic
test methodologies, the fluorescent antibody test is still
the standard for rabies diagnosis globally. The first rapid
diagnostic test in virology has become the oldest!

Robert Emmons Kissling (1923-2013)

Rabies infection, fox, brain,

impression smear, diagnostic
immunofluorescence, ~1965

Goldwasser RA, Kissling RE. Fluorescent antibody

staining of street and fixed rabies virus antigens. Proc Soc
Exp Biol Med. 1958;98:219-223.
Goldwasser RA, Kissling RE, Carski TR, Hosty TS.
Fluorescent antibody staining of rabies virus antigens in
the salivary glands of rabid animals. Bull World Health
Organ. 1959;20:579-588.

Robert Goldwasser
(1918-2012)

CDC Rabies Advisory Committee, 1959: Front (L-R): W.G. Winkler, E.S. Tierkel,
R.K. Sikes, H.N. Johnson, T.F. Sellers. Back: R.L. Parker, R.E. Kissling,
D. Dean, J.H. Steele, J.P. Fox, K. Habel, R. Courter

1958 Robert Kissling, Robert Goldwasser development of rabies immunofluorescence diagnostic method

page 295

Mercedes Weissenbacher
Celia Coto and Armando S. Parodi (1909-1969)

Ignacio Pirosky (1901-1987)

The viral etiology of Argentine hemorrhagic
fever was established in 1958 by two independent
groups. Each isolated the virus from the blood of
patients and organs obtained at autopsies at the
Junn Regional Hospital in the province of Buenos
Aires, Argentina.
Parodi AS, Greenway DJ, Rugiero HR, Frigerio M,
De La Barrera JM, Mettler N, Garzon F, Boxaca
M, Guerrero L, Nota N. Sobre la etiologa del
brote epidmico de Junn. Dia Med. 1958;30:23002301.

Calomys musculinus

Pirosky IJ, Zuccarini EA, Molinelli A, Di Pietro

P, Martini B, Ferreyra LF, Gutman F, Vazquez T.
Virosis hemorrgica del noroeste bonaerense.
Endemoepidmica, febril, enantemtica
y leucopnica. I. La primera inoculacin
experimental al hombre. Orientacion Med.
1959;8:144-148.

Marta Sabattini

1958 Armando Parodi, Ignacio Pirosky, colleagues discovery of Junin virus (Argentine hemorrhagic fever)
page 296

Denis Burkitt was trained as a surgeon at Trinity College, Dublin, and went to Kampala, Uganda first
during WWII. In 1958, he described common jaw tumors in African children. Shortly thereafter,
the tumors were identified as a form of malignant B cell lymphoma, now called Burkitts lymphoma.
Histologically, the lymphomas are composed of morphologically uniform medium sized cells with round
nuclei and abundant basophilic cytoplasm. These cells have an extremely high rate of proliferation.
Burkitts lymphoma is most commonly associated with a complex chromosomal translocation of the c-myc
gene and overexpression of myc protein, but it also is epidemiologically associated with Epstein-Barr virus
(EBV) infection and with malaria in children in equatorial Africa (endemic Burkitts lymphoma). It occurs
sporadically in other geographical areas, where it also occurs among adults. Questions remain about
the role of particular EBV variants rather than ubiquitous virus strains, and the evidence for association
with malaria is weak. Burkitts lymphoma has become the basis of many lessons: (1) The lymphoma is
not amenable to surgery or radiation therapy but the success of chemotherapy (~90% of cases cured) has
influenced all cancer chemotherapy. (2) The involvement of c-myc and the chromosomal translocation
has been a model for understanding the molecular pathogenesis of other lymphomas and leukemias.
(3) The lymphoma was the first human tumor shown to be associated with a virus this inspired the
search for others, some preventable by vaccination (HBV, HPV). (4) EBV was shown to be associated
with nasopharyngeal carcinoma, T cell lymphomas, and other tumors in immunodeficient patients, again
pointing to pathogenetic research themes. (5) The finding that EBV is able to induce proliferation in B cells
via latent genes and probably drives proliferation of Burkitt lymphoma cells has led to yet other discoveries
of neoplastic mechanistics. (6) The finding of the unique geographic distribution Burkitts lymphoma
(often in areas of high incidence of intense malaria infection) has led to the development of a new kind
of geographic medicine. (7) The association of HIV/AIDS and Burkitts lymphoma in Africa has led to
another set of pathogenetic mechanistic questions, all still under investigation.
Burkitt D. A sarcoma involving the jaws in African children. Brit J Surg. 1958;46:218-223.

Denis Parsons Burkitt (1911-1993)

Global distribution of Burkitts lymphoma cases

1958 Denis Burkitt description of Burkitts lymphoma in African children

page 297

CT tomography, brain, PML

Gabriele M. Zu Rhein

Billie L. Padgett
and Duard Lee Walker (1921-2010)

In 1958, at the Massachusetts General Hospital, Karl-Erik strm, Edward Peirson Richardson, Jr. (19181998) and their colleagues described progressive multifocal leukoencephalopathy (PML) as a specific
disease entity after thorough clinical and histopathological observation of two patients with chronic
lymphocytic leukemia and one patient with Hodgkins disease who presented with multiple demyelinating
lesions of the central nervous system and a rapid, fatal outcome. The first clue of a possible viral cause
stemmed from the detection of inclusion bodies in the nuclei of damaged oligodendrocytes; this was
confirmed in 1965 by demonstration of papovavirus-like particles in lesions. The virus was isolated in
1971 from the brain of a patient with Hodgkins disease who died of PML, and it was named JC virus
(JCV) after the patients initials.

JC virus, oligodendrocyte nucleus, PML

strm K-E, Mancall EL, Richardson EP Jr. Progressive multifocal leuko-encephalopathy; a hitherto
unrecognized complication of chronic lymphatic leukaemia and Hodgkins disease. Brain 1958;81:93-111.
Zu Rhein GM, Chou S-M. Particles resembling papovaviruses in human cerebral demyelinating disease.
Science 1965;148:1477-1479.
Padgett BL, Walker DL, ZuRhein GM, Eckroade RJ, Dessel BH. Cultivation of papova-like virus from
human brain with progressive multifocal leucoencephalopathy. Lancet 1971;1:1257-1260.

Demyelination, brainstem, PML

1958 Gabriele Zu Rhein, Duard Walker, Billie Padgett, others progressive multifocal leukoencephalopathy, JC virus
page 298

By the late 1960s, it was known that hematopoietic stem cells from the bone
marrow are disseminated into the bloodstream as T cell precursor cells or as B
cells. The T cell precursors migrate to the thymus, where they differentiate into T
cells (blue). They are then exported to the periphery, where they recirculate from
the blood through thymus-dependent areas of lymphoid tissues (e.g., paracortical
areas of lymph nodes), into the lymph and back to the blood via the thoracic
duct. B cells (red) populate the thymus-independent areas of lymphoid tissues
(e.g., follicles); some recirculate in the same way as T cells. From: Miller JF. The
golden anniversary of the thymus. Nat Rev Immunol. 2011;11:489-495.

Peter Medawar (1915-1987) and James Learmonth Gowans

When James Gowans published his landmark paper on the recirculation of lymphocytes in the rat from blood to lymph and back
again, the field of immunology was still focused on humoral immunity cellular immunology was ill defined. Gowans had become
interested in lymphocytes at the suggestion of Howard Florey and Peter Medawar. Today we know that lymphocyte recirculation
is the main mechanism for dispersing the immunologic response repertoire, for directing lymphocyte subsets to specialized
microenvironments (especially in the thymus) that control their differentiation, and for targeting immune effector cells to the sites of
viral (and microbial) invasion and damage. The continual mixing of cells is particularly important for proper function of the immune
system because cellular activation events often require direct cell-to-cell contact. Inappropriate migration and activation, however, can
contribute to immunopathology and autoimmunity. Gowans and E. Julie Knight found that the output of lymphocytes is about 109/day,
and is kept in homeostatic status by recirculation. Their work was made possible by mastering the difficult technique of thoracic duct
cannulation. The thoracic duct, the terminal lymphatic draining conduit, empties into the superior vena cava. Tracing the fate of RNAlabeled small lymphocytes collected from the thoracic duct and transfused into normal rats, Gowans and Knight found that the main
channel from blood to lymph was within lymph nodes, the lymphoid follicles of the spleen and Peyers Patches of the intestine.
Gowans JL. The recirculation of lymphocytes from blood to lymph in the rat. J Physiol. 1959;146:54-169.
Gowans JL, McGregor DD, Cowen DM. Initiation of immune responses by small lymphocytes. Nature 1962;196:651-655.
Gowans JL, Knight EJ. The route of re-circulation of lymphocytes in the rat. Proc R Soc Lond B Biol Sci. 1964;159:257-282.

1959 James Gowans discovery of lymphocyte recirculation

page 299

People lined up for blocks

for Sabin polio vaccine,
San Antonio, Texas, 1962

Albert Bruce Sabin

(1906-1993)

Herald Rea Cox

(1907-1986)

Hilary Koprowski
(1916-2013)
Derivation of Sabins vaccine viruses

Oral polio vaccine is an attenuated live-virus vaccine, which was developed by passage of
the viruses through cells in culture and animals, thereby producing attenuating mutations.
Such vaccines were developed by Albert Sabin and coworkers and separately by Hilary
Koprowski and Herald Cox. In 1958, an NIH committee evaluated these vaccines for their
ability to induce immunity to polio, while retaining a low incidence of neuropathogenicity
in monkeys. Based on these results, the Sabin strains were chosen for worldwide
distribution. In 1961-1962, monovalent and in 1963 trivalent vaccines were licensed the
latter became the vaccine of choice in the U.S. and most other countries of the world it
was easy to administer, making it more suitable for mass vaccination campaigns. There
was second wave of mass immunizations between 1962 and 1965 about 100 million
Americans (~56% of the population) received Sabin vaccine. The result was a substantial
reduction in the number of polio cases, even from the much reduced levels that followed
the earlier introduction of the Salk vaccine.

1959 Albert Sabin, Herald Cox, Hilary Koprowski, others development of attenuated live-virus polio vaccine
page 300

Kilham rat virus

infection ataxia
(splayed feet) from
virus-induced cerebellar
hypoplasia

Kilhams H-1 virus

negative contrast electron microscopy

Lawrence Kilham (1910-2000)

Lawrence Kilham: When I
started out as a microbiologist or
virus hunter, I wondered how
my other love, studying birds on
early mornings, evenings, and
weekends, would fare. Would my
enthusiasm for birds and animals
cut in on my pursuit of viruses? I
need not have worried. Indeed,
Kilhams book became a key
resource during the West Nile
virus invasion of the U.S.
starting in 1999.

Parvoviruses of rats and mice have been shown to cause (1) fetal death and resorption, (2) neonatal hepatitis,
(3) cerebellar hypoplasia, and (4) severe hemorrhagic encephalopathy, all varying with the species, strain,
age and immune competence of the host animal. Most early experiments, led by Lawrence Kilham, involved
parenteral inoculation of virus into pregnant or neonatal animals, seeking models for virus-induced birth
defects. This work was extended to cats and hamsters, which when infected before or at birth also exhibited
cerebellar hypoplasia. Productive replication of murine parvoviruses (Kilham rat virus, H-1 virus, minute
virus of mice, and perhaps all the autonomously replicating parvoviruses of other species) requires cellular
factors that are expressed only during cell differentiation and division this accounts for the predilection of
these viruses for mitotically active cells and this in turn accounts for the tropism and damage seen in infected
animals. In recent years, the interest in these viruses has changed: they are of major interest because of their
adverse effects on all biomedical research involving rodents. Infection, often not recognized in colonized
rodents, causes immunomodulation (inhibition of lymphocyte proliferation and the generation of cytolytic
T cell activity, etc.), inhibition of the antibody response, hematopoetic disruption, tumor suppression, and
contamination of cultured cell lines any and all confounding much research involving laboratory rodents.
Kilham L, Olivier L. A latent virus of rats isolated in tissue culture. Virology 1959;7:428-437.
Kilham L. Rat virus (RV) in hamsters. Proc Soc Exp Biol Med. 1961;106:825-829.
Kilham L, Margolis G. Fetal infection of hamsters, rats and mice induced with minute virus of mice (MVM).
Teratology 1971;4:43-62.

1959 Lawrence Kilham, others discovery of murine parvoviruses and their role in congenital diseases

page 301

Sydney Brenner

Robert W. Horne (1923-2010)

Negative staining revolutionized our view of the viruses, for the first time allowing details of
virion surfaces [capsomeres, envelopes, projections (spikes, peplomers)] and some internal
features] to be seen. Where would virology be today if the many variations on the negative
staining method had not been available?
Brenner S, Horne RW. A negative staining method for high resolution electron microscopy of
viruses. Biochimica et Biophysica Acta 1959;34:103-110. [The first viruses imaged by the new
method were tobacco mosaic virus and turnip yellow mosaic virus.]
Horne RW, Brenner S. A negative staining technique for high resolution visualization of viruses.
In: Proc Fourth International Conference on Electron Microscopy, vol. 2. Berlin: Springer Verlag;
1960. p. 625. [from the Medical Research Council Unit for Molecular Biology and Electron
Microscopy, Cavendish Laboratory, Cambridge, Great Britain]
Horne RW, Wildy P. Virus structure revealed by negative staining. Adv Virus Res. 1963;10:101-70.
Horne RW, Wildy P. An historical account of the development and applications of the negative
staining technique to the electron microscopy of viruses. J Microsc. 1979;117:103-122.

Human adenovirus 5, negative contrast electron microscopy

1959 Sydney Brenner Robert Horne invention of negative contrast electron microscopy
page 302

In researching the virologists cited in this book, many talents were found other than
those related to their scientific careers. This page, a placeholder for this subject, is
exemplary.
From Alasdair Steven and Wolfgang Baumeister: Graphical representation of
molecular and cellular features is a key form of communication in structural biology,
in which abstract symbols of an economical and visually appropriate kind, pseudocolor coding, and dynamic animations all play their parts. Accordingly, it should
not be surprising that many structural biologistslike traditional biologists before
themare talented artists who also express themselves on non-scientific topics.
[Here we] illustrate the approaches of and pictures by several practicing scientistartistsin this sampling, electron microscopists.
The Swedish training schooners Gladan and Falkon off Bornholm. From an original
oil painting by Robert W. Horne. Robert Hornes interest in marine artwork started
with studies relating to the form, construction and development of early sailing
vessels. His color work began with the use of inks, pens and fine brushes, followed
by water-colors, and finally oil painting. He embarked seriously on marine painting
during the 1970s at the Norwich School of Art and Design. His work includes
detailed research into the plans of historic sailing vessels and their reconstruction in
paintings.

Robert W. Horne (1923-2010)

The two-mast cutter/schooner HMS Pickle bringing news of the naval victory
at Trafalgar in 1805. Water-color painting by Robert W. Horne. Pickle is depicted
being driven hard in a freshening wind as she approaches Falmouth on the south
coast of England. Completing delivery to London required an additional two-anda-half days land journey by the messenger, Lieut. J. Lapotiere, in a post-chaise,
involving 23 staging points.
Steven AC, Baumeister W. The state of the art. J Struct Biol. 2008;163:201-207.

1959 Sydney Brenner Robert Horne invention of negative contrast electron microscopy

page 303

Single-stranded
circular DNA of
X174 bacteriophage.
This was the first DNA
viral genome to be
sequenced, by Fred
Sanger, in 1977.

Albrecht Karl Kleinschmidt

(1916-2000)
Albrecht Kleinschmidt invented the technique for preparing monolayers
of DNA for electron microscopy, in which the DNA molecules are bound
to a film of denatured basic protein (e.g., cytochrome c) on the surface of
an aqueous solution. The DNA molecules are consequently brought from
a three dimensional conformation to a two dimensional one by adsorption
to the protein layer. The DNA-protein complex is then transferred to a
solid surface (e.g., an electron microscope grid containing a collodion film),
dried, and contrasted with heavy metals by shadowing or staining. The
method has also been applied to RNA.
Kleinschmidt, AK, Lang D, Jachters D, Zahn RK. Darstellung und
lngenmessung des gesamten desoxyribonucleinsure-inhaltes von T2bakteriophagen. Biochimica et Biophyisca Acta 1962;61:857-864.
[This paper contains the famous electron micrograph of the DNA of T2
bacteriophage, which was used in many publications, even on the cover of
several molecular biology and microbiology books. It is used here as well.]

Electron micrograph of the DNA of T2 bacteriophage

Original magnification, x100,000

1959 Albrecht Kleinschmidt invention of method for electron microscopic visualization of viral DNA

page 304

Gerald Maurice Edelman

Porters model of 7S antibody (IgG) 1959

Rodney R. Porter (1915-1985)

Contemporary model of IgG

Alfred Nisonoff (1923-2001)

Up to the year 1959, our knowledge of the nature and mode of action of antibody
molecules was vague and incomplete, in spite of a century of research. Gerald
Edelmans and Rodney Porters experimental approaches were quite different:
both first separated the molecule now called IgG (molecular weight 150 kD),
from other serum proteins. Porter then subjected IgG to brief digestion with
the enzyme papain and separated the fragments he obtained two identical
fragments, now called Fab, and one fragment, now called Fc. Edelman dissociated
IgG by reducing its interchain disulfide bonds with mercaptoethanol he
obtained two fractions, one called light chain, the other heavy chain. Alfred
Nisonoff s approach was similar to Porters, only he used the enzyme pepsin he
obtained a single large fragment composed of two Fab-like fragments and one
Fc fragment. Porter finally answered how the fragments went together using
antibodies against each fragment. Porter was then able to build a model of the
molecule, which has since been generally accepted and successively fitted with
additional molecular details. The structure of the other classes of antibody
molecules, IgM, IgA, IgE and the subclasses of IgG were resolved subsequently.
These discoveries incited a major burst of research in immunology, which
immediately spilled over into virological research.

1959 Gerald Edelman, Rodney Porter, Alfred Nisonoff discovery of the molecular structure of antibody molecules

page 305

DNA of
bacteriophage
X174

Robert L. Sinsheimer

Computer model of
bacteriophage X174

From Robert Sinsheimer, Oral History, 1990. California Institute of Technology Archives. (Interview by
Shelley Erwin):
[1957] we got Phi X DNA purified, did the light-scattering measurements on it, established its
molecular weight it wasnt Watson and Crick, and therefore could not be double-stranded. And that
fit with the sedimentation coefficient and the light scattering, which gave now a molecular weight of
1.7 million daltons. All that said it was single-stranded. And then the only question really was, Was this
somehow something we had done in extracting it from the virus? I was able to show spectroscopically and
in other ways that it was a single-strand in the virus itself. So it clearly was single-stranded DNA, and we
wrote this up and published it in the first issue of the Journal of Molecular Biology. And that, of course,
was for a time a sensation. We had never seen a single-stranded DNA. Of course, that immediately also
raised a number of obvious questions of great importance. We had this model for double-stranded DNA,
but how did the single-stranded DNA reproduce? Indeed, how would it even get transcribed? So we
developed ways of labeling the DNA and following it into the cell. And to make a long story short, we
were able to show that the first thing that happened to the single-stranded DNA after it went into the cell
was that it became a complementary-stranded DNA it became a double-stranded molecule. And then,
obviously that could be transcribed. And then, that double-stranded form multiplied as such we call
that a replicative form it multiplied as such for a time, and then later in the infection it started to make
the single strands of DNA. It went back to the single-stranded form to put into the progeny. Not only was
it really novel in itself, but what was really novel were the techniques to follow it into the cell.
Sinsheimer RL. A single-stranded deoxyribonucleic acid from bacteriophage X174.
J Mol Biol. 1959;1:43-53.

1959 Robert Sinsheimer discovery that a virus may have a single stranded DNA genome (bacteriophage X174)
page 306

Yalow RS, Berson SA. Assay of plasma insulin in

human subjects by immunological methods. Nature
1959;184:1648-1649.
Yalow RS, Berson SA. Immunoassay of endogenous
plasma insulin in man. J Clin Invest.
1960;39:1157-1175.

Rosalyn Sussman Yalow (1921-2011)

Solomon A. Berson (1918-1972)

Rosalyn Yalow spent many years at the Veterans Administration Hospital in the Bronx, New York, where with
her collaborator, Solomon Berson, she used radioisotopes in novel ways, first to obtain more accurate estimates
of blood volume, then to study how the thyroid gland and kidneys remove iodine from the blood. They expanded
these studies to globins and other serum proteins, and then to hormones, especially insulin. They discovered that
globulins bind radioactive insulin in the blood of insulin-treated diabetics, and concluded that treatment with
insulin injections immunized patients so that they developed insulin-binding antibodies, which kept the insulin
molecules in the bloodstream. This was the first proof that so small a protein as insulin could stimulate an immune
response. The scientific establishment was reluctant to accept this finding, but when their observations were
confirmed by others the significance of the discovery became clear. Not only had they proven that the immune
system could recognize and respond to smaller molecules than previously thought possible, but they had done so
using a breakthrough technology radioimmunoassay. The development of radioimmunoassays, the first method
for measuring antigen-antibody complexes directly, stimulated a revolution in immunology and also in virology. By
the end of the 20th century, many different radioimmunoassays were in use in viral diagnostics and research. Bersons
death at age 54 precluded his nomination for the Nobel Prize in Physiology or Medicine, which Yalow shared with
Roger Guillemin and Andrew Schally in 1977.

1959 Rosalyn Yalow, Solomon Berson development of radioimmunoassays (RIAs)

page 307

Arthur Kornberg
(1918-2007)

Severo Ochoa de Albornoz

(1905-1993)

In 1955, Arthur Kornberg started to focus on the synthesis

of DNA after learning that Severo Ochoa and his colleagues
had apparently created a synthetic RNA from adenosine
diphosphate (ADP) (the product turned out to be not RNA
polymerase, but polynucleotide phosphorylase). Working with
cell extracts of E. coli, Kornberg found which combinations of
nucleotides and other ingredients resulted in the most rapid
synthesis of DNA. From this, in 1958 he found and purified
DNA polymerase, and with it was able to synthesize DNA
in the lab. Subsequently, Kornberg and his students found
enzymes responsible for DNA repair and others responsible for
the initiation and elongation of DNA molecules. The enzymes
they discovered helped make possible the development of
recombinant DNA technology, i.e., genetic engineering. [In
1967, Kornberg became the first person to synthesize infectious
viral DNA in vitro (bacteriophage X174).] A chance discovery
in 1955 by Marianne Grunberg-Manago, a postdoctoral fellow
in Ochoas lab, was the key to the eventual discovery of RNA
polymerase: she observed that a bacterial extract converted
phosphate substrate into ADP. The responsible enzyme,
purified with Ochoas urging and direction, astonishingly
converted ADP and other nucleoside diphosphates into
RNA-like polymers. The initial hope that this enzyme, named
polynucleotide phosphorylase, might be responsible for the
biosynthesis of RNA was soon dispelled the discovery of
true RNA polymerases, which copy DNA templates with
great specificity using nucleoside triphosphates rather than
diphosphates, settled the matter. Nevertheless, polynucleotide
phosphorylase proved to be of great value, particularly in
deciphering the genetic code. Ochoa (with help from Leon A.
Heppel) employed the enzyme to synthesize a variety of RNAlike polymers, which were then used to identify many of the
nucleotide triplets that encode the amino acids in the synthesis
of proteins. The 1959 award of the Nobel Prize in Physiology or
Medicine to Kornberg and Ochoa was for their discovery of
the mechanisms in the biological synthesis of ribonucleic acid
and deoxyribonucleic acid.
Grunberg-Manago M, Ortiz PJ, Ochoa S. Enzymic synthesis of
polynucleotides. I. Polynucleotide phosphorylase of Azotobacter
vinelandii. Biochim Biophys Acta 1956;20:269-285.
Lehman IR, Bessman MJ, Simms ES, Kornberg A. Enzymatic
synthesis of deoxyribonucleic acid. I. Preparation of substrates
and partial purification of an enzyme from Escherichia coli. J
Biol Chem. 1958;233:163-70.

1959 Arthur Kornberg, Severo Ochoa discovery of mechanisms in the replication of DNA and RNA
page 308

William H. Prusoff (1920-2011)

William H. Prusoff developed the first antiviral drug ever to be (and still) licensed and marketed, first for the topical treatment of herpetic keratitis, and then for fatal or
disabling illnesses such as adult herpetic encephalitis, keratitis in immunocompromised (transplant) patients, and disseminated herpes zoster. This is 5-iodo-2-deoxyuridine
(IUdR), an analog of thymidine. Prusoff first was looking for anti-cancer activity, but a physician, H.E. Kaufman, working in a Boston Hospital, conceived the idea of using IUdR
to treat herpetic keratitis. He first reported animal experiments, showing that the drug was truly therapeutic; it promoted healing of the cornea when applied 2 or even 5 days
after infection the virus load in rabbit corneas after 4 days of treatment was much less than in controls. This led to clinical trials and licensing.
Prusoff is also known for research conducted during the 1980s with his colleague Tai-Shun Lin, on d4T, a potent treatment for HIV/AIDS. Under the trade name Zerit (BristolMyers Squibb), d4T (2-3-didehydro-2-3-dideoxythymidine, a nucleoside analog reverse transcriptase inhibitor) forms part of many drug cocktails used to treat AIDS
worldwide.
Prusoff WH. Synthesis and biological activities of iododeoxyuridine, an analog of thymidine. Biochim Biophys Acta 1959;32:295-296.
Kaufman HE. Clinical cure of herpes simplex keratitis by 5-iodo-2-deoxyuridine. Proc Soc Exptl Biol Med. 1962;109:251-252.
Kaufman HE, Nesburn AB, Maloney ED. IDU therapy of herpes simplex. Arch Ophthalmol. 1962;67:583-591.

1959 William Prusoff development and licensing of the first antiviral drug, 5-iodo-2-deoxyuridine (IUdR)

page 309

William Reeves (1916-2004) Wilbur Downs (1913-1991) William Scherer (1925-1982)

The founding committee,

American Committee on
Arthropod-Borne Viruses
1959
The Committee still provides a forum for
exchange of information among people
interested in arbovirus research. It meets
annually in association with the American
Society of Tropical Medicine and Hygiene.
An executive council is its governing body,
and its work is carried out by subcommittees:
Subcommittee on Arbovirus Laboratory
Safety, Subcommittee for Evaluation of
Arthropod-Borne Status, Subcommittee
on Information Exchange, Subcommittee
on Inter-relationships Among Catalogued
Arboviruses, and Subcommittee on Low
Passage Viruses.

Alexis Shelokov

Richard Taylor (1887-1981)

Telford Work (1921-1995)

1959 William Reeves, others founding of the American Committee on Arthropod-Borne Viruses (ACAV)
page 310

World Food Prize

Walter Plowright and his team at the East African Veterinary

Research Organization at Muguga in Kenya, ~1970. Sitting on
Plowrights left is the Head Laboratory Assistant, Francis Ngugi
Plowright W. The application of monolayer tissue
culture techniques in rinderpest research. II. The use of
attenuated culture virus as a vaccine for cattle. Bulletin
de lOffice International des Epizooties 1962;57:253-257.
Plowright W, Ferris RD. Studies with rinderpest virus in
tissue culture. The use of attenuated culture virus as a
vaccine for cattle. Res Vet Sci. 1962;3:172-182.

Walter Plowright (1923-2010)

Plowright W, Taylor WP. Long-term studies of the

immunity in East African cattle following inoculation
with rinderpest cell culture vaccine. Res Vet Sci.
1967;8:118-128.

Walter Plowright, as a young veterinary pathologist, left his native Great Britain to carry out research in Kenya and
Nigeria, starting in 1950. Working at the East African Veterinary Research Organization at Muguga in Kenya from
1956 to 1971, he adopted the then new cell-culture techniques of John Enders, et al., that had been used to develop
measles vaccine, to produce a live-attenuated rinderpest virus vaccine. Unlike its predecessors, Plowrights tissue
culture rinderpest vaccine (TCRV) could be used safely in all breeds of cattle of any age or health status. It could
be produced very economically and conferred lifelong immunity. Initial field trials (1956 to 1963) showed that the
vaccine was genetically stable, produced no clinical side effects and was easily standardized. This vaccine was used
as the centerpoint in the global rinderpest eradication program (GREP) of the FAO, which succeeded in its goal in
2010. Plowright was recognized with the 1999 World Food Prize for his development of the vaccine that led to the
eradication of the second disease from the world, the first being smallpox.

Walter Plowright demonstrating the growth

of rinderpest virus in cell culture to the
Governor of Kenya, ~1970

1960> Walter Plowright development of the attenuated-live virus rinderpest vaccine used in global eradication

page 311

Maurice Hilleman (1919-2005)

at Walter Reed Army Institute of Research, 1957

SV40 virus

negative contrast electron microscopy

from minimum-exposure study by Robley Williams, 1974

In 1957, after two years of widespread distribution of Salk inactivated polio vaccine, Maurice Hilleman and Benjamin Sweet, then at the Walter Reed Army Institute of
Research, identified the existence of a virus in the rhesus monkey kidney cells being used for vaccine production (the 1:4,000 dilution of formaldehyde used to inactivate
poliovirus did not inactivate this other virus). By 1960, they had characterized the virus, calling it SV40, and had found that it contaminated Sabin attenuated live-virus vaccine
as well. The virus was named for its effect on African green monkey cells, which developed an unusual number of vacuoles, and for the fact that it was the 40th virus they
found contaminating rhesus monkey kidney cells. Soon, SV40 virus antibody was found in the sera of more than half of all individuals that had received Salk vaccine. The virus
became famous when Bernice Eddy, working at the NIH, tested extracts from the monkey kidney cells used to make polio vaccine for possible cancer causing agents the
newborn hamsters she inoculated developed several kinds of tumors similar to those induced by polyoma virus, which she and Sarah Stewart had described earlier. A scandal
arose when she was silenced and prohibited from working on polio vaccine by supervisors who saw political fallout from her findings. Her findings did leak out and Sweet
and Hilleman, now at Merck Research Laboratories, replicated her work. To his credit, Hilleman repeatedly sounded warnings about the risks inherent in the use of monkey
cells for vaccine production. After years of procrastination by industry, polio vaccine production was switched to African green monkey cells, which were free of the virus
(but which were contaminated by other viruses, such as a simian cytomegalovirus). By 1962, it was confirmed that SV40 virus can transform human cells, but the question of
whether the virus does cause cancer in humans has only been answered in the negative by the most recent statistical analyses of epidemiologic/serologic data meanwhile, the
issue has become part of the argument used by activist groups that wish to stop all vaccinations.
Sweet BH, Hilleman MR. The vacuolating virus, SV40. Proc Soc Exp Biol Med. 1960;105:420-427.
Hilleman MR. Discovery of simian virus 40 (SV40) and its relationship to poliomyelitis virus vaccines. Dev Biol Stand. 1998;94:183-190.

1960 Benjamin Sweet, Maurice Hilleman discovery of SV40 virus (simian virus 40) (polyomavirus)
page 312

Vernon Todd Riley

(1914-1982)

Kenneth Edmund Keith Rowson

(1924-2004)

Lactate dehydrogenase-elevating virus (LDH virus) was discovered by Vernon Riley and his colleagues in
1960 during work on plasma enzyme levels in tumor-bearing mice. They found that transplantable tumors
of many types caused a 5-10-fold increase in plasma lactate dehydrogenase (LDH) activity within 3 days of
transplantation and before the tumors were clinically obvious. To produce this dramatic increase in plasma
enzyme level it was not necessary to transplant cells; cell-free plasma from tumor-bearing mice was equally
effective. The raised enzyme levels could be serially transmitted from mouse to mouse and proved to be
caused by a virus which replicated very rapidly, mostly from infection of macrophages. Very high titers of viral
infectivity (109 ID50/ml) are present in the plasma within 24 hours after infection, and a stable viremia at a lower
level (104 ID50/ml) is established after 7 to 10 days. This persists for the remainder of the animals life but does
not cause any obvious disease or reduction in life expectancy. The persistent viremia clearly provides a source
of virus for transmission by bloodsucking ectoparasites. It seems likely that this is the method of cross-infection
by which the virus is maintained in feral mice, a number of which have been found to be infected in Europe,
Australia and the U.S. Kenneth Rowson and Brian Mahy did much to call attention to the confounding, often
inexplicable effect of the presence of contaminating LDH virus in various kinds of experiments employing mice,
in effect giving rise to the universal demand for specific-pathogen-free mice [from Brian W.J. Mahy]
Vernon Riley was an innocent victim of the McCarthy era. He was accused of being a communist, and although
he eventually received restitution and acknowledgment of his innocence, he was forced to leave the National
Cancer Institute. Thanks to the invitation of C.P. Rhodes. then President of the Memorial Sloan-Kettering
Cancer Center, he joined the Center and eventually became head of a Section in the Division of Experimental
Chemotherapy. It is at Sloan-Kettering where Riley did his seminal work on LDH virus.

The discovery of LDH virus had a profound effect on our

understanding of the subtleties of the effects of some
infections, especially on immunological responses and
tumor growth in experimental animals. Its discovery
also had a great effect on how we use experimental
animals in research and how we interpret data from such
experimentation, such that we now expect experimental
animals to be free of all adventitious viruses (even if this is
not always the case).
Riley VT, Lilly F, Huerto E, Bardell D. Transmissible agent
associated with 26 types of experimental mouse neoplasms.
Science 1960; 132:545-547.
Rowson KEK, Mahy BWJ. Lactate dehydrogenase-elevating
virus. J Gen Virol. 1985; 66: 2297-2312.

1960 Vernon Riley, Kenneth Rowson discovery of lactate dehydrogenase-elevating virus (arterivirus)

page 313

Wayne H. Thompson
(1922-2000)

Ralph Owen Anslow

(1915-1998)

In 1960, a 4-year-old girl died of rural encephalitis in a

hospital in La Crosse County, Wisconsin. Four years later,
Wayne H. Thompson, Bernard Kalfayan and Ralph O.
Anslow isolated a novel virus from frozen homogenates
of the childs brain tissue by intracerebral inoculation of
suckling mice. The virus, La Crosse virus, is maintained in
nature through vertical and horizontal transmission cycles
involving the tree-hole breeding mosquito vector, Aedes
triseriatus and primary amplifying hosts (the eastern
chipmunk, Tamias striatus, the gray squirrel, Sciurus
carolinensis, and the fox squirrel, Sciurus niger). Habitat to
support this natural history pattern is found in deciduous
forests widely distributed throughout the central and
eastern United States, with cases reported from more
than 20 states. During an average year, about 75 cases of
encephalitis caused by La Crosse virus are reported to
the CDC. Because the virus is vertically transmitted from
the female mosquito to her eggs, the virus overwinters
in infected mosquito eggs with the high likelihood of
recurrent human disease each summer in highly endemic
areas. Preventive efforts focus primarily on removal of
potential breeding sites such as tree stumps, old tires and
other objects that hold small, stagnant pools of water.

La Crosse virus

suckling mouse brain, thin section electron microscopy

1960 Wayne Thompson, Bernard Kalfayan, Ralph Anslow discovery of La Crosse virus
page 314

Thompson WH, Kalfayan B, Anslow RO.

Isolation of California encephalitis group
virus from a fatal human illness. Am J
Epidemiol. 1965;81:245-253.

Frank Dixon (1921-2008)

Michael Oldstone

Richard Lerner

William Weigle (1927-2001) Joseph Feldman (1916-1995)

LCM virus immune complex glomerulonephritis

1960> Frank Dixon, others founding of immunopathology, and the role of viruses in immune complex diseases

page 315

Cedric Mims

Neal Nathanson

Richard Johnson

Bernard Fields

Gerald Cole

Robert Blanden

Ashley Haase

Diane Griffin

Michael Buchmeier

Rafi Ahmed

Francisco GonzlezScarano

Volker ter Meulen

Kathryn Holmes

Geoffrey Smith

Harriet Robinson

1960> Cedric Mims, Neal Nathanson, others founding of the modern era of viral pathogenesis research

page 316

Robert Bruce Merrifield (1921-2006)

Robert Merrifield received his PhD in chemistry

from the University of California Los Angeles in
1949. He then moved on to the Rockefeller Institute
for Medical Research, where he remained for the
rest of his life. In 1963, he published a seminal paper
that launched the method of solid-state synthesis, in
which reactions are carried out on a solid support
rather than in solution. The centerpiece of his
insight was that important biological molecules
such as peptides and proteins were linear polymers
and that one could thus attach one end to a matrix
during their synthesis. The method was initially used
for the synthesis of peptides and proteins; more
recently, it has found wide use in the synthesis of
oligonucleotides, carbohydrates, and even for the
construction of complicated organic molecules.
Initially, his method of solid-state synthesis received
less-than-enthusiastic support from chemists who
were used to a rigorous analysis of the products from
each step in the long series of reactions used to build
up the molecule of interest. By contrast, the solidstate method did not allow analysis at each step; only
the finished product could be analyzed after it was
cleaved from the support. The success that led to the
acceptance of Merrifields process was his synthesis
in 1969, together with Bernd Gutte, of the 124-amino
acid-long enzyme ribonuclease A. The recovery of
active enzyme with a 78% nuclease-specific activity
left little doubt about the fidelity of his method.
In 1984, Merrifield received the Nobel Prize in
chemistry for his development of methodology
for chemical synthesis on a solid matrix. While
his methods were simple, his grasp of the organic
reaction mechanisms employed in the various
synthesis procedures was deep and innovative.
Merrifield RB. Solid phase peptide synthesis. I.
The synthesis of a tetrapeptide J Am Chem Soc.
1963;85:2149-2154.
Merrifield RB, Stewart JM. Automated peptide
synthesis. Nature 1965;207:522-523.

Robert Merrifield adjusting an early version

of his peptide synthesizer, 1969

Gutte B, Merrifield RB. The synthesis of ribonuclease

A. J Biol Chem. 1971;246:1922-1941.

1960s Robert Merrifield development of solid phase synthesis technology (polypeptides, oligonucleotides, etc.)

page 317

Franois Jacob
(1920-2013)

Jacques Monod
(1910-1976)

Andr Lwoff
(1902-1994)

Jacques Monod, Franois Jacob, in the lab, 1960

Franois Jacob and Jacques Monod built upon concepts derived by Andr Lwoff from studies of bacteriophage lysogeny and conceived the mechanisms responsible for the
transfer of genetic information from the genome to the macromolecule synthetic machinery of the cell. They, as well, conceived the regulatory pathways which regulate the
timing and amount of synthesis of these macromolecules. They developed the operon concept, using as their model the lactose-operon (lac-operon) in which the inducible
enzyme -galactosidase in Escherichia coli is activated and regulated to metabolize lactose. In their experiments the inducer, lactose, served to inhibit the gene that was
regulating the synthesis of -galactosidase. According to their operon model, a set of structural genes in the bacterial genome encode a messenger RNA that is delivered to
ribosomes, which then translate the RNA message and synthesize protein accordingly. Each set of structural genes has its own operator and regulator genes lying upstream to
it the three together represent the operon. The operator gene is the switch that turns on or turns off its set of structural genes, and thus regulates the synthesis of the protein.
In an inducible system, like the lac-operon, the regulator gene encodes a repressor protein. The repressor protein does one of two things: when no lactose is present, the
repressor protein attaches to the operator and inactivates it, in turn, halting structural gene activity and protein synthesis. When lactose is present, the repressor protein binds
to the regulator gene instead of the operator, and thus frees up the operator and permits protein synthesis to occur. With a system such as this, a cell can adapt to changing
environmental conditions, and produce the proteins it needs when it needs them. Extending from the work of Lwoff, bacteriophages were found to contain genetic control
circuits as well, complete with operators, repressors, and structural genes. In its lysogenic state, the bacteriophage remains inactive. When repressor signals are interrupted,
the bacteriophage is activated, starts to grow rapidly and soon lyses the host bacterium and is released to continue its life cycle. When, in 1961, the now-famous Jacob-Monod
operon model was published, Gunther Stent described the paper as one of the monuments in the literature of molecular biology.
Jacob F, Monod J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961;3: 318-356.

1961> Franois Jacob, Jacques Monod, Andr Lwoff discoveries of genetic control of enzyme and virus synthesis

page 318

(Above) In the lac-operon, the operator (structural) gene is blocked by a repressor protein when
there is no lactose present (a). When lactose is present, it acts as an inducer by binding to the
repressor protein, preventing it from attaching to the operator. RNA polymerase can then bind to
the operator and transcribe the mRNA molecule (b). Three proteins are synthesized. When all of the
lactose has been catabolized, the repressor protein binds to the operator shutting down the operon.
(Top, left) Micrograph of ongoing transcription of ribosomal RNA, illustrating growing primary
transcripts. Begin indicates the 3 end of the DNA template strand, where new RNA synthesis
begins; end indicates the 5 end, where primary transcription is almost complete.
(Bottom, left) Micrograph of ongoing translation, synthesis of polypeptides from mRNA running
through ribosomes.
The molecule which later became known as ribosomal RNA was first hypothesized by Franois
Jacob and Jacques Monod. RNA synthesis by RNA polymerase was established in vitro by several
laboratories by 1965; however, the RNA synthesized by these enzymes had properties that suggested
the existence of an additional factor needed to terminate transcription. In 1972, Walter Fiers proved
the existence of the terminating enzyme.
Roger Kornberg won the 2006 Nobel Prize in Chemistry for his studies of the molecular basis of
transcription.

1961> Franois Jacob, Jacques Monod, Andr Lwoff discoveries of genetic control of enzyme and virus synthesis

page 319

Sydney Brenner
in 1960s

Franois Jacob
(1920-2013)

Matthew Meselson
in 1964

For years after the discovery of the structure and mode of replication of DNA, there was no understanding
of how genetic information was conveyed from genes (DNA) to the cytoplasmic sites of protein synthesis.
Research pointed towards RNA involvement and there were several bizarre theories about how this might
worke.g., perhaps chromosomes participated directly in protein production. If so, some DNA would be
found in the cytoplasm; however, such had never been found. Thus, DNA could only be linked to protein
synthesis indirectly. The finding of RNA involvement was found by classical cell fractionation studies, such as
used to unravel all cytoplasmic organelle structure/function details in the 1950s. Most protein synthesis was
associated with the cytoplasmic microsomal fraction, now understood to be the ribosomal fraction.

Ribosomes are one of the wonders of the cellular world

mRNA (magenta) entering and leaving a ribosome; two
tRNAs (red, blue) involved in translation. Sanbonmatsu KY.
Computational studies of molecular machines: the ribosome.
Curr Opin Struct Biol 2012; 22:168-174.

In 1961, Sydney Brenner, Franois Jacob and Matthew Meselson used T4 bacteriophage-infected E. coli to prove the existence of an intermediary between DNA and protein synthesismessenger RNA (mRNA). They showed that when the bacteria were infected by the phage, all bacterial RNA synthesis stopped, replaced by T4 phage RNA synthesis. They
found that T4 viral RNA attached to preexisting bacterial cellular ribosomes and directed viral protein synthesis. Protein synthesis was monitored throughout the viral infection
cycle using radioactive sulfur (35S), which ended up in phage proteins. Bacterial ribosomes were harvested and purifiedradioactivity quickly appeared in newly-made and nascent
protein that was attached to ribosomes. When 35S was administered as a short pulse (pulse/chase), the radioactivity remained associated with the ribosomes for only a brief period.
Later, the radioactivity appeared in the soluble protein fraction. Thus, it was shown for the first time that proteins are synthesized from amino acids on RNA-containing particles
ribosomesand are released from them when completed. At this point it was hypothesized that ribosomal RNA carried genetic information from the nucleus to the cytoplasm
this was proven wrong by several lines of evidence.
Further work, employing heavy isotopes to label nucleic acids of the T4 phage DNA and RNAs, confirmed the nature of the mRNA (complex experiments not reviewed here).
Taken together, these experiments established that: (1) The expression of the T4 viral genes in infected bacteria was associated with the formation of new short-lived virus-specific
RNA molecules (mRNAs). (2) Ribosomes were not involved in T4 viral gene expression except as passive sites of synthesis (factories). The same ribosomes worked in translating all
mRNA molecules, no matter which protein they encoded. (3) The mRNA had a base sequence complementary to the viral DNA, and presumably had been transcribed from it. (4)
The viral mRNA could be isolated complexed to ribosomes, but only for a short period. It followed that these new RNA molecules were indeed the genetic messengers predicted by
Francis Crick in discussions with Brenner and Jacob before their experimentation began. It turned out that this variant RNA, mRNA, had been noted earlier by the Elliot Volkin and
Lazarus Astrachan, but they had not understood the full significance of their finding.
Brenner S, Jacob F, Meselson M. An unstable intermediate carrying information from genes to ribosomes for protein synthesis. Nature 1961;190:576-581.

1961 Sydney Brenner, Franois Jacob, Matthew Meselson discovery of messenger RNA (mRNA)
page 320

Jacques Miller

Miller JFAP. Immunological function

of the thymus. Lancet 1961;2:748-749.
Miller JFAP. The discovery of thymus function and
of thymus-derived lymphocytes. Immunol Rev.
2002;185:7-14.
The thymus is divided into two histologically defined regions, the cortex and the medulla, each of
which contains several different thymic epithelial cell subtypes. In adults, T-cell precursors enter
the thymus at the cortico-medullary junction, and then begin a highly ordered differentiation
program, which is linked to migration through the thymic stroma. Different thymocyte subsets are
therefore found in the four spatially restricted regions of the thymus: Region 1, the cortico-medullary
junction, the site of entry into the thymus and contains uncommitted progenitors, CD4-CD8- doublenegative 1 (DN1) cells; Region 2, where cells differentiate to the DN2 stage, undergo a proliferative
clonal expansion, and lose B- and natural killer (NK) cell potential; Region 3, where T-cell lineage
commitment and the onset of T-cell receptor (TCR) chain rearrangement occurs; Region 4, where
the transition from DN to CD4+CD8+ double-positive (DP) status occurs. DP cells then migrate back
through the cortex and, having differentiated into either CD4+ or CD8+ single-positive (SP) cells, into
the medulla. Positive selection occurs mainly in the cortex, whereas negative selection occurs mainly
in the medulla. SP cells that have completed the differentiation program egress from the medulla to the
periphery. CEC, cortical epithelial cell; MEC, medullary epithelial cell.

1961 Jacques Miller discovery of the role of the thymus in cellular immunity

page 321

Stoker M, Macpherson I.
Syrian hamster fibroblast
cell line BHK-21 and
its derivatives. Nature
1964;203:1355-1357.

Michael George Parke Stoker (1918-2013)

Michael Stoker was the first professor of virology at the University of Glasgow (the first
professorship of virology to be established at a British university), his tenure extending
from 1958 to 1968. It was during this time that he and Ian Macpherson developed the
BHK-21 cell line, one of the most widely used in virology. He also did seminal research on
tumor viruses and rickettsiae. He later became Director of the Imperial Cancer Research
Fund Laboratories, and subsequently President of Clare College, Cambridge. He was made
a CBE in 1974 and was knighted in 1980.
Ian Macpherson also worked with Stoker in the Department of Tumour Virology at the
Imperial Cancer Research Fund, London [now: Cancer Research UK] from 1963 to 1965.
While a faculty member of the Institute of Virology in Glasgow, he worked with Luc
Montagnier among other things, they developed the soft agar method for selectively
growing cancer cells.

BHK-21 cells in culture, stained with multiple fluorophors,

especially showing the cytoskeleton (actin filaments,
microtubules, etc.)

1961 Michael Stoker, Ian Macpherson development of BHK-21 cell line

page 322

Sydney Brenner

Francis Crick (1916-2004)

Crick FH, Barnett L, Brenner S, Watts-Tobin RJ. General nature
of the genetic code for proteins. Nature 1961;192:1227-1232.

In the early 1960s, it was thought that the genetic code consisted of units of 4 nucleotides, so as to code for
20 amino acids. Sydney Brenner postulated a triplet code based upon theoretical grounds. Francis Crick and
Sydney Brenner later wrote: In a long series of complex experiments, we induced mutations in the DNA
of bacteriophage T4. The mutations changed individual bases in the DNA, knocking out the function of a
crucial phage gene (the B cistron of the rII region). When two or four mutations were together, the gene was
still inactive, but when three mutations were put together in the same gene, the gene started to work again.
We concluded that the genetic code is a triplet code (three bases code for one amino-acid), and that the
code is degenerate (an amino-acid may be coded by more than one triplet of bases). Separately, we showed
that the code is non-overlapping. As indicated in the diagram, (a) single nucleotide deletions or insertions
cause frameshifts and single nucleotide insertions compensate for single nucleotide deletions. Two or four
single nucleotide insertions still give frameshifts, but (b) when there are a total of three nucleotide deletions
or insertions the code is left in frame. This work was done with John Griffith, Leslie Orgel, Margaret Leslie
Barnett, and Richard Watts-Tobin.

1961 Francis Crick, Sydney Brenner, others discovery of the triplet coding strategy of DNA (bacteriophage)

page 323

Autoradiograph of replicating DNA

of bacteriophage T2

John Cairns and Salvador Luria

at Cold Spring Harbor Laboratory, 1968

Bacteria infected with bacteriophage T2 were

labeled with tritiated thymidine (3H-TdR) and later
lysed. Insert: the circular phage DNA has two forks.
The segment represented by the double black line
had completed two replications in the presence of
3
H-TdR, whereas the segment represented by the red/
black line had replicated only once in the presence
of the 3H-TdR. The density of grains in the original
autoradiogram was twice as great in the segment
that had completed two cycles of replication in the
presence of 3H-TdR versus one cycle.

Many accounts of the history of molecular biology give the

impression that following the discovery of the double helix
structure of DNA, discovery-followed-discovery in short order.
In fact, as John Cairns stated later, many people found it hard to
imagine that biology could be reduced to something so simple. For
some ten years after the discovery of the DNA double helix much
research was done to link its structure to its functional activities
and its replication. In this setting, John Cairns set out to determine
the nature of the DNA of T2 bacteriophage. Two techniques were
available electron microscopy and autoradiography. Cairns
rejected the high resolution of the electron microscopic approach
because he presumed that intact DNA molecules would not fit
on a specimen carrier (grid). So, he turned to autoradiography,
a technique he had used previously. He grew bacteriophage in
the presence of tritiated thymidine, thereby labeling its DNA. He
affixed the bacteriophage DNA to glass microscope slides and
covered the spread out monolayer with stripping film gelatinbased photographic emulsion sensitive to the beta particles
released by the decay of tritium. The emulsion would record the
image of the DNA molecules after a sufficiently long exposure
(about 2 months). As Cairns admitted, the success of his method,
achieving full extension of DNA molecules was just good luck
(and many replicate experiments). Nevertheless, he found a
sufficient number of DNA molecules to indicate that its fully
extended length was 52m. From this, he calculated that the mass
of T2 DNA as 110x106 daltons. He postulated an uncomplicated
double helix, and a few years later showed that it was circular.
Cairns went on: the Watson-Crick model posed the problem of
how DNA strands could unwind and separate for replication,
without everything getting tangled. At the time, theoretical
considerations of unwinding exercised some of the best minds in
the science. Using the same approach, he showed that the DNA
of the bacteriophage T2 is circular, 700-900m long, and that its
replication produces a fork as the helix separates and each strand is
duplicated unwinding did not seem a problem. From: Witkowski
J. Illuminating life: selected papers from Cold Spring Harbor, Vol 1
(1903-1969). Cold Spring Harbor: Cold Spring Harbor Laboratory
Press; 1999.
Cairns, JP. An estimate of the length of the DNA molecule of T2
bacteriophage by autoradiography. J Mol Biol. 1961;3:756-761.
Cairns, JP. The chromosome of Escherichia coli. Cold Spring
Harbor Symposia on Quantitative Biology 1963;28:43-46.

1961 John Cairns discovery of the nature and length of the circular DNA of the bacteriophage T2

page 324

The first illustration ever published in MMWR, 1985

1961 U.S. Communicable Disease Center publication of CDCs Morbidity and Mortality Weekly Report (MMWR)

page 325

J. Heinrich Matthaei
and Marshall W. Nirenberg (1927-2010)

Har Gobind Khorana (1922-2011)

Robert W. Holley (1922-1993)

The genetic code is the set of rules by which the information encoded in DNA or mRNA is translated into
proteins by living cells. The code defines the relationship between tri-nucleotide sequences (codons), and
amino acids. The vast majority of genes contain exactly the same code/codons, although there are some
variants (e.g., mitochondria employ a genetic code that differs a bit from the standard code). The first
elucidation of a codon was done by Marshall Nirenberg and Heinrich J. Matthaei in 1961. They used a cellfree system to translate a poly-uracil RNA sequence (i.e., UUUUU...) and discovered that the polypeptide
that they had synthesized consisted of only the amino acid phenylalanine. They deduced that the codon
UUU specifies the amino acid phenylalanine. Nirenberg presented his results at the International
Biochemistry Congress in Moscow in 1961; his findings electrified the audience and led to great activity to
work out the full code. Severo Ochoa showed that the poly-adenine RNA sequence (AAAAA...) coded for
the polypeptide poly-lysine and the poly-cytosine RNA sequence (CCCCC...) coded for poly-proline: that
is, the codon AAA specifies the amino acid lysine, and the codon CCC specifies proline. Using different
copolymers and other kinds of translation experiments the remaining codons were then determined,
mostly be the work of Har Gobind Khorana. Shortly afterward, Robert Holley determined the structure
of transfer RNA (tRNA), the adapter molecule that facilitates the translation of mRNA into protein.
Nirenberg devised the simple but elegant diagram included here it shows the relationship between the
four nucleotides of DNA, taken three at a time, and the 20 amino acids commonly found in proteins.

1961> Marshall Nirenberg, Har Khorana, Robert Holley, colleagues deciphering of the genetic code
page 326

Jordi Casals
(1911-2004)

Richard Taylor
(1887-1981)

James Porterfield
(1924-2010)

Robert Shope
(1929-2004)

Charles Calisher

Until the 1980s, all the arboviruses obtained from clinical or field-collected samples that ended up in reference collections derived from rather standardized procedures: (1)
isolation of the virus in mice (infant mice) or cell culture; (2) production of a virus stock; (3) production of antigen(s); (4) preparation of antibody(ies); and (5) deposition of
the virus in reference collections. In the heyday of this activity (~1960-1975), accumulation of such reagents allowed reference centers to identify new viruses, and as well to
determine broad serologic/antigenic relationships and from these data build a classification system. In the course of this work, much information was gathered on phenotypic/
pathotypic characteristics and vector relationships of the viruses and clinical and epidemiologic information on the diseases they caused. All data were linked in a large
catalogue (the latest being Karabatsos N. International catalogue of arboviruses including certain other viruses of vertebrates, 3rd ed. San Antonio: American Society of Tropical
Medicine and Hygiene; 1985). As newer techniques (in particular, polymerase chain reaction and nucleic acid sequencing) were introduced in the 1980s it was remarkable how
well the serologic/antigenic classification system held up the basic structure of what became the taxa of today did not change, but were refined, made more precise. In fact,
the greater emphasis of the earlier arbovirus classification system on the most important human and animal pathogens made it less likely that any would disappear among the
huge numbers of viruses discovered by more modern means, most of which have turned out to be less important in infectious disease practice.
Casals J. Procedures for identification of arthropod-borne viruses. Bull World Health Organ. 1961;24:723-734.
Taylor RM. Purpose and progress in cataloguing and exchanging information on arthropod-borne viruses. Am J Trop Med Hyg. 1962;11:169-174.
Porterfield JS. The basis of arbovirus classification. Med Biol. 1975;53:400-405.
Calisher CH, Shope RE, Brandt W, Casals J, Karabatsos N, Murphy FA, Tesh RB, Wiebe ME. Proposed antigenic classification of registered arboviruses I. Togaviridae,
Alphavirus. Intervirology 1980;14:229-232.
Calisher CH, Karabatsos N. Arbovirus serogroups: definition and geographic distribution. In: Monath TP, editor. The arboviruses: epidemiology and ecology, vol. 1. Boca Raton
(FL): CRC Press; 1988. p. 19-58.
Calisher CH, Karabatsos N, Dalrymple JM, Shope RE, Porterfield JS, Westaway EG, Brandt WE. Antigenic relationships between flaviviruses as determined by crossneutralization tests with polyclonal antisera. J Gen Virol. 1989;70:37-43.
American Committee on Arthropod-borne Viruses, Subcommittee on InterRelationships Among Catalogued Arboviruses. Identification of arboviruses and certain rodentborne viruses: reevaluation of the paradigm. Emerg Infect Dis. 2001;7:756-758.

1961> Jordi Casals, Richard Taylor, Robert Shope, Charles Calisher, others antigenic classification of arboviruses

page 327

The Vero cell line was derived from the kidney of a

normal adult African green monkey (Chlorocebus
aethiops) in 1962, by Yosihiro Yasumura and Yosio
Kawakita, at Chiba University School of Medicine,
Japan. Yasumura wanted a cell line susceptible to
SV40 virus for virus-cancer studies the kidneys
of African green monkeys were commonly and
severely affected by SV40, so this seemed like
a logical starting point. Indeed, the cell line
supported SV40 virus growth to very high titer. The
cell line was supplied to the Chiba Serum Institute,
where it was investigated as a substrate for polio
vaccine production. Yasumura and Kawakitas
paper was not immediately translated into English
and so this incredibly valuable cell line remained
unknown in the west until 1964, when it was taken
to the laboratory of Ned Wiebenga at the NIH
by Bunsiti Simizu, and from there to the Middle
America Research Unit in the Panama Canal
Zone. Its first success there was in the growth of
Machupo virus, the etiologic agent of Bolivian
hemorrhagic fever. Subsequently, the cell line
proved valuable for the cultivation of many viruses:
adenoviruses, herpesviruses, influenza viruses,
paramyxoviruses, measles virus, respiratory
syncytial virus, picornaviruses, poxviruses,
reoviruses, rubella virus, and especially many
arboviruses. The name Vero comes from Verda
Reno meaning green (African green monkey)
and truth in Esperanto.
Yasumura Y, Kawakita Y. Studies of SV40 in tissue
culture. Preliminary steps for cancer research in
vitro. Nihon Rinsho (in Japanese) 1963;21:12011215. Translation in: Simizu B, Terasima T, editors.
Vero Cells Origins, Properties and Biomedical
Applications. Chiba, Japan: Chiba University
School of Medicine; 1988.

Vero cell monolayer, normal (top) and infected with herpes simplex virus,
resulting in a large syncytium (bottom). Phase-contrast green light microscopy

1962 Yosihiro Yasumura, Yosio Kawakita establishment of the Vero cell line
page 328

Earley EM, Johnson KM. The lineage of the Vero,

Vero 76 and its clone C1008 in the United States.
In Simizu B, Terasima T, editors. Vero Cells
Origins, Properties and Biomedical Applications.
Chiba, Japan: Chiba University School of Medicine;
1988.

Lizbeth M. Kraft (1920-2002)

Lizbeth Kraft was a true pioneer, specializing in veterinary microbiology and the diagnosis and prevention of
murine viral diseases. In 1958, she published a seminal paper on the natural history and control of epidemic
diarrhea of infant mice (EDIM, caused by a rotavirus), a disease then prevalent in mouse colonies. The control
concepts contained in this publication led to the invention of the filter-top for mouse cages (early-on referred
to as Kraft tops) that ever since have contributed significantly to the control of murine viral diseases.
In 1962, she described lethal intestinal virus of infant mice (LIVIM); she determined that the agent was
<300m in diameter and was sensitive to heat and ether, but she did not characterize it further. In following
years, several theories were advanced about the nature of the etiologic agent, but it was only in 1979 that
John Hierholzer, Roger Broderson and Frederick Murphy showed that the agent is an enteric strain of mouse
hepatitis virus. Mouse hepatitis virus strains/pathotypes are remarkably diverse the first to be discovered
caused encephalomyelitis in mice it was discovered by F. Sargent Cheever and his colleagues in 1949. Mouse
hepatitis virus represents the most important viral pathogen affecting contemporary mouse-based research.

Mouse hepatitis virus

mouse intestine, thin section electron microscopy

Kraft LM. An apparently new lethal virus disease of infant mice. Science 1962;137:282-283.
Cheever FS, Daniels JB, Pappenheimer AM, Bailey OT. A murine virus (JHM) causing disseminated
encephalomyelitis with extensive destruction of myelin. 1. Isolation and biological properties of the virus. J
Exp Med 1949;90:181-194.
Hierholzer JC, Broderson JR, Murphy FA. New strain of mouse hepatitis virus as the cause of lethal enteritis
in infant mice. Infect Immun. 1979;24:508-522.

1962 Lizbeth Kraft discovery of mouse hepatitis virus (coronavirus)

Mouse hepatitis virus

club-shaped epithelial syncytia at tips of intestinal villi,

H&E

page 329

Frontispiece, Cold
Spring Harbor
Symposium on
Quantitative Biology:
Basic Mechanisms in
Animal Virus Biology

Top row (left-to-right): I. Tamm, A. Lwoff, M. G.P. Stoker, R.

Dulbecco, A. Isaacs; F. Fenner, R. Franklin, D. Baltimore.
Second row: A. Granoff, L. Levintow, B. Roizman; D.L.D. Caspar, C.
Hotchkiss; F.L. Schaffer, B.C. Backus.
Third row: A. lsaacs, G.K. Hirst, H. Noll, P. Wildy; G. Klein, F. Fenner,
L. Frisch; B.R. Burmester, T. Hanafusa, B.L. Seecof.
Bottom row: P. Tournier, R.W. Simpson, W. Bernhard; H. Rubin; L.
Sachs, B. Ephrussi.

Top row: G.K. Hirst, H. Koprowski, H. Noll; Y. Ito; F.K. Sanders, C.

Morgan, P. Cooper, R.W. Schlesinger.
Second row: F.K. Sanders; J.S. Colter, T.S. Work; E. Winocour.
Third row: J.E. Hotchin; H.M. Temin, B. Ephrussi, G. Attardi; W.
Levinson, F. Fenner, A. Chovnick.
Bottom row: R. Franklin, P.I. Marcus, B.S. Spendlove, E. Wecker, D.
Baltimore, A.F. Graham; T. Hanafusa, D. Baltimore, W.K. Joklik, N.
Ledinko, J. Cairns.

The rather prosaic title of the 1962 Symposium belied the extraordinary nature of this meeting. The ability to grow viruses in animal cells in culture
had transformed the field [from the time of the 1953 meeting, which dealt mostly with bacteriophages], producing large amounts of virus for physical
and chemical analysis, as well as providing dynamic systems for the analysis of virus life cycles. Renato Dulbecco marveled that not only was it possible
to devote a whole symposium to viruses, but also that the experiments reported are as sophisticated as those obtained in any other field of biology...
Most of the papers provided descriptions of the various virus-cell combinations being studied. The authors of these papers read like a roll call of those
who would lay the foundations of virology in the coming years There were two papers added to the original program for the meeting. One was a short,
one-page document by Caspar, Dulbecco, Klug, Lwoff, Stoker, Tournier and Wildy proposing and defining a set of terms for use in virology [virion,
capsid, capsomere, nucleocapsid, peplomer, envelope, etc.]. The second was an attempt by Lwoff to devise a standardized and rational classification of
viruses We can see, with the wisdom that comes with hindsight, that this Symposium leaves us with tantalizing glimpses of how research on viruses
was going to progress. From Jan Witkowski, in the prospectus for the volume, Cold Spring Harbor Laboratory Press.

1962 Cold Spring Harbor Symposia on Quantitative Biology: Basic Mechanisms in Animal Virus Biology
page 330

Lwoff A, Tournier P. The classification of viruses.

Annu Rev Microbiol. 1966;20:45-74.

Andre Lwoff: My ambition is to show that the word

virus has meaning Frenchmen, said Paul Valry, deem
possible that a prodigiously diverse ensemble of highly
complex phenomena can be and must be condensed and
finally reduced into a few plain formulae, at the same time
necessary and sufficient. Belonging to an hyperlogical
extrovert nation, I have coined numerous definitions as if
I had really penetrated the essence of things. [So,] I shall
defend a paradoxical viewpoint, namely that viruses are
viruses.
The qualities of the viruses have been used to argue the
question, Are viruses alive? One adroit answer is to
envision viruses, at the edge of life (i.e., in some ways
fulfilling the criteria we use to define life, in some ways not)
note that this notion does not indicate which side of the
edge the viruses sit on!
Lwoff A, Horne R, Tournier P. A system of viruses. Cold
Spring Harb Symp Quant Biol. 1962;27:51-55 and Caspar
DL, Dulbecco R, Klug A, Lwoff A, Stoker MG, Tournier P,
Wildy P. Proposals. Ibid. 1962;27:49-50.
Wilson EB. At the Edge of Life: An Introduction to Viruses.
Report from the National Institute of Allergy and Infectious
Diseases. NIH publication 80-433; 1980.

Andr Lwoff (1902-1994)

Andr Lwoff, Robert Horne, Paul
Tournier:
The world, said Paul Valry, is equally
threatened with two catastrophes: order
and disorder. So is virology. Whether
the proposed system will decrease
or increase the entropy of virology
is not yet clear. The system has not,
however, reached the degree of rigidity
or perfection corresponding to a real
catastrophe.

Paul Tournier ( -2004)

at Cold Spring Harbor, 1962

1962 Andr Lwoff, Robert Horne, Paul Tournier first classification of viruses based upon virion characteristics

page 331

Aaron Klug

Donald Caspar

holding Keplers platonic model of the solar

system from his Mysterium Cosmigraphicum

Caspar DLD, Klug A. Physical principles in the

construction of regular viruses. Cold Spring Harbor
Symposium on Quantitative Biology 1962;27:1-24.

Francis Crick, Donald Caspar, Aaron Klug

and Rosalind Franklin, 1955

Donald Caspar: In 1956, Crick and Watson, at Cambridge, predicted that isometric virus particles should be
constructed from identical subunits arranged with cubic symmetry. From the same laboratory, I reported X-ray
crystallographic data providing the first evidence for icosahedral virus symmetry [tomato bushy stunt virus]. Aaron
Klug and his colleagues in London soon showed that other isometric viruses were icosahedral, and suggested
that some general principle might explain a preference for icosahedral symmetry. I began a collaboration with
Klug in 1958 The problem was to explain how to build the virus shells from a large number of identical units
by repeating the same pattern of contact without the constraint of strict equivalence. A key to our solution was
Klugs recognition of an analogy with Buckminster Fullers geodesic dome designs. In 1962, Klug asked me to
join him in writing a paper for a Cold Spring Harbor Symposium that was intended as an introduction to our
major collaborative paper [We] ended up incorporating much of the theory that the major paper was to have
introduced. They never completed the major paper. We introduced three terms in our Citation Classic that
have been useful for describing a variety of structures: triangulation number, to define the possible icosahedral
surface lattice designs; quasi-equivalence, to describe nearly equivalent bonding of identical units; and selfassembly, to identify assembly processes controlled by the specific bonding of the parts Our quasi-equivalence
theory explained icosahedral symmetry and enumerated the possible designs. The beauty of this theory and the
success of the predictions made it appear that conservation of bonding specificity was a necessity in icosahedral
virus architecture. [An exception was found much later, in Caspars lab polyoma virus assembly does not follow
the original rules.]

1962 Aaron Klug, Donald Caspar discovery of the principles of the icosahedral structure of viruses
page 332

John J. Trentin (1918-2005)

Human adenovirus 5 in paracrystalline array

nucleus of a cell in culture, thin section electron microscopy

From the 1962 National Cancer Institute Annual Report: The finding of John J. Trentin, Yoshiro Yabe and Grant Taylor at Baylor College
of Medicine, that human adenovirus type 12 induces tumors in hamsters (confirmed by Robert Huebner and Wallace Rowe along with the
demonstration that adenovirus type 18 also is oncogenic in hamsters), together with the confirmation of the oncogenic nature of SV40 virus
and the further characterization of its properties in tissue culture (e.g., growth of virus and transformation with chromosomal changes in
human cells in culture), and the delineation of the nature of the papovaviruses (especially by Joseph Melnick, Hilary Koprowski, and Maurice
Hilleman) represent important highlights of ongoing cancer virus research. In fact, these findings were the hottest news in oncology in the
early 1960s. It was thought that the quest for the cause of human cancers was within reach and that prevention and treatment would follow
directly.
Trentin JJ, Yabe Y, Taylor G. The quest for human cancer viruses. A new approach to an old problem reveals cancer induction in hamsters by
human adenovirus. Science 1962;137:835-841.

1962 John Trentin, colleagues discovery of the induction of tumors in hamsters by human adenoviruses

page 333

Albert S. Cosgrove (1922-2002) & Hiram N. Lasher (1920-2012)

Infectious bursal disease virus

negative contrast electron microscopy

In 1957, Albert Cosgrove, while working with Hiram Lasher at the Delaware State Poultry Laboratories, recognized a syndrome
later termed avian nephrosis on a broiler farm near the community of Gumboro, Delaware. The syndrome became known as
Gumboro disease and was characterized by kidney pathology and 1-10% mortality infection also caused immunosuppression
associated with chronic infection. Within three years the disease was recognized throughout the southern United States and
then around the world. The etiologic agent was thought to be a variant of infectious bronchitis virus (coronavirus) and in other
instances it was thought to be a picornavirus or an adenovirus or a reovirus-orbivirus. Studies to characterize the etiologic agent
began in 1962 at the University of Delaware and Auburn University, but it was not until 1976 that H. Nick, D. Cursiefen, and H.
Becht of the Institut fr Virologie, Justus Liebig-Universitt, Giessen, Germany characterized the virus well enough to conclude
that it could not be placed into any recognized taxon. They found that virions were nonenveloped icosahedra, 60nm in diameter,
and had two segments of double-stranded RNA. The virus became the prototype of a new family, the Birnaviridae. The family
now contains many viruses, including several from fish, molluscs, insects and birds.
Cosgrove AS. An apparently new disease of chickens avian nephrosis. Avian Dis. 6:385-389. 1962.
Lasher HN, Davis VS. History of infectious bursal disease in the USA The first two decades. Avian Dis. 1997;41:11-19.
Nick H, Cursiefen D, Becht H. Structural and growth characteristics of infectious bursal disease virus. J Virol. 1976;18:227-234.

1962 Albert Cosgrove, Hiram Lasher discovery of infectious bursal disease virus (the first birnavirus)

page 334

(Right) Rate-zonal vs. density

gradient ultracentrifugation:
1. soluble proteins
2. rough membranes
3. smooth membranes
4. mitochondria
5. nuclei
6. ribosomes and polysomes
7. glycogen
8. RNA
9. poliovirus
10. T3 bacteriophage
11. adenovirus
Norman Anderson developed a
combined rate-zonal / density
gradient ultracentrifugation
system that took advantage of
the unique windows in which
various cell organelles fell and
which could be employed in
separation schemes. He found
that viruses fall in a rather clear
space, the virus window.
Anderson NG. Virus isolation in
the zonal ultracentrifuge. Nature
1963;199:1166-1168.

Norman G. Anderson (1919-2007)

Anderson NG. Zonal centrifuges

and other separation systems.
Science 1966;154:103-112.

Top and side

section views
of a zonal rotor
showing
loading,
separation
of virus from
contaminants,
and unloading

Norman Anderson invented the zonal ultracentrifuge at Oak Ridge National Laboratory. His rotors were large, hollow, bowl-shaped chambers. Density gradient forming media
and virus-containing samples were caused to flow into a rotor spinning at low speed (3,000 rpm) and then it was accelerated to maximum speed to effect separation based on
either sedimentation rate or density (or both). This was followed by deceleration to low speed and recovery by displacement of the gradient as isolated fractions. Anderson
took advantage of the expertise at Oak Ridge derived from uranium enrichment in huge gas centrifuges: We built (and sometimes blew up) a lot of centrifuges, but our
rotors became progressively better at separating biological materials. In the early 1960s, Anderson turned to purifying viruses with his ultracentrifuges in collaboration with
Robert Huebner and his human cancer virus program. Anderson thought that the same approach could be used for the purification of human vaccines: Testing these systems
required large quantities of virus [we obtained] a batch of polio vaccine that did not meet FDA standards As it became clear that no human cancer viruses were being
found around which to design a vaccine purification system, I decided that we should work on an existing vaccine that required better purification. At that time, egg-grown
influenza vaccines contained appreciable amounts of egg proteins We approached Eli Lilly about designing a centrifugal system specifically to purify influenza vaccine. The
result was the K-II continuous-sample-flow-with-banding ultracentrifuge. Use of this centrifuge essentially eliminated vaccination deaths from anaphylactic shock and allowed
vaccination under minimal medical supervision. Almost 40 years later, the K-II is still in use for vaccine manufacture around the world, with minimal modifications.

1962 Norman Anderson invention of rate-zonal ultracentrifugation

page 335

Karl-Eduard Schneweis

Andr J. Nahmias

After the discovery of herpes simplex

virus in 1919, a number of studies
on genital herpes were carried out
by several European virologists.
On the basis of both clinical and
laboratory studies, it was suggested
that what was called herpes febrilis
was caused by a different virus than
herpes genitalis. However, this work
was largely ignored for many years.
It was only in the 1960s that it was
demonstrated conclusively that
most isolates from genitalia differ
antigenically and biologically from
most nongenital strains. Today, the
viruses can be distinguished by a
variety of laboratory techniques.
Viral isolates commonly recovered
from nongenital sites are now
designated herpes simplex virus1
(HSV1) (5 to 10% of genital
infections are caused by HSV1).
This virus causes fever blisters
(cold sores), gingivostomatitis,
keratoconjunctivitis, encephalitis,
Walter Reed Dowdle
and skin eruptions above the waist.
The virus associated primarily with
infections of the urogenital organs
is designated herpes simplex virus
2 (HSV2). The recovery of HSV1
mainly from upper body sites and
HSV2 from lower body sites is most
likely a reflection of the principal
mode of transmission of each virus;
however, recent studies suggest that
the two viruses have also evolved to
match the microenvironment of each
target organ/tissue.
Schneweis KE. Serologische untersuchungen zur typendifferen zierung des
herpesvirus hominis. Z Immunitaetsforsch. 1962;124:24-48.
Dowdle WR, Nahmias AJ, Harwell RW, Pauls FP. Association of antigenic types of
herpesvirus hominis with site of viral recovery. J Immunol. 1967;99:974-980.

Herpes simplex virus, cell culture, thin section electron microscopy

Nahmias AJ, Dowdle WR. Antigenic and biological differences in herpesvirus

hominis. Prog Med Virol. 1968;10: 110-159.

1962> Karl-Eduard Schneweis, Walter Dowdle, Andr Nahmias differentiation of herpes simplex viruses 1 and 2

page 336

William Graeme Laver (1929-2008)

Robert G. (Rob) Webster

Graeme Laver and Rob Websters work with avian influenza viruses can be said to have sprung from a
beach walk, during which they noticed a large number of dead birds along the shoreline. They wondered
whether it was possible that the birds had died from avian flu, and subsequently traveled to a Great Barrier
Reef atoll to collect samples from hundreds of birds. This led to more trips, trips that became famous in
Australian virology circles, and eventually they discovered the genetic link between avian flu and human
flu viruses. They saw the link as also suggesting the possibility that the viruses might reassort and yield
new viruses to which human populations had no immunity. Years later, Webster noted that when they
first found that there was a link, few virologists paid any attention to what they saw as a great danger.
They theorized that pigs and ducks would be fine mixing vessels and then showed that important
reassortment events do occur this way in nature. In recent years the complexity of the reassortment
events, over time and place, has been proven over and over.
Webster RG, Laver WG. Studies on the origin of pandemic influenza. I. Antigenic analysis of A2 influenza
viruses isolated before and after the appearance of Hong Kong influenza using antisera to the isolated
hemagglutinin subunits. Virology 1972;48:433-444; and Laver WG, Webster RG. II. Peptide maps of the
light and heavy polypeptide chains from the hemagglutinin subunits of A2 influenza viruses isolated
before and after the appearance of Hong Kong influenza. Virology 1972;48:445-455.

1962> Robert Webster, Graeme Laver discovery of the link between avian and human influenza viruses

page 337

The 2008 Nobel Prize

in Chemistry was
awarded to
Osamu Shimomura,
Martin Chalfie and
Roger Y. Tsien
for their earlier
discovery and
development
of green fluorescent
protein, GFP

(Below) Neurons, mouse

hippocampus, fluorescing
in many colors via three
GFP-tagged proteins,
allowing tracing of
individual neuronal
processes.Jean Livet, Ryan
Draft, et al.

Osamu Shimomura

Martin Chalfie

Intact brain of mouse infected with recombinant Semliki Forest virus

carrying a GFP marker protein. John Fazakerley, et al.

Roger Y. Tsien

Rabies virus carrying GFP gene used to trace the connections of a

single neuron in mouse brain. K-K Conzelmann.

1962> Osamu Shimomura, Martin Chalfie, Roger Tsien green fluorescent protein and its use as a tagging tool
page 338

Peter J. Gomatos (1929-2012)

Igor Tamm (1922-1995)

Reovirus 3, double-stranded RNA

In the early 1960s, Peter Gomatos and Igor Tamm were studying reoviruses, about which little was known other
than that they were found in the respiratory tracts of humans and animals. In 1963, they made the discovery that the
genetic material of these viruses consists of double-stranded RNA. This was the first description of double-stranded
RNA in any biological system, and Gomatos and Tamm soon found double-stranded RNA in another virus, wound
tumor virus of plants. Shortly thereafter, the laboratories of Albrecht Kleinschmidt and of Aaron Shatkin presented
evidence that the reovirus genome comprises several genome segments. Subsequently, many other viruses causing
diseases in humans, animals, insects, and plants were found by various investigators to have segmented doublestranded RNA as their genetic material and the several genera of the family Reoviridae were thus created.
Gomatos PJ, Tamm I. Animal and plant viruses with double-helical RNA. Proc Natl Acad Sci USA. 1963;50:878-885.
Langridge R, Gomatos PJ. The structure of RNA. Science 1963;141:694-698.

1963 Peter Gomatos, Igor Tamm discovery of double-stranded RNA in a virus (reovirus)

page 339

John Franklin Enders

(1897-1985)

Thomas Chalmers Peebles

(1921-2010)

First International Conference on

Measles Immunization, 1961.
L-R: Samuel Katz, Ann Holloway,
Kevin McCarthy, Anna Mitus,
Milan Milovanovic, John Enders,
Gisele Ruckle, Frederick Robbins,
Ikuyu Nagata.

Following their initial isolation of measles virus in cell culture in 1954, investigators in the laboratory of John Enders at Harvard
conducted the research, development, and initial clinical studies responsible for the licensure in 1963 of a successful attenuated
live-virus measles vaccine. The team included Thomas C. Peebles, Samuel Katz (who spent 12 years in the Enders lab), Kevin
McCarthy, Milan Milovanovic, Anna Mitus, Gisele Ruckle and Ann Holloway. Also in the lab during this period were Carleton
Gadjusek, Ian Gresser and George Miller.
The virus was isolated from blood and throat washings of David Edmonston, age 11, son of a mathematician in Bethesda,
Maryland. Propagation of the virus successively in human kidney cells, human amnion cells, embryonating hens eggs, and
finally chick embryo cell cultures selected for an attenuated variant virus that when inoculated into susceptible monkeys by
Kevin McCarthy proved immunogenic without causing viremia or overt disease (in contrast to an early kidney cell-passaged
virus, which in similar monkeys produced viremia with illness mimicking human measles).
Clinical studies in children by the Enders group and then by collaborating investigators in many sites established the safety,
immunogenicity and efficacy of the vaccine. The Edmonston strain measles virus became the progenitor of vaccines used
throughout the United States and many other countries.
Katz SL. John F. Enders and measles virus vaccinea reminiscence. Current Topics in Microbiology and Immunology
2009;329:3-11.

Samuel L. Katz

1963 John Enders, Thomas Peebles, Samuel Katz, Kevin McCarthy, colleagues development of measles vaccine

page 340

Maurice Hilleman
(1919-2005)

Harry Meyer Jr. (1928-2001)

and Paul Parkman

Stanley Plotkin

Michiaki Takahashi

Vaccine coverage, preschool age children, U.S.

Anne Gershon

Ann Arvin

Walter A. Orenstein

When the U.S. Department of Health and Human Services highlighted the top 10 achievements in public health in the
20th century, vaccination was listed first. This success was the result of major scientific discoveries, collaboration between
the public and private sectors of medicine and public health, initiatives of pharmaceutical companies, and the dedication
of numerous people in the immunization community. The global situation is also worthy of much praise. This subject is
too large to be covered here, but as a foundation subject in the history of virology it deserves much prominence.

1963> Maurice Hilleman, others development, promulgation and commercialization of pediatric viral vaccines

page 341

Wilbur George Downs

(1913-1991)

Thomas H. G. Aitken
(1913-2007)

The Trinidad Regional Virus Laboratory was established in Port of Spain in 1953 by
the Rockefeller Foundation. The labs first director was Wilbur Downs who served
until 1961; he was succeeded by Leslie Spence. Large-scale surveys were conducted,
searching for viruses in humans as well as domestic and wild animals and arthropods.
At the time the lab was established there were a number of common but unidentified
infectious diseases seen in Trinidad; over the years the etiologic agents of many of
these were found. A semi-permanent bush camp was set up at Bush Bush Wildlife
Sanctuary in the Nariva Swamp in southeastern Trinidad and a large tower was built
in the Vega de Oropouche rainforest near Sangre Grande, with platforms at 60, 90
and 120 feet, to the top of the forest canopy. From early on there was a great interest
in bats H. Metivier and J. L. Pawan were early investigators of bat rabies, especially
vampire bat rabies. A government program to control vampire bats by shooting,
netting and trapping started in 1931. This program provided the lab with a steady
supply of correctly identified bats belonging to many different genera and species 58
bat species were recognized in Trinidad at the time. In the course of the bat rabies
research program in the lab, Tacaribe virus was discovered in 1956 and described in the
literature in 1963. It was isolated from Artibeus spp. bats (the genus comprises the fruit
bats of the Americas) and from mosquitoes. It was at first thought to be an arbovirus,
but in an addendum to the first paper it was noted that Robert Shope had just shown
the virus to be antigenically related to Junin virus, the etiologic agent of Argentine
hemorrhagic fever. Tacaribe virus became a charter member of the family Arenaviridae
when it was created in 1970.
Downs WG, Anderson TR, Spence L,
Aitken THG, Greenhall AH. Tacaribe
virus, a new agent isolated from Artibeus
bats and mosquitoes in Trinidad, West
Indies. Am J Trop Med Hyg.
1963;12:640-646.
Tacaribe virus
Vero cells
1960s

Charles R. Anderson
(1915-1984)

Leslie Spence

Wilbur Downs, field work,

Trinidad, 1950s

1963 Wilbur Downs, Charles Anderson, Leslie Spence Tacaribe virus (the first western hemisphere arenavirus)
page 342

From 1928 onward, there were reports from

New South Wales, Australia of outbreaks of
an unusual syndrome marked by rash, painful
arthralgia and arthritis it came to be called
epidemic polyarthritis (a syndrome of pain, skin
eruption and general manifestations in which
painful swelling of the joints predominated).
Fifteen years later, during the Second World
War, several outbreaks of arthralgia and arthritis
were described in the Northern Territory,
Queensland, and islands off the northern coast
of Papua New Guinea. In 1960, Robert Shope
and S. Gray Anderson noted the similarity of
the outbreaks to an outbreak of acute viral
polyarthritis caused by Chikungunya virus in
Tanganyika (now Tanzania) in 1952. Differences
were also noted, particularly the sudden onset of
severe arthritis and fever followed by rash in the
African outbreak, contrasted with the gradual
onset of mild symptoms in the Australian
outbreak, with fever rarely noted and mild when
present. Because Chikungunya was known to
be a group A arbovirus (now a member of the
genus Alphavirus), Shope and Anderson did
serologic studies that in turn indicated that the
causative virus was an unknown alphavirus.
Next, in 1963 Ralph Doherty and his colleagues
at the Queensland Institute of Medical Research
isolated an alphavirus from mosquitoes trapped
beside the Ross River in northern Queensland
and identified it as the virus that Shope and
Anderson had predictedthey named the
new virus Ross River virus. Ever since, it has
been recognized as the cause of outbreaks of
polyarthritis in many locations in Australia and
several south Pacific islands.
Shope RE, Anderson SG. The virus aetiology
of epidemic exanthem and polyarthritis. Med J
Aust. 1960;1:156-158.
Doherty RL, Whitehead RH, Gorman BM,
OGower AK. The isolation of a third group
A arbovirus in Australia with preliminary
observations on its relationship to epidemic
polyarthritis. Aust J Sci. 1963;26:183-184.

Ralph L. Doherty

Robert E. Shope (1929-2004)

The Queensland Institute of

Medical Researchs first facility
was Shed 8, also known as
MacArthurs Morgue, when
it was built for the US Army
during WWII. It was meant to
be temporary, but was used as a
virology lab for 30 years

1963 Ralph Doherty, Robert Shope, colleagues discovery of Ross River virus

page 343

Seiichi Matsumoto (1918-1983)

Rabies virus, mouse neuron, thin section electron microscopy, 1961

After their first description by Adelchi Negri in 1903, the nature of the prominent intracytoplasmic inclusion bodies (Negri bodies) seen in rabies virus infected neurons was
intensely questioned, early on based upon some quite bizarre notions. That they are virus-specific was first shown using immunofluorescence microscopy in 1959. Some of
the first attempts to resolve the question of their nature using electron microscopy also led to bizarre, incorrect notions. This was all settled by the beautiful work of Seiichi
Matsumoto and Kaneatsu Miyamoto, who in the early 1960s studied infections in mice caused by street (Pasteurs term for wild type virus) and fixed (laboratory adapted) virus
strains. They showed that the eosinophilic (pink/magenta) matrix of Negri bodies is made up of masses of excess viral nucleocapsid, at first rather loosely aggregated (hardly
visible by light microscopy) and later densely packed (the classic inclusions seen by pathologists). The basophilic (blue) large and small granules seen within matrices were seen
to be entrapped masses of virus and degenerated cellular organelles. They also studied the ultrastructural pathology of street vs. fixed virus strain infections and showed that
the main difference was in the cellular damage that occurs with fixed viruses, rather than anything fundamentally different in viral morphogenesis. Everyone who has followed
in the study of rabies pathogenesis using the magnification afforded by the electron microscope has marveled at the terrible beauty of the infection, especially in the largest
neurons in the nervous system.
Matsumoto S. Electron microscope studies of rabies virus in mouse brain. J Cell Biol. 1963;19:565-591.
Miyamoto K, Matsumoto S. The nature of the Negri body. J Cell Biol. 1965;27:677-682;
and Comparative studies between the pathogenesis of street and fixed rabies virus infection. J Exp Med. 1967;125:447-456.

1963 Seiichi Matsumoto, Kaneatsu Miyamoto morphology/morphogenesis of rabies virus, nature of the Negri body
page 344

Michael Anthony

Anthony Epstein (abridged): In 1961, Denis Burkitt came to London

and gave his first talk outside Africa at Middlesex Hospital on the new
lymphoma, entitled The commonest childrens cancer in tropical Africa. A
hitherto unrecognized syndrome. I saw the title on a notice board and for
reasons I am to this day unsure about, I attended and was electrified from
the start by the strange nature and rapid progression of the tumour, and
its epidemiology which seemed to show that its distribution depended on
temperature and rainfall. I postulated that a climate-dependent arthropod
vector might be involved and resolved to investigate the tumour for unusual
viruses. I began with discussions with Denis Burkitt; it was agreed he would
make biopsy samples available to me. For almost two years the standard
techniques for virus detection current at the time were applied to the
lymphoma samples with uniformly negative results. I recruited Yvonne
Barr and Bert Achong to join the project in 1963; that year, a biopsy sample
arrived as usual, floating in guinea pig serum transit fluid, but unusual in
that the fluid was cloudy. At this point there was a feeling that we could
leave the lab for the weekend since it was assumed that the specimen was
contaminated. But instead of going home I put a drop of the transit fluid on
a slide and examined it as a wet preparation under the microscope. Rather
than seeing bacteria I was astonished to find that the cloudiness was due
to large numbers of round, viable looking, free-floating tumour cells. The
cells were set up in suspension culture: shortly afterwards the first BLderived continuous cell line designated EB1 grew out. As soon as material
could be spared from the BL-derived cell line, it was processed for electron
microscopy. I was exhilarated to observe unequivocal virus particles in the
very first grid square to be searched. I was extremely agitated in case the
specimen might burn up in the electron beam; I therefore switched off,
walked round the block in the snow wearing only my lab coat, and returned
only when somewhat calmer to take electron micrographs of what I had
seen. I recognized the virus at once as having the morphology of the herpes
group with which I was familiar. There was no means of knowing which
Bert Achong
Epstein
herpesvirus it was, although it did seem extraordinary that a member of the
(1928-1996)
herpes family was producing virions in a cell line but was so biologically
Yvonne Barr
inert as not to be causing destruction of the cultures. It was indeed fortunate
that the work on BL cells and the search for a virus was going forward in
Epstein MA, Achong BG, Barr YM. Virus
a laboratory where the electron microscope, a rather rare tool at the time,
particles in cultured lymphoblasts from Burkitts
was in daily use, as otherwise its biological inertness could well have left the
lymphoma. Lancet 1964;1:702-703.
agent undiscovered. In this connection it is worth noting that EBV appears
Epstein MA. The origins of EBV research:
to be the first virus to have been found solely by electron microscopy.
discovery and characterization of the virus. In:
I rapidly set about reporting the discovery with Dr. Achong and Miss
Robertson ES, editor. Epstein-Barr Virus. Saffron Barr the paper became a Citation Classic in 1979. It became imperative
Walden, UK: Horizon Scientific Press; 2005.
to have our work repeated in some other laboratory; two leading British
herpes virologists were approached but neither was interested. I therefore
Human lymphoid cells, EBV capsid
contacted Werner and Gertrude Henle in Philadelphia, thereby initiating
antigen immunofluorescence,
their long-term contributions to our understanding of EBV.

from Paul Feorino, CDC

1964 Michael Epstein, Bert Achong, Yvonne Barr Epstein-Barr virus and its association with Burkitts lymphoma

page 345

William F. H. Jarrett (1928-2011)

Citation Classic (Current Contents 1990;33:24-25): Feline leukaemia was shown to

be transmissible experimentally in cats using cell-free extracts of lymphosarcoma
tissue from a spontaneous field case This was the first transmission of a spontaneous
mammalian leukaemia. William Jarrett: From the start of this century, it had been
known that fowl leukaemia was transmissible and caused by a virus. Somehow this
finding was not thought to be germane to mammals... I had worked for some years
in human pathology and had become interested in haematopoietic tumours. When I
returned to veterinary hospital work, I was surprised to find so many neoplasms of this
type, and I was prompted to carry out a survey in Glasgow. Surprisingly, this showed
that leukaemia appeared to be about five times more common in cats than in humans.
I therefore cooperated closely with a local veterinarian, Harry Pfaff [We] started to
find as many of these as possible in order to get fresh material for passage, electron
microscopy, and culture. Here we had a stroke of luck. One of his clients was a dear,
but slightly crazy, old lady who lived alone in a large house with 100 cats. She had
taken these in, off the streets, as strays. Her feline philanthropy ran to bed and board,
but not to sex. So she had my friend neuter them all, toms and tabbies alike. This was
tough for the cats but excellent for us as it meant that all of these animals were outbred
and unrelated. In the course of a year or so, he submitted to me eight cases of different
kinds of leukaemia from this household. This was very striking evidence for horizontal
transmission of an infection, and so I decided to set up a transmission experiment
from this source material We had no grant and no other money. Mary F. Stewart, a
veterinary surgeon from Cornell University, had recently settled in Scotland (because of
a man and mountaineering) and had an isolated cottage in the middle of a golf course.
She was keen to look after the cats (for free) As the first year was drawing to a close,
we wondered if we had failed, when suddenly a cat died, but the material was unsuitable
for passage. Shortly afterwards, another cat became ill and this time we diagnosed
leukaemia on a routine blood count. Material was taken from this animal and passaged
again. I was nail-biting for several days as I had recently been given access for the first
time to an electron microscope. Instructed by Elizabeth M. Crawford, my technician
and I prepared the cat material, cut sections, and popped it in the microscope. We
found the virus almost at once, and it was one of the great moments of our lives. Despite
the relative crudity of the techniques at that time, there it was, an incontrovertible
leukaemia virus of perfect morphology, two virions in the first picture showing as much
detail as they ever have since. It is one of my prized possessions still. Soon after that my
colleague Bill Martin and I grew tumour cells in culture and isolated virus from that
After this, I was joined by my brother Oswald, who has since become one of the main
figures in this field. The field study that we immediately carried out showed for the first
time that feline leukaemia-virus infection was widespread in a large city and low on
country farms and that the lower the socioeconomic group the cats were in, the higher
the incidence. But that is another story....
Jarrett WFH, Martin WB, Crighton GW, Dalton RG, Stewart MF. Leukaemia in the cat:
transmission experiments with leukaemia (lymphosarcoma). Nature 1964;202:566-567.
Jarrett WFH, Crawford EM, Martin WB, Davie F. Leukaemia in the cat: a virus-like
particle associated with leukaemia (lymphosarcoma). Nature 1964;202:567-568.

1964 William Jarrett, colleagues discovery of feline leukemia virus

page 346

Karl M. Johnson

Patricia Webb (1925-2005)

Machupo virus

In 1962, Karl Johnson and Ron MacKenzie of the Middle America Research
Unit (a unit of the NIH), located in the Panama Canal Zone, traveled to Bolivia,
and ended up investigating a disease killing people in the village of Magdalena.
They immediately found ten patients in hospital suffering from a hemorrhagic
fever. They returned with a larger research team and with biocontainment
equipment and soon determined that the location of the outbreak was actually
the village of San Jouquin. It was later estimated that there were 470 cases and
192 deaths in the area between 1959 and 1965. The work was cut short when
MacKenzie and Johnson developed hemorrhagic fever; they were treated in the
Canal Zone by a physician who had earlier treated Korean hemorrhagic fever
cases. Upon their recovery and presumption that they were now immune, they
returned and working with Merle Kuns unraveled the source of the virus (food
contaminated by rodent droppings and urine), the reservoir host the rodent
Calomys callosus and the natural history of the virus. Mouse traps proved
to be an effective control measure. Back in the Canal zone, Patricia Webb,
characterized the virus, named Machupo virus, showing that it is a member of
the family Arenaviridae.

Ronald MacKenzie

Johnson KM. Epidemiology of Machupo virus infection. III. Significance of

virological observations in man and animals. Am J Trop Med Hyg.
1965;14:816-818.
Webb PA. Properties of Machupo virus. Am J Trop Med Hyg. 1965;14:799-802.

Scenes from Bolivia, 1963, from Karl Johnson

1964 Karl Johnson, Patricia Webb, others Machupo virus, the etiologic agent of Bolivian hemorrhagic fever

page 347

Michel Bouteille

John L. Sever
Subacute sclerosing panencephalitis

(massed measles virus nucleocapsids in the nucleus of a neuron)

Subacute sclerosing panencephalitis (SSPE), a slowly progressive usually fatal disease, was suspected of having a viral etiology from the outset because the microscopic lesions
seen are rather like those produced by several viral infections. Over the years there were many reports of the isolation of various viruses and several unidentified agents. Then,
in 1965, measles or a measles-like virus was implicated not by virus isolation or immunologic means, but by electron-microscopy: Michel Bouteille and his colleagues, of the
Hpitaux de Paris, Institut National de la Recherche Scientifique, described in the brain of a patient particles that resembled the helical nucleocapsids of morbilliviruses. This
finding was confirmed quickly. In 1967, John Connolly and his colleagues reported the finding of astonishingly high levels of measles virus antibody in the sera of patients
with SSPE, as well as the presence of specific immunofluorescent staining for measles virus antigen in brain. Incontrovertible evidence for the role of measles virus came when
Joseph Baublis and Francis Payne maintained brain cells from a patient in culture and demonstrated the development of syncytia and inclusion bodies, and the presence of
measles antigen. However, antigen and cytopathological changes could be passed only with intact cells, and extra-cellular measles virus could not be recovered until HortaBarbosa and his colleagues passaged the cells multiple times followed by their co-culture with HeLa cells the basis for this result was later found to be a complex pattern of
defectiveness of the virus. PCR techniques and in situ RNA hybridization have in recent years further substantiated the etiologic role of measles virus in SSPE. Genetic studies
have supported epidemiologic evidence that measles vaccine virus does not cause SSPE. When diagnosed early, SSPE is treated with some success with interferon- and
inosine pranobex (mimics immune-stimulating hormones).
Bouteille MC, Fontaine C, Vedrenne CI, Delarue J. Sur un cas dencephalite subaigue a inclusions, fitude anatomo-clinique et ultrastructurale. Rev Neurol. 1965;113:454-458.
Connolly JH, Allen IV, Hurwitz LJ, Millar JHD. Measles-virus antibody and antigen in subacute sclerosing panencephalitis. Lancet 1967;1:542-544.
Sever JL, Zeman W. Measles virus and subacute sclerosing panencephalitis. Neurology 1968;18(Part 2):1-192.

1965 Michel Bouteille, John Sever, others etiology of subacute sclerosing panencephalitis (measles virus)

page 348

Harvey J. Alter

Baruch Blumberg
(1925-2011)

Hepatitis B virus

negative contrast electron microscopy

In 1963, while at NIH working with Harvey Alter, Baruch Blumberg discovered Australia antigen (Au), that is an antigen that precipitated in gel when reacted with antibody
in sera from some transfused patients and hemophiliacs. The antigen was named because the first reaction was with the serum of an Australian aborigine. Blumberg went
to Western Australia to collect and test a large number of additional sera, at the time not knowing why the precipitin band had developed between the sera of a hemophiliac
from New York and an antigen from an aborigine from Australia. He had no set views on where this path might lead and later admitted that he had no reason to think of
an association of Au with hepatitis. Using large numbers of stored serum samples from around the world, epidemiological surveys were done to determine the worldwide
distribution of Au. It was very rare in apparently normal populations from the United States, but common in some tropical and Asian populations. The presence or absence
of Au seemed to be a consistent feature of an individual; if it was present on initial testing, then it was present on subsequent testing however, in a Downs syndrome patient
who had originally been negative the Au antigen was present in a later blood sample. This suggested that Aa might be linked to an infection of some sort. The patient was
followed and found to have developed chronic hepatitis between the first and second test. Blumbergs clinical colleague, Alton Sutnick, wrote in the patients chart: SGOT
slightly elevated! Prothrombin time low! We may have an indication of (the reason for) his conversion to Au+. His prediction proved correct. The diagnosis of hepatitis was
clinically confirmed by liver biopsy, and the team now began to test the hypothesis that Au was associated with hepatitis. By 1967, Blumberg, Kazuo Okochi, Alfred Prince,
Alberto Vierrucci, and colleagues reported that Au is involved in the development of hepatitis B, and by 1968-1970 David S. Dane discovered that the Au antigen is actually a
22nm subunit particle of the virus, HBsAg, and is present in the blood of chronic hepatitis patients in very high titer; the virus itself is present also, but in much lower titer.
Blumberg BS, Alter HJ, Visnich S. A new antigen in leukemia sera. JAMA. 191:541-6, 1965.
Prince AM. An antigen detected in the blood during the incubation period of serum hepatitis. Proc Natl Acad Sci USA. 1968;60:814-821.
Dane DS, Cameron CH, Briggs M. Virus-like particles in serum of patients with Australia-antigen-associated hepatitis. Lancet 1970;1: 695-698.

1965 Baruch Blumberg, Harvey Alter, others discovery of Australia antigen, key to discovery of hepatitis B virus

page 349

David Arthur John Tyrrell (1925-2005)

June Almeida (1930-2007)

Human coronavirus 229E

In 1965, David Tyrrell and M. L. Bynoe isolated a virus, B814, from a person showing typical symptoms of the common cold. This was done by inoculating nasal washings into
human embryonic tracheal organ cultures (no virus growth was seen in usual cell cultures). The presence of an infectious agent was demonstrated by inoculating the medium
from the organ cultures intranasally in human volunteers; colds were produced in most of the subjects. At about the same time, D. Hamre and J. J. Procknow propagated in
tissue culture a virus with unusual properties from throat washings obtained from medical students with colds it was called 229E. And, while working in the laboratory of
Robert Chanock at the NIH, Kenneth McIntosh and his colleagues recovered several strains of virus, the series called OC and OC43 chosen as representative, from human
respiratory tract washings by using a technique similar to that of Tyrrell and Bynoe. Then, June Almeida and David Tyrrell did electron microscopy on samples from organ
cultures infected with B814, 229E and OC43 viruses and found particles in each that resembled infectious bronchitis virus of chickens, mouse hepatitis virus and transmissible
gastroenteritis virus of swine. This new group of viruses was named coronavirus. It was June Almeidas suggestion: corona denoting the crown-like appearance of the virion
surface projections the group is now the family Coronaviridae.
Tyrrell DA, Bynoe ML. Cultivation of viruses from a high proportion of patients with colds. Lancet 1966;1:76-77.
Almeida JD, Tyrrell DA. The morphology of three previously uncharacterized human respiratory viruses that grow in organ culture. J Gen Virol. 1967;1:175178.
Kahn JS, McIntosh K. History and recent advances in coronavirus discovery. Ped Inf Dis J. 2005;24:223-227.

1965 David Tyrrell, June Almeida, others discovery of human coronaviruses (B814, 229E, OC43)

page 350

Joseph L. Melnick (1914-2001)

Wallace P. Rowe (1926-1983)

The discovery of adeno-associated viruses (AAVs) is yet another example of, chance favoring the prepared
mind. In 1965, R. Wayne Atchison at the University of Pittsburgh was researching the viral etiology of
human cancers and while checking by electron microscopy an adenovirus stock before injecting it into
hamsters noticed large numbers of small particles intermixed with typical adenovirus virions. Since the small
particles were uniform in appearance and size (as small as 18nm), and even had a hexagonal shape indicative
of icosahedral structure, Atchison presumed they were a virus. Since the particles were smaller than any
previously known virus, he knew his discovery would be novel if it were a virus. Soon, Atchison and research
groups led by Wallace Rowe at NIH and Joseph Melnick at Baylor recognized that the small particles, which
they initially thought to be subunits of adenoviruses, were actually parvoviruses, now members of the genus
Dependovirus, family Parvoviridae. AAVs are replication-defective and dependent upon helper functions
provided by adenoviruses or herpesviruses for productive replication in mammalian cells. Immediately after
their discovery, AAVs were also isolated from humans, but no association with disease has ever been found.
Atchison RW, Casto BC, Hammon M. Adenovirus-associated defective virus particles. Science 1965;49:754756.
Melnick JL, Mayor HD, Smith KO, Rapp F. Association of 20-millimicron particles with adenoviruses. J Bact.
1965;90:271-274.

Hoggan MD, Blacklow NR, Rowe WP. Studies of small DNA viruses found in various adenovirus preparations:
Adeno-associated virus virions and two adenovirus virions
physical, biological and immunological characteristics. Proc Natl Acad Sci USA. 1966;55:1467-1472.
negative contrast electron microscopy

1965 Wayne Atchison, Joseph Melnick, colleagues discovery of adeno-associated viruses (parvoviruses)

page 351

Robin C. Valentine (1928-1968)

Helio Pereira (1918-1994)

In the early 1960s investigation of viruses was based primarily on their immunological properties. Helio Pereira
at the National Institute of Medical Research at Mill Hill, London, Erling Norrby at the Karolinska Institute in
Stockholm and Harry Ginsberg at Columbia University in New York City were at the forefront of research on
adenoviruses and their antigens. The relationship of each antigen to the icosahedral structure of the viruses
was not apparent and it was only when the isolated antigens were examined by negative contrast electron
microscopy that their nature was understood. At Mill Hill, this work was done by the great microscopist, Robin
C. Valentine. Similar work was soon done by Norrby at the Karolinska Institute. One of the antigens, the group
specific antigen, was tied to the major subunit of the virion capsid, the hexon; another was tied to fibers with
one small and one large knob at either end; and the third was tied to fibers with a small knob at one end only.
At first, it was not apparent whether these fibrous structures were derived from virions or not. The key to the
puzzle lay in the visualization for the first time of adenovirus virions at such resolution as to show fibers with
knobs protruding from each of the twelve vertices of the icosahedral structure. The three antigens were renamed
hexon, penton and fiber, respectively. Tragically, Robin Valentine (1928-1968) died a few years later. Pereira and
his colleagues crystallized the hexon this was the first animal virus protein to be crystallized and studied it
by x-ray crystallography. Today, the adenovirus virion is seen as a very complex structure, built from about 40
different polypeptides, but there are still mysteries, especially concerning structures in the interior of the virion.
Valentine RC, Pereira HG. Antigens and structure of the adenovirus. J Mol Biol. 1965;13:13-20.

Erling Norrby

Adenovirus virions
negative contrast
electron microscopy

Norrby E. Structural and functional diversity of adenovirus capsid components. J Gen Virol. 1969;5, 221-236.

1965> Robin Valentine, Helio Pereira, Erling Norrby discovery of adenovirus structural / functional elements
page 352

Bernard Roizman

Graphic art from Russell Knightly; used with permission

Herpes simplex virus infection of a cell

Bernard Roizman, of the University of Chicago, has been a leader in virology research since the 1960s. At first, he pioneered in herpes simplex virus (HSV) biology, and as time
went by he came to be recognized as the leading authority in nearly every area of HSV research. One of Roizmans most important early contributions was the identification of
a viral gene that is responsible for HSV neurovirulence. He was one of the first investigators to apply molecular tools to epidemiological studies of viral disease; in doing so he
became responsible for the widely used (and sometimes abused) term, molecular epidemiology. He did this in a study of an outbreak of HSV infection in a newborn nursery, a
setting where infection is often associated with severe disease. This was done using restriction endonuclease cleavage profiles (fingerprinting) of isolates to trace the spread of
virus from one infant to another. This was based on the observation that no two epidemiologically unrelated isolates of HSV had identical restriction enzyme profiles. Roizman
and his colleagues showed that the nursery outbreak was caused by two different virus strains derived from two different adults. Roizmans work then turned to molecular
virology, per se; he determined the structure of the viral DNA and defined the basic molecular mechanisms of HSV replication. His laboratory also conducted extensive
investigation into the identification of many of the more than 90 HSV genes and the functions of the viral proteins encoded by them. As this information mounted, he was able
to study the mechanisms by which HSV causes disease and the mechanisms involved in the host response to acute infection and chronic recrudescent infection.
Buchman TG, Roizman B, Adams G, Stover BH. Restriction endonuclease fingerprinting of herpes simplex virus DNA: a novel epidemiological tool applied to a nosocomial
outbreak. J Infect Dis. 1978;138:488-498.
Roizman B, Buchman TG. The molecular epidemiology of herpes simplex viruses. Hosp Pract. 1979;14:95-104.

1965> Bernard Roizman seminal studies of the molecular biology and replication of herpes simplex virus

page 353

Kendall Smith

In 1960, Peter Nowell, created a new field, that of

cellular immunology; he made the startling discovery
that an extract from the kidney bean containing a
molecule termed phytohemagglutinin (PHA) could
stimulate lymphocytes to divide. He would never
have made his discovery had he not absentmindedly
left PHA-separated plasma enriched for leukocytes
in the incubator over a weekend. PHA transformed
small lymphocytes into large blastic cells and
stimulated their division. The mechanism responsible
for this remained obscure until 1965, when there
were two reports of an activity found in the culture
media of stimulated lymphocytes that promoted their
proliferation. Termed blastogenic factor by Shinpei
Kasakura and Louis Lowenstein, and Julius Gordon
and Lloyd D. McLean, this activity was due to IL-2, but
it would be another 15 years before this activity was
ascribed to a single molecule. Although IL-2 was the
first soluble, endogenous cytokine to be discovered it
was not named IL-1; but that is a long story, too long
for here. Subsequent to the discovery of IL-2, numerous
reports confirmed the basic observation; however,
further progress awaited Steven Gillis and his colleagues
and their first success in growing T cells in the lab in
1978. They found that T cells use up the IL-2 in the
medium and as they run out they rapidly die. After
being purified by Kendall Smith and his colleagues, the
IL-2 molecule was found to be a variably glycosylated
MW15,500 protein. IL-2 was also the first cytokine to be
cloned and expressed from a cDNA library. Today, there
are more than 50 human cytokines, and many more in
other animals.
Nowell PC. Phytohemagglutinin: an initiator of mitosis
in cultures of normal human leukocytes. Cancer
Research 1960;20:462-468.
Kasakura S, Lowenstein L. A factor stimulating DNA
synthesis derived from the medium of leukocyte
cultures. Nature 1965;208:794-795.

IL-2

Gordon J, Maclean LD. A lymphocyte-stimulating factor

produced in vitro. Nature 1965;208:795-796.
Gillis S, Ferm MM, Ou W, Smith KA. T cell growth
factor: parameters of production and a quantitative
microassay for activity. J Immunol. 1978;120:2027-2032.

1965> Shinpei Kasakura, Kendall Smith, others discovery and characterization of the first cytokine, IL-2

page 354

Leonard Hayflick

WI-38 cell: fluorescence

image of a cell stained
with multi-spectral
probes (tubulin, nucleic
acid and Golgi markers)
[ATCC CCL-75]

Leonard Hayflick began his career in cell culture science with a

post-doctoral fellowship at the University of Texas Medical Branch at
Galveston under the distinguished cell biologist Charles M. Pomerat;
he then spent several years at the Wistar Institute in Philadelphia
with his colleague, Paul Moorehead, where in the early 1960s they
developed the first human diploid cell strains, including the most
widely used of these, WI-38. In 1962, Hayflick discovered that,
contrary to the belief of the day, cultured normal human and animal
cells have a limited capacity for replication. This discovery, known
as the Hayflick limit, overturned a dogma that existed since Alexis
Carrels work early in the 20th century that claimed that normal
cells were immortal and would proliferate endlessly in culture.
Hayflick demonstrated for the first time that mortal (normal) and
immortal (malignant) mammalian cells were different. Hayflick:
A cell strain is a population of cells derived from animal tissue,
subcultivated more than once in vitro, and lacking the property
of indefinite serial passage while preserving the chromosomal
karyotype characterizing the tissue of origin. Conversely, a cell line
is a population of cells derived from animal tissue and grown in
vitro by serial subcultivations for indefinite periods of time with a
departure from the chromosome number characterizing its source.
Hayflick produced the first oral polio vaccine made in a continuously
propagated cell strain. WI-38 is now also used for the production
of rubella, measles, varicella-zoster, mumps, rabies, adenovirus and
hepatitis A virus vaccines. Over one billion people have received
vaccines produced in WI-38 or derivative cells. Hayflick: I received
phone calls from the White House during the Reagan and first Bush
administrations from staffers saying, We finally tracked you down.
We understanding that you are the one who developed the cell
strain derived from an aborted human fetus, and we want to confirm
something we simply cannot believe is going on. And that is that
vaccines have been produced in those cells and are available for sale
in the United States. And my reply was, Where the hell have you
been for the past twenty years?
Hayflick L, Moorhead PS. The serial cultivation of human diploid
cell strains. Exp Cell Res. 1961;25:585-621. [This paper describes the
isolation and characterization of 25 strains of human fetal diploid
fibroblasts, WI-1 to WI-25.]
Hayflick L, Plotkin SA, Nobton TW, Koprowski H. Preparation of
poliovirus vaccines in a human fetal diploid cell strain. Am J Hyg.
1962;75:240-258.
Hayflick L. The limited in vitro lifetime of human diploid cell strains.
Exp Cell Res. 1965;37:614-636. [This paper describes studies with
WI-26, WI-38 and WI-44.]

1965 Leonard Hayflick development of human diploid cell strains (WI-1 through WI-44)

page 355

Lac repressor protein tetramer

Mark Ptashne

Walter Gilbert

When Walter Gilbert and Mark Ptashne began their careers, Franois Jacob and Jacques Monod had just advanced the theory of gene regulation by repressors. Identifying
repressors became a Holy Grail of molecular biology. An understanding of repressors would reveal how genes are turned on and off in response to hormones, growth
factors, drugs, and other environmental signals. Many scientists searched for repressors, including Jacob and Monod, but in the end Walter Gilbert, working on the lac
operon repressor of Escherichia coli, and Mark Ptashne working on the repressor of bacteriophage , found the first repressors. They showed that repressors are proteins that
specifically bind to a small stretch of DNA and control the expression of the related upstream structural gene(s). Ptashne continued to work on gene regulation and soon made
his most notable discovery: he unraveled the molecular basis of the lambda switch, explaining how the repressor and another regulatory protein interact with the DNA of the
bacteriophage to switch genes on or off in response to environmental signals. Upon infecting its host bacterial cell, bacteriophage decides between two pathways, a lytic
pathway or a lysogenic pathway. When the switch is ON, the virus multiplies exponentially and kills the bacterial cell. When the switch is OFF, lysogeny occurs: the DNA of
the bacteriophage inserts itself into the chromosome of the host bacterium and remains quiescent until the switch is reversed by signals from the environment. Elucidating
the lambda switch opened the field of transcriptional regulation as it is known today and provided the first general model to explain the switching between developmental
pathways that occurs not only in viral and bacterial infections, but also during embryogenesis and cancers in higher organisms, including humans. Shortly after his work on
repressors. Gilbert left the field of gene regulation and went on to develop a new technique for sequencing DNA, for which he received a Nobel Prize in 1980.
Gilbert W, Muller-Hill B. Isolation of the Lac repressor. Proc Natl Acad Sci USA. 1966;56:1891-1898.
Ptashne M, Hopkins N. The operators controlled by the Lambda phage repressor. Proc Natl Acad Sci USA. 1968;60:1282-1287.
Meyer BJ, Kleid DG, Ptashne M. Lambda repressor turns off transcription of its own gene. Proc Natl Acad Sci USA. 1975;72:4785-4789.

1966> Mark Ptashne, Walter Gilbert identification of repressor genes (bacteriophage, lac operon of E. coli)

page 356

Peter Wildy
(1920-1987)

Frank Fenner
(1914-2010)

Richard Ellis Ford Matthews

(1921-1995)

1966 Peter Wildy, Frank Fenner, Richard Matthews, others International Committee on Taxonomy of Viruses

page 357

The History of Virus Taxonomy

The earliest experiments involving viruses were designed to separate them from microbes that could be seen in the light microscope and that usually could be cultivated
on rather simple media. In the experiments that led to the first discoveries of viruses at the turn of the 20th century a single physicochemical characteristic was measured,
ultrafilterability. No other physicochemical or other measurements were made at that time, and most studies of viruses centered on their ability to cause infections and
diseases. The earliest efforts to classify viruses, therefore, were based upon perceived common pathogenic properties, common organ tropisms, and common ecological and
transmission characteristics. It was not until the 1930s that evidence of the structure and composition of virions began to emerge. This prompted Frederick Bawden (1941) to
propose for the first time that viruses be grouped on the basis of shared virion properties. Among the first taxonomic groups constructed on this basis were the herpesvirus
group (1954), the myxovirus group (1955), the poxvirus group (1957), and several groups of plant viruses with rod-shaped or filamentous virions (1959). In the 1960s, there was
an explosion in the discovery of new viruses. Prompted by a rapidly growing mass of data, several individuals and committees independently advanced classification schemes.
The result was confusion over competing, conflicting schemes, and for the first but not the last time it became clear that virus classification and nomenclature are topics that
give rise to very strongly held opinions. Against this background, in 1966 the International Committee on Nomenclature of Viruses (ICNV) was established at the International
Congress of Microbiology in Moscow. The committee became the International Committee on Taxonomy of Viruses (ICTV) in 1973. At that time, virologists already sensed
a need for a single, universal taxonomic scheme, that is that all viruses should be classified in a single system. Nevertheless, there was much dispute over the taxonomic system
to be used. Andr Lwoff, Robert Horne and Paul Tournier (1962) argued for the adoption of an all-embracing scheme for the classification of viruses into subphyla, classes,
orders, suborders, and families. Descending hierarchical divisions were to be based, arbitrarily and monothetically, based upon nucleic acid type, capsid symmetry, presence
or absence of an envelope, etc. Opposition to this scheme was based upon its arbitrariness in deciding the relative importance of virion characteristics to be used and upon
the argument that not enough was
known about the characteristics
of most viruses to warrant an
elaborate hierarchy. Nevertheless,
except for its fancy nomenclature,
the Lwoff-Horne-Tournier scheme
was accepted and evolved in the
1970s into todays system. As
genomic sequencing has replaced
many earlier criteria for placing
viruses in taxa, little change
has been necessary this may be
taken as a tribute to the wisdom
of the founders in basing taxa on
very carefully considered data.
As Frank Fenner said in 1974, Taxa
are often built on relationships
that we would like to believe (from
an evolutionary standpoint), but
are unable to prove. At its meeting
in Mexico City in 1970, the ICTV
approved the first two families, 24
floating genera and 16 plant virus
groups. Today, the ICTV keeps
track of more than 6,000 viruses,
1991 Executive Committee of the International Committee on the Taxonomy of Viruses (ICTV)
in about 2,000 taxa (species, genus,
Seated (L to R): C. Calisher, S. Ghabrial, A. Jarvis, F. Murphy, C. Fauquet.
family, and order), all based on
Standing : M. van Regenmortel, R. Goldbach, B. Mahy, C. Pringle, H. Pereira, L. Berthiaume, G. Martelli, E. Carstens, J Maniloff,
evolutionary relationships.
J. Strauss, M. Summers, M. Mayo, A. Gibbs, H. Ackermann, D. McGeoch, A. Della Porta, M. Horzinek.

1966 Peter Wildy, Frank Fenner, Richard Matthews, others International Committee on Taxonomy of Viruses
page 358

William Hadlow

Photo: Rocky Mountain Laboratories/NIAID/NIH

D. Carleton Gajdusek (1923-2008)

Photo from 1957, New Guinea

Michael Alpers

Photo from 1972, New Guinea

In 1957, Carleton Gajdusek went to New Guinea from Melbourne, Australia where he was working with Macfarlane
Burnet (1899-1985); he was to set up a study on child development and disease. Gajdusek met Vincent Zigas, district
medical officer, who introduced him to a strange neurological disorder, known as kuru, which was found only
among the Fore people, who lived in isolation in the eastern highlands (kuru is the Fore word for shiver or shake).
People with kuru stumbled and twitched and were belligerent, prone to mirth, grinning, and shrieking. They died
within months. Later studies of the Fore people indicated that the disease was transmitted by ritual cannibalism, in
which women and children ate the brains of those who had died of kuru. After the ritual was abandoned, the disease
slowly disappeared. Gajdusek spent almost a year living with the Fore, recording 200 cases of kuru and collecting
tissue samples for study when he returned to the United States in 1958. At the NIH, he found that victims had many
central nervous system lesions similar to the spongiform lesions seen in patients with Creutzfeldt-Jakob disease.
William Hadlow, a veterinary pathologist, by chance saw an exhibition on kuru at the Wellcome Museum and wrote
to Gajdusek, saying how similar the lesions were to those in sheep and goats with scrapie. Hadlow wrote: In my
letter to the editor of Lancet, I pointed out the striking similarity of kuru and scrapie and suggested that kuru, like
scrapie, might be a transmissible disease expressed after a long incubation period. Gajdusek, Clarence (Joe) Gibbs
and Michael Alpers built a primate facility at Patuxent, Maryland and inoculated nonhuman primates, especially
chimpanzees, with brain tissue. After an incubation period of two years, the chimpanzees became ill and died,
revealing the same spongiform lesions as seen in the human disease. Eventually, other species of nonhuman primates
also developed the same disease, confirming the infectious nature of the etiologic agent the kuru prion.
Hadlow WJ. Scrapie and kuru. Lancet 1959; 2:289-290.

Frank Macfarlane Burnet (1899-1985)

Clarence Joseph Gibbs (1924-2001)
Photo from 1971, Patuxent Primate Center

Gajdusek DC, Gibbs CJ Jr, Alpers M. Experimental transmission of a kuru-like syndrome to chimpanzees. Nature
1966;209:794-796.
Gajdusek DC, Gibbs CJ Jr, Alpers M. Transmission and passage of experimental kuru to chimpanzees. Science
1967;155:212-214.

1966 Carleton Gajdusek, Clarence Gibbs, William Hadlow transmission of the kuru prion to non-human primates

page 359

On an early morning in March 1963, Maurice

Hilleman was scheduled to travel to South America,
but his five-year-old daughter, Jeryl Lynn, woke him up
early. She had a high fever and he recognized that she
had mumps. After comforting her, he first drove to his
lab to get cotton swabs, then returned home to obtain
a throat specimen from Jeryl Lynn, and then went back
to the lab to inoculate cell cultures with the material.
Later that morning, he flew to South America and
when he returned he found that he had indeed isolated
mumps virus from his daughter. This virus, called
the Jeryl Lynn strain, was attenuated by cell culture
passage and then used in the first mumps vaccine;
derivatives of it are still used today.
In the United States, the incidence of mumps
decreased from >100 cases per 100,000 population
in most years in the prevaccine era (before 1967) to
10 cases per 100,000 population in 1977. After the
1989 institution of a 2-dose measles, mumps, rubella
(MMR) vaccine schedule, the number of reported
mumps cases further decreased to 1 case per 100,000
population in 1992 and to 0.1 case per 100,000
population in 2001.
Buynak EB, Hilleman MR. Live attenuated mumps
virus vaccine. 1. Vaccine development. Proc Soc Exp
Biol Med. 1966;123:768-775.

Eugene Buynak and Maurice Hilleman

in the lab at the Merck Institute for Therapeutic Research

Mumps virus: Top, N protein

(turquoise) in cytoplasm of a cultured
cell, multiple fluors. Bottom, Vero cells,
thin section electron microscopy
Maurice Hillemans daughter, Kirsten, being vaccinated against
mumps by Robert Weibel while her sister, Jeryl Lynn, looks on

1966 Eugene Buynak, Maurice Hilleman development of mumps vaccine

page 360

History of Rubella Vaccine Development

Abridged from Plotkin SA. History of rubella vaccines and the recent history of
cell culture. In: Plotkin SA, editor. History of vaccine development. New York:
Springer; 2011. p. 219-232.

By the late 1940s the discovery by Norman McAlister Gregg (18911966) that rubella virus caused congenital rubella syndrome was widely
appreciated, while at the same time large rubella epidemics continued
to occur worldwide. Efforts to cultivate rubella virus, however, proved
very difficult and it was not until 1961 that there was any success.
Thomas Weller cultivated the virus in human amniotic cells, but the
cytopathic effect was subtle and took a long time to become evident.
Paul Parkman and Malcolm Artenstein cultivated the virus in African
green monkey kidney (AGMK) cells, using a secondary interferon assay
(an ECHO 11 virus challenge of cell cultures) to confirm rubella virus
replication in the absence of cytopathic effect.

Stanley A. Plotkin (photo from ~1979)

By 1966, Parkman and Harry Meyer had serially passaged the virus
77 times in AGMK cells, and had conducted clinical trials with their
attenuated live-virus vaccine candidate (HPV-77) with satisfactory
results. This was made available to Merck and Philips-Roxane, but at
this point it was found that the vaccine seed stock was contaminated
with a simian cytomegalovirus, so Maurice Hilleman at Merck adapted
the virus to duck embryo cells (HPV77-DE5) and Philips Roxane
adapted it to dog kidney cells (HPV77-DK12), thereby eliminating the
cytomegalovirus. At the same time, scientists at Smith-Kline serially
passaged the virus in rabbit kidney cells, producing a vaccine virus
strain they named Cendehill. The first of these vaccines was licensed in
1969, but in the general population they caused an unacceptable rate of
adverse reactions, especially in adult women, or low immunogenicity,
and in turn each was withdrawn from the market.
Fortunately, as these first vaccines unraveled, Stanley Plotkin made
an isolate from the kidney of a rubella-infected fetus by passage first
through human embryonic kidney cells and then between 1964 and
1967 through 25 passages in the human diploid cell strain (HDCS)
WI-38, all at reduced temperature (attenuation by cold adaptation).
This became the vaccine virus RA27/3; clinical trials showed good
immunogenicity, safety and efficacy. The vaccine was first licensed in
the UK in 1970, and shortly thereafter in other European countries. It
took until 1978 for the U.S. FDA to license the vaccine, largely because
of an ongoing debate over the safety of human diploid cells as vaccine
substrates. Merck started producing the vaccine in the U.S. while other
companies continued production in other countries. Today, RA27/3
is the only rubella vaccine used in most countries, nearly always as a
component of MMR (measles, mumps, rubella) vaccine. In 2005, CDC
announced that rubella was no longer endemic in the U.S.

1966 Stanley Plotkin, Paul Parkman, Harry Meyer, Maurice Hilleman development of rubella vaccine

page 361

Rudolf Siegert (1914-1988)

Werner Slenczka

Gustav Martini (1916-2007)

Marburg virus, rhesus macaque liver

Siegert R, Shu HL, Slenczka W, Peters D,
Mueller G. Zur tiologie einer unbekannten,
von affen ausgegangenen menschlichen
Infektionskrankheit. Dtsch Med Wochenschr.
1967;92:2343-2370.
Gordon Smith CE, Simpson DIH, Bowen ETW,
Zlotnik I. Fatal human disease from vervet
monkeys. Lancet 1967; 2:1119-1121.

Robert E. Kissling

Roslyn Q. Robinson

(~1931-1981) with Senator Ted Kennedy

Frederick A. Murphy

Kissling RE, Robinson RQ, Murphy FA, Whitfield

SG. Agent of disease contracted from green
monkeys. Science 1968;160:888-890.

1967 Rudolf Siegert, Werner Slenczka, Robert Kissling, Frederick Murphy, others discovery of Marburg virus
page 362

Marburg Virus and the Beginnings of Civilian High-Containment Laboratories

In 1967, 31 cases of hemorrhagic fever, with seven deaths, occurred among laboratory workers in Germany and Yugoslavia
who were processing kidneys from African green monkeys [Cercopithecus (now Chlorocebus) aethiops] that had been
imported from Uganda. A new virus was isolated from patients and monkeys in three laboratories: that of Rudolf Siegert and
colleagues in Marburg, Charles E. Gordon Smith and colleagues at the Microbiological Research Establishment at Porton
Down, and Robert Kissling and colleagues at the U.S. Centers for Disease Control (CDC). It was named Marburg virus.
Zoonotic transmission occurred in circumstances where there was close contact between humans and monkeys or monkey
tissues (kidneys, cell cultures derived from kidneys) in the absense of personal protective equipment or safe practices.
From the beginning, it was realized that Marburg virus was dangerous, calling for the best biocontainment of the day. At
CDC, a mobile containment lab was borrowed from the National Institutes of Health; it was housed in a truck trailer (an
18-wheeler), which was set up in the parking lot at CDC. The containment level provided was about what would be called
BSL-2+ today. The main safety feature was that only three people would be at risk, Robert Kissling, Roslyn Robinson and
Frederick Murphy, and each was experienced in safe practices for working with dangerous pathogens. Specimens were
obtained from David Simpson and Gordon Smith (originating from G. May, Frankfurt and W. Hennessen, Marburg); these
were used to develop diagnostic reagents (antigens, antisera, virus stocks) and for morphology, pathology and ultrastructural
pathology studies. Papers were published, complementing the excellent work done by the German and British virologists.
This Marburg virus episode of 1967 was the impetus for the development of permanent biocontainment facilities at CDC and
elsewhere. The model was the first glove port cabinet line, built in the late 1960s at the U.S. Army Medical Research Institute
for Infectious Diseases (USAMRIID) at Fort Detrick, Maryland. It was for military biological warfare defense purposes. At
CDC, a glove port cabinet line lab was constructed in a tin building; it was opened in 1968. Completion of the lab coincided
with the emergence of Lassa fever in West Africa. Work on Lassa virus at
Yale University (Yale Arbovirus Research Unit) was terminated after one
person died and another nearly died. Thomas Monath and others at CDC
set to work to develop diagnostic systems and unravel the natural history
of Lassa virus. Work expanded from an emphasis on diagnostics to the
pathogenesis of several viral hemorrhagic fevers this even involved the
use of small monkeys. All this put a great strain on the glove port lab .
A second lab was completed at CDC in 1979 it housed both glove port
cabinet lines and the first space suit (positive pressure biocontainment
suit) laboratory, invented by Karl Johnson. He stated in an interview: As
technology developed, and the number of instruments grew, it became
prohibitive to build huge stainless steel glove port lines. His inspiration
came from a colleague, Peter Jahrling. Whereas all the dangerous lab work
at USAMRIID was done in glove port cabinet lines, animals were held in a
negative pressure room serviced by technicians wearing rubber suits with
masks connected to an air hose. One day, Jahrling needed to make some
frozen section tissue slides and could not get the freeze-microtome into a
glove port cabinet. So, he put on one of the animal room suits and brought
the tissues and microtome into the animal room. Johnson saw this: I
watched him doing that, and when I got back to the CDC, I sat down with
the engineers and said, We need to make a suit lab well wear suits,
and well have coiled hoses in the ceiling for air. All BSL-4 laboratory
development since then has followed upon this insight.

Karl M. Johnson

Above: James Lange and Herta Wulff working

through glove ports in CDC Building 8, 1970s
Right: LuAnne Elliott in the first positive
pressure biocontainment suit, invented by
Karl Johnson at CDC, 1977

1967> U.S. Communicable Disease Center development of civilian high-containment virology laboratories

page 363

Kates JR, McAuslan BR. Messenger RNA

synthesis by a coated viral genome. Proc Natl
Acad Sci USA. 1966;57:314-320.
Kates JR, McAuslan BR. Poxvirus DNAdependent RNA polymerase. Proc Natl Acad Sci
Joseph R. Kates
Brian R. McAuslan
USA. 1967;58:134-141.
Early on, enzymes required for viral DNA synthesis were found to be induced in vaccinia virus-infected cells. DNA polymerase activity was identified in 1962, and in the
following year Brian McAuslan and others reported the presence of thymidine kinase and deoxyribonuclease. These enzymes are present in uninfected cells but they are
located in the nucleus. Because vaccinia virus DNA synthesis occurs in the cytoplasm, the question arose as to whether the virus-induced enzymes are different. McAuslan
showed that the thymidine kinases in uninfected and infected cells are different. These observations were compatible with the idea that the enzymes required for viral
DNA synthesis are encoded by the virus, but it was difficult at the time to see how their transcription from viral genes could take place in the cytoplasm of infected cells.
In eukaryotic cells, synthesis of mRNA, which is transcribed from chromosomal DNA, requires RNA polymerase II and this enzyme is located in the nucleus. In a paper
published in 1967, Joseph Kates and McAuslan showed that virus-specific mRNA is not synthesized de novo and must be present at the time of infection. All available evidence
pointed to the presence of RNA polymerase within the vaccinia virus virion. Thirty years ago this was difficult to conceive because virions were thought to be metabolically
inert and no virion-associated enzyme had previously been described in any animal virus. It was at this stage that Kates and McAuslan published their study on the ability of
vaccinia virus cores to synthesize virus-specific RNA. Kates and McAuslans discovery played a seminal role in studies which led to the detection of virion-associated enzyme
activities in other viruses. Attempts to infect cells with nucleic acid recovered from some RNA viruses proved unsuccessful and the possibility began to be considered that their
genomes may be negative-sense. If so, a transcriptase would be required to act on the genomic RNA to produce a positive sense, complementary copy. Because a mammalian
enzyme with such activity does not exist, it was considered that it must be brought into the host cell by the infecting virus, that is, the transcriptase would have to be a virionassociated enzyme. Such an RNA-dependent RNA transcriptase was found, in 1968, to be present in reovirus virions (by Aaron Shatkin and Jean Sipe), and two years later in
vesicular stomatitis virus virions (by David Baltimore and Alice Huang), and, in 1971, in influenza virions (by David Bishop, Jack Obijeski and Robert Simpson). Of course, in
1970 an RNA-dependent DNA polymerase was found in retrovirus virions (by Howard Temin and David Baltimore) this enzyme, reverse transcriptase, became so famous
that in some ways the other virion-associated transcriptases became lost in time.

1967 Joseph Kates, Brian McAuslan discovery of viral DNA-dependent RNA polymerase (vaccinia virus)
page 364

Peter M. Biggs

Jzsef Marek
(1868-1952) who
first described the
disease named for
him in 1907

Ben Roy Burmester (1910-2009)

The complex pathogenesis of Mareks disease in chickens,

characterized by persistent virus infection, unique virus shedding
in feather follicle dander, and lethal T cell lymphomas

Mareks disease is a highly contagious viral neoplastic disease of

chickens. The disease is characterized by the presence of T cell
lymphomas as well as the infiltration of nerves and organs by
lymphocytes. The discovery of Mareks disease virus involves more
individuals than most of the other discoveries described so far in
this book it resembles more the discoveries of recent times. The
discovery began with evidence provided by Peter Biggs and Jim
Payne at the Houghton Poultry Research Station, England, that
Mareks disease and avian lymphoid leukosis were separate entities.
Next was their demonstration that the agent was cell-associated.
These experiences were shared with Ben Burmester and his
colleagues at the USDA Regional Poultry Laboratory, East Lansing,
Michigan, with whom Biggs and Payne had a close relationship.
Next, Tony Churchill was recruited to join Biggs and his Houghton
Group to provide greater expertise in cell culture techniques.
Using the knowledge of the cell-associated nature of the infectious
agent, he inoculated chick kidney cell cultures with tumor tissue
from Mareks disease infected chickens he found a cytopathic
effect proof of the replication of a virus in the cells required
back-titration in chickens. From the character of the cytopathic
effect in cell culture (syncytia, intranuclear inclusions), the etiologic
agent was suspected of being a herpesvirus; Robert Dourmashkin,
of the Imperial Cancer Research Fund, Mill Hill, London, carried
out electron microscopic studies and, indeed, found a herpesvirus.
Biggs initiated correspondence with Robert Huebner at the National
Cancer Institute, who expressed disbelief that a herpesvirus could
be oncogenic - he felt strongly that all cancers were associated with
retrovirus oncogenes. Eventually, Huebner became convinced.
In 1967, Biggs visited East Lansing he told Burmester and his
colleagues about the latest findings and learned that they had also
had success in finding the virus, using duck embryo fibroblast cell
cultures. Keyvan Nazerian did the electron microscopy in East
Lansing and found the same herpesvirus. Proof that the herpesvirus
was the causative agent of Mareks disease was difficult because of its
cell-associated nature; conclusive evidence was the demonstration
by the Houghton group that an attenuated variant of the virus
protected chickens from Mareks disease. Mareks disease virus
together with the closely related herpesvirus of turkeys now form the
genus Mardivirus in the subfamily Alphaherpesvirinae of the family
Herpesviridae.
Churchill AB, Biggs PM. Agent of Mareks disease in tissue culture.
Nature 1967;215:528-530.
Biggs PM. History of Mareks disease. In: Hirai K, editor. Mareks
Disease. Berlin: Springer; 2001. p. 1-24.

1967 Peter Biggs, Anthony Churchill, Ben Burmester, others discovery of Mareks disease virus (herpesvirus)

page 365

Jacob V. Maizel, Jr.

Ulrich K. Laemmli

The separation of proteins from complex mixtures, one of the

major challenges in biochemistry in the early 20th century, was first
advanced by Arne Tiselius, who made electrophoretic separations
in unstable liquid columns. In following years, proteins separated by
their electrical charge were stabilized in place using various supports
such as starch blocks, polyacrylamide gels, etc. These methods could
only resolve proteins having distinctive isoelectric points, but it soon
became apparent that most of the mixtures of interest comprised
proteins with closely overlapping isoelectric points. Jacob Maizel
and his colleagues, at Albert Einstein Medical Center in New York,
had been working with protein separations for some time when a
method of solubilizing chloroplasts using urea and SDS (sodium
dodecyl sulfate, an anionic detergent) was seen in the literature by
Bill Joklik, and was incorporated into the polyacrylamide gels in
Maizels ongoing attempts to separate the proteins of poliovirus.
The separation principle in SDS-gel electrophoresis is not protein
net charge, but is sieving in the polyacrylamide gel matrix due to
the unfolded extended conformation of proteins in the presence of
the detergent and urea. This soon led Maizel and his colleagues to
their breakthrough studies, moving from poliovirus, to adenovirus,
and then to reovirus virion proteins. As initially employed, the
method gave sharper bands than earlier methods, and although
there was a sense that the observed resolution was to some degree
correlated with molecular weight, this had not yet been established.
This correlation was finally resolved using known molecular weight
proteins as markers in the same or parallel gels indeed, there was
an inverse correlation between mobility and protein molecular
weight. The next advance was the development of discontinuous
buffer systems and stacking gels by Ulrich Laemmli that further
enhanced the sharpness of protein bands. Polyacrylamide gel
electrophoresis (PAGE) became one of the most enabling methods in
the modern era of protein chemistry and molecular virology. The first
SDS-polyacrylamide gel in the literature was of poliovirus proteins
(the graph here is from the 1965 paper) the sliced gel showed
quite clearly that the virus mRNA was directing the synthesis of four
capsid proteins plus at least ten noncapsid proteins.
Summers DF, Maizel JV Jr, Darnell JE Jr. Evidence for virus-specific
noncapsid proteins in poliovirus infected HeLa cells. Proc Natl Acad
Sci USA. 1965;154:505-513.

The four poliovirus virion proteins, labeled with

14
C; cell extracts treated with SDS and urea and
electrophoresed in 10% polyacrylamide gel.
y-axis: molecular weight, semi-log scale; x-axis:
relative migration.

Maizel JV Jr. SDS polyacrylamide gel electrophoresis. Trends

Biochem Sci. 2000;25:590-592.
Laemmli UK. Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 1970;227:680-685.

1967 Jacob Maizel Jr., Ulrich Laemmli development of SDS polyacrylamide gel electrophoresis of proteins
page 366

Bovine ephemeral fever is an arthropod-borne viral disease

of cattle and several wildlife species, characterized by short
duration, fever, stiffness and disinclination to move, but usually
with complete recovery. Its economic impact derives from loss
of condition and milk production, and abortions. It was first
recognized in southern Africa, and then in Egypt, Australia and
Japan. In recent years it has been found across large regions
of Africa, Asia and Australasia. The etiologic agent was first
characterized by Barnard van der Westhuizen at the Veterinary
Research Institute at Onderstepoort, South Africa, and then by
Yoshio Tanaka, Yuji Inaba and their colleagues at the National
Institute of Animal Health of Japan. It is a rhabdovirus that
is transmitted by Culicoides spp., Culicine and Anopheline
mosquitoes and perhaps other arthropods. Inapparent infections
occur in Cape buffalo, hartebeest, waterbuck, wildebeest, deer,
and probably other wildlife species.

van der Westhuizen B. Studies on bovine ephemeral fever.

I. Isolation and preliminary characterization of a virus from
natural and experimentally produced cases of bovine ephemeral
fever. Onderstepoort J Vet Res. 1967;34:29-40.
Ito Y, Tanaka Y, Inaba Y, Omori T. Electron microscopic
observations of bovine epizootic fever virus. Nat Inst Animal
Health Tokyo Quart. 1969;9:35-44.
Holmes IH, Doherty RL. Morphology and development of
bovine ephemeral fever virus. J Virol. 1970;5:91-96.

Barnard van der Westhuizen

Unlike the other rhabdoviruses, which are bulletshaped, bovine ephemeral fever virus and other members
of the genus Ephemerovirus are usually rather conical in
shape.

Murphy FA, Taylor WP, Mims CA, Whitfield SG. Bovine

ephemeral fever virus in cell culture and mice. Archiv fr die
Gesamte Virusforschung 1972;38:234-249.

Bovine ephemeral fever virus, thin section and negative contrast electron microscopy

1967 Barnard van der Westhuizen, Yuji Inaba, Yoshio Tanaka, colleagues discovery of bovine ephemeral fever virus

page 367

Aaron J. Shatkin (1934-2012)

Jean D. Sipe
In the models, the polymerase
3 (red) is anchored to
the inner surface of the
icosahedral core shell, making
contact at the five-fold vertex
position. The polymerase is
oriented with its transcript
exit channel facing a small
channel through the inner
capsid shell, suggesting that
nascent RNA is passed to the
exterior for translation.

One of the central problems in virology during the 1960s was

determining the mechanism by which viruses initiate their
replicative functions. Reoviruses were especially mysterious in this
regard because of their dsRNA genomes; dsRNAs were known to
be inactive as messengers (mRNAs). The first clue came from the
work of Kates and McAuslan, showing that vaccinia virus contains
an RNA polymerase this solved the conceptual problem. Shortly
thereafter Aaron Shatkin and Jean Sipe and Joseph Borsa and Angus
Graham showed that reoviruses contain a dsRNA-dependent RNA
transcriptase (polymerase). But, the story became much more
complicated as time went by. The polymerase (3 polymerase) of
reoviruses functions within the confined interior of the viral core
particle, which is only partially degraded upon entering the host cell
cytoplasm. Within the core particle, replication requires recruitment
of one capped, positive-sense strand for each of the 10 RNA genome
segments, along with an equal number of RNA-dependent RNA
polymerase molecules. Synthesis of minus-sense strands and then
full complements of positive strands accompanies this process (the
3 polymerase molecules switch mode to drive each synthetic step).
Nascent positive-strand RNA is capped and methylated by other
core-associated enzymes and then serves as mRNA for translation
of viral proteins it also serves as genomic positive strands for
packaging into virions. The structure of the reovirus core shows
that 3 polymerase molecules lie just inside the inner capsid shell
and close to five-fold vertex positions, where there are RNA exit
pores. Ten of the twelve five-fold exit positions are associated with
a polymerase molecule, probably with a single genome segment
dedicated to each. [Perhaps all 12 five-fold exit sites are used
by Colorado tick fever virus, which is a member of the family
Reoviridae and has 12 genome segments!] This organization suggests
that during transcription the template (minus-sense) strand of the
genomic RNA is pulled through the polymerase, which remains
more or less in place, and that the transcript, as it emerges from
the polymerase, is fed into the pore for delivery outside. It has been
considered that the polymerase would have to stay tethered at the
pore for this scheme to work
Shatkin AJ, Sipe JD. RNA polymerase activity in purified reoviruses.
Proc Natl Acad Sci USA. 1968;61:1462-1469.
Borsa J, Graham AF. Reovirus: RNA polymerase activity in purified
virions. Biochem Biophys Res Commun. 1968;33:895-901.
Tao Y, Farsetta DL, Nibert ML, Harrison SC. RNA synthesis
in a cagestructural studies of reovirus polymerase 3. Cell
2002;111:733-745.

1968 Aaron Shatkin, Jean Sipe discovery of dsRNA-dependent RNA polymerase (reovirus)
page 368

Werner Henle (1910-1987)

Gertrude S. Henle (1912-2006)

Diagnostic blood film: differential >50% lymphocytes, 10%

atypical lymphocytes (large, irregular nuclei)

Werner and Gertrude Henle were early pioneers in Epstein-Barr virus (EBV) research. They had worked with Anthony Epstein in 1964, helping to establish the relationship
between the virus and Burkitts lymphoma. By 1966, they had developed an immunofluorescent test to detect EBV viral antigens, which not only facilitated Burkitts lymphoma
research, but had also linked the virus to nasopharyngeal carcinoma. In 1968, the Henles identified EBV as the causative agent of infectious mononucleosis (IM). As Werner
Henle wrote in his Annotated Bibliography: The answer to our search [for an illness caused by EBV] came quite unexpectedly right in our laboratory when one of our
technicians reported ill in August 1967. She was seronegative for EBV and had been a frequent, more or less willing, donor of leukocytes for EBV transmission/transformation
studies carried out by Volker Diehl, a German postdoctoral fellow. Our technician returned to work five days later but developed under our eyes a rubella-like rash. Her
physician had considered either rubella or IM as probably diagnoses. We obtained blood from her for serological tests as well as yet another leukocyte culture, thinking more
of rubella than IM. The heterophil antibody test, however, turned out to be positive. The rash was thus not due to rubella but to ampicillin which she had received and which
for unknown reasons can be the cause of a rash in IM patients. We noted next with astonishment that our technician had developed antibodies to EBV since the last time
we tested her and observed in due course her leukocytes, which formerly never survived in culture, grew this time into a permanent lymphoblastoid cell line with a small
percentage of EBV-producing cells. These were the first exciting clues that EBV was the long sought cause of IM. It was fortunate that we never took our summer vacation in
August since in our absence this event would probably have passed unnoticed. The Henles then found that 100% of 42 serum samples from IM patients had EBV antibodies. In
a subsequent collaboration with James Niederman and Robert McCollum, prospective studies of college students clearly demonstrated that cases of IM were confined to those
individuals lacking EBV antibodies prior to their illnesses. Such antibodies appeared regularly in association with the disease and persisted for years or for life.
Henle G, Henle W, Diehl V. Relation of Burkitts tumor-associated herpes-type virus to infectious mononucleosis. Proc Natl Acad Sci USA. 1968;59:94-101.
Diehl V, Henle G, Henle W, Kohn G. Demonstration of a herpes group virus in cultures of peripheral leukocytes from patients with infectious mononucleosis. J Virol.
1968;2:663-669.

1968 Werner Henle, Gertrude Henle association of Epstein-Barr virus with infectious mononucleosis

page 369

Peter Wildy
(1920-1987)

Joseph L. Melnick
Nils Oker-Blom
(1914-2001)
(1919-1995)
The Four Founding Fathers

Victor Zhdanov
(1914-1991)

Gathering for the Opening Session

First International Congress for Virology
Helsinki, 1968 (536 registrants from 39 countries)

1968 Peter Wildy, Joseph Melnick, Nils Oker-Blom, Victor Zhdanov the First International Congress for Virology

page 370

Meeting of the Executive

Committee and Advisory Council
at the Second International
Congress for Virology, Budapest,
1971

International Congress for

Virology

Place and Year of

Congress

Chairman of Congress

President of ICTV
& Editor(s) of Report

First Congress

Helsinki, 1968

P Wildy, J Melnick,
N Oker-Blom,
V Zhdanov

P Wildy

Second Congress

Budapest,1971

J Melnick, P Wildy,
N Oker-Blom

Third Congress

Madrid, 1975

P Wildy, J Melnick,
N Oker-Blom

F Fenner

Fourth Congress

Hague, 1978

P Wildy,
J van der Want,
E Norrby, J Melnick

R Matthews

Fifth Congress

Strasbourg, 1981

J van der Want

R Mathews

Sixth Congress

Sendai, 1984

F Murphy

Seventh Congress

Edmonton, 1987

E Norrby

R Francki, C Fauquet,
D Knudson, F Brown

Eighth Congress

Berlin, 1990

M van Regenmortel

Ninth Congress

Glasgow, 1993

B Mahy

Tenth Congress

Jerusalem, 1996

R Pettersson

Eleventh Congress

Sydney, 1999

J Almond

Twelfth Congress

Paris, 2002

R Compans

M van Regenmortel,
C Fauquet, D Bishop,
E Carstens, M Estes,
S Lemon, J Maniloff, M Mayo,
D McGeoch, C Pringle,
R Wickner

Thirteenth Congress

San Francisco, 2005

H Klenk

L Ball, C Fauquet, M Mayo,

J Maniloff, U Desselberger

F Murphy, C Fauquet,
D Bishop, S Ghabrial,
A Jarvis, G Martelli,
M Mayo, M Summers

Fourteenth Congress

Istanbul, 2008

G Smith

E Carstens

Fifteenth Congress

Sapporo, 2011

K Nagata

J Mackenzie

Sixteenth Congress

Montreal, 2014

T Dermody

E Carstens

Participants, clockwise from left: H. von

Magnus (Denmark); F. Fenner (Australia);
A.J. Rhodes (Canada); P. Wildy (U.K.);
E. Farkas (Hungary); N. Oker-Blom
(Finland); J.L. Melnick (USA); E. Norrby
(Sweden); G. Ivanovic (Hungary); L. Hirth
(France); J.H. Subak-Sharpe (U.K.);
V. Rennick (USA)

1968 Peter Wildy, Joseph Melnick, Nils Oker-Blom, Victor Zhdanov The First International Congress for Virology

page 371

The Beginnings of the International Congresses for Virology

by Joseph L. Melnick (abridged)

How did the International Congresses for Virology begin? This question has been
asked repeatedly, so I was asked to write this article for the record. Virologists had been
playing key roles in the affairs of the International Association of Microbiological Societies
many years prior to 1968, the year of the first Virology Congress in Helsinki. They included
Thomas M. Rivers (1939-47), Macfarlane Burnet (1953-58), Andre Lwoff (1962-70), and
Victor M. Zhdanov (1970-74), each of whom served, in the years indicated, as President of
the Executive Committee, and Sven Gard, who served as President of the 7th Congress for
Microbiology in 1958.

Among virologists who attended Congresses of the International Association of
Microbiological Societies (IAMS), in which virological reports were interspersed among
presentations oriented toward a variety of other disciplines, it became clear that it would
be desirable to have a separate Section of the IAMS devoted to Virology. But in order to be
recognized by the IAMS, it was necessary to spring fully grown from the sea, as Aphrodite
perhaps not as beautiful a body, but a functioning one nevertheless. Thus during the
IAMS Congress in Moscow in 1966, a small revolutionary group of workers felt that the
time was ripe for the International Virologists of the World to unite. The group included
Peter Wildy, Victor Zhdanov, Joseph Melnick, and Nils Oker-Blom. The affairs of the
IAMS at that time were run by a bureaucracy whose main interest had been focused on
Bacteriology, and the Microbiology Congresses failed to provide sufficient opportunities
for virologists to meet and to discuss their rapidly developing discipline.

...At the IAMS Congress in Moscow virologists were again disappointed: sessions
devoted to Virology were few and far-apart, so that most fruitful virologic discussions
were held impromptu, often at street corners while waiting for the Congress busses-which
ran on infrequent and irregular schedules. Our group of revolutionaries proposed to the
Executive Committee of the IAMS that the virologists be allowed to form a Section within
the IAMS. And if we were a Section, why not meet separately, preferably at different times
from the large congresses? The response to our petition was that virologists rightfully
belonged among microbiologists, and could not be a separate body-and furthermore, that
no significant virologists group existed, so our request was without substance.

The Helsinki Congress in 1968 came into being with the aid of an International
Committee that was recruited from 39 countries. In attendance at that Congress were 536
virologists, each of whom, in accordance with the admission regulations established by
the International Committee, had to have contributed at least 3 significant publications
to the worlds virologic literature. The fact that so many persons qualified for attendance
indicated how large and significant the literature of our science had already become.

I had the pleasure of serving as Secretary-General of the Helsinki Congress.
In my opening remarks I said: Now, this week, by our meetings and discussions of our
laboratory work, we shall reaffirm our existence. Speaking not only for myself but also for
each of the participants, I wish to express the appreciation that is owed to Professors Peter
Wildy, Victor Zhdanov, and Nils Oker-Blom, with whom I have had the pleasure of serving
on the Convening Committee, to the 39 members of the International Committee, and
particularly to Professor Oker-Blom and his colleagues, to the University of Helsinki...

At the end of the weeks program the following resolutions were adopted... (1) Be
it resolved that the members of the First International Congress for Virology petition the

IAMS to proceed at once to form a section on Virology, to include all branches of the
science, and to arrange for future and regular International Congresses for Virology.
(2) Until such time as the IAMS establishes a Section in Virology with the responsibility of
holding International Congresses for Virology, the Secretary-General of the International
Congress for Virology is requested to continue in office and to proceed with arranging for
the Second International Congress for Virology in 1971...

The scene now shifts to Mexico City in 1970. In view of the fact that virologists
provided firm evidence of their existence by having held a successful independent
Congress in Helsinki, and faced with the real possibility of virologists withdrawing from
the IAMS to form their own organization, the delegates to the 10th International Congress
for Microbiology modified their statutes and agreed to the formation of a Section on
Virology. The virologists present at the Mexico City Congress met and organized their
Section. The elected officers were Joseph L. Melnick (Chairman), Peter Wildy (ViceChairman), Nils Oker-Blom (Secretary) and the elected Advisory Council consisted
of S. Gaidamovich (USSR), J. Hidaka (Japan), L. Hirth (France), E. Norrby (Sweden),
A. J. Rhodes (Canada), F. Fenner (Australia). The delegates voted to hold the Second
International Congress in 1971 in Budapest... aided by a Hungarian Host Committee:
G. Ivanovics, E. Farkas, and I. Dmk.

The Budapest Congress was a huge success, with 984 virologists from 45
countries participating. Included in the Scientific Program of the Congress were five
plenary sessions on topics of general interest, and thirty specialized workshops.

At the opening ceremony of the VIIIth International Congress for Virology in
Berlin in 1990, the Congress paid tribute to the four founding fathers. Unfortunately, Peter
Wildy and Victor Zhdanov were no longer with us. I was privileged to be present but Nils
Oker-Blom had previously committed himself to be lecturing in Nairobi.

I would like to close these reminiscences with a quotation on the subject of
international virology from Peter Wildy, who was one of the leading figures in 20th
century virology, and who played a key role in founding the International Congresses:
Something must be said about International Virology. Up until 1966, virologists were
provided for by meetings held from time to time in different countries under the auspices
of the International Association of Microbiological Societies. These meetings steadily
grew in size and dullness and virologists became more and more disinterested. In 1966,
four mutineers (ringleader: J. Melnick) decided to run an International Virology meeting
independently. The meeting was successful and followed by other more comprehensive
programmes in Budapest, Madrid, The Hague and Strasbourg and since then in Sendai,
Edmonton, and Berlin. Several points have become clear. First, it is important for the
health of virology and for the interests of virologists that meetings be held separately from
others dealing with microbiological disciplines, in order to attain the maximum chance
of personal encounter. Second, the breadth of virology is great enough to sustain varied
combinations of interest. Indeed, considerable ingenuity is required to ensure mutually
fruitful exchanges. Third, the heterogeneity of virologists is such that it has often been
questioned whether it is wise to continue with such large congresses. So far, I believe they
have been a powerful force of good communication. I hope that they remain so.

I believe that the Virology Congresses continue to be a powerful force of good
communication and I believe that they will be so in the future.
Melnick JL. The beginnings of the international congresses for virology.
Arch Virol. 116;293-300:1991.

1968 Peter Wildy, Joseph Melnick, Nils Oker-Blom, Victor Zhdanov The First International Congress for Virology

page 372

Kathleen Danna

Daniel Nathans (1928-1999)

Hamilton Smith

Werner Arber

In the early 1960s, Werner Arber and Daisy Dussoix, a PhD student, found that bacteria can distinguish between their own DNA and
the foreign DNA of an invading bacteriophage they found that the bacterial DNA is protected by methylation and the foreign DNA is
cleaved and further degraded by specific enzymes. The enzymes doing this cleavage were called restriction and modification enzymes,
and are now known as restriction endonucleases. Arber provided the theoretical framework for all future work and he also isolated the
first enzyme, EcoBI. Hamilton Smith, Kent Wilcox and Thomas Kelly verified Arbers hypothesis by purifying both bacterial restriction
and modification enzymes and showing that they specifically cut DNA and methylated the DNA, respectively. Starting in 1971, Daniel
Nathans and Kathleen Danna pioneered in the application of restriction enzymes to the study of the DNAs of several organisms, starting
with that of simian virus 40 (SV40). They used the restriction enzyme endonuclease R, discovered by Smith and Wilcox to produce
specific fragments of SV40 DNA and showed that these fragments could be nicely separated from one another by polyacrylamide gel
electrophoresis. [We now know that the original preparation of endonuclease R contained two enzymes, the one characterized by
Smith, HindII, the second HindIII the SV40 cleavage pattern was the product of both enzymes.] It was Nathans who made the key
intuitive leap that cleavage patterns could be used to produce a physical map of SV40 DNA, as well as indicating the origin of replication
and the location of specific genes. These pioneering studies set the stage for modern recombinant DNA technology and molecular
biology the photograph of the first published gel provided immediate visualization of just how powerful the combination of restriction
endonucleases and gel electrophoresis would be. Today, more than 3,600 restriction enzymes are recognized, representing more than
250 specific cleavage patterns more than 600 of these enzymes are available commercially.

Thomas J. Kelly

Arber W, Dussoix D. Host controlled

modification of bacteriophage
lambda. J Mol Biol. 1962;5:18-36.
Smith HO, Wilcox KW. A restriction
enzyme from Hemophilus
influenzae. I. Purification and
general properties. J Mol Biol.
1970;51:379-391.
Danna K, Nathans D. Specific
Cleavage of simian virus 40 DNA
by restriction endonuclease of
Hemophilus Influenzae. Proc Natl
Acad Sci USA. 1971;68:2913-2917.

1968> Werner Arber, Hamilton Smith, Daniel Nathans, others discovery of restriction endonucleases

page 373

Robert J. Huebner (1914-1998)

Symbols for oncogenes are three lowercase
characters in italics. These may be preceded
by a c or v in roman type indicating
whether the origin was chromosomal or viral.
E.g., scr, c-src, v-src. Symbols for the protein
products of oncogenes are three characters in
roman type with the initial letter capitalized.
E.g., Src. However, one often sees all three
letters capitalized, e.g., SRC, or all lower
case, e.g., src the convention has not been
followed very well.

George Todaro

In 1969, Robert Huebner and George Todaro proposed the oncogene

hypothesis they proposed that normal cells must contain genes related
to those found in retroviruses, since viral proteins could often be found
in cells of apparently uninfected animals, especially chickens and mice.
Such genes were believed to be transmitted vertically, integrated into the
chromosomes of germ-line cells, and expressed in response to a variety of
stressors. Since some retroviruses were known to be highly oncogenic in
animals, it was also proposed that tumor-inducing genes, e.g., retroviruses
genes (viral oncogenes) might also be transmitted through the germ line as
a consequence of ancient infection. Activation of these endogenous viral
oncogenes by substances we recognize as carcinogens chemicals, radiation,
other viruses could serve to initiate a neoplastic process. Although their
hypothesis that most cancers were caused by expression of retroviral genes
was not correct, their work did lead to the identification of the first retroviral
oncogene, src, and to the realization that these viral genes were derived from
functional cellular genes or proto-oncogenes. They proposed that cellular
proto-oncogenes are precursors of transforming cancer genes. In 1981,
Huebner wrote (abridged): The viral oncogene theory was inescapable
given the years of cumulative evidence culminating in our 1969 report
in PNAS [It was our view] that cancer was the result of derepression
of inherent species-specific endogenous oncogenes often linked in mice
and chickens with endogenous retroviruses. Genetic abnormalities or
deficiencies, environmental carcinogens, and oncogenic viruses were thus
inducers of such oncogenes. Another key factor was our observation that the
endogenous leukemia and sarcoma viruses in chickens, mice, rats, and cats
actually carried movable oncogenes, since src genes could be rescued from
primary hosts and transmitted as transforming oncogenes to cultured cells
of a wide variety of animal hosts, including humans The viral oncogene
hypothesis expressed a new concept in which it was postulated that all
carcinogenesis was mediated through activation of an endogenous host cell
gene present in every vertebrate cell. Recently, it became apparent that the
oncogene determinants of cancer represented also the required determinants
for immunoprevention. Vaccines and IgG preparations incorporating
immunity to src and leu gene expressions have recently been used to prevent
virtually all types of cancers in mice and rats, including cancers produced
by chemicals and DNA tumor viruses. These successful immunoprevention
studies, we believe, provide excellent models for development of comparable
prevention of cancer in man. Of course, in the years since Huebner and
Todaro conceived this grand hypothesis much has changed much of the
hypothesis has been proven wrong and the promise of immunoprevention
of human cancers by vaccines against any retrovirus antigen is still a work in
progress.
Huebner RJ. Todaro GJ. Oncogenes of RNA tumor viruses as determinants of
cancer. Proc Natl Acad Sci USA. 1969;64:1087-1094.

1969 Robert Huebner, George Todaro development of the viral oncogene hypothesis
page 374

Sonja Buckley (1919-2005)

Jordi Casals-Ariet (1911-2004)

Lassa hemorrhagic fever field research station, Nigeria, 1972

Thomas Monath, Jordi Casals, Kent Campbell, Verne Newhouse, Mission Hospital Nurses

Lassa virus, human liver, massive

hepatocellular necrosis

1 plastic-embedded section, polychrome stain

Events leading to the discovery of Lassa virus in 1969 were dramatic.
Two nurses in a mission hospital in Lassa, Nigeria, died but one
was first flown to a larger hospital in Jos. There, an attending nurse
also became very ill; she was evacuated to Columbia-Presbyterian
Hospital in New York. She survived. Clinical specimens were sent
to the Yale Arbovirus Research Unit in New Haven, where Lassa
virus was isolated by Jordi Casals and Sonja Buckley. Later, while
working with the virus, Casals became very ill and was successfully
treated with convalescent serum from the surviving nurse; however,
a laboratory technician at Yale died. Work at Yale was terminated
and shifted to CDC where Thomas Monath and colleagues further
characterized the virus, unraveled its natural history and prevalence
and discovered its reservoir host, the multimammate rat, Mastomys
(Praomys) natalensis. Today, Lassa hemorrhagic fever is recognized
as an important disease in West Africa.
Buckley SM, Casals J. Lassa fever, a new virus disease of man from
West Africa. Am J Trop Med Hyg. 1970;19:680-691.

1969 Sonja Buckley, Jordi Casals-Ariet discovery of Lassa virus, Lassa hemorrhagic fever

page 375

Charles A. (Chuck) Mebus

Epizootic diarrhea of newborn calves has for centuries been a

plague of cattle husbandry. The disease attacks calves within
four or five days of birth, causing at times significant mortality
a 30% loss is not uncommon. Attempts at experimental
transmission of the disease had been made from time to time,
but these had given equivocal results, probably because the
etiologic agent(s) is ubiquitous, so that controls as well as
inoculated animals became ill or did not because of maternal
immunity. Charles Mebus, Marvin Twiehaus and their colleagues
first succeeded in transmitting infection in a reproducible way
by inoculating gnotobiotic calves by intraduodenal tube (and
later by feeding) with bacteria-free filtrates of diarrheal feces.
Calves developed diarrhea usually within 24-48 hours, and
65nm diameter virus particles were found in large numbers in
their feces. The virus was described as reovirus-like; the name
rotavirus was coined in 1973 by Thomas Flewett. The virus
was adapted to grow in cell culture, and extensive pathology
and pathogenesis studies were done using histopathology
and frozen-section immunofluorescence of intestinal tissues.
Thomas Flewett, one of the discoverers of human rotaviruses
stated: In the early days of virology, workers on human, animal,
insect and plant viruses were all acquainted with each other and
knew each others work. But with proliferation of laboratories,
diversification of interests, and multiplication of journals, this
happy state has not existed for some time. Oblivious of the work
of Mebus and his colleagues in Nebraska or not appreciating
its importance, groups in 1973 in Birmingham, Melbourne and
Toronto were independently searching for virus particles in feces
or intestinal biopsies of children. this story is continued under
the year 1973, when Ruth Bishop, Ian Holmes, Thomas Flewett,
Peter Middleton and their colleagues discovered the first human
rotavirus.
Mebus CA, Underdahl NR, Rhodes M, Twiehaus M. Calf
diarrhea (scours): Reproduced with a virus from a field outbreak.
Univ Nebraska Res Bull. 1969;233:1-16.

Rotavirus

(top) purified virus, negative contrast

electron microscopy
(bottom) virions assembling at edge
of an intracytoplasmic inclusion
body (virus factory), cell culture, thin
section electron microscopy

Mebus CA, Underdahl NR, Rhodes MB, Twiehaus MJ. Further

studies on neonatal calf diarrhea virus. Proc Annu Meet US
Anim Health Assoc. 1969;73:97-99.
Mebus CA, Kono M, Underdahl NR, Twiehaus MJ. Cell culture
propagation of neonatal calf diarrhea (scours) virus. Can Vet J.
1971;12: 69-72.
Flewett TH, Woode GN. The rotaviruses: brief review. Arch
Virol. 1978;57:1-23.

1969 Charles Mebus, Norman Underdahl, Marvin Twiehaus discovery of bovine rotavirus (the first rotavirus)

page 376

Knowing is not enough; we must apply.

Willing is not enough; we must do.
Goethe
Motto of the Institute of Medicine
U.S. National Academy of Sciences

Members of the Board on Medicine,

as it became the Executive Committee
of the Institute of Medicine in 1970:

Albert Einstein

on the grounds of the National Academy of Sciences

Robert J. Glaser, M.D., President pro tem

John R. Hogness, M.D., first President
Walsh McDermott, M.D.
Joseph S. Murtaugh
Ivan L. Bennett, Jr., M.D.
Samuel M. Nabrit, Ph.D.
Charles G. Child III, M.D.
Irvine H. Page, M.D.
Julius H. Comroe, Jr., M.D.
Henry W. Riecken, Ph.D.
John T. Dunlop, Ph.D.
Dwight L. Wilbur, M.D.
Walter A. Rosenblith, Ing. Rad.
Rashi Fein, Ph.D.
Lucile P. Leone, M.A.
Bryan Williams, M.D.
Irving M. London, M.D.
Adam Yarmolinsky, LL.B.
Colin M. MacLeod, M.D.
Alonzo S. Yerby, M.D.
Eugene A. Stead, Jr., M.D.

1970 Irvine Page, others founding of the Institute of Medicine of the U.S. National Academy of Sciences

page 377

Robert Ellis Shope (1929-2004)

From the time of Pasteur, rabies virus had been singular antigenically,
genetically and taxonomically singular. In fact, when the African viruses that
were to become the members of the genus Lyssavirus, family Rhabdoviridae,
that is the rabies-like viruses, were first isolated there was no notion of their
relationship to rabies virus. The first two of these viruses were discovered in
arbovirus field projects and so were expected to be members of one of the taxa
housing the arboviruses. L.R. Boulger and James Porterfield isolated a virus from
Eidolon helvum fruit bats at Lagos Island, Nigeria, in 1956; they called it Lagos
bat virus, a possible arbovirus, and distributed it widely to reference centers in
the hope of finding out more about its relationship to other arboviruses. Graham
Kemp isolated Mokola virus from organs of Crocidura spp. shrews in 1968,
although its reservoir host species is still unknown. Later two strains of Mokola
virus were isolated from cases of central nervous system disease in Nigerian
children. Lagos bat virus and Mokola virus were sent to the Yale Arbovirus
Research Unit in 1969,where Robert Shope, using all the classic arbovirus
identification methods confirmed that the two viruses behaved like other
ungrouped arboviruses and that the two viruses cross-reacted by complementfixation testing. Shope had been collaborating with Frederick Murphy at the
Centers for Disease Control and Prevention trying to classify the rhabdoviruses
of vertebrates (quite a few had been identified by electron microscopy by this
time). The two viruses were found to be rhabdoviruses, but more so, they were
identical to rabies virus in their appearance in infected cells (intracytoplasmic
inclusion bodies Negri bodies and virions budding in association with these
inclusion bodies quite unlike other rhabdoviruses such as vesicular stomatitis
viruses). Murphy telephoned Shope with this exciting news and suggested that
the two viruses be tested against rabies virus this was done immediately and
a serologic relationship was found. A few years later, in South Africa, Courtney
Meredith read about the rabies-related viruses and thought of them when a virus
he isolated from the brain of a man bitten on the lip by a bat did not react in the
rabies immunofluorescence test, although the patient had died of what appeared
to be typical rabies. This virus was found to be another rabies-like virus; it
was named Duvenhage virus. Since then, several other viruses that, except for
political sensitivities, might be called rabies virus have been isolated in Europe,
including the United Kingdom, and Australia. Recently, four viruses genetically
quite distant from rabies virus have been found in bats in central and southeast
Asia. The genus Lyssavirus is rather well populated now, after having only one
member for 66 years.
Shope RE, Murphy FA, Harrison AK, Causey OR, Kemp GE, Simpson DI, Moore
DL. Two African viruses serologically and morphologically related to rabies
virus. J Virol. 1970;6:690-692.

Lagos bat virus, hamster brain

thin section electron microscopy Tignor GH, Murphy FA, Clark HF, Shope RE, Madore P, Bauer SP, Buckley SM,

Frederick A. Murphy

Meredith CD. Duvenhage virus: morphological, biochemical, histopathological

and antigenic relationships to the rabies serogroup. J Gen Virol. 1977;37:595-611.
Shope RE. Rabies-related viruses. Yale J Biol Med. 1982;55:271-275.

1970> Robert Shope, Frederick Murphy, others discovery of rabies-like viruses (the genus Lyssavirus)
page 378

Frank Fenner (1914-2010)

David Ogilvie White (1931-2004)

Frank Fenner, later recalling book writing in 1969-1970: I had always felt somewhat guilty being called a Professor, having
never given a course of lectures (I was never invited to lecture to students in the faculties of the university), and now I was never
likely to. So I thought that I might occupy the spare time I had, always early in the morning before anyone else arrived and at
irregular times on most days, to write a textbook on virology for medical students. I persuaded David White, who was by then
Professor of Microbiology at the University of Melbourne, who I knew to be a first-class teacher of virology, to be a co-author.
The first edition, entitled Medical Virology, 390 pages long, was published by Academic Press in 1970. It received excellent
reviews and sold very well. M.A. Epstein, writing in Nature, said: Despite the risk of writing a rave notice, I am still filled with
wonder on reflecting on the seeming ease with which extremely complicated topics have been reduced to an orderly survey of
the basic facts involved, together with suitable explanations of their significance. This book can be used, therefore, both as an
introductory textbook and as a very reliable and solid reference book. More than 25,000 copies of the first edition were sold.

Medical Virology, First Edition, 1970:

Frank Fenner, David O. White.
Medical Virology, Second Edition, 1976:
Frank Fenner, David O. White.
Medical Virology, Third Edition, 1986:
David O. White, Frank Fenner.
Medical Virology, Fourth Edition, 1994:
David O. White, Frank Fenner.

Fenner F. Nature, nurture and chance. The lives of Frank and Charles Fenner.
Canberra: Australian National University ePress; 2006.

1970 Frank Fenner, David White publication of book, Medical Virology (currently four editions)

page 379

David Baltimore
Baltimore D, Franklin RM. A new ribonucleic acid
polymerase appearing after mengovirus infection of
L-cells. J Biol Chem. 1963;238:3395-3400.
Baltimore D, Huang AS, Stampfer M. Ribonucleic acid
synthesis of vesicular stomatitis virus, II. An RNA
polymerase in the virion. Proc Natl Acad Sci USA.
1970;66:572-576.

Alice S. Huang

Viral RNA-dependent RNA polymerases were

discovered in the early 1960s in studies of
mengovirus and poliovirus when it was observed
that these viruses were not sensitive to actinomycin
D, a drug that inhibits cellular DNA directed
RNA synthesis. This lack of sensitivity suggested
that there was a virus specific enzyme that could
copy viral RNA from an RNA template. It was
found that in these positive-sense RNA viruses
this enzyme is encoded in the virion RNA and is
transcribed and translated directly. The advantage
of this for the positive sense RNA viruses is that
there is no intermediate stage, so replication can
be quick and often very productive. However,
purified genomic RNA from viruses like vesicular
stomatitis viruses (VSV) is non-infectious, and
this observation led to the discovery that the
genomic RNA is complementary to its mRNA
these are the negative-sense RNA viruses. David
Baltimore, Alice Huang and Martha Stampfer
discovered the gene encoding the RNA-dependent
RNA polymerase and the polymerase itself in
1970. The transcription and replication scheme of
viruses like the VSVs first requires production of
complementary strand RNAs, which are then used
as mRNAs. Even so, there are quite a few differences
in the transcription, translation and replication
schemes employed by the many negative-sense RNA
viruses. In 1973, Alice Huang also suggested that
the virion polymerase of VSV serves its distinctly
dual functions as transcriptase and replicase in
complex fashion. Recently, it has been found that
there are two RNA-dependent RNA polymerases in
VSV, each a multiprotein complex, each containing
the viral L and P proteins (the polymerase), but
each containing different cellular proteins. Thus,
the mechanism for carrying out the two different
functions, transcription and replication, seems
to have evolved in the simplest way using two
distinct enzymatic complexes rather than some
mysterious single complex that would have to shift
from one function to the other.

1970 David Baltimore, Alice Huang discovery of RNA-dependent RNA polymerase in vesicular stomatitis virus
page 380

Extracellular mature C particles

thin section electron microscopy

Baltimore D. RNA-dependent DNA polymerase
in virions of RNA tumour viruses. Nature
1970;226:1209-1211.

Howard M. Temin (1934-1994)

David Baltimore

Temin HM, Mizutani S. RNA-dependent DNA

polymerase in virions of Rous sarcoma virus.
Nature 1970;226:1211-1213.

Through many experiments done over many years, the principles by which genetic information is transferred were demonstrated. DNA provided the information that was
transcribed into mRNA, which was then used for translation into proteins. DNA>mRNA>proteins the central dogma. However, in 1970, it was shown that the central
dogma did not cover everything. In 1961, Howard Temin began gathering evidence that was inconsistent with the central dogma. Temin, working on Rous sarcoma virus
(RSV), felt that the best explanation for the often dormant behavior of the virus was a proviral non-replicating state in infected cells. Since RNA is notoriously unstable,
Temin proposed that the RNA genome of RSV is converted into a DNA provirus. He amassed data suggesting that RSV-transformed cells have a DNA complementary to
RSV genomic RNA. Separately, David Baltimore had been studying the replication of vesicular stomatitis virus (VSV) and found that the its genome is complementary to
its mRNA thus was born the idea of negative sense viruses. One puzzle was how a negative sense virus can initiate infection its RNA must either be copied by a host
cell enzyme or the virus must enter the cell with its own RNA-dependent RNA polymerase. In 1970 Baltimore, with Alice Huang and Martha Stampfer, found the virionassociated polymerase. This led directly to Baltimores discovery of reverse transcriptase; he hypothesized that other viruses must also solve the problem of initiating infection
by employing a virion-associated polymerase. After being lucky enough to obtain a large amount of Rauscher murine leukemia virus, Baltimores search for an RNA-dependent
DNA polymerase took only a few days. He found that the polymerase activity was ribonuclease (RNase)-sensitive and actinomycin D-resistant, suggesting that the virionmediated DNA synthesis employed an RNA not a DNA template. As with VSV, the virion-associated polymerase allowed the Rauscher virus genome to serve as the template
for nucleic acid synthesis without having first to make new protein(s). Temin and Satoshi Mizutani came to the same conclusion at the same time actually, the experiments
done by the two groups to identify the template for the viral DNA synthesis, the virion RNA, were remarkably similar. The polymerase was soon referred to by the editors of
Nature as reverse transcriptase and the viruses as retroviruses. Back-to-back papers were published with great fanfare, and soon a broader impact of the discovery became
evident: the ability to convert mRNA into DNA permitted creation of cDNA libraries, the enzyme became crucial in cloning technologies and the rise of the biotechnology
industry, and 15 years later the enzyme was at the heart of research into the cause of AIDS and the discovery of human immunodeficiency virus (HIV).

1970 Howard Temin, David Baltimore, colleagues discovery of the reverse transcriptase of retroviruses

page 381

1--D-ribofuranosyl-1,2,4-triazole-3-carboxamide [Ribavirin]

Ribavirin

(brand names: Copegus, Rebetol, Ribasphere, Vilona and Virazole)

Roland K. Robins (1926-1992)

and an unnamed technician

Sidwell RW, Huffman JH, Khare GP, Allen LB, Witkowski JT,
Robins RK. Broad-spectrum antiviral activity of virazole: 1--Dribofuranosyl-1,2,4-triazole-3-carboxamide [Ribavirin]. Science
1972;177:705-706.

Ribavirin was discovered in 1970 as part of a systematic search for antiviral and antitumor activity in
synthetic nucleosides at ICN Pharmaceuticals, Inc. (later Valeant Pharmaceuticals International) by
laboratory director Roland K. Robins, chemist Joseph T. Witkowski (1901-1986) and their colleagues. This
was inspired in part by discovery in the 1960s of the antiviral activity of naturally-occurring purine-like
nucleoside antibiotics. The earlier compounds were too toxic to be clinically useful, but they served as the
starting point for pharmaceutical chemists interested in antivirals and antimetabolic chemotherapeutic
agents. In 1972, it was reported that ribavirin was active against several RNA and DNA viruses in cell
culture and/or in animals, all without undue toxicity. Experimental data indicated that the drug might
be useful in treating influenza, hepatitis B, polio, measles, canine distemper, smallpox, West Nile virus
infection, dengue and a number of viral hemorrhagic fevers, including Lassa fever, yellow fever, CrimeanCongo hemorrhagic fever, and hantavirus pulmonary syndrome fever. Ribavirin also exhibits activity
against some DNA viruses in cell culture. Although ribavirin protected mice against mortality from
influenza A and B viruses, in human trials results were mixed and FDA did not approve its use. In 1980,
ICN was allowed to market ribavirin, in inhalant form, for respiratory syncytial virus infection in children,
but it was used for this indication only infrequently. By the time oral ribavirin was finally approved by
the FDA in 1998 as part of a combination treatment (with interferon- derivatives) for hepatitis C, the
original patents had expired, and ribavirin had become essentially a generic drug. Ribavirin is a prodrug,
which when metabolized becomes an analogue of purine nucleotides used in the replication of RNA. How
it affects viral replication was unknown for many years and this affected its wider approval, but in recent
years it has been found that a major mode of action is as a hypermutagen, inducing lethal mutations in
the RNA-dependent RNA polymerases central to the replication of RNA viruses. There are still important
impediments to ribavirin use: clinical treatment requires high drug dosage, which leads to significant side
effects. The thrust of ongoing research is to combine ribavirin with other drugs, thereby allowing use of a
smaller dose.

1970 Roland Robins, Joseph Witkowski, colleagues synthesis of ribavirin, the first broad spectrum antiviral drug
page 382

Joseph R. Kates

James E. Darnell Jr.

After the discovery of poly(A) tails on viral

mRNAs in 1970 by Joseph Kates, the same tails
were found on most mRNAs of eukaryotes.
James Darnell and his colleagues, as well
as several other groups, studied these in
eukaryotic cells and found that they served
several functions: the tails promote processing
and export of mRNAs from the nucleus,
facilitate translation, and protect the mRNAs
from degradation. It was found that nearly
all eukaryotic mRNAs possess a poly(A) tail
(50-300 nucleotides in length) at their 3 end.
Most of the eukaryotic mRNAs are posttranscriptionally polyadenylated in the nucleus.
However, most positive sense RNA viruses
replicate in the cytoplasm, and so have evolved
their own mechanisms to acquire their polyA
tails: they either encode a 3 poly(A) sequence
on the end of their genomic RNA (positive
strand) and/or a 5 poly(U) sequence on the
negative sense RNA used as their replicative
intermediate, or they employ reiterative
copying (stuttering) on short 3 (U) sequences
on their negative sense RNA strand. Poly(A)
tails and other terminal additions to RNAs are
so important that cells have even evolved repair
mechanisms to assure that damage is not fatal
the RNA viruses enjoy such repair afforded by
the host cell that they are usually committed to
destroy.

Kates J. Transcription of vaccinia virus genome and the occurrence of polyriboadendylic acid
sequences in mRNA. Cold Spring Harbor Symposium on Quantitative Biology 1970;35:743-752.
Darnell JE Jr, Wall R, Tushinski RJ. An adenylic acid rich sequence in mRNA of HeLa cells and its
possible relationship to reiterated sites in DNA. Proc Natl Acad Sci USA. 1971;68:1321-1325.

Edmonds M, Vaughan MH, Nakazato H. Polyadenylic acid sequences in the heterogenous nuclear
RNA and rapidly labeled poly-rRNA of HeLa cells: possible evidence for a precursor relationship.
Proc Natl Acad Sci USA. 1971;68:1336-1340.
Lee SY, Mendecki J, Brawerman G. A polynucleotide segment rich in adenylic acid in rapidly
labeled poly-rRNA composed of mouse sarcoma 180ascites cells. Proc Natl Acad Sci USA.
1971;68:1331-1335.

Viral mRNA circularization at the ribosome enhances

translation. The poly(A)-binding protein (PABP)
interacts with the translation initiation factor eIF4G
and the 5 cap-binding protein eIF4E, brings the 5
and 3 ends of the mRNA together.

1970 Joseph Kates James Darnell Jr., others discovery of the 3 poly(A) tail on many viral and cellular mRNAs

page 383

Ornithodoros spp

Phlebotomus papatasi

Walter Plowright (1923-2010)

Robert B. Tesh

Aedes triseriatus

Douglas M. Watts

Of all the infectious agents, the Rickettsiae and the viruses have been most successful in employing vertical transmission in their arthropod hosts in their quest for longterm survival. One reason is that Rickettsiae and certain viruses have developed a capacity for persistent cellular infection without disturbing host functions too much. In
many cases the viruses that are capable of transovarial (or transstadial) transmission in their arthropod vectors use this mechanism as a secondary or backup life cycle, for
use when their primary horizontal transmission cycle fails. For example, it has long been thought (but is largely unproven) that transovarial virus carriage might explain the
overwintering of certain arboviruses in areas where adults cannot survive. Some viruses do, however, employ vertical transmission in their primary life cycle. The history of
our understanding of the transovarial transmission phenomenon is long, complex and difficult to assign attributions. The table included here (abridged from the review by
Burgdorfer and Varma) illustrates this. As long ago as 1906 there was an unconfirmed report that yellow fever virus was occasionally transmitted to human volunteers by
transovarially infected Aedes aegypti mosquitoes. Transovarial infection of ticks by certain viruses was the first to be considered important, but here the older literature is not
often included in databases. The more modern literature based upon more modern laboratory methods gives precedent to the proof of the transovarial transmission of (1)
African swine fever virus by ticks; (2) La Crosse virus by mosquitoes, and (3) vesicular stomatitis viruses by phlebotomine flies (sandflies).
Daubney R, Hudson JR. Nairobi sheep disease. Parasitology 1931;23:507-524.
Chumakov MP. Studies on virus encephalitides. 6. Transmission of tick-borne encephalitis to the offspring in Ixodidae ticks and the question of
natural reservoirs of this infection. Med Parazitol Parazitar Bolezni. 1944;6:38-40 (In Russian).
Benda, R. The common tick, Ixodes ricinus as a reservoir and vector of tick borne encephalitis. I. Survival of the virus (strain B3) during the
development of the tick under laboratory conditions. J Hyg Epidemiol Microbial Immunol (Prague). 1958;2:314-330.
Blakovi D, ehek J. Ticks as virus vectors in Eastern Europe. In: Maramorosch K, editor. Biological transmission of disease agents. New York:
Academic Press; 1960. p. 192-196/
ehek J. Transovarial transmission of tick-borne encephalitis virus by ticks. Acta Virol. 1962;9:220-226.
Florio L, Miller MS, Mugrage ER. Colorado tick fever. Isolation of the virus from Dermacentor andersoni in nature and a laboratory study of the
transmission of the virus in the tick. J Immunol. 1950;64:257-263.
Avakian AA, Lebedev AD. Natural focalization of haemorrhagic fevers. J Microbial Moscow. 1955;4:20-26 (In Russian, English translation:
NAMRU 3-T 147).
Plowright W, Perry CT, Pierce MA. Transovarial infection with African swine fever virus in the argasid tick, Ornithodorus moubata porcinus
Walton. Res Vet Sci 1970;11:582-584.
Tesh RB, Chaniotis BN, Johnson KM. Vesicular stomatitis virus (Indiana serotype): transovarial transmission by phlebotomine sandflies. Science
1972;175:1477-1479.
Watts, DM, Pantuwatana S, DeFoliart GR. Transovarial transmission of La Crosse virus (California Encephalitis Group) in the mosquito, Aedes
triseriatus. Science 1973;182:1140-1141.
Burgdorfer W, Varma GR. Transstadial and transovarial development of disease agents in arthropods. Ann Rev Entomol. 1967;12:347-376.

Date

Virus / Arthropod

Discoverers

1931

Nairobi sheep disease virus / tick Rhipicephalus appendicutatus

R. Daubney, J.R. Hudson

1944>

Tick-borne encephalitis viruses / tick Ixodes persulcatus, I. ricinus

M.P. Chumakov, et al.; R. Benda, et al.; D. Blakovi, J. ehek

1950

Colorado tick fever virus / tick Dermacentor andersoni

L. Florio, M.S. Miller, E.R. Mugrage

1955

Omsk hemorrhagic fever virus / tick Dermacentor reticulates

A.A. Avakian, A.D. Lebedev

1960

Crimean-Congo hemorrhagic fever virus / Hyalomma marginatum

D. Blakovi, J. ehek

1970

African swine fever virus / Ornithodoros spp

W. Plowright, C.T. Perry, M.A. Pierce

1972

Vesicular stomatitis virus (Indiana) / sandfly Phlebotomus papatasi

R.B. Tesh, B.N. Chaniotis, K.M. Johnson

1973

La Crosse virus / mosquito Aedes triseriatus

D.M. Watts, S. Pantuwatana, G.R. DeFoliart

1970> Walter Plowright, Robert Tesh, Douglas Watts, others transovarial transmission of viruses by arthropods
page 384

Salmi AA, Grnroos JA, Halonen PE. Mixed

haemadsorption with herpes simplex virus. Ann Med Exp
Biol Fenn. 1968;46:103-108.
Halonen PE, Murphy FA, Fields BN, Reese DR.
Hemagglutinin of rabies and some other bullet-shaped
viruses. Proc Soc Exp Biol Med. 1968;127:1037-1042.

Direct examination of clinical specimens

Histopathology, ultrastructural pathology

Detection of viruses by electron microscopy

Detection of viral antigens

Herrmann KL, Halonen PE, Stewart JA, Casey HL,

Ryan JM, Hall AD, Caswell KE. Evaluation of serological
techniques for titration of rubella antibody. Am J Public
Health Nations Health. 1969;59:297-304.

Enzyme immunoassay (EIA, ELISA, TR-FIA)

Sarkkinen HK, Halonen PE, Arstila PP. Comparison of

four-layer radioimmunoassay and electron microscopy for
detection of human rotavirus. J Med Virol. 1979;4:255-260.

Immunohistochemistry

Sarkkinen HK, Halonen PE, Arstila PP, Salmi AA.

Detection of respiratory syncytial, parainfluenza type
2, and adenovirus antigens by radioimmunoassay and
enzyme immunoassay on nasopharyngeal specimens from
children with acute respiratory disease. J Clin Microbiol.
1981;13:258-265.
Meurman OH, Hemmil IA, Lvgren TN, Halonen PE.
Time-resolved fluoroimmunoassay: a new test for rubella
antibodies. J Clin Microbiol. 1982;16:920-925.

Pekka Eljas Halonen (1927-2001)

Viral Diagnostic Methods, 1970-1980s

Calisher CH, Meurman O, Brummer-Korvenkontio

M, Halonen PE, Muth DJ, 1985. Sensitive enzyme
immunoassay for detecting immunoglobulin M antibodies
to Sindbis virus and further evidence that Pogosta disease is
caused by a western equine encephalitis complex virus.
J Clin Microbiol. 1985;22:566-571.

Radioimmunoassay

Immunofluorescence

Immunochromatography
Detection of viral nucleic acids

Polymerase chain reaction (PCR)

Hybridization (in situ, Southern blot, dot-blot)

Oligonucleotide fingerprinting,

Restriction endonuclease mapping

Virus isolation

Virus isolation in cultured cells

Virus isolation in animals or chick embryos

Serology

Enzyme immunoassay (EIA, ELISA, IgM-EIA)

Serum neutralization assay

Immunoblotting (western blotting)

Indirect immunofluorescence assay

Hemagglutination-inhibition assay
Complement-fixation assay

Rapid viral diagnostic methods, applicable to many virus infections, made great strides in the 1970s and 1980s. This followed an era when most viral diagnostic tests were
done in public (state or city) laboratories, an era when results were only available weeks after submission of specimens. Often, the tests used centered on virus isolation and
identification, and on paired-sera serology. Needless to say, such test results had little or no clinical relevance, but were said to have public health purposes (surveillance,
outbreak investigation, etc.). Starting in Scandinavia, especially in Turku, Finland, this changed when better diagnostic technologies became available and were married
to systems for rapid testing and reporting; usually results reached clinicians caring for hospitalized patients, especially children, on the same day as specimen submission.
Diagnostic tests became part of the standard-of-care, and were relied upon in the real-time decisions of clinicians treating seriously ill patients. This era started with
immunofluorescence tests for viral antigens in respiratory diseases and was followed by radioimmunoassays and enzyme immunoassays. When tests were found to have
similar sensitivity and specificity, simplicity and rapidity became the bases for choosing methods and reagents. Pekka Halonen was the leader of this movement, which grew in
1977 into the The European Group for Rapid Laboratory Viral Diagnosis. Halonens influence was straightforward: Its the diagnosis, not the method To be useful the tests
must be rapid, specific, high-throughput Whatever method is best in a given circumstance with a given virus must be used The methods will keep changing Indeed, the
introduction of monoclonal antibody reagents and antigens produced by recombinant DNA technologies in the 1980s did keep changing the methods. In the United States,
this wave of progress in clinical virology never quite took hold and by the turn of the century clinical diagnostic labs were doing less and less virus diagnosis except by the use
of kit tests much of this failing was due to the cost-recovery system. The continuing revolution in methods, now usually based in virus genomic sequencing and very high
throughput automated equipment, is still not linked to clinical care in the United States or in many other developed countries.

1970> Pekka Halonen, others linkage of rapid virus diagnostics to clinical care, especially pediatric clinical care

page 385

Some Nonhuman Primate Viruses:

Seymour S. (Sy) Kalter (1918-2007)

Richard L. Heberling

There are about 200 species of nonhuman primates comprising four basic
groups, and viruses have been isolated from each. Approximately thirty species
have been used in medical research, with ten being used most commonly:
1. Prosimians (lemurs, aye-ayes, bushbabies, tarsiers) are primitive animals
and are not widely used in research.
2. Old World monkeys are the macaques (most commonly used are the rhesus
macaque, Macaca mulatta, and the cynomologous or long-tailed macaque,
Macaca fascicularis), baboon (Papio spp), patas monkey (Erythrocebus
patas) and vervet or African green monkey (Chlorocebus aethiops).
3. New World monkeys are the marmosets (Callithrix spp), squirrel monkey
(Saimiri sciureus), spider monkey (Ateles spp), and owl monkey (Aotus spp).
4. Apes. The chimpanzee (Pan troglodytes) is the only ape that has been used
in research and this is coming to a close.

Seymour Kalter and Richard Heberling

founded the NIH / WHO Collaborating
Center for Reference and Research
in Simian Viruses, at the Southwest
Foundation for Research and Education,
San Antonio, Texas, in 1970.

Adenoviruses (numerous types)

B virus
Benign epidermal monkeypox virus
Callitrichid hepatitis (LCM) virus
Cytomegaloviruses
Ebola virus
Encephalomyocarditis virus
Epstein-Barr virus
Hepatitis A virus
Hepatitis B virus
Hepatitis C virus
Herpes simplex virus 1
Herpes simplex virus 2
Herpesvirus ateles
Herpesvirus saimiri
Herpesvirus tamarinus
Marburg virus
Measles virus
Molluscum contagiosum virus
Monkeypox virus
Papillomaviruses
Rabies virus
Simian agent (SA8 virus)
Simian foamy viruses
Simian hemorrhagic fever virus
Simian hepatitis A virus
Simian immunodeficiency viruses
Simian varicella virus
SV40 virus
Tanapox virus
Yabapox virus
Yellow fever virus

Kalter SS, Heberling RL. Comparative virology of primates. Bacteriol Rev. 1971;35:310-364.
Kalter SS, Heberling RL. Primate viral diseases in perspective. J Med Primatol. 1990;19:519535.
Kalter SS, Heberling RL, Cooke AW, Barry JD, Yian PY, Northam WJ. Viral infections of
nonhuman primates. Lab Animal Sci. 1997;47:461-467.

1970> Seymour Kalter, Richard Heberling development of nonhuman primate virology

page 386

Janet S. Butel

Butel JS, Lednicky JA. Cell and molecular

biology of simian virus 40: implications for
human infections and disease. J Natl Cancer
Inst. 91:119-134, 1999.

Keerti Shah

Poulin DL, DeCaprio JA. Is there a role for SV40

in human cancer? J Clin Oncology 24:4356-4365,
2006.
Shah KV. SV40 and human cancer: A review of
recent data. Int J Cancer 2006;120:215-223.

Simian vacuolating virus (SV40), cell culture

The question whether SV40 virus can cause human tumors

has been one of the most highly controversial topics
in cancer research during the last 40 years. The debate
began with the discovery of SV40 virus as a contaminant
of poliovirus vaccines used in >100 million children and
adults between 1955 and 1963. Concerns were reinforced
by the finding that SV40 virus transforms human cells
and induces tumors in animal models. SV40 DNA has
been found in a variety of human cancers (mesotheliomas,
brain tumors, osteosarcomas, non-Hodgkin lymphomas);
however, the question remained whether these were linked
causally to the presence of SV40 virus. In 2002, the Institute
of Medicine published a report entitled Immunization
Safety Review: SV40 Contamination of Polio Vaccine and
Cancer, which concluded that: The biological evidence is
strong that SV40 is a transforming virus (able to transform
normal cells into malignant cells); the biological evidence
is of moderate strength that SV40 exposure could lead to
cancer in humans under natural conditions; the biological
evidence is of moderate strength that SV40 exposure from
polio vaccine is related to SV40 infection in humans; and
concerns about exposure to SV40 through inadvertent
contamination of polio vaccines are significant because of
the seriousness of cancers as the possible adverse health
outcomes and because of the continuing need to ensure and
protect public trust in the nations immunization program.
Others, especially anti-vaccine activists went much
further, condemning all or most current antiviral vaccines.
However, several more recent studies indicate that older
conclusions were flawed most studies describing the
recovery of SV40 DNA sequences from tumors have
not been reproducible. Contamination with laboratory
SV40 plasmids was identified as a possible source of false
positive results, and low-level immunoreactivity of human
sera to SV40 was likely the result of cross-reactivity with
antibodies to the SV40-related human polyomaviruses BK
and JC viruses. SV40 immunoreactivity in patients with
suspect tumors was no greater than that in controls. In
epidemiologic studies, the increased incidence of some of
the suspect tumors in the 1970s and 1980s was not related
to the risk of exposure to SV40-contaminated vaccines. In
summary, although many people were exposed to SV40
by polio vaccination the most recent evidence does not
support the conclusion that SV40 has contributed to the
development of human cancers.

1970s> Janet Butel, Keerti Shah, others controversy whether SV40 virus (in polio vaccines) causes brain tumors

page 387

Gertrude Belle Elion (1918-1999) George H. Hitchings (1905-1998)

Richard J. Whitley

In 1944, Gertrude Elion was hired as an assistant to George Hitchings in Burroughs-Wellcomes research labs. Her first assignments
were in purine chemistry. In the late 1950s, advances in biosynthesis pointed Hitchings and Elion to applications for their research
they invented rational drug design, replacing the trial-and-error approach of the day. They developed drugs based on purine analogs /
antagonists. In 1959, they were awarded a patent for 2-amino-6-mercaptopurine (6-MP; Purinethol) and methotrexate, the first major drugs
to treat leukemia. In 1962, they were awarded a patent for azathioprine (Imuran), the first drug used to suppress the immune rejection of
transplanted organs, especially kidneys. Next, Elion developed allopurinol (Zyloprim), used to treat gout. Finally, in the 1960s Elion and
Hitchings developed a screening program for antiviral drugs, which resulted in the discovery of acyclovir (Zovirax) in 1974. A preclinical
investigation brought the drug to clinical trials in 1977 and the first form of the drug, for topical use against herpes keratitis, was made
available in 1982. Their early antiviral drug work was done before the structure of DNA and the anabolic pathways involved in the utilization
of purines for nucleic acid synthesis had been imagined. The work of Elion and Hitchings opened a wide field of laboratory and clinical
research as several prominent clinical virologists explored the application of rationally selected candidate drugs: Richard Whitley, Lawrence
Corey and others advanced the use of acyclovir and derivative drugs in the treatment of the various diseases caused by the herpesviruses.
Hitchings GH, Elion GB, Falco EA, Russell PB, VanderWerff H. Studies on analogs of purines and pyrimidines. Ann NY Acad Sci.
1950;52:1318-1335.
Elion GB, Furman PA, Fyfe JA, de Miranda P, Beauchamp L, Schaeffer HJ. Selectivity of action of an antiherpetic agent,
9-(2-hydroxyethoxymethyl) guanine. Proc Natl Acad Sci USA. 1977;74:57165720.
Whitley RJ, Soong S-J, Dolin R, Galasso GJ, Chien LT, Alford CA. Adenine arabinoside therapy of herpes simplex encephalitis: National
Institute of Allergy and Infectious Diseases Collaborative Antiviral Study. N Engl J Med. 1977;297:289-294.

1970s> Gertrude Elion, George Hitchings, others rational drug design, acyclovir for herpesvirus infections
page 388

Radioimmunoassays (RIAs) were first described in 1960 by Rosalyn

Yalow and Solomon Berson and immediately caught the imagination
of many investigators; over the next decade many specific assays were
developed, including several for viral diagnostics. In many laboratories
around the world special facilities were built for safely working with
the amounts of radioisotopes required for the labeling of antigens
or antibodies, but safety concerns persisted. In the early 1970s, the
idea of using enzyme labels was met with skepticism: how could so
large a molecule as an enzyme be attached to an antigen or antibody
without sterically hindering the immunochemical reaction between
antigen and antibody? Peter Perlmann (1919-2005) and Eva Engvall
at Stockholm University in Sweden and Anton Schuurs and Bauke
van Weemen at the Research Laboratories of NV Organon, Oss,
The Netherlands, independently invented enzyme immunoassays
(EIAs / ELISAs), the former first measuring IgG in rabbit serum
with alkaline phosphatase as the reporter label, the latter first
quantifying human chorionic gonadotropin concentrations in urine
with horseradish peroxidase as the reporter label. In the early 1970s,
blood-bank screening for viral diseases such as hepatitis B (HBsAg)
was done either by (semi)automated RIA or nonradioactive but rather
cumbersome hemagglutination tests. In 1976, Organon Teknika
developed and marketed a highly successful EIA system for hepatitis
B surface antigen (HBsAg), in 96-well microtiter plates. This became
the first commercially produced EIA. Other diagnostic tests followed,
most prominently EIAs for human immunodeficiency virus antibodies
in the 1980s.
Engvall E, Perlmann P. Enzyme-linked immunosorbent assay
(ELISA). Quantitative assay of immunoglobulin G. Immunochemistry
1971;8:871-874.
van Weemen BK, Schuurs AHWM. Immunoassay using antigenenzyme conjugates. FEBS Letts. 1971;15:232-236.

Eva Engvall, Anton Schuurs, Peter Perlmann, Bauke van Weemen

The first EIA kit,

HEPANOSTIKA test
kit for hepatitis B
(HBsAg)
Organon Teknika,
The Netherlands

There are many different types of EIAs / ELISAs for the detection of
viral antigens or antibodies in serum or other clinical specimens. One
of the most common is the sandwich ELISA, in which the moiety (say
an antibody) to be detected is sandwiched between an antigen fixed
on the surface of the wells of a 96 well plate and a chromogenicallyconjugated anti-Ig, which is added last. Common chromogens
are alkaline phosphatase and horseradish peroxidase. Common
chromogen substrates, each yielding a different color, are ABTS: 405410nm (purple); TMB: non-stopped 620-650nm (red), stopped 450nm
(green, as shown here); OPD: non-stopped 450nm (green), stopped
490nm (blue); pNPP: 405-410nm (purple), and BluePhosTM: 595650nm (yellow).

1971 Eva Engvall, Bauke van Weemen, colleagues development of enzyme-linked immunoassays (EIAs, ELISAs)

page 389

David Baltimore

I:
II:
III:
IV:
V:
VI:
VII:

dsDNA viruses (Adenoviruses, Herpesviruses, Poxviruses)

(+) sense ssDNA viruses (Parvoviruses)
dsRNA viruses (Reoviruses)
(+) sense ssRNA viruses (Picornaviruses, Togaviruses)
(-) sense ssRNA viruses (Orthomyxoviruses, Rhabdoviruses)
ssRNA-RT viruses (Retroviruses)
dsDNA-RT viruses (Hepadnaviruses)

David Baltimore: The world of animal viruses appears to offer an unfathomable diversity, but as the molecular biology of the replication of many viruses has been studied, a
pattern of behavior has emerged. The viruses can be divided into classes, each of which has its own method of transmitting its genetic information from one generation to the
next and its own style of expressing its genetic information. Although in some cases the data are still fragmentary, it is possible to outline these systems and to place them in a
formal scheme. Viruses have evolved to the point where they are concerned mainly with two processes: duplication of their genetic material and controlled expression of the
information inherent in their genetic material.... There are two end products of the viral genetic system: new genetic material and messenger ribonucleic acid (mRNA). The one
process which all viruses must perform is mRNA synthesis. The central role of mRNA derives from the fact that viruses use cellular ribosomes and soluble factors to translate
their mRNA. Therefore, the job of the virus is almost finished once it has made mRNA, except for certain minor alterations in the functioning of the translation machinery.
One important aspect of the transcriptional systems of animal viruses has emerged during the last few years: the role of virion-associated nucleic acid polymerases. The initial
discovery of the virion-associated DNA-dependent RNA polymerase of vaccinia virus, followed a year later by the discovery of the double-stranded RNA-dependent RNA
polymerase of reovirus, opened up a new concept of how viruses carry out their initial transcriptional processes. The finding that vesicular stomatitis virus and Newcastle
disease virus both have virion-associated RNA-dependent transcriptases has led us to see such enzymes as the rule rather than the exception. Finally, the existence of an
RNA-dependent DNA polymerase in the virions of the RNA tumor viruses has led us to the concept that, whenever the first function performed by virion nucleic acid after its
introduction into the cell is the transfer of its information to another nucleic acid, the enzyme responsible for this transfer is likely to be found in the virion. Viruses which
have a double-stranded DNA genome can express and duplicate their genetic material by processes which are at least formally identical to those used by cells. Viruses with
other types of genomes require special systems for replication and transcription. These processes make up the viral genetic system, and it is possible to group viruses according
to general properties of their genetic systems.
Baltimore D. Expression of animal virus genomes. Bacteriol Rev. 1971;35:235-241.

1971 David Baltimore system of virus categorization based on the replicative path to mRNA
page 390

In 1971 Theodore Diener and his colleagues (William B. Raymer, Muriel OBrien, others) showed that the
causative agent of potato spindle tuber disease was the potato spindle tuber viroid (PSTVd), which is a
130,000 dalton (341-364 nt, varying with strain this is at least three orders of magnitude smaller than
even the most diminutive virus) single-stranded circular RNA molecule that has no coat protein, and which
does not code for any protein. Despite their small size, and therefore extremely limited genetic information,
viroids are replicated autonomously (requiring no helper virus) in the nucleus or chloroplasts of plant cells
by host RNA-dependent RNA polymerase. In some ways, viroids resemble transposons, but taxonomically
they have been accorded their own taxon. The painstaking work leading to these conclusions took six
years, particularly since the discovery had to stand up against anti-dogma skeptics. One key was the
development of a bioassay for the PSTVd viroid quantitating the infectious moiety was difficult because
of the very slow development of the disease in potatoes, often taking several years to become manifest.
The new bioassay was based on infection of tomatoes, where plants become dramatically stunted in a few
weeks. Overall, the discovery brought about a revolution in plant virology as more and more diseases
were found to be caused by viroids presently, there are at least 53 viroid diseases, some commercially
important, including cadang-cadang, a devastating disease of commercial coconut palm plantations in
several parts of the world.
It has been proposed that human hepatitis D virus (hepatitis delta virus, HDV) may have originated as a
viroid. Evidence in support of this stems from the fact that both HDV and viroids exist as single-stranded,
linear and closed circular RNAs that have rod-like structures. Likewise, both HDV and viroids contain
RNA sequences that can form catalytically active structures, that is ribozymes. During replication, these
catalytic RNAs are required in order to produce unit length copies of the genome. Neither HDV nor viroids
encode their own polymerase; instead, their replication requires a host polymerase that can utilize RNA
as a template. RNA polymerase II has been implicated as the polymerase responsible for the replication
of HDV. Normally RNA polymerase II utilizes DNA as a template and produces mRNA. Consequently, if
HDV indeed utilizes RNA polymerase II for its replication, it would be the only known pathogen capable of
using a DNA-dependent polymerase as an RNA-dependent polymerase.

Theodore Otto Diener

Diener TO, Raymer WB. Potato spindle tuber virus: a plant virus with properties of a free nucleic acid.
Science 1967;158:378-381.
Diener TO. Potato spindle tuber virus. IV. A replicating, low molecular weight RNA. Virology
1971;45:411-428.
Diener TO. Discovering viroidsa personal perspective. Nat Rev Microbiol. 2003;1:75-80.
Taylor JM. Hepatitis delta virus. Virology 2006;344: 71-76.

Secondary structure of potato spindle tuber viroid, 341-364 nucleotides, varying with strain.
Nucleotides in central area that are common to most viroids are highlighted in yellow/red
Potatoes, infected and uninfected with
potato spindle tuber viroid (PSTVd)

1971 Theodore Diener discovery of viroids (infectious naked RNA molecules)

page 391

Sylvia D. Gardner

Anne Field (1936-2007)

BK virus was discovered at the Virus Research Laboratory, Public Health Laboratory Service, Collindale, London,
during a study by Sylvia Gardner of cytomegalovirus infection in kidney transplant recipients. Urine collected from
a patient after transplantation was found to contain numerous cells bearing viral inclusions. Electron microscopy
by Anne Field demonstrated virus particles that resembled papillomaviruses. Inoculation of the urine into rhesus
monkey kidney cells and human embryonic kidney cells produced a cytopathic effect and confirmed that the
virus was not a papillomavirus. The new virus and named BK virus after the initials of the patient from whom it
was isolated. Epidemiological studies at Collindale in the 1970s and 1980s showed that up to 90% of some human
populations became infected by BK virus by adulthood. Following transplantation, 10% to 60% of allograft recipients
excreted virus in their urine. However, viral infection was usually asymptomatic or associated with only transient graft
dysfunction. The first case of clinically significant BK virus interstitial nephritis was diagnosed by a needle biopsy of
an allograft in 1993, after which BK virus-associated interstitial nephritis was reported from most kidney transplant
centers around the world. In recent years, an etiological role of BK virus in human kidney cancers has been advanced,
but the evidence is disputed and the view most experts is that there is not an etiologic association.
Gardner SD, Field AM, Coleman DV, Hulme B. New human papovavirus (BK) isolated from urine after renal
transplantation. Lancet 1971;1:1253-1257.

Renal allograft biopsy, BK virus nephropathy:

(top) intranuclear inclusion in tubular epithelial
cell; (bottom) electron microscopy of inclusion
showing that it is comprised of virions arranged in
paracrystalline array

1971 Sylvia Gardner, Anne Field, colleagues discovery of BK virus, etiologic agent of kidney and bladder disease
page 392

Thottapalayam virus has been isolated from Suncus murinus (Asian house
shrew, musk shrew) in India, Afghanistan, Pakistan, India, Sri Lanka, Nepal,
Bhutan, Burma, China, Taiwan, and Japan. This is the only hantavirus not
isolated from a rodent; whether the virus is naturally harbored by this
insectivore host or represents spillover from a rodent reservoir host is
unknown, but some evidence suggest the virus co-evolved with this host.
Carey DE, Reuben R, Panicker KN, Shope RE, Myers RM. Thottapalayam
virus: a presumptive arbovirus isolated from a shrew in India. Indian J Med
Res 1971;59:1758-1760.
Song J-W, Baek L-J, Schmaljohn CS, Yanagihara R. Thottapalayam virus, a
prototype shrew-borne hantavirus. Emerg Infect Dis. 2007;13:980-985.

Donald E. Carey

Jin-Won Song

Geographic distribution of Suncus murinus

(blue = native; red = introduced)

Robert E. Shope (1929-2004)

Richard Yanagihara

Suncus murinus (Asian house shrew, musk shrew)

1971 Donald Carey, Jin-Won Song, Robert Shope, Ric Yanagihara, others Thottapalayam virus (the first hantavirus)
page 393

Bert Geoffrey Achong (1928-1996)

Michael Anthony Epstein

Simian foamy virus 1

cell culture, thin section electron microscopy

Simian foamy viruses (SFVs) have been reported in 1-4% of persons
occupationally exposed to nonhuman primates in zoos, primate centers,
and laboratories, in North America and in Europe. More recently, naturally
acquired SFV infections were described in hunters in Cameroon and in
a person who had had contact with Macaca fascicularis macaques in
Indonesia. About 70-90% of non-human primates born in captivity are
infected with SFVs, but no disease has been associated with these infections.
Achong BG, Mansell PW, Epstein MA, Clifford P. An unusual virus in
cultures from a human nasopharyngeal carcinoma. J Natl Cancer Inst.
1971;46:299-307.
Heneine W, Switzer WM, Sandstrom P, Brown J, Vedapuri S, Schable CA,
Khan AS, Lerche NW, Schweizer M, Neumann-Haefelin D, Chapman LE,
Folks TM. Identification of a human population infected with simian foamy
viruses. Nat Med. 1998;4:403-407.

Thomas Folks

Paul Luciw

Herchenrder O, Renne R, Loncar D, Cobb EK, Murthy KK, Schneider J,

Mergia A, Luciw PA. Isolation, cloning, and sequencing of simian foamy
viruses from chimpanzees (SFVcpz): high homology to human foamy virus
(HFV). Virology. 1994;201:187-199.

1971 Bert Achong, Michael Epstein, Paul Luciw, Thomas Folks, others simian foamy virus infection in humans
page 394

George Martin Baer (1936-2009)

Franz Steck (1932-1982)

The concept of vaccinating wildlife rabies reservoir host species against rabies was developed by
George Baer and his colleagues at the U.S. Centers for Disease Control in 1971. They did extensive
containment-housed animal experiments to show safety and efficacy; however, because of regulatory
entanglements they were not allowed to do field tests. Two veterinarians from the Swiss Rabies
Center, Franz Steck and Alexander Wandeler, used the method to immunize foxes against rabies; they
experimented with encapsulating vaccine and placing capsules in chicken heads. When unsuspecting
foxes ate the chicken heads, they broke open the capsules and were immunized. Starting in 1978, the
Swiss started dropping vaccine-containing chicken heads in the Rhone Valley by helicopter, but after
Steck was killed in a helicopter crash other methods of distribution were devised. The method was
a great success and within a few years Switzerland was free of rabies (the fox was the only reservoir
host present in the country). The method, improved over the years in several ways, was taken up by
other European countries and finally in Canada and the U.S., where the variety of rabies reservoir
host species again called for modifications of bait delivery, but where again there has been great
success in reducing wildlife rabies and consequent transmission to domestic animals and humans.
Baer GM, Abelseth MK, Debbie JG. Oral vaccination of foxes against rabies. Am J Epidemiol.
1971;93:487-490.
Steck F, Wandeler A, Bichsel P, Capt S, Hafliger V, Schneider L. Oral immunization of foxes against
rabies: laboratory and field studies. Comp Immunol Microbiol Infect Dis. 1982;5:165-171.

Alexander Wandeler

(above) Rabies, Animals,

Europe, 1983 after 30 years
of conventional rabies control
(left) Rabies, Animals,
Europe, 2008 after 26
years of bait-delivered oral
vaccination of foxes

1971 George Baer, Franz Steck, Alexander Wandeler, colleagues oral vaccination of wildlife against rabies

page 395

Albert Z. Kapikian

The discovery of Norwalk virus is the first instance of bypassing classical tissue-culture and
experimental animal virology in necessary favor of direct virology or particle virology, as
described Albert Kapikian. In 1969, his lab at the NIH turned to the study of the etiology of acute
nonbacterial (viral) gastroenteritis, where it was already known from human volunteer studies
involving the feeding of bacteria-free stool ultrafiltrates, that virus(es) were involved as important
etiologic agents. Newer techniques developed for the detection of fastidious viruses, such as organ
culture, were tried by research groups in the U.S., the United Kingdom and Japan, still without
success. In 1970, at NIH fecal ultrafiltrates from four gastroenteritis outbreaks were studied in
human volunteers one of the outbreaks occurred in an elementary school in Norwalk, Ohio in
1968. During a 2-day period, 50% of the students and teachers developed gastroenteritis, along
with 32% of contacts. Standard lab studies failed to reveal an etiologic agent. An ultrafiltrate from
a secondary case in the Norwalk outbreak was administered orally to 3 volunteers, 2 of whom
developed gastroenteritis. Minimal virus characterization studies were conducted using this
ultrafiltrate. Then, everything changed: Kapikian had studied in the lab of Anthony Waterson and
June Almeida at the Royal Postgraduate Medical School, University of London, and had brought the
immunoelectron microscopy (IEM) technique back to the NIH, where still the Norwalk agent could
not be cultivated. After 20 months of developmental work, Kapikian examined Norwalk agent stool
ultrafiltrates, using a volunteers convalescent serum as the source of antibody. He stated, glistening
aggregates of nonenveloped, antibody-coated 27-nm, virus-like particles, which resembled
rhinoviruses, were visualized. [The virus is now known to be 35-39nm in diameter.] It was clear that
further studies were needed to determine the significance of this finding this was done by showing
that volunteers who became ill developed a serologic response to the 27-nm particles visualized
by IEM. An antibody-rating system was developed, in which dilutions of pre- and post-challenge
serum were reacted with the Norwalk agent ultrafiltrate and examined by negative contrast electron
microscopy, that is by IEM. When not coated by antibody the icosahedral virions appear to have a
Star of David pattern on their surface; when heavily coated by antibody virion structure is almost
totally obscured. The final proof involved testing paired acute and convalescent sera from patients
who developed naturally occurring illness during the outbreak in Norwalk, Ohio it was reasoned
that it would be highly unlikely that the newly discovered 27-nm particle was an adventitious agent
if the naturally occurring cases developed an antibody response to it. Success here, with negative
controls used in blinded observations of IEM reactions, was a major link in establishing the etiologic
association of the virus and the disease. Soon, the same method showed that there were several
distinct Norwalk-like viruses. During the early 1990s, cloning and sequencing of Norwalk-like
viruses led to the development of sensitive molecular assays [e.g., reverse transcription-polymerase
chain reaction (RT-PCR), nucleotide hybridization probes, and enzyme-linked immunosorbent
assays (ELISA) based on recombinant-DNA expressed viral antigens]. Using these assays, it was
shown that Norwalk-like viruses are the cause of a high percentage of foodborne and waterborne
gastroenteritis outbreaks. The viruses are now classified as belonging to the genus Norovirus, family
Caliciviridae.
Kapikian AZ, Wyatt RG, Dolin R, Thornhill TS, Kalica AR, Chanock RM. Visualization by immune
electron microscopy of a 27nm particle associated with acute infectious nonbacterial gastroenteritis.
J Virol. 1972;10:1075-1081.

Norwalk virus, without and with antibody (IEM)

Kapikian AZ. The discovery of the 27-nm Norwalk virus: an historic perspective. J Infect Dis.
2000;181S2:S295-302.

1972 Albert Kapikian, colleagues discovery of Norwalk virus (the first human calicivirus, the first norovirus)
page 396

Paul Berg

By the mid-1960s, several members of Paul Bergs team at Stanford University, including
Dale Kaiser, had trained at the Pasteur Institute and followed the research of Franois Jacob
and Jaques Monod closely. In a 1965 presentation, Kaiser suggested that the mechanisms at
work in bacteriophage might have analogues in mammalian tumor viruses such as SV40
virus. Berg was fascinated by the implications: could viruses be used to study gene regulation
in mammalian cells, as they had been in bacterial cells? He felt that the understanding of
gene expression in bacteria was becoming clear, but there was still little known about gene
expression in higher life forms. Eager to explore such fresh territory, Berg spent several years
preparing for the work that would lead to the development of recombinant DNA technologies.
One immediate problem was that the DNA of obvious vectors SV40 and polyoma viruses
was so small that it was unlikely that it could pick up genes from other sources. To overcome
this limitation, Berg and his team set out to combine SV40 DNA with a plasmid found in E.
coli. The plasmid consisted of phage DNA together with three genes from E. coli. By splicing
the plasmid into the SV40 DNA, they created a recombinant DNA molecule that had both
the ability to enter a mammalian cell and the ability to carry new genes from other sources
when it did so. The technique that Berg and his team devised for splicing two DNA molecules
together utilized an array of enzymes: first, they cut open the circular SV40 and plasmid
DNAs. Working from the knowledge that phage DNA strands have cohesive or sticky ends
that allow complimentary base pair bonding, they created their own sticky ends using another
enzyme to add strings of thymine (T) and adenine (A) nucleotides to the ends of each DNA.
Then finally, the A and T strands of the two DNAs were annealed using DNA polymerase,
ligase, and other enzymes. This complex procedure, resulting in the first recombinant DNA
molecule, was described by Berg, David Jackson, and Robert Symons in 1972. Because such
a revolutionary technique was considered by some to create unpredictable occupational or
environmental hazards, Berg did not insert the recombinant DNA molecule into a bacterium
rather, within a few years he took the leading role in organizing the scientific community to
address the issue of recombinant DNA safety (he served as chair of the National Academy of
Sciences Committee on Recombinant DNA Molecules and in 1975 organized the Asilomar
Conference on Recombinant DNA). Within a year of Berg and his teams publication, Stanley
Cohen, Herbert Boyer, and others had discovered that a restriction enzyme, EcoR1, would
create the necessary sticky ends on almost any DNA molecule, which greatly simplified the
recombination process and made it possible to splice together any DNA from any source this
was the beginning of genetic engineering and the biotechnology industry.
Jackson DA, Symons RH, Berg P. Biochemical method for inserting new genetic information
into DNA of simian virus 40: circular DNA molecules containing lambda phage genes and the
galactose operon of Escherichia coli. Proc Natl Acad Sci USA. 1972;69: 2904-2909.
Berg P, Mertz JE. Personal reflections on the origins and emergence of recombinant DNA
technology. Genetics 2010;184:9-17.

Method used by Jackson, Symons and Berg

in 1972 for constructing SV40-dvgal 120
recombinant DNA in vitro

1972 Paul Berg, Dale Jackson, Robert Symons development of recombinant-DNA technology

page 397

Stanley Norman Cohen

Herbert Boyer

Stanley Cohens molecular cloning diagram

In 1972, Stanley Cohen from Stanford University and Herbert Boyer from the University of California San Francisco met in Honolulu at a U.S.-Japan conference on bacterial
plasmids. Boyer gave a paper on the nature of the cleavage of DNA carried out by the restriction enzyme EcoRI, and Cohen gave a paper on his procedure for transfecting E.
coli with plasmid DNA. They made a now-famous late evening excursion to a delicatessen in Waikiki for pastrami sandwiches and immediately began planning collaborative
experiments. The work involved Cohens plasmids and Boyers EcoRI enzyme, with analysis of recombinant products in each laboratory. Within four months they and their
team members had a breakthrough the first success involved one of Cohens plasmids, pSC101, a tetracycline-resistance gene of E. coli, which they transferred from one
strain of E. coli to another. They soon moved on to more complicated cloning experiments. They joined tetracycline-resistant plasmids with kanamycin-resistant plasmids
and inserted them into E. coli. Next, they showed that genes could be transferred between species: a Staphylococcus aureus plasmid was spliced into E. coli plasmids and then
inserted into E. coli. They then cloned genes from a South African toad (Xenopus laevis) into E. coli. They wrote three landmark papers in 1973 and 1974, which revolutionized
biological research and launched the multi-billion dollar biotechnology industry.
Cohen SN, Chang AC, Boyer HW, Helling RB. Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci USA. 1973;70:3240-3244.
Chang AC, Cohen SN. Genome construction between bacterial species in vitro: replication and expression of Staphylococcus plasmid genes in Escherichia coli.
Proc Natl Acad Sci USA. 1974;71:1030-1034.
Morrow JF, Cohen SN, Chang AC, Boyer HW, Goodman HM, Helling RB. Replication and transcription of eukaryotic DNA in Escherichia coli.
Proc Natl Acad Sci USA. 1974;71:1743-1747.

1973 Stanley Cohen, Herbert Boyer application of recombinant DNA technology, start of genetic engineering
page 398

Maxine Singer, Norton Zinder (1928-2012), Sydney Brenner, Paul Berg

at Asilomar, 1973

Time Magazine cover art, 1977

Doomsday: Tinkering With Life

It is one of the lowliest of natures creatures, a rod

-shaped beastie less than a ten-thousandth of an
inch long [Escherichia coli]... Yet this tiny parcel
of protoplasm has now become the center of a
stormy controversy that has divided the scientific
community, stirred fearsoften farfetchedabout
tampering with nature, and raised the prospect of
unprecedented federal and local controls on basic
scientific research.
CLASSIFICATION OF INFECTIOUS MICROORGANISMS BY RISK GROUP
Following upon the Asilomar Conference of 1973
Risk Group 1

Agents that are not associated with disease in healthy adult humans.

Risk Group 2

Agents that are associated with human disease which is rarely serious and for which preventive or
therapeutic interventions are often available.

Risk Group 3

Agents that are associated with serious or lethal human disease for which preventive or therapeutic
interventions may be available (high individual risk but low community risk).

Risk Group 4

Agents that are likely to cause serious or lethal human disease for which preventive or therapeutic
interventions are not usually available (high individual risk and high community risk).

After Stanley Cohen, Herbert Boyer, and their colleagues

succeeded with the first cloning experiments in 1973, concerns
over biosafety led to a request that the National Academy of
Sciences address the situation. In response, Paul Berg convened
a meeting of seven scientists, which produced an open letter
to the scientific community (published in Science) requesting a
moratorium on recombinant DNA research until the hazards of
such research could be assessed by an international conference of
scientists, and procedures developed for containment of possible
hazards. This provoked a wide range of commentary, as well as
protests, but the voluntary moratorium was universally observed.
The first conference was held at the Asilomar Conference Center
near Monterey in 1973. Although no problems arising from
working with recombinant infectious agents were uncovered,
several recommendations were made; the proceedings were
published by the Cold Spring Harbor Laboratory Press (Alfred
Hellman, Michael N. Oxman, Robert Pollack, editors). The
second conference was organized by Berg, Maxine Singer, and
Richard Roblin, and held at the Asilomar Conference Center,
this one in 1975. There were about 140 participants; it was
recommended that the moratorium on experiments be lifted
and replaced with guidelines governing such research. In 1976,
the NIH issued its first set of Guidelines for Research Involving
Recombinant DNA. As the guidelines took hold, research
under seemingly appropriate containment recommenced with
a bang. The first principle for dealing with potential risks was
that containment facilities should be made an essential part
of experimental design. A second principle was that the level
of containment should match the estimated risk as closely as
possible varying risk levels were identified (table). Biological
barriers to limit the spread of recombinant DNA were also
included in the guidelines; these included fastidious bacterial
hosts that were unable to survive in natural environments and
equally fastidious vectors (plasmids, bacteriophages, viruses).
Hellman A, Oxman MN, Pollack R, editors. Biohazards in
biological research. Proceedings of a conference held at the
Asilomar conference center, Pacific Grove, California, January 2224, 1973. New York: Cold Spring Harbor Laboratory Press; 1973.
Berg PD, Baltimore D, Boyer HW, Cohen SN, Davis RW, et al.,
Letter: potential biohazards of recombinant DNA molecules.
Science 1974;185: 303.
Berg PD, Baltimore D, Brenner S, Roblin RO, Singer MF.
Summary statement of the Asilomar conference on recombinant
DNA molecules. Proc Natl Acad Sci USA. 1975;72:1981-1984.

1973, 1975 Paul Berg, others Asilomar conferences and proceedings: Biohazards in Biological Research

page 399

Robert H. Purcell, Albert Z. Kapikian, Stephen M. Feinstone

Hepatitis A virus

negative contrast electron microscopy

Albert Kapikians success in discovering and characterizing Norwalk virus as a major cause of acute gastroenteritis provided the impetus and methodology for seeking other
previously undetected human viral pathogens over time, he developed immune-electron microscopy (IEM) to a fine art. Using the method in carefully controlled studies
(samples from human volunteer studies and outbreaks, IEM results recorded from blinded/coded reactions, etc.), Kapikian, Robert Purcell and Stephen Feinstone, at the NIH,
succeeded in visualizing and identifying the virus responsible for hepatitis A. Prior to this, even the diagnosis of acute viral hepatitis was problematic; diagnosis was largely
based upon clinical and clinical chemistry evidence, but the overlap between what turned out to be hepatitis B and then hepatitis C virus infections was confounding. In
addition, in the 1960s, a number of unclassified, so-called candidate hepatitis viruses were isolated from patients with acute hepatitis, all of which proved inconsequential.
Further, a number of agents belonging to well-known virus families were also isolated from patients with hepatitis, again just confounding. Prior to 1973, it was known that
hepatitis A was transmissible by oral ingestion of ultrafiltered stool extracts from infected patients. Thus, the IEM method was employed on stool ultrafiltrates, not serum or
other clinical specimens. In the first experiments, ultrafiltered stool specimens from hepatitis A infected volunteers were incubated with dilutions of convalescent serum from
a hepatitis A patient. The 27nm virus was found in two of four specimens from subjects suffering acute illness but in none of four controls. Large numbers of experiments
followed, with stool specimens and convalescent serum from other sources (including Saul Krugmans Willowbrook State Hospital and outbreaks at Holy Cross College and
in American Samoa). The work was quickly confirmed by other investigators, including several working on food-borne acute hepatitis outbreaks. The same stool specimens
reactive by IEM infected chimpanzees and marmosets, with the same 27nm virus particles appearing in their stools.
Feinstone SM, Kapikian AZ, Purcell RH. Hepatitis A: detection by immune electron microscopy of a virus-like antigen associated with acute illness.
Science 1973;182:1026-1028.

1973 Stephen Feinstone, Albert Kapikian, Robert Purcell discovery of hepatitis A virus
page 400

Human rotavirus, varying degrees of stain penetration

into the three-layered virion

Ruth F. Bishop

Ian H. Holmes (1931-2010)

In 1973, Ruth Bishop, at the Royal Childrens Hospital in

Melbourne, was studying childhood diarrhea and concluded
that a nonbacterial infectious agent was probably to blame. She
teamed up with Geoff Davidson, Ian Holmes and Brian Ruck; by
negative contrast electron microscopy they found a previously
unknown virus in the first bowel biopsy sample examined and
in many following samples. When their finding was published,
confirmation came from around the world and soon the virus
was recognized as the most common cause of severe diarrhea in
infants and young children worldwide.

Bishop RF, Davidson GP, Holmes IH, Ruck BJ. Virus particles in epithelial cells of
duodenal mucosa from children with acute non-bacterial gastroenteritis. Lancet
1973;1:1281-1283.

Thomas Flewett (1922-2006)

Albert Z. Kapikian

Global distribution of rotavirus mortality. Each dot = 1000 deaths

~527,000 rotavirus-related deaths per year in children <5 years of age

1973 Ruth Bishop, Ian Holmes, Thomas Flewett, Albert Kapikian, others discovery of human rotaviruses

page 401

Joseph Sambrook

Phillip A. Sharp
Sharp PA, Sugden B,
Sambrook J. Detection of
two restriction endonuclease
activities in Haemophilus
parainfluenzae using analytical
agarose-ethidium bromide
electrophoresis. Biochemistry
1973;12:3055-3063.
Aaij C, Borst P. The gel
electrophoresis of DNA.
Biochim Biophys Acta
1972;269:192-200.

Most of the critical developments in DNA, RNA and protein electrophoresis

occurred in a brief spurt around 1971-1973. The first major development was
SDS- polyacrylamide gel electrophoresis, which was most useful in separating
smaller molecules such as proteins. For separating gene-sized fragments of DNA,
agarose gel electrophoresis was developed by Joe Sambrook, Bill Sugden and Phillip
Sharp at Cold Spring Harbor Laboratory. This method separated DNA molecules
ranging from several hundred nucleotides in length to over 10,000 nucleotides.
The phosphate groups in the DNA backbone are negatively-charged, giving DNA
molecules an overall negative charge; smaller molecules migrate through the pores
of the agarose gel faster than larger molecules. Initially, agarose gels were run in
tubes, tapered at the end to stop the slippery agarose from sliding out, and then
later on vertical slabs, using special frames to prevent slippage and intricate combs
to allow the even loading of samples. These gels were, in turn, replaced by the
horizontal slab gels, which are most common today. Early on, radioactive DNA
fragments were analyzed by autoradiography and scintillation counting of gel slices;
then, Piet Borst and Cees Aaij at the University of Amsterdam introduced the
use of the fluorescent dye, ethidium bromide (added to the conductive medium),
which binds to DNA and fluoresces when illuminated with ultraviolet light. The
size of DNA fragments were and still are determined by comparison with markers
(DNA fragments of known sizes). Viruses were among the first DNAs studied by
this method, for example adenovirus DNA, as studied by Michiaki Takahashi,
Takeo Ogino and Koichi Baba. When viral DNAs were cut with various restriction
enzymes, complete restriction maps of viral genomes followed quickly.

(Left) One of many agarose gel

electrophoresis formats
(vertical slab format)
(Below) Ethidium bromide
stained agarose gel showing
separated DNA fragments

Borst P. Ethidium DNA

agarose gel electrophoresis:
How it started. IUBMB Life
2005;57:745-747.

Piet Borst

Takahashi M, Ogino T, Baba K.

Separation of adenovirus DNAs
of different molecular length
by agarose-gel electrophoresis.
Biken J. 1968;11:69.

1973 Joseph Sambrook, others development of agarose gel electrophoresis of DNA / ethidium bromide staining
page 402

Zanvil A. Cohn (1926-1993) and Ralph A. Steinman (1943-2011)

Adherent dendritic cell and macrophage

(scanning electron microscopy, colorized)

Dendritic cells were discovered in 1973 by Ralph Steinman and Zanvil

Cohn at Rockefeller University. At the time, they were trying to understand
the induction of immune responses in mice. Steinman noticed unusual cells
in the spleen that acted like macrophages but looked nothing like them.
Naming them dendritic cells, for their branching tree-like shape, they were
initially unaware of their significance. However, over the next several years
Steinman discovered that these cells were the central players in triggering
the immune response dendritic cells, not macrophages, are the main
T cell-stimulating cells. In the 1990s, he found that immature dendritic
cells position themselves at sites that are most vulnerable to invasion by
pathogens skin, respiratory tract, intestines and genital organs. They also
localize in the T-cell areas of the spleen and lymph nodes. When stimulated
by exposure to a pathogen, these cells undergo an incredible maturation
process, increasing antigen-capture and antigen-presenting capacities
and increasing cytokine signalling to T lymphocytes. Today, dendritic cell
research is central to subjects as diverse as autoimmune disease, cancer and
viral vaccine development.
Steinman RM, Cohn ZA. Identification of a novel cell type in peripheral
lymphoid organs of mice. J Exp Med. 1973;137:1142-1162.

1973> Ralph Steinman, Zanvil Cohn discovery of dendritic cells and their role in antigen presentation

page 403

Councilman Morgan
(1920-1990)

Ari Helenius

Multiple portals of virus entry into mammalian cells. (1) Clathrin-mediated entry
(vesicular stomatitis virus), (2) fusion-entry (HIV), (3) macropinocytosis-mediated
entry (vaccinia virus, HIV), (4) phagocytosis-like-mediated entry (herpes simplex
virus), (5) phagocytosis-mediated entry (bacteria), (6) caveolin-mediated entry
(simian virus 40)

There is a very large literature on virus entry and its routes and mechanisms; it is impossible to attribute primacy because over the years so many different approaches
were used to study phenomena from electron microscopy to molecular genetics. Both non-enveloped and enveloped viruses share the same main steps and routes of
virus entry, which begin with attachment to cell-surface receptors and end with the delivery of the viral genome into the cell cytoplasm. The mechanisms underlying this
are now known to be quite complex and diverse. After binding to cellular receptors, viruses use two main routes to enter the cell: endocytic and non-endocytic routes. The
endocytic route is usually accomplished by transport in clathrin-coated vesicles or pits, but non-clathrin-coated pits, macropinocytotic vesicles and caveolae are also used.
The non-endocytic route involves directly crossing the plasma membrane at neutral pH. Some viruses use both pathways (e.g., HIV). The complex processes are regulated
and mediated by membrane proteins once viral and cellular membranes come into close proximity. For both enveloped and non-enveloped viruses, entry into cells involves
important conformational changes of viral entry proteins and/or host-cell receptors, which are induced by low endosomal pH. Viruses may leave endosomes by penetration
(for non-enveloped viruses) or fusion (for enveloped viruses). After entry into the host cell, many viruses are transported through the cytoplasm as nucleoprotein complexes
using nuclear localization signals on the viral nucleoprotein complex to target entry into the nucleus. Elucidation of the molecular mechanisms and the dynamics of the
conformational changes driving virus entry are key to our understanding of virus tropism and pathogenesis, and are key to development of entry inhibitors and novel vaccine
immunogens much contemporary research is being done in this area.
Miyamoto K, Morgan C. Structure and development of viruses as observed in the electron microscope. XI. Entry and uncoating of herpes simplex virus. J Virol. 1971;8:910-918.
Helenius A, Kartenbeck J, Simons K, Fries E. On the entry of Semliki forest virus into BHK-21 cells. J Cell Biol. 1980;84:404-420.
Dimitrov DS. Virus entry: molecular mechanisms and biomedical applications. Nat Rev Microbiol. 2004;2:109-122.

1973> Councilman Morgan, Ari Helenius, others discovery of the variety and complexity of virus entry mechanisms
page 404

Daniel Nathans (1928-1999)

Kathleen J. Danna

Autoradiogram of 14C-labeled SV40 DNA

from Nathans and Dannas classic paper
depicting 11 distinct HINDII/III restriction enzyme
fragments of virus DNA as separated by
SDS-polyacrylamide gel electrophoresis

Modern SV40 genome map

In the late 1960s, Daniel Nathans colleagues, Bernard Roizman

and Robert Wagner, left The Johns Hopkins University School
of Medicine, and he decided to redirect his research to the
animal virology niche they left unoccupied after some
thought he selected the simplest DNA tumor virus, SV40. Even
though the SV40 genome was known to be only about 5,000
base pairs of double-stranded circular DNA, its ability to grow
lytically in monkey cells and to cause permanent tumorigenic
transformation of rodent cells suggested that there was much
more to learn. Nathans spent a sabbatical in 1969 with Leo Sachs
and Ernest Winocour at the Weitzman Institute in Israel learning
how to grow and assay SV40 virus. While there, he received
a letter from his colleague, Hamilton Smith, describing an
enzymatic activity by the bacterium Hemophilus influenzae that
degraded DNA from foreign cells but did not degrade its own
DNA. Smith mentioned evidence that this enzyme cleaved DNA
at specific nucleotide sequences. Nathans immediately realized
the implications of this discovery, that restriction enzymes might
be used to dissect the genome of SV40 virus and show something
about how the virus works. Nathans returned to Hopkins with
radiolabeled SV40 DNA in his luggage, and he set about testing
his ideas. Using Smiths H. influenzae restriction enzyme to cut
SV40 virus DNA (actually, the cleavage activity was due to a
mixture of two restriction enzymes), Nathans and his graduate
student Kathleen Danna cleaved SV40 DNA into 11 specific
pieces. These results were published in their 1971 classic paper in
the Proceedings of the National Academy of Sciences. Immediately,
it was understood that a genome map should be developed
structural genes, early and late genes, genes affecting host cell
tumorigenic transformation, the origin of replication, etc., needed
to be mapped. Together with Danna and George Sack, a medical
fellow, Nathans determined the specific order of each fragment
in relation to the others and constructed the first map of a viral
genome. This map was constructed by isolating overlapping
partial digestion products and determining their constituents.
Their identification of the origin of SV40 DNA replication was the
first genetic signal to be positioned on a eukaryotic viral genome.
Danna K, Nathans D. Specific cleavage of simian virus 40 DNA
by restriction endonuclease of Hemophilus influenzae. Proc Natl
Acad Sci USA. 1971;68:2913-2917.
Danna K, Sack GH Jr, Nathans D. Specific studies of simian virus
40 DNA. VII. A cleavage map of the SV40 genome. J Mol Biol.
1973;78: 363-368.

1973 Daniel Nathans, others completion of restriction enzyme map of a viral genome (SV40 virus)

page 405

Peter C. Doherty

Rolf M. Zinkernagel

From Robert Blanden (John Curtin School of Medical Research website, 1996): In 1973 it was known that the major histocompatibility gene complex (MHC) encoded the antigens responsible for stimulating a response by T lymphocytes that led to the
rejection of tissue grafts exchanged between incompatible individuals. However, since grafting was an artificial event performed by
transplantation surgeons and experimental immunologists, the real biological function of the MHC was a mystery. Rolf Zinkernagel
and Peter Doherty solved the mystery. The experiments that they performed made it clear that when T lymphocytes recognized
and killed a virus-infected cell, they were recognizing not only a component of the virus but simultaneously also recognizing major
histocompatibility molecules on the surface of the infected cell. Thus, the major histocompatibility antigen molecules were a means
of focusing T cell recognition on cell surfaces so that they could perform the essential functions leading to elimination of infected
cells and recovery from viral infection. Following the precedent set by Zinkernagel and Doherty, other workers showed that the
basic principles discovered by them also applied to the mechanisms by which a subset of T lymphocytes helped B lymphocytes to
produce antibodies.... Over two decades later, much of the molecular detail of T cell recognition and the cell biology connected with
immune responses mediated by T lymphocytes has been worked out, but these aspects of immunology still constitute an enormous
proportion of current immunological research. Doherty and Zinkernagels discovery has been relevant to clinical medicine as well.
It relates both to efforts to strengthen the immune response against invading viruses and microorganisms and the means to combat
certain forms of cancer (via vaccines, stimulants of innate immunity), and to efforts to diminish the effects of autoimmune reactions.
Zinkernagel RM, Doherty PC. Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngenic
and semiallogeneic system. Nature 1974;248:701-702.
Doherty PC, Zinkernagel RM. A biological role for the major histocompatibility antigens. Lancet 1975;1:1406-1409.

The classic experiment of Zinkernagel

and Doherty demonstrating that
antigen recognition by cytotoxic T
lymphocytes (CTLs) exhibits major
histocompatibility complex (MHC)
restriction. H-2k haplotype mice were
infected with LCM virus to induce
CTLs specific for the virus; several days
later their spleen cells (which included
CTLs) were isolated and incubated
with LCM-infected target cells of the
same or different haplotype that were
intracellularly labeled with 51Cr and
either infected or not with LCM virus.
As measured by release of 51Cr into the
culture supernatant, the CTLs only
killed syngeneic virus-infected target
cells here H-2k haplotype LCMinfected target cells. Later studies with
congenic and recombinant congenic
mouse strains showed that the CTLs
and the virus-infected target cells
must share class I MHC molecules on
their surfaces, encoded by the K or
D regions of the MHC. Thus, antigen
recognition by CD8+ CTLs is class I
MHC restricted.

1974 Peter Doherty, Rolf Zinkernagel discovery of how the cellular immune system recognizes virus-infected cells

page 406

Csar Milstein (1927-2002)

Georges Khler (1946-1995)

The history of the development of monoclonal antibodies suffers from two issues that went unresolved for many years: first, there are multiple, conflicting claims of primacy,
and second, there is great criticism that the key discovery, with its great commercial value, was not protected by patents. In the 1970s, it was known that the B-cells that
proliferate in multiple myelomas produce antibodies with unique (and unknown) specificities, but it was not possible to produce identical antibodies specific to a given antigen.
Despite the controversy over primacy, the work of Csar Milstein and Georges Khler from the Medical Research Council Laboratory of Molecular Biology at Cambridge
was found to be so crucial, so innovative that they were awarded the 1984 Nobel Prize in Physiology or Medicine. Their key idea was to fuse cultured immortal myeloma
cells with healthy antibody-producing B-cells the resulting fused cells were called hybridomas. Milstein was experienced in culturing mouse myeloma cells that produced
immunoglobulins of unknown specificities; determining these unknown specificities was a major research focus in his and many other labs. Khler, a postdoc in Milsteins
lab, carried out the fusions of myeloma cells with antibody-producing B-cells (spleen cells from immunized mice). The resulting hybridomas combined two attributes: the
immortal properties of the myeloma cells, and the specificity of the antibody produced by the B-cells. The first hybridoma (designated P3-X63 Ag8) and the next two produced
monoclonal antibodies against different antigens of sheep red blood cells, a good choice due to the simplicity and sensitivity of available assays. For the first time, large
quantities of a pure, specified antibody could be produced. The discovery revolutionized immunology, and the infectious disease sciences including virology, and generated
many clinical and diagnostic applications. The last sentence of their first paper in Nature states, Such cultures could be valuable for medical and industrial use. To some
extent, for some years after the discovery the failure by the British Medical Research Council to protect this value with patents overshadowed the significance of the original
research, but today the scholarly merit of the discovery is fully recognized.
Khler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:495-497.
Tansey EM, Catterall PP. Monoclonal antibodies: a witness seminar in contemporary medical history. Med Hist. 1994; 38: 322-327.
Milstein C. The hybridoma revolution: an offshoot of basic research. BioEssays 1999;21:966-973.

1974 Georges Khler, Csar Milstein development of monoclonal antibodies

page 407

Pioneering studies by Vigdis Torsvik, Wolfram Zillig and their colleagues identified the
first viruses of archaeal organisms [although the Archaea resemble Bacteria in their
cellular structure and genome organization, their DNA replication, transcription and
translation machineries and several other qualities are unique they represent a separate
kingdom, with several phyla]. Although initial studies suggested that the viruses were
similar to tailed bacteriophage like T2, further research revealed morphologies and
genomes indicative of the need to create at least seven new families. Some of the viruses
are rod-shaped, some with claw-like end structures, others are icosahedral, some with
large turrets or spikes at vertices, while yet others are fusiform or lemon-shaped or
bottle-shaped. All but one of the viruses contain double-stranded DNA genomes, but
their genomes contain few recognizable genes suggesting that they might have unique
evolutionary origins.
Torsvik T, Dundas ID. Bacteriophage of Halobacterium salinarium. Nature 1974;248:680681.
Zillig W, Reiter W-D, Palm P, Gropp F, Neumann H, Rattenberger M. Viruses of Archaea.
In: Calender R, editor. The Bacteriophages. New York: Plenum Press;1988. p. 517-558.
Prangishvili D, Forterre P, Garrett RA. Viruses of the Archaea: a unifying view. Nature
Rev Microbiol. 2006;4:837-848.

Wolfram Zillig (1925-2005)

Vigdis Torsvik

David Prangishvili

Patrick Forterre

Lawrence CM, Menon S, Eilers, BJ, Bothner B, Khayat R, Douglas T, Young MJ.
Structural and functional studies of archaeal viruses. J Biol Chem. 2009;284: 1259912603.

Some of the viruses of Archaea, each representing a unique taxon

1974 Wolfram Zillig, Vigdis Torsvik, Ian Dundas discovery of viruses of Archaea, many with odd morphologies
page 408

Harald zur Hausen

In a famous paper from 1842, Rigoni Stern

analyzed death certificates of women in Verona
and noted a high frequency of cervical cancer
in married women, widows and prostitutes,
but rare occurrence in virgins and nuns. He
concluded that the development of this type of
cancer is related to sexual contact. Attempts
to link cervical cancer to an infectious agent
failed until 1974, when after 10 years of effort
Harald zur Hausen and his colleagues provided
the first proof of the etiologic role of human
papillomaviruses (HPVs). These studies were
grounded in anecdotal reports of rare malignant
conversion of genital warts into squamous cell
carcinomas. In 1982, zur Hausen, Lutz Gissmann
and Ethel-Michelle de Villiers found HPV 6 DNA
in biopsies from three invasive tumors using
technology that led further to the quantitative
association of other specific virus genotypes
with cervical carcinoma and other carcinomas
(anogenital carcinomas, head and neck cancers).
Then, together with his collaborators, zur Hausen
identified HPV16 and HPV18 in cervical cancers.
Today, it is known that 99.7 % of cervical cancers
are a result of human papillomavirus 16 and
18 infections this is the case, even though
there are approximately 138 distinctive human
papillomaviruses (as discriminated by PCR and
full genome sequencing). Over 5% of all human
cancers, globally, can be attributed to HPVs. zur
Hausens research led directly to the development
of HPV vaccine, which was introduced in 2006.
zur Hausen H, Meinhof W, Scheiber W,
Bornkamm GW. Attempts to detect virusspecific DNA in human tumors. I. Nucleic acid
hybridizations with complementary RNA of
human wart virus. Int J Cancer. 1974;13:650-656.
zur Hausen H. Condylomata acuminata and
human genital cancer. Cancer Res. 1976;36:794.

Global cervical carcinoma mortality

Drst M, Gissmann L, Ikenberg H, zur Hausen H.

A papillomavirus DNA from a cervical carcinoma
and its prevalence in cancer biopsy samples from
different geographic regions. Proc Natl Acad Sci
USA. 1983;80:3812-3815.

1974 Harald zur Hausen, colleagues discovery of association of human papillomaviruses and cervical carcinoma

page 409

Hans Gelderblom

Porcine circovirus, negative contrast electron microscopy

Porcine circovirus was first recognized as a contaminant of the widely used pig kidney cell line PK-15 in 1974 in Germany and
described as picornavirus-like. The virus, and now all its fellow members of the family Circoviridae, is very small (the smallest viruses
replicating autonomously in eukaryotic cells 16-18nm in diameter), nonenveloped, with single stranded DNA (~1,800 nt) forming
a circular genome. Initially, the virus from the cell line did not induce disease in pigs but in the 1990s strains of the virus were
associated with a newly-emerged disease syndrome in pigs described as postweaning multisystemic wasting syndrome (PMWS).
Further, many other circoviruses were discovered, all pathogens: chicken anemia virus, beak and feather disease virus of parrots,
columbid circovirus of pigeons, goose circovirus, canary circovirus. Genetic analyses strongly suggest that porcine circovirus, which
has become a global problem, is of relatively recent origin, having recently switched hosts from birds to swine. In 2010, porcine
circovirus was found to be a contaminant of human rotavirus vaccines, but since the virus is not known to cause disease in humans
or other animals the vaccines have been left on the market.
Tischer I, Rasch R, Tochtermann G. Characterization of papovavirus- and picornavirus-like particles in permanent pig kidney cell
lines. Zbl Bakt Hyg I Abt Orig A. 1974;226:153-167.
Tischer I, Glederblom H, Vettermann W, Koch MA. A very small porcine virus with circular single stranded DNA. Nature
1982;295:64-66.
Tischer I, Mields W, Wolff D, Vagt M, Griem W. Studies on epidemiology and pathogenicity of porcine circovirus. Arch Virol.
1986;91:271-276.

1974 Ilse Tischer, Hans Gelderblom discovery of porcine circovirus (the first circovirus)
page 410

Yvonne Edna Cossart

Anne Field (1936-2007)

The typical slapped cheek rash of fifth

disease (also called erythema infectiosum)

Yvonne Cossart graduated in medicine and then science from the University of Sydney in 1957-1959 and then completed her training as a pathologist at the Royal Postgraduate
Medical School in London. She was, by good luck during a class excursion, introduced to the virologists at the Central Public Health Laboratories at Colindale where there
was a vacancy. She took the job and stayed for 14 years, mostly working on viral hepatitis. In the mid-1970s, she noted an anomalous reaction of a normal blood donors
serum in an immunoprecipitation assay for hepatitis B (the anomaly occupied position 19 in plate B). When Cossart excised the precipitate and examined it by negative
contrast electron microscopy she saw ~25nm icosahedral virus particles this was the first human parvovirus, named parvovirus B19. Using the same immuneprecipitation
technique, antibodies to parvovirus B19 were found in a high proportion of normal adults. The most common illness caused by parvovirus B19 was identified a few years later,
during outbreaks in the United Kingdom of fifth disease (also called erythema infectiosum), a highly contagious childhood exanthem long suspected of having a viral cause.
Fifth disease takes its name from an old list of common childhood exanthems, named in the order of when they were first reported: measles, scarlet fever, rubella, Dukes
(or fourth) disease, fifth disease, and roseola infantum or exanthem subitum or sixth disease. [Most experts question the validity of fourth disease.] Parvovirus B19 has also
been implicated in several other diseases in 1981, John Pattison and his colleagues linked the virus to a severe complication of sickle cell disease in children, called transient
aplastic crisis (an aplastic anemia).
Cossart YE, Field AM, Cant B, Widdows D. Parvovirus-like particles in human sera. Lancet 1975;1:72-73.
Pattison JR, Jones SE, Hodgson J, Davis LR, White JM, Stroud CE, Murtaza L. Parvovirus infections and hypoplastic crisis in sickle-cell anaemia. Lancet 1981;1:664-665.
Anderson MJ, Lewis E, Kidd IM, Hall SM, Cohen BJ. An outbreak of erythema infectiosum associated with human parvovirus infection. J Hyg (London) 1984;93:85-93.

1975 Yvonne Cossart, Anne Field, colleagues discovery of parvovirus B-19 and its association with fifth disease

page 411

C. Richard (Dick) Madeley

Human astrovirus 1, negative contrast electron microscopy

Astroviruses were discovered in 1975 by C. Richard Madeley and Bonnie P. Cosgrove, using electron microscopy to examine diarrheal stool
samples from infants. The appearance of vrisions was unique and immediately distinguished them from other viruses seen in diarrheal
stools their appearance also suggested their name about 10% of the 28-35nm virions show a characteristic five- or six-pointed star
motif on their surface. Since the viruses (there are at least eight serogroups of human astroviruses) were not initially cultivable in cell
cultures, diagnosis relied heavily on electron microscopy enzyme-linked immunosorbent assay (ELISA)-based kits have now simplified
diagnostics and epidemiologic research. Several studies have found them to be the second most important cause of symptomatic
viral enteric infection in the young, worldwide (after rotavirus). Human astroviruses have also been associated with disease in the
immunosuppressed; the viruses are a common cause of acute diarrhea in patients infected with human immunodeficiency virus (HIV), and
prolonged astrovirus shedding has been observed in bone marrow transplant recipients. Astroviruses have also been found in the feces of a
variety of animals: cattle, sheep, pigs, cats, dogs, turkeys and chickens, ducks, mice, cheetahs, sea lions, dolphins and bats. Strikingly, more
than 100 genetically distinct astroviruses have been detected in various bat species.
Madeley CR, Cosgrove BP. Letter: Viruses in infantile gastroenteritis. Lancet 19;2:124-125, 1975.

1975 C.Richard Madeley, colleagues discovery of human astroviruses (the first astroviruses)
page 412

Aaron J. Shatkin (1934-2012)

Bernard Moss

Capping of mRNA is an early posttranscriptional event, unique to eukaryotes, that strongly influences subsequent processing, nuclear export, stability, recognition by
ribosomes, and translation of mRNA. Accordingly, viruses of eukaryotes, whether they reside in the nucleus or cytoplasm, must solve the capping problem for efficient
replication. The 5 cap (also termed an RNA 7-methylguanosine cap), which is a modified guanine nucleotide, is added to the 5 end of eukaryotic messenger RNAs shortly
after the start of transcription. The 5 end of the mRNA being synthesized is bound by a cap-synthesizing complex associated with RNA polymerase. This enzymatic complex
catalyzes the multi-step chemical reactions that are required for mRNA capping. The discovery of 5 capping of viral mRNAs followed a long, complex path, with the earliest
work done in Japan. It was greatly hindered by the primitive state of nucleic acid sequencing at the time. In 1968, K-I. Miura spent time at the NIH with Aaron Shatkin working
on reovirus genomic RNA. He continued this work when he returned home to the National Institute of Genetics in Mishima, and brought Yasuhiro Furuichi and others into
the lab to pursue not only 5 capping but also 3 polyadenylation. However, it was several years before the phenomenon of capping became understood. This story was reviewed
by Furuichi and Aaron Shatkin in 2000. Understanding of the enzymatic mechanistics followed much later, dependent upon the great advances in sequencing in recent years.
Miura K-I, Watanabe K, Sugiura M, Shatkin AJ. The 5-terminal nucleotide sequence of the double-stranded RNA of human reovirus. Proc Natl Acad Sci USA. 1974;71:39793983.
Furuichi Y, Morgan M, Muthukrishnan S, Shatkin AJ. Reovirus mRNA contains a methylated, blocked 5-terminal structure: m7G(5)ppp(5) GmpCp. Proc Natl Acad Sci USA.
1975;72:362-366.
Wei CM, Moss B. Methylated nucleotides block 5-terminus of vaccinia virus mRNA. Proc Natl Acad Sci USA. 1975;72:318-322.
Furuichi Y, Shatkin AJ. Viral and cellular mRNA capping: Past and prospects. Adv Virus Res. 2000;55:135-184.

1975 Aaron Shatkin, Bernard Moss, colleagues discovery of 5 cap on messenger RNAs (vaccinia virus, reovirus)

page 413

Edwin Mellor Southern

Edwin Southern developed the southern blot technique in 1973 while at Edinburgh University,
but his paper was not published until 1975 because the Journal of Molecular Biology didnt
think it was interesting or significant. He spent two more years performing experiments to
show that his method was important. The method is used for the detection of specific DNA
sequences in DNA samples, say the genomic DNA of viruses or reverse-transcribed DNA
of RNA viruses. Other blotting methods (i.e., Western blot, Northern blot) employ similar
principles, but are used to detect RNAs and proteins they have been named playing off
Southerns name. Southern blots were also used for genetic fingerprinting (e.g.,paternity
testing, forensic testing) prior to the development of microsatellite markers (RFLPs) for
these purposes. The concept of the Southern blot was also used in the creation of microarray
technology. When double-stranded DNA molecules are separated into single strands, by heat
denaturation, and then allowed to reanneal, the efficiency of hybridization between any two
strands is dependent upon their sequence complementarity. This is the central principle upon
which the Southern blot and many other DNA detection techniques are based. In order to
detect whether a specific sequence, such as particular viral gene, is present in a DNA sample, a
probe is made, consisting of the strand complementary to the sequence of interest. The probe
contains a label to indicate the presence of the target sequence. More specifically, Southerns
technique is carried out by first cutting the DNA specimen into manageable fragments
by digestion with restriction endonucleases. Then, the restriction fragments generated
are separated by agarose gel electrophoresis. Next, they are denatured with a strong alkali
solution. The DNA fragments are now single-stranded but remain in the gel matrix in their
original position as determined by their size. Prior to Southerns technique, DNA fragments
were not immobilized in the gel and were frequently lost during the detection process. The
beauty of Southerns technique is that the single-stranded fragments are transferred onto
a more robust membrane where they can be permanently immobilized. It is this transfer
that is the blotting step. The agarose gel is placed in contact with a membrane (originally
nitrocellulose, now nylon) and a blotting paper is overlaid; the ends of the blotting paper are
placed in a strong salt solution and a mass of dry highly absorbent paper is placed on top of
the membrane and weighted down. The salt solution is drawn up by capillary diffusion into
the blotting paper and then passed through the gel and membrane into the absorbent paper.
As the solution passes between the gel and the membrane, it carries the single-stranded DNA
and vertically transfers it to the membrane where it is immobilized. Large DNA fragments are
transferred inefficiently and may move horizontally leading to blurred bands in the final result;
in order to reduce this, DNA fragments are nicked by exposing the membrane to UV light.
Negatively charged DNA sticks to the positively charged membrane but cannot pass through
it. The fragments may be permanently immobilized in the membrane by baking or crosslinking with UV light. Probes (usually about 750 bp in length) are chosen with high DNA
binding efficiency they may be a cDNA clone, a genomic DNA fragment, an oligonucleotide
of a known sequence that is complementary to the target DNA, etc., and may be labeled,
usually with 32P or with digoxigenin, biotin, or fluorescent-tagged nucleotides. Once the probe
has hybridized with the target sequences on the membrane, detection of radio-labeled probes
involves exposure of the membrane to X-ray film; detection of other labeled probes usually
involves various light-generating methods.
Southern EM. Detection of specific sequences among DNA fragments separated by gel
electrophoresis. J Mol Biol. 1975;98:503-517.

1975 Edwin Southern development of southern blotting

page 414

Karl M. Johnson

Patricia A. Webb (1925-2005)

Ebola virus
negative contrast
electron microscopy,
from the first cassette,
among the first few
micrographs taken,
the day the virus was
discovered at CDC,
1976

Johnson KM, Lange JV, Webb PA, Murphy FA. Isolation and partial
characterization of a new virus [Ebola virus] causing acute hemorrhagic
fever in Zaire. Lancet 1977;309:569-571.
Murphy FA, van der Groen G, Whitfield SG, Lange JV. Ebola and
Marburg virus morphology and taxonomy. In: Pattyn SR, editor. Ebola
virus haemorrhagic fever. Amsterdam: Elsevier; 1978.

Frederick A. Murphy

Guido van der Groen

Pattyn SR, editor. Ebola virus haemorrhagic fever. Proceedings

of an international colloquium on ebola virus infection and other
haemorrhagic fevers held in Antwerp, Belgium, 6-8 December, 1977.
Amsterdam: Elsevier; 1978.

1976 Karl Johnson, Patricia Webb, Frederick Murphy, Guido Van der Groen, others discovery of Ebola virus

page 415

The Discovery of Ebola Virus, 1976

The story of the discovery of Ebola virus starts with a history of the development of
biocontainment facilities at the U.S. Centers for Disease Control. After working on
Marburg virus in 1967, in the midst of the lethal outbreaks in Europe it became clear that
CDC needed permanent biocontainment facilities, so plans were drawn up for a glove-box
containment lab. It was constructed in a tin building, the Maximum Security Laboratory,
otherwise known as Building 8, located adjacent to the main viral diseases laboratory
building and was opened in 1968. Completion of the Building 8 facility coincided with
the emergence of Lassa fever in West Africa. Work on Lassa virus at Yale University
(Yale Arbovirus Research Unit) was terminated after one person died and another, the
famous virologist, Jordi Casals, nearly died. Immediately, I was put in charge and worked
with Tom Monath and Bill Gary conducting the experiments that began to unravel the
natural history of Lassa virus. Tom Monath later moved to West Africa and discovered the
reservoir host of the virus, the rodent, Mastomys natalensis, and showed how prevalent
the human infection was. The Building 8 Laboratory became a part of my Viral Pathology
Branch, in the Division of Viral Diseases. The work expanded from an emphasis on
diagnostics to work on the pathogenesis of the viral hemorrhagic fevers. Washington
Winn and David Walker later developed animal models of several of the highly pathogenic
human viruses, including Lassa virus. By 1976, Karl Johnson and Patricia Webb were
scheduled to take over the Maximum Security Laboratory and a new facility. The latter
was another tin building, a gift from the NIH it had never been used and delay followed
delay in completing it, all the while Karl becoming more and more frustrated [it was not
completed until 1977 this was to be the first high containment lab to make use of space
suits (positive pressure personnel protection suits), invented by Karl Johnson].
In the meantime, in October 1976, Ebola virus appeared while we were still using the old
Maximum Security Laboratory (MSL / Building 8), but at least we had Karl and Patricia
Webb with all their experience and wisdom, and we had the great technical skills of Bill
Gary, Jim Lange and Herta Wulff. There were two nearly simultaneous epidemics of
hemorrhagic fever in Zaire (now Democratic Republic of Congo) and the Sudan. The
outbreak in the Sudan was as dramatic as that in Zaire, but it is the latter that stands out
in my memory as if it were yesterday. We heard that the Zaire epidemic was centered at
a missionary hospital in the village of Yambuku and seemingly all the staff of the hospital
had died. The whole region was being quarantined by the Zaire army and there were
rumors of devastation over a large area. CDC sent a team into the area and they found
that things were bad enough, but not quite as bad as the rumors. Between the Zaire and
Sudan epidemics there were 550 cases of severe hemorrhagic fever and 430 deaths. The
CDC team was delayed by a regional quarantine in getting out with their specimens. On
October 11, specimens were received in Atlanta and Patricia Webb used all of her acumen
to save the day. When she opened the package she found that the tubes containing blood
and tissues from patients were broken. Instead of taking the whole box to the autoclave,
she carefully (wearing a single pair of surgical gloves, surgical gown and mask) squeezed
out a drop of fluid from the surrounding cotton packing material. She inoculated this into
Vero cell cultures, along with a double dose of antibiotics.
Two days later the cells showed traces of cytopathology, so she gave me a few drops of the
supernatant fluid from the cultures. Wearing the same sort of garb (surgical gown, mask,

gloves) and working in a hood (CDCs old Biological Safety Cabinet, BSC, quite primitive
by todays standards), I prepared specimens for examination by negative contrast electron
microscopy. I put the electron microscopy grids in a sealed box, told the staff not to go
near the hood room where I had worked and trundled off to the electron microscope lab.
What I saw raised the hairs on the back of my neck! I saw telltale long filaments and was
sure it was Marburg virus. I was the only one still at CDC who had worked on Marburg
virus when it appeared in 1967. I had also done a project on Marburg virus in 1977 with
David Simpson and his colleagues from Porton Down, the British high containment
lab, so I thought I really knew enough about this unique virus. I shut down the electron
microscope and went back to the room in which I had prepared the specimen. I cloroxed
the hell out of the hood where I had done the preparation, carried my discard pan with
gown and gloves, etc., to the autoclave and ran it myself. Then I went back to the electron
microscope and called Karl and Patricia to come and take a look. I shot a cassette of
pictures and with wet negatives (not good for the enlarger) I made prints, which were
available within minutes.
Along with Karl I carried these dripping prints to the office of the director of the CDC
(I cannot remember why we did not stop at other offices in the chain of command).
It was a dramatic moment: the director, David Sencer, was sitting at the far end of the
very long table in the conference room along with a guest. He said hed be with us in a
moment. As we gathered at the other end of the table, others started coming in and it
got a bit noisy as plans were discussed about what to do next. When the noise got out
of hand, David Sencer and his guest joined us. The dripping prints seemed to say it all.
Marburg virus was the only virus that looked like the virus particles in the pictures.
As we sat at the table discussing the ramifications of our findings, Patricia came in and
whispered in Karls ear. He turned and repeated her findings: Patricia had done a twoway cross immunofluorescent antibody test, using the newly isolated virus, a reference
Marburg virus, serum from one of the surviving Zaire patients and a reference Marburg
antiserum. The two did not cross: the virus causing the epidemic in Zaire was something
new. We went across the hall and David Sencer called James Gear, director of the national
laboratory in Johannesburg, South Africa, the most senior public health infectious disease
figure in Africa. Plans for a team to go to Zaire were formulated. Incredible excitement,
based in sound reasoning and experienced planning, dominates my memory. David
Sencers guest, who joined in the discussion as if he were an old hand turned out to be the
then junior Senator from Georgia, Sam Nunn.
Within days, Karl was appointed leader of a WHO international field team and left with
a large international entourage for Kinshasa and Yambuku. Patricia and I and our staffs
continued to work for the next several months on what was soon to be named Ebola virus.
The rest of the story is well described in Richard Prestons book, The Hot Zone.
One of the dripping electron micrographs from that first cassette was the image that
became rather famous. It is the shepards crook image of an Ebola virion that is now
all over the Internet, the image that high school students often still ask me for. Let me
add one prideful caption in this regard: years later my dear friend Karl Johnson, chatting
with a group of old friends, said, Fred is not really a scientist, he is just a photographer, a
photographer who uses very big, very expensive cameras. So true!
Frederick A. Murphy, from his unpublished personal family memoir, 2004.

1976 Karl Johnson, Patricia Webb, Frederick Murphy, Guido Van der Groen, others discovery of Ebola virus

page 416

J. Michael Bishop and Harold E. Varmus

Varmus HJ, Bishop JM, Vogt P. Appearance of virus-specific DNA in mammalian cells
following transformation by Rous sarcoma virus. J Mol Biol. 1973;74:613-626.
Stehelin D, Varmus HJ, Bishop JM, Vogt P. DNA related to the transforming gene(s) of
avian sarcoma viruses is present in normal avian DNA. Nature 1976;260:170-173.
Spector D, Varmus HJ, Bishop JM. Nucleotide sequences related to the transforming gene
of avian sarcoma virus are present in DNA of uninfected vertebrates. Proc Natl Acad Sci
USA. 1978;75:4102-4106.

Between 1970 and 1984, Harold Varmus and J. Michael Bishop set out to
resolve one of the great contemporary challenges in biomedical research: to
understand how retroviruses cause cancer. During the 1960s, Howard Temin had
hypothesized that retroviruses made double-stranded DNA copies of themselves
(termed proviruses), which could be inserted into the DNA of infected cells;
he suggested that proviruses implanted oncogenes in the DNA of host cells,
resulting in cancer. This was the era of Robert Huebners virogene-oncogene
hypothesis, that is, that all forms of cancer arise from the activation of oncogenes
embedded in normal cells through infection of germline cells by retroviruses.
Varmus and Bishop set out to add experimental detail to these hypotheses: to
detect proviruses in infected cells and to track these proviruses in time and
place. They chose Rous sarcoma virus (RSV), a retrovirus that triggers cancer
in chickens and carries the first known viral oncogene, src. They set out first to
demonstrate the presence in the genomic DNA of chicken cells sequences that
derived from the RSV src oncogene a needle in a haystack in the days before
restriction mapping and gene cloning. Varmus devised a radioactive molecular
probe that could identify src genes amid the mass of host cell DNA. Working
with Dominique Stehelin, they made the surprising discovery that the RSV src
oncogene was nearly identical to a sequence in the normal cellular DNA of several
different species of birds. Then, working with Deborah Spector, they found a
src proto-oncogene in fish as well as in several mammals, including mice, cows,
and humans. They hypothesized that the many different forms of cancer all arise
from a common genetic mechanism involving specific genes present in normal
cells of all species. When these normal cellular genes undergo mutations, the
result can be cancer. Their work refocused thinking on the ultimate origins of
cancer, directing attention away from the viral genome to the host genome. The
fact that src sequences may be found in the normal genome of a wide range of
species and had been there through evolutionary species divergence indicated
that src originated in normal cells; it was captured from the genome of a host
cell by an invading retrovirus far in the evolutionary past in an event known as
viral transduction. The process by which retroviruses capture host cell protooncogenes damages these genes in ways that can turn them into full-fledged
oncogenes, capable of inducing malignant growth directly when the virus infects
a new host. The fact that the cellular version of src (c-src) survived through many
stages of evolution also indicated that it must perform a vital function, most likely
in directing the growth and development of cells, although its precise role was not
at first understood. From all this, Varmus and Bishop also reasoned that src was
the archetype of an array of genes that give rise to cancer, and that the oncogenes
of other retroviruses likely also stemmed from cellular proto-oncogenes. More
than a hundred proto-oncogenes have since been found. Finally, Varmus and
Bishop proposed that cancer can ensue when so-called tumor suppressor genes,
genes that control the expression of proto-oncogenes, themselves undergo
mutation or are deleted, leaving the latter free to divide unhindered. It is thus the
behavior of both proto-oncogenes and tumor suppressor genes that drives the
malignant growth of cancer cells.

1976 J. Michael Bishop, Harold Varmus discovery of the cellular origin of retroviral oncogenes

page 417

Rudolf Rott (1926-2003)

Christoph Scholtissek

Influenza virus and host cell

After influenza viruses were found to have a segmented genome, reassortants obtained in the laboratory became invaluable tools to address many important problems in
influenza virology. The polygenic nature of influenza virus pathogenicity was unraveled over several years, initially by the work of Kenneth Fraser, Edwin Kilbourne and others,
and then in great depth by Rudolf Rott and Christoph Scholtissek, working in the veterinary faculty (Fachbereich Veterinrmedizin, der Justus-Liebig-Universitt), Giessen,
Germany. The latter made reassortants between highly virulent and avirulent strains of avian influenza virus, exchanging separately each of the eight genome segments of each
virus strain. The virulence of the progeny viruses they obtained varied in complex patterns. They showed that some genes worked better together than others, that there was an
optimal combination of genes, an optimal gene constellation, which favored viral survival in nature and determined virulence. They recognized the preeminent role of the viral
hemagglutinin (and its enzymatic cleavability and consequent activation) and the related role of the viral neuraminidase, but they also found that the three parts of the virion
polymerase complex played important roles in determining the virulence of reassortants. Importantly, Rott and Scholtissek initiated the distinction of the two key phenotypic
characteristics of influenza (and all other)viruses virulence (the relative pathogenicity of each virus strain) and transmissibility (the relative capacity of each virus strain to
cause epidemics). To this, we may add other phenotypic characteristics of various strains that are important, such as ease of adaptation and virus yield for vaccine production,
ease of evasion of host immune mechanisms, etc.
Scholtissek C, Harms CE, Rohde W, Orlich M, Rott R. Correlation between RNA fragments of fowl plague virus and their corresponding gene functions.
Virology 1976;74:332-344.
Rott R, Orlich M, Scholtissek C. Attenuation of pathogenicity for fowl plague virus by recombination with other influenza A viruses nonpathogenic for fowl: Nonexclusive
dependence of pathogenicity on hemagglutinin and neuraminidase of the virus J Virol. 1976;19:54-60.

1976 Rudolf Rott, Christoph Scholtissek concept that an optimum constellation of genes determines virulence
page 418

Criteria for Proof of Disease

Causation 1976

A unified concept appropriate for viruses

as causative agents of disease based on the
HenleKoch postulates, by Alfred S. Evans,
1. Prevalence of the disease is significantly higher
in subjects exposed to the putative virus than in
those not so exposed.
2. Incidence of the disease is significantly higher
in subjects exposed to the putative virus than in
those not so exposed (prospective studies).

Springer-Verlag
1993

3. Evidence of exposure to the putative virus is

present more commonly in subjects with the
disease than in those without the disease.
4. Temporally, the onset of disease follows
exposure to the putative virus, always
following an incubation period.
5. A regular pattern of clinical signs follows
exposure to the putative virus, presenting a
graded response, often from mild to severe.

Alfred S. Evans (1918-1996)

Plenum
Fourth Edition
1997

6. A measurable host immune response, such as

an antibody response and/or a cell-mediated
response, follows exposure to the putative
virus. In those individuals lacking prior
experience, the response appears regularly, and
in those individuals with prior experience, the
response is anamnestic.

7. Experimental reproduction of the disease

follows deliberate exposure of animals to the
The classic concept of necessary criteria to prove the causation of an infectious disease was elaborated by Jakob Henle (1809putative virus, but nonexposed control animals
1885), and his student Robert Koch (1843-1910), and his student Friedrich Loeffler (1852-1915), in 1884-1890 the Henleremain disease free. Deliberate exposure may
Loeffler-Koch postulates. The postulates worked well for some bacterial diseases (e.g., anthrax, tuberculosis), but even Koch
be in the laboratory or in the field, as with
realized that they did not for other bacterial diseases (e.g., typhoid, cholera). In the latter case, he thought that fulfilling just
sentinel animals.
some of the criteria was satisfactory. Thomas Rivers (1888-1962) reviewed the postulates in regard to viral diseases in 1937
and found them wanting. He published a refined set of postulates, which were then edited by several other virologists as
8. Elimination of the putative virus and/or its
the technologies of virology advanced, but seemingly at each step along the way new viral diseases came along that defied
vector decreases the incidence of the disease.
the current postulates (e.g., mononucleosis and Epstein-Barr virus, AIDS and HIV; cervical carcinoma and certain human
papillomaviruses). The next major step was taken by Alfred Evans, who in 1976 revisited the postulates and proposed a set 9. Prevention or modification of infection, via
immunization or drugs, decreases the incidence
that included more solid epidemiologic criteria (list). This set of postulates became widely used for many years, up until the
of the disease.
need to add criteria based in the new, central role of molecular biology in all etiologic research.
10. The whole thing should make biologic and
Evans AS. Causation and disease: the Henle-Koch postulates revisited. Yale J Biol Med. 1976;49:175-195.
epidemiologic sense.
Evans AS. Causation and disease: a chronological journey. Am J Epidemiol. 1978;108:249-258.

1976 Alfred Evans criteria for proof of viral disease causation: the Henle-Koch postulates revisited again

page 419

The 1976 Swine Flu Episode (the Swine Flu Fiasco)

David J. Sencer (1924-2011)

In February 1976, Private David Lewis, aged 18, collapsed and died at Ft. Dix, New Jersey. Five additional patients
developed respiratory illness in the next week (eventually there were 155 similar cases). Samples were sent to
the Influenza Branch at CDC; it was quickly determined that the illness in the soldiers had been caused by an
H1N1 virus, that is a swine influenza virus, then thought to be similar to the virus that caused the 1918-19
pandemic in which 500,000 Americans died. The implications of the discovery were complicated by a number of
unfounded scientific inferences about the virus, but in any case it was suggested that there was the possibility of
an extremely severe flu epidemic and many deaths the following winter and that it was crucial to do something
quickly, namely mount an emergency vaccination program. These inferences first developed in the Influenza
Branch, then in the CDC directors office (David Sencer, director), then in the Office of the incoming Secretary
of the Department of Health, Education and Welfare (Joseph Califano, secretary designate) and finally in the
White House (Gerald Ford, president). At the same time, many informal meetings held in the hallways and
offices of the Division of Viral and Rickettsial Diseases of CDC, and elsewhere, urged caution. The failure of
the virus to take off, to infect more soldiers at Ft. Dix, was seen as similar to other circumstances when swine
influenza had been seen in the past: in such instances there were a few human infections, often with serious
clinical presentation, but nothing more. Such virologic and epidemiologic chatter was more removed from the
political scenes being played out at high levels than most virologists realized. In any case, President Ford made a
decision to initiate a massive immunization program. A group of well-known scientists supported the program
to vaccinate all 196 million Americans. Albert Sabin and Jonas Salk, who were not normally the best of friends,
appeared with President Ford on television. Congress approved funds totaling $135 million and 150 million
doses of vaccine were prepared, 40-45 million doses of which were used. Within weeks, cases of Guillain-Barre
syndrome were seen and vaccination was stopped a total of 4,181 claims were filed, amounting to $3.2 billion
[but by 1993 only $93 million had been paid out]. Articles in the Atlanta Constitution of those days describe the
rest of the story: David Sencer [the director of CDC] was at the peak of his powers, 52 years old, with a proven
track record When Jimmy Carter was inaugurated, he made Joseph Califano Secretary of Health, Education,
and Welfare. Califanos first act was to call Sencer to Washington and fire him The public reason was the
failed swine flu program and associated costs... Sencer, along with almost all proponents of the [vaccination]
program, argued that dollars were less important than lives. That is the crux of the problem. That is why Sencer
was fired That is how the CDC became a political beast rather than a scientific institution After hearing
about Sencers removal, Dr. Alexander Langmuir of Harvard University and former Chief Epidemiologist at CDC
said, This is the politicization of the CDC, and every important man is going to leave as fast as he can find a
new job. From that day on, the CDC changed from an effective, professional organization dedicated to the
control of communicable diseases into an outlet for political agendas, social engineering, and the manipulation
of public opinion The Atlanta Constitution eventually softened its tone and came to support CDC through
thick and thin, in keeping with the sense of civic pride in CDC that still pervades Atlanta. At a national level,
the episode, often called the Swine Flu Fiasco, affected all public vaccination programs and perhaps all public
health infectious disease programs, driving future leaders to a more conservative, less risk-taking, view of their
responsibilities.
Top FH Jr, Russell PK. Swine Influenza A at Fort Dix, New Jersey (January- February 1976). IV. Summary and
speculation. J Infect Dis. 1977;136 Suppl:S376-380.
Sencer DJ, Millar JD. Reflections on the 1976 swine flu vaccination program. Emerg Inf Dis. 2006;12:29-33.

President Gerald Fords swine flu shot, 1976

Neustadt RE, Fineberg HV. The swine flu affair - decision-making on a slippery disease. Special report; 1978.
Available online at http://www.nap.edu/catalog.php?record_id=12660.

1976 U.S. Government threat of swine flu epidemic, national emergency vaccination program

page 420

The 3,569 nucleotide genome of the

bacteriophage MS2, sequenced by Walter Fiers
and his colleagues in 1976

Walter Fiers

Frederick Sanger

1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961
1021
1081
1141
1201
1261
1321
1381
1441
1501
1561
1621
1681
1741
1801
1861
1921
1981
2041
2101
2161
2221
2281
2341
2401
2461
2521
2581
2641
2701
2761
2821
2881
2941
3001
3061
3121
3181
3241
3301
3361
3421
3481
3541

gggtgggacc
tctttagcga
gtttgacctg
gttcgcgttt
aactggactc
tctccgtatt
agtgggtcat
gcacgctcct
cagaacgttg
aaccttggtg
attgcgctcg
taccttgccc
gagttgcagt
acgaaggttc
atcaagttaa
tcgcgacgta
ctaggtatct
gactggctcc
tacatgtcag
ccctacgggt
cgaggggtac
gtccatacct
ccggagtttg
ctggcgacgt
ctaactcgcg
atcgcaaata
tagagcttcc
tcgctacgaa
gaaacccgat
ttcaaacatg
atcttcctcg
gcggtgatcc
caaagcatcc
ctaagctacg
acaaatcctt
acctcctctc
ctatggggca
ttaccccccg
gacacgcggt
ttacagttcc
tgtacctcca
atctgaatga
ttgcgacgat
ttctcccacc
atggcgagac
tagagtccat
gaaccatagg
tggaggcact
tctttcgcga
tcaagaaacc
ggggagttgt
cccaggtgcc
gcccgcccac
cccgtacctc
agcatgacag
tgaagtccgc
tccctcagga
cggggtgggt
ggcgggcttc
taactagctg

cctttcgggg
gacgctacca
tgcgagcttt
acgcggacgg
ccggtcgttt
cacggggggc
cgtggggtcg
gctacagcct
cgaaccgggc
ttgctttagc
tgaaggcgta
taaacgaaga
tcggttggtt
accttcaaga
atggccgtct
tcgtgatatg
tgaacccact
tacctgtagg
gaacagttac
ggactgtgga
aatccgtatg
tagatgcgtt
aagcatggct
gactgtcgcc
ttcacaggct
caccatcaaa
tgtagccgca
ttccgactgc
tccctcagca
aggattaccc
cgatctttct
gcacagtgac
gaccttaggt
ggaggcgaat
gtcatgggat
tggctaccga
caagttgcag
cgctctgaga
ccgctataac
gaagaataat
gaaaggggtc
tcaatcgatc
agacttatcg
tgagctatat
gatacgatgg
gatattctgg
catctacggg
tgcctactac
gagctgcggc
tgttgacaat
cggaggtatg
ttcgatgttt
ggcagtctcg
gggtttccgt
tggccgctac
cggcgtgcgt
gtgtgggcca
gtgctcgaaa
ggcccaggga
cttggctagt

tcctgctcaa
tggctatcgc
tagtaccctt
tgagactgaa
taactcgact
gttaagtgtc
cccgtacgag
cttccctgta
gtcgaccgaa
agaggccagg
cactgccgct
tcgaaagttt
accactaatg
gtttcttcct
gtcgtatcca
gttttacata
aggtatagtg
taacatgctc
tgacgtaata
gagacagggc
gccaacaact
agcattaatc
tctaacttta
ccaagcaact
tacaaagtaa
gtcgaggtgc
tggcgttcgt
gagcttattg
atcgcagcaa
atgtcgaaga
ctcgaaattt
gactttacag
tctggtaatg
gatcggtgcg
ccggatgttt
tcgtcgttgt
gatgcagcgc
gcggctctat
gagtcatatg
aaaatagatc
ggtgccttta
aaccagcttc
tctgcatccg
tcatatctcg
gaactatttt
gcaatagtca
gacgatatta
ggtttcaaac
gcgcactttt
ctcttcgccc
tcagatccac
ttcggtggga
gtatatacca
cttgctcgta
atagcgtggt
attatgcgca
gcgagctctc
gagcacgggt
cctccccttg
taccaccca

cttcctgtcg
tgtaggtagc
gatagggaga
gataactcat
ggggccaaaa
acatcgatag
gagaaagccg
agccagaact
gtcctgcaaa
tcgacagcct
cgtcgcggta
cgatcaaaac
agtgatatcc
atgagagccg
gctgcaaact
aacgatgcac
tgggaaaagg
gagggcctta
acgggtgagt
actgctaagg
ggcgcgtacg
aggcaacggc
ctcagttcgt
tcgctaacgg
cctgtagcgt
ctaaagtggc
acttaaatat
ttaaggcaat
actccggcat
caacaaagaa
accaatcaat
caattgctta
acgaggcgac
gtcagataaa
tacaaaccag
ttgggcaatg
cttacaagaa
tggtccgaga
aatttaggct
gggctgcctg
tcagacgccg
tggctcagca
attccatctc
atcgtatccg
ccacaatggg
aagcgaccca
tatgtcccag
cgaatctccg
accgtggtgt
ttatgctgat
gcctttacaa
cggacctcgc
agactccgta
tcgctcgaga
tccatactgg
cttcggagtg
ctcggtagct
ccgcgaaagc
aagagagggc

agctaatgcc
cggaattcca
acgagacctt
tctctttaaa
cgaaacagtg
atcaaggtgc
gtttcggctt
tgacttacat
aggtcaccca
cacaactcgc
attggcgcca
acgtggccgg
agggtgcata
tacgtcaggt
tccagacaac
gtttggcatg
tgcctttctc
cggcccccgt
ccatcataag
cccaaatctc
taaagtctcc
tctctagata
tctcgtcgac
ggtcgctgaa
tcgtcagagc
aacccagact
ggaactaacc
gcaaggtctc
ctactaatag
gttcaactct
tgcttctgtc
cttaagggac
ccgtcgtacc
tagagaaggt
catccgtagc
cacgttctcc
gttcgctgaa
ccaatgtgcg
cgttgtaggg
taaggagcct
gctcaaatcc
gggcagcgta
cgatcgcctg
ctcacactac
aaatgggttc
aatccatttt
tgagattgca
taaaacgttc
cgatgtcaaa
attgaatcgg
ggtgtgggta
tgccgactac
tgggcggcta
acgcaagttc
aggtgaagtc
gctaacgccg
gaccgaggga
ggtggctcca
ccgggattct

atttttaatg
ttcctaggag
cgtcccctcc
atatcgttcg
gcactacccc
ctacaagcga
ctccctcgac
cgaagtgccg
gggtaatttt
gacgcaaacc
ggcgctccgc
caggtggttg
tgagatgctt
cggtactaac
gtgcaacata
gttgtcgtct
attcgttgtc
gggatgctcc
cgttgacgct
agccatgcat
tttctcgatg
gagccctcaa
aatggcggaa
tggatcagct
tctgcgcaga
gttggtggtg
attccaattt
ctaaaagatg
acgccggcca
ttatgtattg
gctactggaa
gaattgctca
ttagctatcg
ttcttacatg
cttattggca
aacggtgcct
caagcaaccg
ccgtggatca
aacggagtgt
gatatgaata
gttggtatag
gatggttcgc
gtgtggagtt
ggaatcgtag
acgtttgagc
ggtaacgccg
ccccgtgtgc
gtgtccgggc
ccgttttaca
ctacggggtt
cgactctcct
tacgtagtca
ctcgcggata
ttcagcgaaa
accgacagta
gttcccacat
cccccgtaaa
ccgaaaggtg
cccgatttgg

In 1976, Walter Fiers and his large team at the Laboratory of Molecular Biology of the University of Ghent were
the first to determine the nucleotide sequence of a virus, the single-stranded RNA bacteriophage MS2. In 1971,
Ray Wu had published the sequence of 12 nucleotides of the cohesive ends of the DNA of bacteriophage; this is
considered to be the first DNA sequence ever determined. The first DNA viral genome to be sequenced was that of
the double-stranded DNA bacteriophage -X174 its 5,368 base pair genome was sequenced by Frederick Sanger
and his team in 1977, using the enzymatic method Sanger had developed in 1975. Several surprising features were
identified, including genes that partially overlap one another and the first clues that different organisms might have
slightly different codon usage patterns. The Sanger enzymatic sequencing method went on to become the basis for
all later sequence methods, including todays automated, high-throughput methods.
In 1978, Walter Fiers and his team also reported the
Fiers W, Contreras R, Duerinck F, Haegeman G, Iserentant D, Merregaert J, Min Jou W, Molemans F, Raeymaekers complete sequence of the genome of SV40 virus5,224 bp:
A, Van den Berghe A, Volckaert G, Ysebaert M. Complete nucleotide-sequence of bacteriophage MS2-RNA W. Fiers, R. Contreras, G. Haegeman, R. Rogiers, A. Van
primary and secondary structure of replicase gene Nature, 1976;260:500-507.
de Voorde, H. Van Heuverswyn, J. Van Herreweghe, G.
Sanger F, Air GM, Barrell BG, Brown NL, Coulson AR, Fiddes CA, Hutchison CA, Slocombe PM, Smith M.
Nucleotide sequence of bacteriophage phi X174 DNA. Nature 1977;265: 687-695.

Volckaert & M. Ysebaert. Complete nucleotide sequence

of SV40 DNA. Nature 273:113-120, 1978.

1976-1977 Walter Fiers, Frederick Sanger complete sequencing of viral genomes (bacteriophages MS2 and 174)

page 421

Phillip A. Sharp

Richard J. Roberts

By the 1970s the physical structure of the gene was firmly established from work on bacteria. The sequence of the DNA, the RNA, and the
protein were thought to be colinearly organized and expressed what was true for bacteria must be true for higher forms of life. However,
more and more evidence began to appear that implied that this might not be universal. This led Phillip Sharp at MIT and Richard Roberts
at Cold Spring Harbor Laboratory and their colleagues (Louise Chow, Richard Gelinas, Thomas Broker, Susan Berget, Claire Moore, Arnold
Berk, Tim Harrison) to compare the complementarity of sequences expressed as cytoplasmic mRNAs during the late stage of adenovirus
infection with the viral DNA from which it was transcribed. Hybridization of the mRNA encoding the adenovirus virion hexon protein
to a restriction endonuclease fragment of the adenovirus genome generated a structure which by electron microscopy was seen to have
three different length loops of viral DNA. That is, the single mRNA molecule corresponded to at least three well-separated segments in the
DNA molecule. Roberts and Sharp came to the conclusion that the genetic information in the gene was discontinuously organized in the
genome. The discovery immediately led to intensive research by which the phenomenon was found to be present also in other viruses and in
vertebrate cells. The discovery of split genes and RNA splicing also led to the concept of introns and exons, terms coined by Walter Gilbert.
Berget SM, Moore C, Sharp PA. Spliced segments at the 5 terminus of adenovirus 2 late mRNA. Proc Natl Acad Sci USA. 1977;74:31713175.
Chow LT, Gelinas RE, Broker TR, Roberts RJ. An amazing sequence arrangement at the 5 ends of adenovirus 2 messenger RNA. Cell
1977;12:1-8.

Richard Roberts and Phillip

Sharp made their discovery
of split genes in adenovirus
when they examined a hybrid
between a viral mRNA and its
template DNA in the electron
microscope. They observed that
the mRNA was not encoded
co-linearly in the DNA Instead,
loops of unhybridized DNA
(A, B, C) were seen. The
interpretation was that mature
messenger RNA was derived
from discontinuous segments
(exons) in the viral DNA. The
intervening sequences (introns
A, B, C) are excised during
mRNA maturation.

1977 Phillip Sharp, Richard Roberts, colleagues discovery of RNA splicing and split genes (adenovirus)
page 422

D. A. Henderson

Frank Fenner (1914-2010)

Isao Arita

WHO
Smallpox
Eradication
Programme

In 1958 Victor Zhdanov, Deputy Minister of Health of the USSR, called upon the World Health Assembly to undertake a global
program to eradicate smallpox; the proposal was accepted in 1959. At this point, 2 million people were dying from smallpox every year.
At first, progress was disappointing, so in 1967 the WHO formed a smallpox eradication unit, under the leadership D.A. Henderson,
with Isao Arita serving as the senior WHO staff member. The unit established a network of consultants who assisted in setting up
programs in all countries where smallpox was still present. The program centered on surveillance of cases (the most important failing
in most countries), the isolation of cases and the vaccination of everyone who lived close by cases (ring vaccination). Early on,
vaccine was provided by the United States and the Soviet Union, but by 1973 more than 80% of all vaccine was produced in developing
countries. The last major European outbreak occurred in 1972 in Yugoslavia. By the end of 1975, the disease persisted only in the Horn
of Africa Ethiopia and Somalia, where the program faced incredible challenges: poor roads and transport, civil war, famine, and many
refugees in transit. The last naturally occurring case of variola minor was diagnosed in Ali Maow Maalin, in Merca, Somalia, in 1977.
The last naturally occurring case of variola major had been detected in Rahima Banu, in Bangladesh, in 1975. In 1977, Frank Fenner
was named the chairman of the Global Commission for the Certification of Smallpox Eradication eradication was certified in each
country based upon intense on-the-ground searching for cases. It was Fenners great honor to announce to the World Health Assembly
in 1980 that global eradication had been achieved. His announcement was accepted unanimously by the World Health Assembly the
same day. He later said that making this announcement stood out most among his many achievements.
Fenner FD. Henderson DA, Arita I, Jeek , Ladnyi ID. Smallpox and its eradication. Geneva: World Health Organization; 1988.

Ali Maow Maalin (1954-2013), Merka, Somalia,

the last known natural case of smallpox

1977 D.A. Henderson, Isao Arita, Frank Fenner, many others global eradication of smallpox

page 423

Walter Gilbert
Sangers End-labeled DNA
Sequencing Method

Single-stranded DNA is mixed with

a primer and split into four aliquots,
each containing DNA polymerase,
four dideoxyribonucleotide
triphosphates and a modified
nucleotide replication terminator.
Each reaction proceeds until a
replication-terminating nucleotide
is added. The mixtures are loaded
into separate lanes of a gel and
electrophoresis is used to separate
the DNA fragments. The sequence
of the original DNA strand is
inferred from the results.

Frederick Sanger

The first DNA sequences were obtained in the early 1970s

using laborious methods based on two-dimensional
chromatography. Then in 1975-77, Frederick Sanger at the
University of Cambridge invented a partial cleavage of endlabeled DNA method and independently Walter Gilbert
and Allan Maxam at Harvard invented a chain termination
(dideoxy) method, both of which initiated the sequencing
revolution, even though it was the Sanger method that later
became the method of choice. Sangers method employs
dideoxynucleotide triphosphates (ddNTPs) as DNA replication
terminators this requires use of modified nucleotides
(dideoxyNTPs) that when incorporated terminate DNA strand
elongation. These ddNTPs are radioactively or fluorescently
labeled. The DNA sample is divided into four separate
sequencing reactions, each conting only one of the four chainterminating dideoxynucleotides (ddATP, ddGTP, ddCTP, or
ddTTP). This results in DNA fragments of varying length,
which are separated by size by polyacrylamide electrophoresis
(with a resolution of just one nucleotide); the DNA bands
are then visualized by autoradiography or UV light, and the
DNA sequence can be directly read off the X-ray film or gel
image. It is fluorescent dye-labeled ddNTPs that are read in
automated optical systems, the first invented by Leroy Hood,
that was the key in the first high-throughput machines.
Today several different methods are employed producing
thousands or millions of sequences at once (massively parallel
signature sequencing, polony sequencing, pyrosequencing,
reversible dye-terminator sequencing, sequencing by ligation,
ion semiconductor sequencing, DNA nanoball sequencing,
sequencing by hybridization, etc.).
Maxam AM, Gilbert WA. A new method for sequencing DNA.
Proc Natl Acad Sci USA. 1977;74:560-564.
Sanger F, Coulson AR. A rapid method for determining
sequences in DNA by primed synthesis with DNA polymerase.
J Mol Biol. 1975;94: 441-448.
Sanger F, Nicklen S, Coulson AR. DNA sequencing with chainterminating inhibitors. Proc Natl Acad Sci USA. 1977;74:
5463-5467.

1977 Walter Gilbert, Frederick Sanger development of the technology for rapid sequencing of DNA
page 424

In 1977, Mario Rizzetto and his colleagues in Torino,

Italy, while examining liver biopsies from individuals
infected with hepatitis B virus (HBV), discovered by
immunofluorescence a previously unrecognized nuclear
antigen that was subsequently shown to be a specific
marker of a novel human pathogen, hepatitis delta virus
(HDV). As discovered by John Gerin and his colleagues,
HDV is a unique defective RNA virus with similarities to
viroids, the subviral pathogens of higher plants. It is the
smallest animal virus (~1,679 nucleotides) and the only
RNA animal virus to possess a circular RNA genome.
For its replication the virus requires the helper function
of hepatitis B virus (HBV), which provides the essential
coat protein for virion assembly. Both superinfection
and coinfection of HBV carriers with HDV results in a
greater likelihood of experiencing liver failure in acute
infections and a rapid progression to liver cirrhosis,
with an increased chance of developing liver cancer
in chronic infections. In combination with hepatitis B
virus, hepatitis D has the highest mortality rate of any
the hepatitis infection ~20%. There are currently an
estimated 15 million HDV carriers worldwide, but there
has been a dramatic decline in the incidence has been
observed in the last decade, likely as a result of HBV
vaccination programs and improved socioeconomic
conditions.

Mario Rizzetto

John L. Gerin
Global Prevalence of HDV in HBsAg Positive People

Rizzetto M, Canese MG, Arico S, Crivelli O, Bonino F,

Trepo CG, Verme G. Immunofluorescence detection of a
new antigen/antibody system (d /anti-d) associated with
hepatitis B virus in liver and serum of HBsAg carriers.
Gut 1977;18:997-1003.
Hess G, Shih JW, Kaplan PM, Gerin JL. The
demonstration of subtype (D or Y)-specific determinants
on the surface of the presumed hepatitis B virus. J
Immunol. 1977;119:1542-1544.
Rizzetto M, Shih JW, Gocke DJ, Purcell RH, Verme
G, Gerin JL. Incidence and significance of antibodies
to delta antigen in hepatitis B virus infection. Lancet
1979;2:986-990.

Hepatitis delta virus

HBV proteins, blue
HDV proteins, green/red
HDV RNA, yellow

Rizzetto M, Purcell RH, Gerin JL. Epidemiology of HBVassociated delta agent: geographical distribution of antidelta and prevalence in polytransfused HBsAg carriers.
Lancet 1980;1:1215-1218.

1977 Mario Rizzetto, John Gerin, colleagues discovery of hepatitis delta virus (the only deltavirus)

page 425

Scott B. Halstead

Dengue infection can be asymptomatic or present in two clinical syndromes, dengue fever and dengue
hemorrhagic fever/dengue shock syndrome (DHF/DSS). Plasma leakage, hemorrhage and thrombocytopenia
characterize the latter. DHF/DSS is the result of the interaction of several virus and host factors. Some dengue
virus genotypes have more potential to produce DHF/DSS than others, but in addition, several host factors
interplay with virus factors: these include age (children at higher risk than adults), race, underlying chronic
diseases, nutritional status, sex and particular allelic variants of genes that encode cellular receptors. However,
secondary infection is considered the main risk factor for DHF/DSS this concept was first published by Scott
Halstead in 1977. He proposed that preexisting low-level dengue virus antibodies can modulate subsequent
infection by enhancing the infection of mononuclear phagocytes he called this antibody-dependent
enhancement (ADE), which he went on to show can suppress the cellular immune response and thereby increase
the extent of the infection and can generate inflammatory cytokines that contribute to the development of severe
disease. Single-serotype natural infections result in lifelong immunity to the infecting serotype but only shortterm cross-protection against heterotypic serotypes. There is also evidence to suggest that over time there is a
continuous selection of neutralizing-antibodies with increasing homologous reactivity and concurrent decrease
in heterotypic reactivity. Studies in Thailand of first-time infected infants born to dengue-immune mothers and
children who had experienced a mild or primary asymptomatic dengue infection and then became secondarily
infected by a different dengue serotype had a 15-80 times higher probability of developing DHF/DSS than
other infants and children in one study, 99% of DHF/DSS cases had heterotypic antibodies to the dengue
serotype causing the severe disease. The cellular and molecular mechanisms acting in ADE are complex and to
some extent still mysterious; many studies are centered on how antibody-virus complexes are internalized into
mononuclear cells via their Fc receptor, resulting in infection of a higher number of target cells, which in turn
may lead to even more virus production.
Halstead SB, ORourke EJ. Dengue viruses and mononuclear phagocytes. I. Infection enhancement by nonneutralizing antibody. J Exp Med. 1977;146:210-217.
Halstead SB, ORourke EJ. Antibody-enhanced dengue virus infection in primate leukocytes. Nature
1977;265:739-741.
Halstead SB. In vivo enhancement of dengue virus infection in rhesus monkeys by passively transferred antibody.
J Infect Dis. 1979;140:527-533.

1977 Scott Halstead discovery of immune enhancement in dengue hemorrhagic fever and dengue shock syndrome
page 426

Darwin provided the first unifying theory of the nature life on Earth and drew a famous diagram expressing the
universal tree of life. The basis for constructing this tree of life was fundamentally and dramatically changed in
1977 by the work of Carl Woese and his colleagues. Morphologic determinants of relationships among species
that were in place from the days of Linnaeus were replaced by similarities of mRNA gene sequences of related
organisms. Today, the simple tree concept has been adjusted by our knowledge of the occurrence of horizontal
gene transfer, but in most published works the three-branched unrooted tree has not been adjusted by the addition
of the viruses. Several reasons have been advanced to argue against adding viruses to the universal tree of life
[from Vincent Racaniello, http://www.virology.ws/2009/03/19/viruses-and-the-tree-of-life/ summary of the
argument of David Moreira and Purificacin Lpez-Garca]: (1) Viruses are not alive; (2) Viruses are polyphyletic
they did not evolve from a common ancestor; (3) There are no ancestral viral lineages, common genes, common
proteins; (4) Viruses do not have a structure derived from a common ancestor (e.g., cell membrane); (5) Viral
metabolic genes originated from cells; (6) Viral translation genes originated from cells, again by horizontal gene
transfer; (7) Viruses are gene robbers, not gene inventors (although they are major gene suppliers); etc.
Evidence of an ancient origin of viruses is controversial because of horizontal virus gene movement; people who
wish to see the viruses added to the universal tree of life cite recent studies that indicate that the viruses infecting
all three domains, Bacteria, Archaea and Eucarya, have a very ancient origin. One notion is that there are at least
two large DNA sequence-spaces on Earth, one represented by capsid-encoding viruses and another by ribosomeencoding cells. Despite their probable distinct evolutionary origin, both sequence-spaces were and are connected
by intensive two-way gene transfer. It remains to be seen just how the viruses would be added to the universal tree
of life, except as a fourth domain (a domain without a root).
Woese CR, Fox GE. Phylogenetic structure of the prokaryotic domain: the primary kingdoms. Proc Natl Acad Sci
USA. 1977;74:5088-5090.

Carl Richard Woese (1928-2012)

Fox GE, Stackebrandt E, Hespell RB, Gibson J, Maniloff J, Dyer TA, Wolfe RS, Balch WE, Tanner RS, Magrum
LJ, Zablen LB, Blakemore R, Gupta R, Bonen L, Lewis BJ, Stahl DA, Luehrsen KR, Chen KN, Woese CR. The
phylogeny of prokaryotes. Science 1980;209:457-463.
Moreira D, Lpez-Garca P. Ten reasons to exclude viruses from the tree of life. Nat Rev Microbiol. 2009;7:306-311.

Darwins notebook

The Virosphere by Claude Fauquet: taxonomy, but few implied

phylogenetic relationships among the families of viruses

1977 Carl Woese, colleagues recasting of The Tree of Life, phylogeny based on mRNA gene sequences

page 427

Ho Wang Lee

Apodemus agrarius, the field mouse that

is the reservoir host of Hantaan virus, the
etiologic agent of Korean hemorrhagic fever
(hemorrhagic fever with renal syndrome)

Pyung Woo Lee

Karl M. Johnson

Hemorrhagic fever with renal syndrome (HFRS) is the contemporary name for a group of clinically similar illnesses
that occur throughout the Eurasian landmass, with many older and local names: Korean hemorrhagic fever, epidemic
hemorrhagic fever, nephropathia epidemica, et al. Although these diseases were recognized in Asia for centuries, HFRS first
came to western attention when approximately 3,200 cases occurred from 1951 to 1954 among United Nations forces in
Korea. It was not until 1978 that the rodent reservoir for the etiologic agent of Korean hemorrhagic fever was found by Ho
Wang Lee, Pyung Woo Lee and Karl Johnson, who demonstrated that patient sera reacted with antigen in frozen sections
of lung of wild-caught Apodemus agrarius field mice and that the virus in these rodents could be passed from rodent to
rodent. The virus, Hantaan (HTN) virus, was grown in cell culture in 1981, thereby providing opportunity to study this
pathogen systematically. Many related viruses are associated with the syndrome, representing a significant gradient in
pathogenicity; generally, the less virulent variants are seen in northern Europe and the most virulent in eastern Asia but
there are exceptions mortality rates vary from 0.1% to 5-10%. Approximately 150,000 to 200,000 cases of severe HFRS,
involving hospitalization, are reported each year throughout the world, with more than half in China. Russia and Korea also
report hundreds to thousands of cases each year.
Lee HW, Lee PW, Johnson KM. Isolation of the etiologic agent of Korean hemorrhagic fever. J Infect Dis. 1978;137:298-308.
Johnson KM. Nephropathia epidemica and Korean hemorrhagic fever: the veil lifted? J Infect Dis. 1980;141:135-136.
Johnson KM. The discovery of hantaan virus: comparative biology and serendipity in a world at war. J Infect Dis.
2004;190:1708-1710.
Lee HW. Hemorrhagic fever with renal syndrome in Korea. Rev Infect Dis. 1989;Suppl 4:S864-76.

1978 Ho Wang Lee, Pyung Woo Lee, Karl Johnson Hantaan virus, hemorrhagic fever with renal syndrome

page 428

Leland E. (Skip) Carmichael

Max J. G. Appel

Canine parvovirus type 2 (CPV) was first recognized by Leland Carmichael and Max Appel and their colleagues in 1978; the
virus appeared almost simultaneously in the United States, Australia and Europe and spread worldwide within two years.
The highly contagious virus causes cardiac and intestinal disease with high mortality especially in puppies. At first the canine
virus was thought to be a direct host-range extension mutant of feline panleukopenia virus (FPLV), but later research proved
that the canine virus evolved from an unknown carnivore parvovirus and was the result of accumulated mutations over the
preceding 10 years, mutations which affected its capacity to infect dogs, and to spread and cause disease. In contrast to other
carnivore parvoviruses, CPV continued to evolve rapidly after its emergence. Both a high mutation rate and positive selection
of mutations in the capsid gene appear to be the driving force for such rapid evolution. The original antigenic/genetic type
disappeared more than two decades ago, being replaced by a succession of new antigenic/genetic variants that have also spread
worldwide. The phylogenetic pathway of this evolution has been studied in detail by Colin Parrish and his colleagues. Each new
variant has required reformulation of the very effective vaccines.

Colin R. Parrish

Appel MJG, Cooper BJ, Greisen H, Carmichael LE. Status report: canine viral enteritis. J Am Vet Med Assoc. 1978;173:15161518.
Parrish CR, CarmichaelLE. Antigenic structure and variation of canine parvovirus type-2, feline panleukopenia virus, and mink
enteritis virus. Virology 1983;129:401-414.
Carmichael LE. An annotated historical account of canine parvovirus. J Vet Med Series B. 2005;52:303-311.

1978 Leland Carmichael, Max Appel discovery and characterization of canine parvovirus (CPV-2)

page 429

The history of RNA virus reverse genetics, now

usually defined as the ability to recover or rescue
infectious virus from complementary DNA
(cDNA), can be traced back to the report from
Charles Weissmanns lab on the recovery of the
RNA bacteriophage, Q, from cloned cDNA:
Taniguchi T, Palmieri M, Weissmann C. Q
DNA-containing hybrid plasmids giving rise to
QB phage formation in the bacterial host. Nature
1978;274:223-228.

Charles Weissmann

Synthesis of a hybrid plasmid containing a

complete DNA copy of the Q genome. Q
RNA extracted from plaque-purified phage
was used as template for the synthesis of Q
minus strands by Q replicase. Q RNA and
purified Q minus strands were elongated with
AMP residues by polyA polymerase and used
as templates for reverse transcriptase, with
oligo dT as primer. The minus and plus cDNA
copies were hybridized and the incomplete
duplex was repaired with DNA polymerase.
Plasmid PCRI was cleaved with EcoRI and the
ends were trimmed back with 5 exonuclease.
The double-stranded Q DNA and the
PCRI DNA were elongated with dAMP and
dTMP residues, respectively, using terminal
nucleotidyl transferase. The two components
were hybridized and transfected into E. coli.

Before the advent of recombinant DNA and DNA sequencing technologies, genetic analysis of viruses involved random isolation and characterization of mutants, and rough
mapping of genes affected. With the availability of full genomic sequences for virtually all of the prototypical members of each virus family, genetic analysis can be conducted
with a reasonably complete foreknowledge of the genetic structure of the virus, the individual gene(s) of interest and their general function(s). Mutations can be deliberately
engineered in genes to further study their function(s). Termed reverse genetics, this process has come to dominate the genetic analysis of viruses. Reverse genetic analysis
involves two distinct manipulations: (1) the construction of a given mutation (not reviewed further here), and (2) the method for incorporation of the mutation into the virus of
interest. The principles governing these manipulations are dependent on the structure of the viral genome of interest and the strategy of its replication, but there are common
themes. For DNA or RNA viruses with relatively small genomes and for which the nucleic acid is infectious, incorporation of a given mutation into virus is a straightforward
matter of constructing the desired mutation in a full-length genomic clone in a prokaryotic vector, then transfecting cells with the mutant nucleic acid. For example,
engineered mutations have been constructed in both picornaviruses and polyomaviruses using full-length genomes. For DNA viruses that either are too large or complex to
be manipulated as a full-length clone or that contain genomes that are not infectious, incorporation of mutations into the viral genome can be accomplished via homologous
recombination and transfection of cells with the recombinant genome. For example, this has been used successfully to construct engineered mutations in vaccinia virus and
herpes simplex virus. For RNA viruses where the genomic nucleic acid is not infectious, protocols for incorporation of mutations into virus become more complex and tailored
to particular viruses. For example, construction of influenza virus recombinants requires co-transfection of multiple plasmids expressing the wild-type and mutant genomic
RNA segments plus several influenza replication proteins, all transcribed from an RNA polymerase promoter. From: Conditt, R. Principles of virology. In: Knipe D, Howley
PM, editors. Fields virology. Philadelphia: Wolters Kluwer, Lippincott Williams & Wilkins; 2007.

1978 Charles Weissmann, colleagues first reverse genetics experiment (bacteriophage Q)

page 430

Anthony Peter Waterson

(1923-1983)

Lise Slver Wilkinson (1924-2012)

Waterson AP,
Wilkinson L.
An introduction
to the history
of virology.
Cambridge:
Cambridge
University
Press;1978.

The 1978 book, An Introduction to the History of Virology, by Anthony Waterson and Lise Wilkinson, is unique. It came at a time just after the emergence of virology as a
distinct scientific discipline, and yet at a time when there were still tugs from the other microbiological disciplines that virology rejoin the fold. The book played a significant
role in bolstering the conviction of virologists to press on, to continue to build an independent community and to continue to build links with other new biological sciences:
cell biology, molecular biology, structural biology, et al. The authors were in just the right place to capture this perspective: Anthony Waterson was a professor in the
Department of Virology at the Royal Postgraduate Medical School, University of London, and had worked with many leading virologists characterizing many human viral
pathogens. Lise Wilkinson was a distinguished historian, at the time a fellow in the same department, and also associated with the Wellcome Institute for the History of
Medicine. Over the years, she wrote many articles on aspects of the history of virology. The following is abridged from the introduction to the book:
[Virologys] relatively recent origin means that the sources are copious and, on the whole, accessible, even if they are somewhat diffusely spread. Very much has happened
in a short time, so that the rate and acceleration of growth of knowledge have been so fast that the direction and flow of thought have been easy to follow. The origins are
various, but easy to discern: botany, plant pathology, human and veterinary medicine, and especially that activity known as hygiene on the continent of Europe and as
bacteriology in Great Britain and the United States, whether by doctors or veterinarians, have all contributed.... It was in fact a consideration of virology as an offshoot of
bacteriology, and an experience of the difficulties of working in a young subject under the terms of reference of an old, which supplied much of the stimulus to initiate the
current study.... The very element of near impossibility with which the early virologists were faced makes the subject a fertile one for the historian. The principal difficulties
were those intrinsic to the study of very small biological particles, and the pioneers had little in their armamentarium. However, techniques did emerge. Centrifuges grew in
speed and power, and microscopes were developed until their magnification went beyond what the specimen required.... The underlying theme of this book is the evolution
of the present concept of a virus. This present-day concept is essentially a chemical one, but that is not to imply that it is divorced from the mainstream of biological thinking;
[it is a rather good] vantage point from which to look back, so that we can better look forward. It is therefore essentially apocalyptic, in the proper sense of the word, i.e.
revelatory. It is possible to study the virological scientists of the past, and to know in the light of present knowledge exactly what they were studying, even though this was
not revealed to them in their day. It is, in other words, the story of the progressive unveiling of the nature of the virus particle. This history is therefore essentially, and indeed
deliberately, conceptual. It is a study of ideas and concepts, and the inter-reactions between these, on the one hand, and experiment and technique, on the other. That it is
inextricably interwoven with the administrative, the technical and the personal goes without saying, and to compose this history involved in itself the administrative, the
technical and the personal. A catalogue may be compiled by a computer, but stories can be told only by a person.... Individual virologists figure in these pages, but it is their
thinking as much as their doing with which we have been concerned, because the lesser (doing) is included in the greater (thinking). This is, above all, a story. It is to be
hoped that, as a story, it will be read with enjoyment no less than, as a history, it will be studied with interest.

1978 Anthony Waterson, Lise Wilkinson publication of the book, An Introduction to the History of Virology

page 431

Steven C. Harrison

James M. Hogle

The first determination of the atomic structure of a virus was of tomato bushy stunt
virus determined to 2.9 resolution it was published by Stephen Harrison and his
colleagues in 1978. Solving the structure of the icosahedral plant viruses was a major
achievement, but it wasnt clear that it had a profound impact on virologists at the
time. However, it did mean that the structure of other viruses, animal viruses, might
also be determined. Picornaviruses seemed the obvious next goal. One reason was that
poliovirus had been crystallized by Carlton Schwerdt and Frederick Schaffer in 1955.
Harrison tells the following story: Apparently Schaffer had crystallized the Mahoney
strain of polio virus and his wife was going to bring it to England in her pocketbook.
The custom agent asked her what it was and when she said it was poliovirus, he said:
Lady, you cant bring poliovirus into England. She responded: But it is crystalline.
The custom agent must have realized the importance of this as he let her pass.
Then, Jim Hogle came to Harrisons lab to learn crystallography, planning to solve
the structure of polio virus. Hogle had been a graduate student at the University of
Wisconsin where he came under the influence of Roland Rueckert. Rueckert was
responsible for connecting Hogle with Harrison for the eventual goal of solving the
structure of polio virus and also for influencing Michael Rossmann to tackle the
rhinoviruses. In 1985, Hogle and his collaborators solved the structure of poliovirus
to a resolution of 2.9. One factor in achieving this goal had been that, because of the
vaccines, it was possible to work with the virulent Mahoney strain which gave more
stable crystals than the attenuated (Sabin) strains.
Harrison SC, Olson AJ, Schutt CE, Winkler FK, Bricogne C. Tomato bushy stunt virus
at 2.9 resolution. Nature 1978;276:368-373.
Hogle JM, Chow M, Filman DJ. Three-dimensional structure of poliovirus at 2.9
resolution. Science 1985;229:1358-1365.
Rossmann MG, Arnold E, Erickson JW, Frankenberger EA, Griffith JP, Hecht HJ,
Johnson JE, Kamer G, Luo M, Mosser AG, Rueckert RR, Sherry B, Vriend B. Structure
of a human common cold virus and functional relationship to other picornaviruses.
Nature 1985;317:145-153.

tomato bushy
stunt virus

Marie Chow

poliovirus

Michael G. Rossman

1978> Steven Harrison, James Hogle, others structure of viruses (tomato bushy stunt virus, poliovirus, rhinovirus)
page 432

Many viruses initially infect epithelial cells at mucosal body surfaces; epithelial
cells are polarized, having distinct apical and basolateral plasma membranes.
These membrane domains are exposed to very different physiological environments; apical membranes face the luminal environment, whereas basolateral
membranes abut underlying tissue strata. The two domains exhibit distinct
profiles of proteins and lipids, which allow cells to carry out diverse surfacespecific functions. David Sabatini and Enrique Rodriguez-Boulan first showed
that viruses discriminate between these membrane domains as sites for viral
assembly and directional budding in epithelial cells in culture. This polarized
localization of viral proteins (e.g., glycoproteins and matrix proteins) and
cellular moieties associated with viral budding was later shown to be of importance in understanding viral transmission patterns, and therefore disease
spread in nature. In cell culture, for example, influenza A virus, Sendai virus,
SV5, and measles virus bud preferentially from apical membranes, whereas
vesicular stomatitis and Marburg viruses bud almost exclusively from basolateral cell surfaces. Budding from the apical surface might favor restriction
of infection to the epithelium, as is the case with influenza and parainfluenza
viruses, whereas budding from the basolateral surface might favor viral access
to underlying tissue and systemic infection, as is the case with Marburg virus.
This correlation holds true in vivo in many instances, but there are exceptions.
For many viruses that exhibit polarized budding, it has been found that the viral glycoproteins are targeted intrinsically to the same membrane from which
virus buds. However, the exceptions are important: some viral glycoproteins
have been found to sort intrinsically to the membrane opposite from the one
that is used for virus budding. For example, a major portion of Marburg virus
glycoprotein is transported to the apical cell surface, despite the fact that
Top: Apical and basal plasma membrane domains of a MDCK cell in monolayer culture release of infectious virus is from the basolateral cell surface. Measles virus
infected for 10 hours with influenza A virus. Bottom: Similar culture infected for 9 hours infection is similar, suggesting that other factors in addition to the viral glycoproteins are important for selection of viral assembly sites in polarized cells.
with vesicular stomatitis virus. Thin section electron microscopy.
From Boulan and Sabatini, PNAS, 1978
Study of this phenomenon in experimental animal models of viral diseases has
proven complex; indeed, in the pathogenetic progression of infection from
entry site, to major target sites, to exit/transmission sites, a virus may bud
preferentially from different cell membrane domains, including intracytoplasRabies virus infection, fox submandibular
mic organelle membranes and the nuclear envelope. Much more needs to be
salivary gland: virions budding from the
done to unravel this complexity in viral infections as they occur in nature.
lateral canalicular plasma membrane
(part of apical plasma membrane domain) Boulan ER, Sabatini DD. Asymmetric budding of viruses in epithelial
of two mucogenic epithelial cells. This
monlayers: a model system for study of epithelial polarity. Proc Natl Acad Sci
delivers virus into the furthest upstream USA. 1978;75:5071-5075.
channel of the salivary duct system.
Compans RW. Virus entry and release in polarized epithelial cells. Curr Top
Further downstream in the major
Microbiol Immunol. 1995;202:209-219.
salivary ducts extraordinary numbers of
virions can accumulate. In reservoir hosts, Schmidt AP, Lamb RA. Assembly and budding of negative strand RNA
such as the fox, virus titers in saliva at the
viruses. In: Kawaoka Y, editor. Biology of negative strand RNA viruses: The
time of peak transmissibility may reach
power of reverse genetics. Current topics in microbiology and immunology,
106 ID50 per ml. Thin section electron
vol. 283. Berlin: Springer; 2004. p. 174-176.
microscopy

1978> David Sabatini, Enrique Rodriguez-Boulan, Richard Compans directional budding from polarized cells

page 433

R. Palmer Beasley (1936-2012)

Lu-yu Hwang

Countries with most liver cancer

Hepatocellular
carcinoma

classic arrangement of tumor

cells in trabecular and acinar
patterns. H&E

The relationship between hepatitis B virus

(HBV) and primary hepatocellular carcinoma
(HCC) was first noted in the 1970s, when it
was found that HBV antigenemia corresponded
geographically with HCC. The prevalence of
antigenemia was found to be higher in cases of
HCC than in controls in both high- and lowincidence areas for HCC. The epidemiologic
data supporting this causal relationship were
brought together in 1978 by Wolf Szmuness. This
was greatly advanced in 1981 by the prospective
study of Palmer Beasley and his colleagues in
which the risk of HCC over a 5-year period in
persons with persistent HBV antigenemia was
found to be 217 times greater than in people
without antigenemia. This relationship was
further strengthened by laboratory studies
demonstrating HBV DNA sequences integrated
into the chromosomal DNA of tumor cells, and
then by similar findings in studies of animals
infected with their own HBV-like viruses
(woodchuck hepatitis virus, Pekin duck hepatitis
virus). All this, in turn, greatly motivated the
drive for global HBV vaccination, since there
are still ~700,000 new cases of HCC annually,
worldwide [the fifth most common cancer in
men (523,000 cases, 7.9% of the total number
of cancers reported) and the seventh in women
(226,000 cases, 6.5% of total)]. This opportunity
has been turned into a global success story.
Blumberg BS, Larouz B, London WT, Werner
B, Hesser JE, Millman I, Saimot G, Payet M. The
relation of infection with the hepatitis B agent
to primary hepatic carcinoma. Am J Pathol.
1975;81:669-682.
Szmuness W. Hepatocellular carcinoma and
the hepatitis B virus: evidence for a causal
association. Prog Med Virol. 1978; 24:4069.
Beasley RP, Hwang LY, Lin CC, Chien CS.
Hepatocellular carcinoma and hepatitis B virus:
A prospective study of 22,707 men in Taiwan.
Lancet 1981;318:1129-1133.
Wolf Szmuness (1919-1982)

1978 Wolf Szmuness, Palmer Beasley, others relation of hepatitis B virus infection with hepatocellular carcinoma
page 434

Lynn W. Enquist

Enquist LW, Madden MJ, Schiop-Stanley P,

Vande Woude GF. Cloning of herpes simplex
type 1 DNA fragments in a bacteriophage
lambda vector. Science 1979;203:541-544.

Abstracted from the 1979 paper by Enquist, Madden, Schiop-Stanley and Vande Woude:
Our approach to analysis of the large, complex DNA genome of herpes simplex virus type 1 (HSV1) was to insert DNA fragments into a coliphage vector and to grow
these hybrids in Escherichia coli. Only in this way were we able to pick out single large DNA fragments from the complicated mixture and amplify them in quantities suitable
for detailed study. To insert HSV1 DNA into the vector, we were required by the NIH Guidelines on Recombinant DNA to use the most stringent biological and physical
containment available: P4 combined biological and physical containment. The basic procedure for our experiments was as follows: the vector DNA was prepared and cleaved
with the restriction endonuclease EcoR1. Fragments of the cleaved vector DNA were separated and mixed with EcoR1-cleaved HSV1 DNA and joined by means of T4 DNA
ligase. The ligated DNA was then packaged into phage particles. The recombinant phage particles were used to infect disabled E. coli K12 and lysates containing recombinant
phage DNA were concentrated and the DNA extracted a certified treatment of this product effectively inactivated the recombinant phage and bacteria. After treatment
with ribonuclease and extraction with phenol the DNA was frozen for subsequent analysis. The vector is particularly suited for insertion of EcoR1 fragments; however,
fragments less than about 1 kbp or more than 15 kbp theoretically cannot be inserted into the vector because of size limitations of the phage head and limits of the packaging
mechanism. When HSV1 DNA was cleaved by EcoR1, about 15 fragments were obtained unfortunately, most of the fragments approached or exceeded the theoretical 15kbp size capacity of the vector. We sought to improve our chances of inserting large fragments that were close to the theoretical size limit by a procedure in which recombinant
DNA is added to a mixture of partially assembled virions, and the added DNA is encapsidated to form viable phage particles. In this procedure the larger DNA molecules are
preferentially packaged over smaller ones. Represented among the recombinant DNA molecules studied were fragments from about 50% of the HSV1 genome. The ability to
make large quantities of specific HSV DNA fragments allowed us to make structural and transcriptional analyses that could never be done before. The identification of viral
proteins by the hybrid arrest method was also simplified using the highly purified HSV DNA fragments.

1979 Lynn Enquist, colleagues cloning viral DNA fragments into phage vector (herpes simplex virus)

page 435

David P. Lane

Arnold J. Levine

The protein p53 was discovered in 1979 by Lionel

Crawford, David Lane, Lloyd Old, and their co-workers.
Over the next ten years, the gene for p53 was found to act
as a tumor suppressor gene. Its role in the cell is to prevent
cell transformation leading to cancer. About 60 % of
human cancers contain mutations in the p53 gene. It now
appears that the function of the p53 protein is to respond
to damage to DNA, by either moving the cell into a lethal
apoptotic program or permitting cellular repair processes
to act prior to duplication of the DNA and cell division. In
this way, the p53 protein, sensing DNA damage, minimizes
mistakes in the genetic information and eliminates
the rise of genetically altered cells that lead to cancer.
Lane and Levine and their colleagues used antibodies
specifically reactive to SV40 large T antigen to show that
immunoprecipitation from transformed cells resulted in
the recovery of not only SV40 large T antigen itself but also
a cellular protein having an approximate molecular weight
of 53 kDa. Based on its size, this protein became known
as p53. Although this finding was significant in providing
the first evidence that the products of DNA tumor virus
oncogenes function through physical interactions with
cellular proteins, a decade of additional research was
required before the enormous effect of the p53 discovery
would be fully comprehended. The central importance of
p53 is further underscored by the fact that, in addition to
SV40 large T antigen, oncoproteins encoded by other DNA
tumor viruses, such as human papillomaviruses and human
adenoviruses, have similarly evolved to bind and inactivate
p53 in cells.
Lane DP, Crawford LV. T antigen is bound to a host protein
in SV40-transformed cells. Nature 1979;278:261-263.
Linzer DI, Levine AJ. Characterization of a 54K dalton
cellular SV40 tumor antigen present in SV40-transformed
cells and uninfected embryonal carcinoma cells. Cell
1979;17:43-52.

p53 tetramer
bound to
the major
groove of
DNA

DeLeo AB, Jay G, Appella E, Dubois GC, Law LW, Old LJ.
Detection of a transformation-related antigen in chemically
induced sarcomas and other transformed cells of the
mouse. Proc Natl Acad Sci USA. 1979;76:2420-2424.
Javier RT, Butel JS. The history of tumor virology. Cancer
Res. 2008;68:7693-7706.

1979 David Lane, Arnold Levine, others discovery of p53 tumor suppressor protein in SV40-transformed cells
page 436

George R. Stark
The western blotting method originated in the laboratory of George Stark at Stanford University. It is used to detect specific proteins in a given sample, say a cell or tissue
homogenate or purified virus extract. It uses gel electrophoresis to separate native or denatured proteins by their length and their conformation. The proteins are then
transferred to a membrane (typically nitrocellulose, nylon or polyvinylidene fluoride - PVDF), where they are detected using antibodies specific for the target protein. The
detection step was at first autoradiography using radiolabeled secondary antibody, but today the most common methods involve use of (1) a horseradish peroxidase-linked
secondary antibody to cleave a bound chemiluminescent reagent, with the luminescent reaction product detected with a CCD camera (the image may be analyzed by
densitometry to estimate the amount of target protein present newer software allows further data analysis such as molecular weight analysis); or (2) a fluorescently labeled
secondary antibody, with the reaction product detected by a CCD camera. Fluorescence is considered to be among the most sensitive detection methods for blotting analysis.
The name western blot was given to the technique by W. Neal Burnette and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin
Southern. Detection of RNA by similar means is termed northern blotting.
Renart J, Reiser J, Stark GR. Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen
structure. Proc Natl Acad Sci USA. 1979;76: 3116-3120.
Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci
USA. 1979;76:4350-4354.
Burnette WN. Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection
with antibody and radioiodinated protein A. Anal Biochem 1981;112:195-203. (Citation Classic)

1979 George Stark, others western blotting: transfer of electrophoresed proteins from gel and detection with antibody

page 437

Peste des petits ruminants is one of the most serious diseases of domestic
and wild small ruminants; it is caused by a virus related to rinderpest. The
disease had long been considered caused by rinderpest virus or a variant
adapted to small ruminants. The first suggestion that this is not the case
dates to 1942 when L. Gargadennec and A. Lalanne working in in Cte
dIvoire observed that the disease was not transmissible from small ruminants to in-contact cattlethis led them to conclude that the disease was
caused by a novel virus. Proof of this came only in 1979, when Paul Gibbs,
William Taylor and their colleagues showed that the virus is a distinct
member of the genus Morbillivirus, family Paramyxoviridae. For many
years the disease was thought to be limited to West Africa, but rather
recently its distribution has been shown to extend across Asia as well as
Africa much of the data underpinning this realization stemmed from the
diagnostics done in association with the global rinderpest eradication program. Because of its great economic impact and associated restrictions on
animal and product movements, peste des petits ruminants virus has been
considered the next target for global eradication, following only smallpox
and rinderpest. An effective single-dose vaccine has been available since
the 1980s so regional elimination seems feasible, yet the lesson of just how
difficult it was to eradicate rinderpest will test international political will.
Gargadennec, L. & Lalanne, A. (1942). La peste des petits ruminants.
Bulletin des Services Zoo Techniques et des Epizooties de lAfrique
Occidentale Francaise 5, 16-21.

E. Paul J. Gibbs

William P. Taylor

Gibbs EP, Taylor WP, Lawman MJ, Bryant J. Classification of peste des
petits ruminants virus as the fourth member of the genus Morbillivirus.
Intervirology. 1979;11:268-274.

Global distribution of the four peste des petits ruminants virus lineages. Background
color and stripes represent the last identified lineages in the country. Albina E, Kwiatek
O, Minet C, Lancelot R, Servan de Almeida R, Libeau G. Peste des petits ruminants, the
next eradicated animal disease? Vet Microbiol. 2013;165:38-44.

1979 Paul Gibbs, William Taylor, colleagues peste des petits ruminants virus shown to be a distinct morbillivirus
page 438

Robert A. Swanson (1947-1999) & Herbert W. Boyer

Sculpture at Genentechs South San Francisco headquarters

allegorically depicting the first meeting of Robert Swanson
and Herbert Boyer at Churchills Bar in 1976

In 1976, Robert Swanson and Herbert Boyer created the biotechnology industry over a couple of beers at Churchills Bar in San Francisco. Swanson, then just 29, was a venture
capitalist who saw the promise in recombinant DNA technology. Boyer, a 40-year-old biochemistry and biophysics professor at the University of California San Francisco,
had co-developed the technology with Stanley Cohen. After being put off by Cohen, Swanson cold-called Boyer, stopped by his lab, and the two retreated to the bar to sketch
out a business plan. To turn recombinant DNA technology into a business, Boyer and Swanson incorporated under the name Genentech, Inc. Putting down $500 apiece, they
capitalized their new business, to seek practical uses for Boyer and Cohens engineered protein technology. Swanson raised money for staff and labs, and in a two-year, roundthe-clock effort, Genentech staff successfully created their first product: human insulin, which won Food & Drug Administration approval in 1982. In 1980, Genentech went
public and was listed on the New York Stock Exchange its stock leapt from $35 a share to a high of $88 after less than an hour on the market. The offering raised a spectacular
$38.5 million and made its founders multi-millionaires The event was one of the largest stock run-ups ever. Today, Genentech, which has more than 11,000 employees, is a
wholly-owned subsidiary of Roche it was purchased in 2009 for approximately $46.8 billion.

1980 Robert Swanson, Herbert Boyer Genentech, the first biotech company listed on the NY Stock Exchange

page 439

Bernard J. Poiesz

Robert C. Gallo

Adult T-cell leukemia (ATL) was identified as a distinct clinical entity by Kiyoshi Takatsuki,
Junji Yodoi and Takashi Uchiyama and their colleagues in Japan in 1976; this was based on the
unique geographic distribution of the disease as well as its distinctive clinical, immunological
and histopathological features. Isao Miyoshi established cell lines from peripheral lymphoid cells
of ATL patients, which exhibited extreme chromosomal abnormalities. In 1980, its causative
agent, human T-lymphotropic virus type I (HTLV-I) was identified by Bernard Poiesz, Robert
Gallo and their colleagues in a cell line derived from a patient with cutaneous T cell lymphoma
they used reverse transcriptase assays and electron microscopy to prove the presence of the
virus. In addition, the close linkage between ATL and HTLV-I was demonstrated in 1981 by
Yorio Hinuma, Mitsuaki Yoshida and their colleagues, by the presence of antibody against viral
antigens in the sera of patients with ATL. Thus, HTLV-I (now HTLV-1) was the first retrovirus
shown to be associated with human disease. Today, HTLV-1 is known to infect 10 to 20 million
individuals worldwide, and the virus is known to be associated not only with ATL, but also
with an inflammatory disease termed HTLV-associated myelopathy (HAM) or tropical spastic
paraparesis (TSP) as well as other neurologic diseases. In 1981, David Golde described an
unusual immortalized T-lymphocyte cell line grown from spleen tissue of a patient with hairycell leukemia. Gallo suggested that a new retrovirus could be present, but because the cell line
was producing a valuable lymphokine and had been patented, access was denied. Later, Golde
decided to collaborate anyway and HTLV-2 was isolated.

HTLV-1 T cell lymphoma, skin

Takatsuki K, Uchiyama T, Sagawa K, Yodoi J. Adult T cell
leukemia in Japan. In: Seno S, Takaku F, Irino S, editors.
Topics in hematology. Amsterdam: Excerpta Medica;
1977. p. 73-77.

Mitsuaki Yoshida

Yorio Hinuma

Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD,
Gallo RC. Detection and isolation of type C retrovirus
particles from fresh and cultured lymphocytes of a patient
with cutaneous T-cell lymphoma. Proc Natl Acad Sci
USA. 1980;77: 7415-7419.
Hinuma Y, Nagata K, Hanaoka M, Nakai M, Matsumoto
T, Kinoshita KI, Shirakawa S, Miyoshi I. Adult T-cell
leukemia: antigen in an ATL cell line and detection of
antibodies to the antigen in human sera. Proc Natl Acad
Sci USA. 1981;78:6476-6480.
Yoshida M, Miyoshi I, Hinuma Y. Isolation and
characterization of retrovirus from cell lines of human
adult T-cell leukemia and its implication in the disease.
Proc Natl Acad Sci USA. 1982;79:2031-2035.

David Golde (1940-2004)

Kalyanaraman VS, Sarngadharan MG, Robert-Guroff M,

Miyoshi I, Golde D, Gallo RC. A new subtype of human
T-cell leukemia virus (HTLV-II) associated with a T-cell
variant of hairy cell leukemia. Science. 1982;218:571573.

1980 Bernard Poiesz, Robert Gallo, Mitsuaki Yoshida, David Golde, others Human T-lymphotropic viruses 1 & 2
page 440

The National Center for Infectious

Diseases was created at CDC in 1980
by Walter Dowdle, who served as its
founding director. For the first time, it
brought together all resources needed to
support the mission of infectious disease
prevention and control: lab sciences,
epidemiology, field programs, etc. It was
very successful, but was terminated in
2004 as the CDC director reorganized,
forming smaller, easier to manage units.

James M. Hughes

Walter R. Dowdle Frederick A. Murphy

The Board of Scientific Counselors, National Center for Infectious

Diseases, Centers for Disease Control and Prevention (1987)
Front row L to R): Joseph Jones (chairman, The Robert Woodruff Foundation), Jeff
Davis (state epidemiologist, Wisconsin Department of Health), J. Mehsen Joseph
(director of laboratories, Maryland Department of Health), Mary Ann Danello
(assistant commissioner, FDA), Walter Bowie (dean, School of Veterinary Medicine,
Tuskegee University), Bernard Fields (chairman, Department of Microbiology and
Molecular Genetics, Harvard Medical School). Back row: Frederick A. Murphy
(director, National Center for Infectious Diseases, CDC), David Fraser (president,
Swarthmore College), John Bennett (director, Clinical Mycology Laboratory, NIAID,
NIH), Lee Hand (director, Division of Infectious Diseases, Emory University School
of Medicine), Rebecca Rimel (executive director, The Pew Charitable Trusts), Scott
Halstead (director, Health Sciences, The Rockefeller Foundation), Carlos Lopez
(infectious disease specialist, Northside Hospital, Atlanta), D. A. Henderson (dean,
Johns Hopkins School of Hygiene and Public Health), Joseph Losos, director
general, Laboratory Centre for Disease Control, Canada), John LaMontagne
(deputy director, NIAID, NIH), Gail Cassell (chair, Department of Microbiology,
University of Alabama, Birmingham). Not present for this photo: Glenn Close
(actor, and president, Trillium Production Company), Stanley Falkow (professor,
Stanford University School of Medicine), Harlyn Halvorson (president, Marine
Biological Laboratory, Woods Hole), George Hill (associate dean for research and
graduate studies, Meharry Medical College), John Maupin (executive vice president,
Morehouse School of Medicine), Phillip Russell (commander, U.S. Army Medical
Research and Development Command), Laurence Foster (state epidemiologist,
Oregon Department of Health).

The only three directors of the NCID / CDC

1980 Walter Dowdle, Frederick Murphy James Hughes National Center for Infectious Diseases at CDC

page 441

Elizabeth S. Williams (1951-2004)

Stuart Young (1925-2003)

Chronic wasting disease (CWD) was first identified as a clinical syndrome of unknown etiology
in mule deer (Odocoileus hemionus) in 1967 in the wildlife facility of Colorado State University.
Biologists conducting natural history and nutritional studies on captive deer observed clinical signs
(progressive weight loss, behavioral changes, listlessness, lowering of the head, repetitive walking in
set pattern, death) and thought these were associated with stresses of captivity, nutritional deficiencies,
or intoxication. Only after 13 years of substantial disease incidence were the veterinary pathologists
Elizabeth Williams and Stuart Young invited to seek a pathological diagnosis. They immediately
identified the disease as a spongiform encephalopathy, that is a prion disease. Shortly thereafter, CWD
was recognized among captive mule deer and in Rocky Mountain elk (Cervus elaphus) in a facility in
Wyoming. The first CWD case in a free-ranging cervid was found in 1981 in a Rocky Mountain elk in
Rocky Mountain National Park, Colorado. The first affected mule deer diagnosed in a wild population
was found in 1984 very near the deer pens where the disease was first observed in 1967. It has since
also been recognized in several other species in zoos and wild animal parks: greater kudu (Tragelaphus
strepsiceros), Arabian oryx (Oryx leucoryx), white-tailed deer (Odocoileus virginianus), black-tailed deer
(Odocoileus hemionus columbianus), et al. CWD in the game farm industry has become a significant
problem in several regions of Canada and the United States (map) and control (test and slaughter)
programs are in place in many affected areas, so far without any real sense of success.

Mule deer brain, immunohistochemical

stain for PrP protein

Williams ES, Young S. Chronic wasting disease of captive mule deer:

a spongiform encephalopathy. J Wildl Dis. 1980;16:89-98.
Williams ES, Young S. Spongiform encephalopathy of Rocky
Mountain elk. J Wildl Dis. 1982;18:465-471.

1980 Elizabeth Williams, Stuart Young discovery that chronic wasting disease of deer and elk is a prion disease
page 442

In 1967, Carl Woese, Francis Crick, and Leslie Orgel

suggested that RNA could act as a catalyst or enzyme.
This idea was based upon the discovery that RNA can
form complex secondary structures. The first ribozymes
were discovered in the 1980s by Thomas Cech and his
colleagues (especially Benjamin Stark) at the University of
Colorado, who were studying RNA splicing in the ciliated
protozoan Tetrahymena thermophila, and Sidney Altman
and his colleagues (especially Ryszard Kole), working at Yale
University, who were working on a bacterial RNase complex.
Cechs ribozyme was found when trying to purify an enzyme
responsible for a very complex self-splicing reaction they
found that an intron was spliced out during the maturation
of the enzyme in the absence of any added cell extract. As
much as they tried, Cech and his colleagues could not identify
any protein associated with the splicing reaction. Eventually,
they found that the intron portion of the immature RNA was
excised by a complex process self-accomplished by the RNA.
Altmans ribozyme, located in the RNA component of the
RNase P complex, was found to be responsible for conversion
of a precursor tRNA into an active tRNA. They found that
the enzyme that facilitated this was RNase-P and that it
contained RNA in addition to protein and that the RNA was
an essential component of the active enzyme in fact, the
RNA subunit could catalyze the cleavage of precursor tRNA
into active tRNA in the absence of any protein component.
The discovery of an enzymatic role of RNA came as a
complete surprise, falling outside the central dogma of
DNA>RNA>protein, in which RNAs were thought to only
act in transcription and translation. Among other things, the
discovery reinvigorated the hypothesis of the RNA World,
that is that the earliest forms of life may have relied solely on
RNA to store genetic information and to catalyze chemical
reactions. After Cechs and Altmans discovery, many other
examples of self-cleaving RNA or catalytic RNA molecules
were found today, about 100 RNA ribozymes are known. It
is now possible to make ribozymes that will specifically cleave
RNA molecules of interest. For example, a ribozyme has been
designed to cleave the RNA of HIV. If such a ribozyme was
made by a cell, all incoming virus particles would have their
RNA genome cleaved by the ribozyme, which would prevent
infection this idea is being actively pursued in regard to
other viral pathogens as well.

Sidney Altman

Thomas R. Cech
Stark BC, Kole R, Bowman EJ, Altman S. Ribonuclease
P: an enzyme with an essential RNA component. Proc
Natl Acad Sci USA. 1978;75:3717-3721.

Extreme oversimplification of the action of

a ribozyme cleaving an RNA molecule at a
specific sequence site

Kole R, Altman S. Properties of purified ribonuclease

P from Escherichia coli. Biochem. 1981;20:1902-1906.
Kruger K, Grabowski PJ, Zaug AJ, Sands J, Gottschling
DE, Cech TR. Self-splicing RNA: autoexcision and
autocyclization of the ribosomal RNA intervening
sequence of Tetrahymena. Cell 1982;31:147-157.
Cech TR. RNA splicing: three themes with variations.
Cell 1983;34:713-716.

1980 Thomas Cech, Sidney Altman, colleagues discovery of the catalytic properties of RNA, the ribozyme concept

page 443

Gary Larsons, The Far Side first appeared

in the San Francisco Chronicle in 1980
and ran until 1995 when he retired. Many
different sciences claim Larson as their
own, but the virologic/microbiologic/
entomologic sciences seem to make this
claim most personally. Over the years,
it was rare to find a virology lab that
did not have Far Side cartoons taped to
the fridge or the professors door. They
are perhaps less ubiquitous today, but
they are still there in many labs, as the
Xerox machine keeps cranking them out
for each new generation. It is not that
Larson was a specialist or had a favorite
species to anthropomorphize; rather, it
seemed as if his themes carried over from
one field of science (and scientists) to
others. Virologists have smiled over all
of Larsons cartoons featuring microbes;
arbovirologists have smiled even more
because of Larsons great use of surreal
insects/arthropods (e.g., The unexpected
dangers of being an insect) and
veterinary virologists have had it the best
as Larsons cows flash through memories.
Edward O. Wilson described Larson
as the madcap sage of the biological
sciences and credits the cartoonist with
broadcasting an important insight, that
Nature is part of us and we are part of
Nature, that humans are just one species
among many, just one small part of life
on planet Earth. Larson published 4,337
Far Side cartoons, which were collected
into 19 books in 17 languages, and
printed on innumerable greeting cards,
coffee mugs, et al. He was honored by
having named after him a biting louse of
owls, Strigiphlus garylarsoni, a butterfly
from Ecuador, Serratoterga larsoni, and
a beetle, genus name Garylarsonus. It
seems proper to include his work in this
historical perspective on medical and
veterinary virology.

Gary Larson

* For 14 years (1964-1978) the Viral Pathology Branch was

the name of my lab at the CDC in Atlanta. However, I

have no idea how Gary Larson chose the name on this, his
greatest cartoon (at least its my notion of his greatest).
Alas, I never met him. Today, a greatly expanded version of
the lab at CDC, the Infectious Diseases Pathology Branch,
is directed by the distinguished pathologist, Sherif Zaki.

**

As I was preparing to give a lecture in Mexico, I asked my son,

John, to translate six Far Side cartoon captions into Spanish for use
as PowerPoint slide transitions. He, saying that captions are quite
idiomatic, spent an evening with several friends and a few beers to get
them perfect. They refused to keep Betty. She became Paquita. In
any case, Larson knew that only female mosquitoes take blood meals.

1980> Gary Larson The Far Side, a wonderful fixture in virology labs everywhere
page 444

James Curran

Donald Francis

Harold Jaffe

Kevin De Cock

Paul Volberding

Helene Gayle

Jay Levy

These physician-epidemiologists, and many others, did the first outbreak

investigations and natural history studies on the disease that emerged in 1981,
eventually called acquired immunodeficiency syndrome (AIDS)

1981 MMWR reports of Pneumocystis carinii pneumonia in Los Angeles and Kaposi sarcoma in New York AIDS

page 445

The Early Years of the HIV/AIDS Epidemic/Pandemic

Key reference: Curran JW, Jaffe HW. AIDS: the Early Years and CDCs Response. MMWR Supp. 2011;60:64-69.
The first two MMWR articles calling attention to cases among homosexual men of
Pneumocystis carinii pneumonia (PCP) in Los Angeles and Kaposi sarcoma in New
York are credited to astute clinicians and recognition of increased requests for the
orphan drug used to treat PCP. Immediately, additional cases of other life-threatening
opportunistic infections (OIs) were reported in the same populations in other U.S.
cities. In the summer of 1981 CDC formed a Task Force and began surveillance and
epidemiologic investigations. This involved developing a surveillance case definition,
partly to unravel the basis for the severe, often fatal cellular immune immunosuppression. Later, the WHO employed a modified case definition for use in settings with limited diagnostic capacity. Within 6 months, it was clear that a new, highly concentrated
epidemic of immunosuppressive life threatening illness was occurring, but for several
years its incidence was underestimatedjust the tip of the iceberg was seen, partly
because the long incubation period and varying clinical course were not appreciated.
In 1982, CDC conducted a national case-control study that found that case-patients
tended to be much more sexually active than controls; another study showed that
many case-patients had had sexual contact with other case-patientsstrongly suggesting that the new syndrome was caused by a sexually transmissible infectious
agent. Nonetheless, whether because of competing hypotheses or merely denial, many
scientists and the public were skeptical of the infectious agent causation theory. Then,
people in several states with severe hemophilia A were reported to have died from
PCP with unexplained immunosuppression (they had received clotting factor concentrates derived from pooled human plasma). Soon, cases were reported among infants,
female sex partners of men at high risk for the syndrome and people who had received
blood transfusions. All this provided further evidence that the syndrome was caused
by an infectious agent that could be transmitted by blood and from mother to child,
as well as through homosexual and heterosexual contact. The syndrome was then
reported among Haitian migrants to the U.S. During this time, given the nature of the
risk groups and the underestimation of the incidence, it received relatively little attention from the mainstream media, the public, and politicians.
By 1983, however, it was clear that others were at risk for the disease, and what had
been complacency turned into serious concern, even panic. There was concern for
physicians and other persons caring for AIDS patients, laboratory workers, et al.
Recommendations for the prevention of AIDS based on the epidemiologic data were
developed two years before the cause of the syndrome was discovered and before
diagnostic testing of individuals and screening of blood donations were possible.
Nevertheless, the recommendations had a positive effect and illustrate the importance
of epidemiologic investigation in understanding and preventing new diseases, even in
the absence of an identified etiology.
The causative virus, human immunodeficiency virus (HIV), was discovered by Luc
Montagnier, Francois Barre-Sinoussi, Jean-Claude Chermann and their colleagues at
the Institut Pasteur in May 1983. Additional proof of causality was reported by Robert

Gallo and his colleagues at the NIH in 1984. In 2008, Barre-Sinoussi and Montagnier
were awarded the Nobel Prize in physiology or medicine for their discovery.
The availability of laboratory reagents and techniques to identify HIV led to rapid
advances in understanding the transmission, natural history, and spectrum of illness.
It is not possible to detail this large subject here. It was shown that the virus persists
in infected asymptomatic individuals for years and that seropositivity is equivalent to
infection and infectiousness. This understanding allowed for more targeted prevention efforts and paved the way for the usage system for the first effective antiviral
drugs. Eventually, serologic testing (which evolved into a massive enterprise with
much regulation and oversight) also allowed better estimates of the extent of the
epidemicfor example, it became clear that as many as 200,000-300,000 gay men had
already been infected in the U.S. at the time of the first case reports in 1981; projections of the number of AIDS cases in future years went up dramatically. In 2013, CDC
estimated that >1M people in the U.S. were infected with HIV (21% were unaware of
their infection). The annual number of new HIV infections was estimated at 53,600
and the number of deaths at 18,000.
By 1984, case reports described AIDS in Africa, first in Zaire (now the Democratic
Republic of Congo), then across the continent, then across Asia. While AIDS prevalence peaked and then declined in the U.S. and other developed countries, it rose to
catastrophic levels in many developing countries, especially in sub-Saharan Africa.
Since the start of the epidemic/pandemic ~75 million people have been infected with
HIV and ~36 million have died of AIDS-related illnesses. The peak prevalence was
reached in ~2001; since then prevalence has
Global Summary of the AIDS Pandemic, 2012
fallen by 33%. In 2012, ~9.7 million people
Number of people infected with HIV
infected with HIV in low-and-middleTotal
35.3 million
income countries had access to antiretroAdults
32.1 million
viral therapy, representing 61% of those
Women
17.7 million
eligible for treatment under 2010 WHO
Children (<15 years)
3.3 million
AIDS newly infected in 2012
guidelines and 34% of those eligible under
Total
2.3 million
the 2013 WHO guidelines. TuberculosisAdults
2.0 million
related deaths in people infected with HIV
Children (<15 years)
260,000
have fallen by 36% since 2004, but this still
AIDS deaths in 2012
remains the leading cause of death among
Total
1.6 million
people infected with HIV. US$ 18.9 billion
Adults
1.4 million
Children (<15 years)
210,000
was available in 2012 from all sources for
the global AIDS response, but the amount
estimated to be needed by 2015 is US$ 2224 billionit seems eminently clear that
this, one of the most significant emergent
diseases ever, still requires a paradigmatic
breakthrough, a safe, effective preventive
UNAIDS, 2013
vaccine for all people at risk.

1981> Acquired immunodeficiency syndrome (AIDS) and human immunodeficiency virus (HIV)
page 446

Vincent Racaniello

Racaniello
and
Baltimores
poliovirus
cloning
strategy

David Baltimore

Eckard Wimmer

In 1981, in two separate sets of experiments conducted in two laboratories, a poliovirus

infectious clone was constructed and the complete genomic RNA sequence was determined
both were firsts for a virus of vertebrates. The infectious clone was made by Vincent Racaniello
and David Baltimore at MIT and the sequencing was done by the same individuals and
independently by Eckard Wimmers team at the State University of New York, Stony Brook
the team included Naomi Kitamura, Bert Semler, Paul Rothberg, Glenn Larsen, Cheryl Adler,
Andrew Dorner, Emilio Emini, Ronnie Hanecak, James Lee, Sylvie van der Werf and Carl
Anderson.
Racaniello V, Baltimore D. Cloned poliovirus complementary DNA is infectious in mammalian
cells. Science 1981;214:916-919.
Kitamura N, Semler BL, Rothberg PG, Larsen GR, Adler CJ, Dorner AJ, Emini EA, Hanecak R,
Lee JJ, van der Werf S, Anderson CW, Wimmer E. Primary structure, gene organization and
polypeptide expression of poliovirus RNA. Nature 1981;291:547-553.

Structural model of poliovirus

Racaniello VR, Baltimore D. Molecular cloning of poliovirus cDNA and determination of the
complete nucleotide sequence of the viral genome. Proc Natl Acad Sci USA. 1981;78:4887-4891.

1981 Vincent Racaniello, David Baltimore, Eckard Wimmer infectious clone and complete sequence of poliovirus

page 447

Gerd Binnig and Heinrich Rohrer

The scanning tunneling microscope (STM) is an instrument for

imaging surfaces at the atomic level. Its development in 1981
earned its inventors, Gerd Binnig and Heinrich Rohrer, the
Nobel Prize in Physics in 1986. For an STM, good resolution
is considered to be 0.1nm lateral resolution and 0.01nm depth
resolution. With this resolution, individual atoms within materials
are routinely imaged and manipulated. A literature search of
papers published over the past 10 years reveals that most uses of
STM have been in materials sciences, with only a few innovative
approaches having been applied to biological materials: for
example, quite a few STM images of various bacteriophages and
tobacco mosaic virus have been published. Further, some viral
proteins have been imaged, such as the reverse transcriptases of
human immunodeficiency virus (HIV1) and of Moloney murine
leukemia virus. However, even though very high resolution has
been achieved, it has been difficult to interpret images obtained
by STM relative to other high resolution methods such as negative
contrast transmission electron microscopy and cryoelectron
microscopy and even harder to interpret findings relative to X-ray
crystallography data. Seemingly, more work is needed.

Scanning tunneling electron microscope

Binnig G, Rohrer H, Gerber Ch,
Weibel E. Surface studies by
scanning tunneling microscopy.
Phys Rev Lett. 1982;49:57-61.

Scanning tunneling
microscopy image of
tobacco mosaic virus

1981 Gerd Binnig, Heinrich Rohrer development of the scanning tunneling electron microscope
page 448

Don C. Wiley (1944-2001)

John J. Skehel

Ian A. Wilson
In 1981, Ian Wilson, Don Wiley and John Skehel
published the atomic structure of the influenza
hemagglutinin to a resolution of 3 this was the
first depiction of the structure of a viral peplomer
protein. Two years later, the structure of influenza virus
neuraminidase was solved to a resolution of 2.9 by
Joseph Varghese, Graeme Laver and Peter Coleman.
Wilson IA, Skehel JJ, Wiley DA. Structure of the
haemagglutinin membrane glycoprotein of influenza virus
at 3 resolution. Nature 1981;289:366-373.

Various depictions of the ectodomain of

the influenza virus hemagglutinin trimer

1981 Don Wiley, John Skehel, Ian Wilson, others visualization of the structure of the influenza virus hemagglutinin

page 449

The beginning of research on the ras (RAt Sarcoma) oncogene family can be
traced back to 1964 when Jennifer Harvey observed that a preparation of the
rat leukemia virus that now bears her name induced sarcomas in new-born
rodents. Werner Kirsten found the same thing with a similar virus preparation
that now bears his name. Three additional viruses were later shown to carry
ras oncogenes. By the late 1970s, it was known that retroviral oncogenes could
rapidly transform cells, and that specific retroviruses had acquired these genes
from the genomes of the mammalian and avian cells that they had infected.
It was therefore proposed that mutations in the cellular homologues of these
genes could transform cells in the absence of any viral involvement, and that
this occurred in a substantial proportion of human cancers. In 1982, Robert
Weinberg, Michael Wigler and Mariano Barbacid each independently cloned
ras, the first oncogene, from bladder carcinoma cell lines. This cloned cellular
gene (c-ras) had the same transforming properties as the oncogene from the
viruses (v-ras). It was found that the oncogenes in question were the cellular
homologues of ras genes from Harvey and Kirsten rat sarcoma viruses. In 1982,
Weinberg, Wigler and Barbacid discovered that a single amino-acid change
in the Ras protein glycine to valine at position 12 was responsible for the
oncogenic effect. Subsequent research has shown that this change alters the
structure of the Ras protein to make it constitutively active. The family of
normal Ras proteins are involved in cellular signal transduction, and mutations
in the gene that encoded them in turn causes cell growth, differentiation,
dysregulated cell division and long-term cell survival. ras oncogene signaling
can lead to oncogenesis and cancer. Activating mutations in ras are found in 2025% of all human tumors and up to 90% in certain specific tumor types.
Shih C, Weinberg RA. Isolation of a transforming sequence from a human
bladder carcinoma cell line. Cell 1982;29:161-169.

Robert A. Weinberg and Michael H. Wigler

Goldfarb M, Shimizu K, Perucho M, Wigler M. Isolation and preliminary

characterization of a human transforming gene from T24 bladder carcinoma
cells. Nature 1982;296:404-409.
Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nature
Rev|Cancer 2003;3:7-13.

Mutations in
ras signaling
pathways
associated
with human
diseases

Ribbon model of
H-Ras protein
structure

1981 Robert Weinberg, Michael Wigler discovery and cloning of the first human tumor-derived oncogene, h-ras
page 450

Fred Murphy
Council

Milt Zaitlin
Council

Dorothy Horstman
(1911-2001) Council

Bill Joklik
President

David Bishop
Secretary-Treasurer

Max Summers
Council

Harry Ginsberg
(1917-2003)
Vice President

The Founding Officers

and Council Members
1981

Bob Haselkorn
Council

Priscilla Schaeffer
(1943-2010) Council

1981 Bill Joklik, Harry Ginsberg, others founding of the American Society for Virology

page 451

Founding of the American Society for Virology

[abridged from: Joklik WK, Grossberg SE. How the American Society for Virology was founded. Virology 2006;344:250-257.]:

1966:

Ninth International Congress of Microbiology (ICM), Moscow dissatisfaction with virology venues at ICMs led to planning for a virology congress.

1968:

First International Congress for Virology, Helsinki a grand success.

<1980: Virology was one of the Divisions of the American Society for Microbiology (ASM) dissatisfaction with virology venues at ASM meetings.
1980:

Ad hoc discussions were held as to whether to found a free-standing American Society for Virology. Requests to the ASM by its Virology Division for travel funds to
the Fifth International Congress of Virology in Strasbourg in 1981 were denied.

1981:

Bill Joklik sent an organizational planning letter to Peter Vogt, Purnell Choppin, Bob Wagner, David Baltimore, Tom Merigan, Juli Youngner, Norton Zinder, Harry
Ginsberg, Al Kaplan, Lee McLaren, Fred Murphy, Fred Rapp and Walter Schlesinger, laying out general planning ideas. The reaction to this letter was
overwhelmingly positive.

1981:

A public meeting of 40 virologists was held on 9 June 1981 in Chicago at which a resolution was passed overwhelmingly to found a virology society 9 June 1981 is
the birthday of the American Society for Virology.

1981:

An Organizing Committee was constituted both to design an administrative structure and to organized the first annual meeting of the society in August 1982 at
Cornell University. More than 650 virologists were invited to become charter members and attend the Ithaca meeting. The members of the interim Organizing
Committee were: David Baltimore, Purnell Choppin, Harry Ginsberg, Bob Haselkorn, Dorothy Horstman, Bill Joklik, Tom Merigan, Fred Murphy, Bernard
Roizman, Max Summers, Peter Vogt, Bob Wagner, Julius Youngner, Milt Zaitlin and Norton Zinder.

1982:

The first annual meeting of the American Society for Virology was held at Cornell University, locally organized by Milt Zaitlin, and attended by more than 500
virologists (with the society already having about 1,000 paid-up members). The scientific program, organized by the interim president Bill Joklik, consisted of four
symposia and 27 workshops: the topics of the symposia were: Genome Structure and Expression (Norton Zinder, David Baltimore, Jan Kaper); Transformation and
Persistence (Mike Bishop, George Miller, Mark Ptashne); Mechanisms of Infection (Abner Notkins, Bill Robinson, Tom Monath); and Epidemiology and Ecology
(Peter Palese, Max Summers, Karl Johnson).

At the first business meeting of the Society the following were elected:
President
Bill Joklik
President-Elect
Harry Ginsberg
Councilors
Fred Murphy, Milton Zaitlin, Dorothy Horstman, Max Summers, Bob Haselkorn, Priscilla Schaeffer
Secretary-Treasurer David Bishop

At the first business meeting the following committee appointments were made:
Meetings & Program Committee
Ken McIntosh, Chairman, Skip Carmichael, Bob Goodman, Bill Joklik, Jonathan King, Bill Rawls, Max Summers
Membership Review Committee
Al Wood, Chairman, Brian Derbyshire, Harry Ginsberg, Michael Gottesman, Nancy Hopkins, Tom Merigan, Andy

Nahmias, Helen Revel, Hugh Robertson, Hans Storz, Milt Zaitlin
Charter and Bylaws Committee
Bernard Fields, Chairman, Bob Haselkorn, Dorothy Horstman, Jack Morris
Finance Committee
George Miller, Chairman, David Bishop, Fred Murphy, Fred Rapp, Ed Scolnick
>1982: By 2010, the Society had 3,543 members, 614 of whom were from 44 countries outside the USA, including 183 from Canada. Successful annual meetings have been
held every summer since 1982.

1981 Bill Joklik, Harry Ginsberg, others founding of the American Society for Virology
page 452

The development of the first recombinant vaccine for humans, hepatitis B surface antigen (HBsAg) expressed
in yeast, began with the discovery by Baruch Blumberg, Barbara Werner, Manfred Bayer, and Lawrence Loeb of
particles smaller than whole virions in HBsAg-positive blood. Experiments showed that these particles could
induce protective immunity. Blumberg and Irving Millman proposed that a vaccine could be made from HBsAg
particles obtained from the blood of hepatitis B carriers. A patent for this concept was filed in 1969. Maurice
Hilleman and colleagues at Merck recognized the importance of the concept and by 1971 they obtained a license to
develop a hepatitis B vaccine made from HBsAg purified from blood. In 1980, Wolf Szmuness and others showed
that the vaccine provided more than 90 percent protection against hepatitis B and had no adverse effects. In 1981,
the serum-derived vaccine was made available for general use. Production of the hepatitis B subunit vaccine in large
quantities was hampered by the need for the blood of hepatitis B carriers and the realization that such blood could
be contaminated with other viruses. In 1977, William Rutter, Pablo Valenzuela and their colleagues at the University
of California San Francisco obtained material containing hepatitis B virus from Merck. After cloning the hepatitis
B virus and obtaining the genetic sequence of HBsAg, Rutter and his colleagues explored a variety of different
biological systems in which to produce the particles using recombinant techniques. They were unsuccessful using
bacteria. Then, in 1980 and 1981, Rutter collaborated with Benjamin Hall and colleagues, of the University of
Washington, who had developed a model system using yeast cells. Rutter and Hall successfully produced pure
HBsAg particles from recombinant yeast cells. Rutter and colleagues then founded Chiron Corporation, in part
to develop the HBsAg vaccine through a contractual relationship with Merck and also to develop other medical
therapies using recombinant techniques. At Merck, Hilleman used the recombinant yeast-derived HBsAg to make
an improved version of a hepatitis B vaccine. This vaccine was the first recombinant vaccine licensed for use in
humans by the FDA this occurred in 1986. There is another side to this story, not covered here: there have been
many precedent-setting law suits involving the recombinant yeast technology and its ownership, and beyond this,
legal questions over the patenting of products, processes and concepts, each of which has fundamentally changed
the entire biotechnology industry.

William Rutter (center) and his former students,

Edward E. Penhoet of the University of California
Berkeley (left) and Pablo Valenzuela of the
University of California San Francisco (right).
They developed the first recombinant vaccine
for humans, hepatitis B surface antigen (HBsAg),
expressed in yeast. They then founded Chiron
Corporation, now part of Novartis International AG.

Edman JC, Hallewell RA, Valenzuela P, Goodman HM, Rutter WJ. Synthesis of
hepatitis B surface and core antigens in E. coli. Nature 1981;291:503-506.
Valenzuela P, Medina A, Rutter WJ, Ammerer G, Hall BD. Synthesis and assembly
of hepatitis B virus surface antigen particles in yeast. Nature 1982;298:347-350.
Hilleman MR. Yeast recombinant hepatitis B vaccine. Infection 1987;15:3-7.

HBsAg as vaccine substrate, produced in yeast

(Saccharomyces cerevisiae)
negative contrast electron microscopy

1981 William Rutter, Pablo Valenzuela, colleagues first recombinant human vaccine, Hepatitis B (HBsAg in yeast)

page 453

The term prion (pronounced pree-on) was

coined in 1982 by Stanley Prusiner; it is derived
from protein and infection. Tikvah Alper and her
colleagues in London had earlier suggested that
the scrapie agent of sheep, which had been studied
for many years, might lack nucleic acid, which
usually can be degraded by ultraviolet or ionizing
radiation. When extracts of scrapie-infected brains
were irradiated, the extracts retained their ability to
transmit the disease. Prusiner and his colleagues at
the University of California San Francisco purified
the scrapie infectious agent and showed that it
consisted solely of a specific protein prion protein
(PrP, a 35kD membrane glycoprotein). Normal prion
protein (PrPC) consists primarily of alpha helices
where the amino acid backbone twists into a specific
kind of spiral; the abnormal form (PrPSc, for scrapie),
however, contains beta sheets where the amino acid
backbone is fully extended. The prion diseases of
humans include: several types of Creutzfeldt-Jakob
disease (CJD); kuru, a now-extinct disease of New
Guinea natives; and several familial neurologic
diseases. The most common animal prion diseases
are: scrapie, an important disease of sheep; bovine
spongiform encephalopathy (BSE); chronic wasting
disease (CWD) of deer and elk; and others that
follow cross-species transmission from scrapie or
BSE. All of the prion diseases are invariably fatal,
always with a very long incubation period, always
with characteristic brain lesions (vacuoles in
neurons, progressing to spongiform degeneration
usually with associated with amyloid plaques. The
most amazing thing about the prion diseases is that
the infectious agents, the prions, are propagated
Stanley B. Prusiner
and transmitted solely by misfolded proteins
when the abnormal form of prion proteins (PrPSc)
Prusiner SB. Novel proteinaceous infectious particles cause scrapie. enter a healthy animal they induce a refolding of
Science 1982;216:136-144.
normal prion proteins (PrPC), which in turn act as a
template causing the misfolding of more and more
Committee on Transmissible Spongiform Encephalopathies.
Advancing prion science: guidance for the national prion research normal protein in a chain reaction. The abnormal
prion proteins are extremely stable and resistant
program. Washington: National Academies Press; 2003.
to normal proteolysis. They accumulate in neural
Colby DW, Prusiner SB. Prions. Cold Spring Harb Perspect Biol.
tissue and cause cellular damage and death by as yet
2011;3:a6833.
unknown means.

Bovine spongiform encephalopathy, bovine

brain, vacuoles in neurons, progressing to
florid spongiform degeneration and neuronal
degeneration. H&E.

Mode of production of PrPC in neurons and the

accumulation of PrPSc as scrapie-associated
fibrils and plaques

Molecular models of normal prion protein

(PrPC) and the abnormal prion protein (PrPSc)

1982 Stanley Prusiner concept of the prion the etiologic agents of the spongiform encephalopathies
page 454

Goad WB. GenBank - and

its promise for molecular
genetics. Los Alamos
Science 1983;9(Fall):52-61.
GenBank Overview: http://
www.ncbi.nlm.nih.gov/
genbank/

Walter Goad (1925-2000)

In 1979, 30 molecular biologists and computer scientists meeting at the Rockefeller University agreed on the necessity to create a national, computerized database. The impetus
was the explosive growth in the number of known DNA sequences and the promise of producing biological knowledge by analyzing and comparing them a database seemed
indispensable. It took almost three years for the NIH to come up with a funding scheme; Walter Goad and his colleagues were awarded a $2 million, 5-year grant in 1982 and
began building the Los Alamos National Laboratory (LANL) Sequence Library, which was re-christened GenBank, the Nucleic Acid Sequence Data Bank. Goad was a physicist
who had spent 1970-1971 with Francis Crick at the MRC Laboratory of Molecular Biology at Cambridge University; Crick lent strong support to the idea of an international
data resource. From Goads paper in Los Alamos Science in 1983: It was clear that just accumulating the data in a computer was not particularly interesting. What was
interesting were questions about organizing, managing, and analyzing the data. The first job we faced was collecting the sequence data. This was straightforward although
physically very demanding. We worked very hard just to get caught up with the data that was in existence when we started. Our estimate of what needed to be added was
based on scanning some journals and looking through lists of titles. Unfortunately, the estimate was low because the titles of many articles with sequences in them didnt reveal
much. Most of the sequences were on the order of a thousand bases long, but the longest the entire genome of the lambda bacteriophage contains about 50,000 bases.
My outlook was that mysticism about life was being crowded out by the greater joy of knowledge thanks to molecular genetics and molecular biology in general. There is,
after all an immense difference between speculating about the way things might work and knowing how they do work. Today, GenBank, which is managed by NIH and its
National Library of Medicine, in collaboration with the DNA Data Bank of Japan and the EMBL Nucleotide Sequence Database in the United Kingdom, supports some 40,000
users searches per day, accessing its 22 billion item library for many purposes, not the least of which concerns virology.

1982 Walter Goad, others development of GenBank

page 455

Stunting of growth (a) and death (b) of newborn mice infected with LCM
virus (strain Armstrong), and reversal of stunting by transplantation of cells
secreting growth hormone. (b) Without hormone replacement the mice die.
From Michael Oldstone and colleagues paper in Nature, 1984.
Oldstone MBA, Sinha YN, Blount P, Tishon A, Rodriguez M, von Wedel R, Lampert PW. Virus-induced alterations in
homeostasis: Alterations in differentiated functions of infected cells in vivo. Science 1982;218:1125-1127.
Rodriguez M, von Wedel RJ, Garrett RS, Lampert PW, Oldstone MBA. Pituitary dwarfism in mice persistently
infected with lymphocytic choriomeningitis virus. Lab Invest. 1983;49:48-53.

Michael B.A. Oldstone

Oldstone MBA, Rodriguez M, Daughaday WH, Lampert PW. Viral perturbation of endocrine function: Disorder of
cell function leading to disturbed homeostasis and disease. Nature 1984;307:278-280.
Oldstone MBA. Viral alteration of cell function. Scientific Amer. 1989;260:42-48.

Early in his career, Michael Oldstone and his colleagues showed that, contrary to established dogma, in persistent virus infections the host was not tolerant but made specific
antiviral immune responses usually ineffective in clearing infection but nevertheless affecting its course, outcome and pathogenesis. This immune response often caused
tissue damage and disease, the phenomenon of immunopathology. Discoveries in this area were made originally with lymphocytic choriomeningitis (LCM) virus and murine
retroviruses, but later were extended to measles virus and other infections including several in humans. Buildings on this work, they showed that certain antibodies to some
viruses recognized similar amino acid sequences or motifs found in host/cell proteins this cross-reactivity, referred to as molecular mimicry, has been shown to be important
in several viral diseases. They also explored the details of how viruses cause immunosuppression and abrogate normal host immunologic surveillance, again determining
the pathogenetic role of this phenomenon in viral persistence and chronic disease. Another mechanism discovered by Oldstone and his colleagues by which persistent virus
infection can produce disease involves infection effects upon the differentiation or functions of cells, thereby altering physiological homeostasis and normal functioning of key
organs and tissues. This was termed interference with cellular luxury functions, but many of the functions found to be affected are hardly luxuries. For example, they showed
that persistent noncytopathic LCM virus infection of the anterior lobe of the murine pituitary gland affects the production of growth hormone, causing growth retardation,
hypoglycemia and death. This finding of a noncytopathic virus causing endocrine dysfunction has led to exploration of the possible viral etiology of other endocrine,
neurotransmitter, cytokine and inflammatory diseases this subject, as applied to human diseases, especially neurologic diseases, is an important work-in-progress.

1982 Michael Oldstone, colleagues concept of virus damage to cellular homeostasis and luxury functions

page 456

Gerald N. Woode (1934-2013)

Marian C. Horzinek

Bovine torovirus
negative contrast electron microscopy

In 1979, Gerald Woode and his colleagues visualized an unusual virus in feces from a calf with acute enteritis its morphology was distinct from that of known viruses. The
calf was from a farm near Breda, Iowa (hence the name Breda virus now, bovine torovirus), where >50% of calves developed diarrhea during the first 20 days of life. In
following years, similar viruses were identified in calves from other states. After Woodes initial report, morphological and antigenic similarities of Breda virus and an equine
virus from Berne, Switzerland (Berne virus, now, equine torovirus), were noted. The latter virus was first described by Marianne Weiss, Franz Steck and Marian Horzinek
in 1983. Because of their similarities, the two viruses were grouped together as the toroviruses, in a provisional family, the Toroviridae. The term torovirus, suggested by
Horzinek, refers to the unique kidney-shape of the viral nucleocapsid and often the virion itself torus is Latin for the doughnut-shaped molding that some columns have in
their bases. Today, based also on common genomic organization and replication strategy, the toroviruses form a genus in the family Coronaviridae. The genus now includes
toroviruses of other animals, and in 1984 torovirus-like particles were detected in feces from children with gastroenteritis by Graham Beards and his colleagues. The human
virus(es) have since been found in many countries.
Woode GN, Reed DE, Runnels PL, Herrig MA, Hill TH. Studies with an unclassified virus isolated from diarrheic calves. Vet Microbiol. 1982;7:221-240.
Weiss M, Steck F, Horzinek MC. Purification and partial characterization of a new enveloped RNA virus (Berne virus). J Gen Virol. 1983;64:1849-1858.
Horzinek MC, Weiss M. Toroviridae: a taxonomic proposal. Zentralbl Veterinarmed B. 1984;31:649-659.
Beards GM, Green J, Hall C, Flewett TH, Lamouliatte F, du Pasquier P. An enveloped virus in stools of children and adults with gastroenteritis that resembles the Breda virus
of calves. Lancet 1984;1:1050-1052.

1982> Gerald Woode, Marian Horzinek, Graham Beards, others discovery of bovine, equine, human toroviruses

page 457

Hepatitis E virus, negative contrast electron microscopy

Mikhail Surenovich Balayan

The self-sacrifice of Russian virologist, Mikhail Balayan, bordered on the extreme. In 1983, Balayan was
investigating an outbreak of non-A, non-B hepatitis in a central Asian part of the Soviet Union. Though
he wanted to bring samples back to his Moscow laboratory (in the Institute of Poliomyelitis and Viral
Encephalitides, Russian Academy of Medical Sciences), he lacked refrigeration. So, he made a shake of
yogurt and an infected patients stool, drank it, went back to Moscow, and waited. When he became
seriously ill a few weeks later, he started collecting and analyzing his own stool samples. In these he found
a new virus that produced liver injury in laboratory animals and could be seen by electron microscopy.
It looked like hepatitis A virus, but he showed that it was not, because he already had antibodies against
hepatitis A virus and these did not react with the new virus. Subsequently, in 1990, Gregory Reyes and his
colleagues at Genelabs Technologies, Inc. cloned and sequenced the genome of the virus in collaboration
with Balayan and Daniel Bradley and his colleagues at the Centers for Disease Control and Prevention.
The virus was named hepatitis E virus. It was found in succeeding years to be the cause of major epidemics
of severe hepatitis with high mortality in pregnant women. Further, it was found to be zoonotic with
reservoirs in swine and other animals.
Balayan S, Andjaparidze AG, Savinskaya SS, Ketiladze ES, Braginsky DM, Savinov AP, Poleschuk
VF. Evidence for a virus in non-A, non-B hepatitis transmitted via the fecal-oral route. lntervirology
1983;20:23-31.

1983 Mikhail Balayan discovery of hepatitis E virus

page 458

Francoise Barr-Sinoussi

Luc Montagnier

Jean-Claude Chermann

Much has been written about the discovery of human immunodeficiency virus 1 (HIV1); the full story cannot be repeated here, but the discovery of the virus itself at the
Institut Pasteur and how this was made public can be. After AIDS was first clinically recognized in Los Angeles and New York, several research groups sought its etiology, most
without success. Clinicians in Paris asked for etiologic research to be done and in 1982 Jean-Claude Chermann and Franoise Barr-Sinoussi working with Luc Montagnier
and other colleagues responded. Tissues from patients were cultured and then searched for viruses, including retroviruses [using a reverse transcriptase (RT) assay]. One RT
assay turned positive after three weeks of culture, but this was associated with the death of the cells, not characteristic of the retroviruses of the day. The virus was named
lymphadenopathy-associated virus (LAV); it was studied by electron microscopy and seen to be a lentivirus (now a genus in the family Retroviridae). At first it was not known
if the virus was the cause of AIDS; nevertheless, in 1983 its description was published in Science. For some time it was said that the team led by Montagnier had discovered
the virus, but the team led by Robert Gallo of NIH had proven that the virus was the cause of AIDS. Later, it came out that a highly selected set of human sera from AIDS
patients together with controls, put together by Donald Francis and his colleagues at the U.S. Centers for Disease Control and Prevention (CDC), had been tested in 1983 by
Montagnier and his colleagues and found to strongly support an etiologic association between the virus and the disease. Sets of sera from lymphadenopathy patients in France
reacted similarly. Early in 1984, Chermann was invited to speak at a retrovirus conference in Park City, Utah Chermann presented serologic data based on antigen produced
from LAV virus-infected human leukocytes and sera from AIDS patients and controls. After a few minutes, the room went silent. For the first time it was realized that the virus
had been found. Afterward, the audience cheered and also welcomed the news that a blood-test would soon be available. Two day later, Chermann presented the same data
at CDC in Atlanta the largest auditorium was jammed, and again silence was followed by cheers. The name HIV1 was chosen in 1986 by third parties to end the dispute
between the French teams name, LAV, and American teams name, HTLV3. Patent rights disputes and discovery primacy issues went on far into the 1990s.
Barr-Sinoussi F, Chermann J-C, Rey F, Nugeyre MT, Chamaret S, Gruest J, Dauguet C, Axler-Blin C, Brun-Vezinet F, Rouzioux C, Rozenbaum W, Montagnier L. Isolation of a
T-lymphotropic retrovirus from a patient at risk for acquired immunodeficiency syndrome (AIDS). Science 1983;220:868-871.
Brun-Vezinet F, Barr-Sinoussi F, Saimot AG, Christol D, Montagnier L, Rouzioux C, Klatzmann D, Rozenbaum W, Gluckman JC, Chermann J-C. Detection of IgG antibodies
to lymphadenopathy-associated virus in patients with AIDS or lymphadenopathy syndrome. Lancet 1984;1:1253-1256.

1983 Francoise Barr-Sinoussi, Luc Montagnier, Jean-Claude Chermann human immunodeficiency virus 1 (HIV1)

page 459

Marvin Carruthers

Andre F. Marion and Sam H. Eletr

ABI 380A, 1983

ABI 394 (4-columns), 1986
ABI 3948, 1998
Applied Biosystems, Inc., Oligonucleotide (DNA) Synthesizers

In 1955, the first chemical synthesis of DNA was

accomplished by A.M. Michelson and Alexander Todd.
Subsequently, this contribution was recognized by a Nobel
Prize to Todd. Next, Har Gobind Khorana and his colleagues
showed how a DNA sequence could be assembled via
chemical means, now known as the phosphodiester method.
In 1976, Khorana with his 19 co-workers reported on the
synthesis of a 126-residue long DNA. This project took
8 years; today the same product can be made in an hour
using an automated DNA synthesizer. In the mid-1970s,
the first solid-phase synthesis of DNA was performed in
the laboratories of Hubert Kster, Michael Gait and Keiichi
Itakura. Solid-phase synthesis is the dominant method used
today. The specific chemistry used today, employing amidite
chemistry, was developed in 1981 by Marvin Caruthers and
Mark Matteucci. DNA strands are formed by the sequential
addition of activated nucleotides to an immobilized starter
DNA molecule. The product remains attached to an
insoluble support until the final release step. This solidphase approach was key to the development of automated
oligonucleotide synthesis machines. In 1981, two engineers
from Hewlett Packard, Andre Marion and Sam Eletr,
commenced operations of Applied Biosystems Inc. (ABI);
their first instrument was a peptide synthesizer, but by 1983
their second instrument, the Model 380A DNA Synthesizer,
entered production and was an instant success. It was a
crucial factor in the advance of technologies requiring
DNA probes and primers, the latter especially for use in
polymerase chain reaction (PCR) technology, which in turn
was crucial for large DNA sequencing projects. By 1986, ABI
marketed its 394 DNA Synthesizer, which could make four
different oligonucleotides simultaneously. Advances were
made continuously, such that today instruments, such as
the ABI 3948 with 96 parallel reaction capillaries, can run
continuously in automated fashion yielding the very large
numbers of oligonucleotides that are needed to support
huge genome sequencing programs, such as the Human
Genome Project. Several other companies make competing
machines.
Matteucci MD, Caruthers MH. Studies on nucleotide
chemistry IV. Synthesis of deoxyoligonucleotides on a
polymer support. J Amer Chem Soc. 1981;103; 3185-3191.
Caruthers MH. Gene synthesis machines: the DNA
chemistry and its uses. Science 1985;230:281-285.

1983 Marvin Carruthers, others, Applied Biosystems, Inc. development of commercial DNA synthesis technology
page 460

Andrew W. Murray

Jack W. Szostack

Construction of a yeast artificial chromosome (YAC)

Yeast (Saccharomyces cerevisiae) artificial chromosomes (YACs) are vectors used to clone DNA fragments larger than 100 kb, up
to a maximum size of 1Mb to 3Mb. YACs are useful for the physical mapping of complex genomes and for the cloning of large
genes. First described in 1983 by Andrew Murray and Jack Szostak, YACs are artificially constructed so that they are rather like
chromosomes they contain (1) telomeres, located at each chromosome end to protect the DNA from degradation by nucleases;
(2) a centromere, the attachment site for mitotic spindle fibers, required to duplicate the chromosome when yeast cells divide;
(3) replication origin sequences needed for chromosome replication in yeast cells; and (4) other specific sequences such as
selectable markers that allow the easy isolation of yeast cells that have taken up the artificial chromosome and recognition sites
for restriction enzymes. YACs are built using an initial circular plasmid, which is typically broken into two linear molecules using
restriction enzymes; DNA ligase is then used to ligate a sequence or gene of interest between the two linear molecules, forming
a single large linear piece of DNA. In some instances, this DNA is re-circularized. YACs and other yeast plasmid vectors have
another advantage in that they can be used to express eukaryotic proteins that require posttranslational modification.
Murray AW, Szostak JW. Construction of artificial chromosomes in yeast. Nature 1983;305:189-193.
The Nobel Prize in Physiology or Medicine of 2009 was awarded to Elizabeth H. Blackburn, Carol W. Greider and Jack W.
Szostak for the discovery of how chromosomes are protected by telomeres and the enzyme telomerase.

1983 Andrew Murray, Jack Szostack yeast artificial chromosomes (YACs) vectors to clone large DNA fragments

page 461

Some Vaccinia Virus Recombinant Vectored Virus Vaccine Candidates

Pathogen

Experimental Animal
Species Protected

Rabies virus

Raccoons, foxes, dogs

G, N

Hepatitis B virus

Chimpanzees

HBsAg

Measles virus

Herpes simplex virus

Murine cytomegalovirus
Epstein-Barr virus

Human influenza viruses

Dengue viruses

Yellow fever virus

Japanese encephalitis virus

Respiratory syncytial virus
Human papillomaviruses

Mice, rats, dogs

Mice, guinea pigs

Mice

Cottontop tamarins

Hamsters, ferrets, mice

Mice
Mice

Mice, swine

HA, F, N
GB, GD

NS pp89
G 340

HA, NA

PreM, E, NS1, NS2

PreM, E, NS1, NS2ab

PreM, E, NS1

Cotton rats, chimpanzees M2 ,G

Mice, rats

E6, E7 oncoproteins

Lassa virus

Guniea pigs

G, N

Rinderpest virus

Rabbits, cattle

Human parainfluenza viruses Monkeys

Vesicular stomatitis viruses

Mice, cattle

Peste d petits ruminants virus Goats

Equine herpesviruses

Hamsters

Equine influenza viruses

Horses

Pesudorabies virus

Bovine leukemia virus

Bovine papillomavirus

Bernard Moss

Antigen

Classical swine fever virus

Mice, swine
Sheep
Rats

Swine

Venezuelan encephalitis virus Mice, monkey, horses

Sendai virus

Mice

Friend leukemia virus

Mice

Avian Influenza viruses

HIV 1 and 2

Chickens
Macaques, chimpanzees

HA, F, N
G, N

HA, F
HA, F

G13, G14

G50, GII, GIII

HA

E, G51

Early proteins
G55, G33
E1, E2, N

HA, F, N, M
HA

Gag

G, gag, pol, protease

Basic scheme for vaccinia vectored vaccines

Despite having first been developed in 1983 by Bernard Moss of NIH, Enzo Paoletti of the New York State Department of Health and their colleagues, there are no viral
vectored vaccines currently on the market for use in humans. However, several are in various stages of development and approval for use. One impediment has been the
conservatism of the regulatory authorities in several countries, especially the FDA in the U.S. Recombinant vaccinia viruses, that is vaccinia vectored vaccine viruses, have been
shown to protect animals against diseases of veterinary importance, including vesicular stomatitis virus and rinderpest in cattle, pseudorabies virus in swine, and influenza
viruses in chickens. A recombinant vaccinia virus expressing rabies virus glycoprotein has been successfully administered in bait form as a wildlife vaccine in both the United
States and Europe. Other poxviruses have also been tested as vectors for veterinary applications. Examples include a raccoon poxvirus vector to protect raccoons against rabies
virus; a capripoxvirus vector for cattle against rinderpest virus; a swinepox virus vector for pigs against pseudorabies virus; a fowlpox vector for chickens against influenza
viruses, Newcastle disease virus, and infectious bursal disease virus; and canarypox virus for cats against feline leukemia virus.
Smith GL, Mackett M, Moss B. Infectious vaccinia virus recombinants that express hepatitis B virus surface antigen. Nature 1983;302:490-495.
Paoletti E, Lipinskas BR, Samsonoff C, Mercer C, Panicali D. Construction of live vaccines using genetically engineered poxviruses: biological activity of vaccinia virus
recombinants expressing the hepatitis B virus surface antigen and the herpes simplex virus glycoprotein D. Proc Natl Acad Sci USA. 1984;81:193-197.
Quinnan GV Jr. Vaccinia viruses as vectors for vaccine antigens. Proceedings of a workshop, 1984, Chevy Chase, Maryland. New York: Elsevier North Holland; 1985.
Draper SJ and Heeney JL. Viruses as vaccine vectors for infectious diseases and cancer. Nature Reviews Microbiology 2010;8:62-73.

1983 Bernard Moss, Enzo Paoletti, others recombinant vaccinia virus as vector for viral vaccines (hepatitis B virus)

page 462

Jacques Dubochet

Freiberg AN, Sherman MB, Morais MC, Holbrook MR, Watowich

SJ. Three-dimensional organization of Rift Valley fever virus
revealed by cryoelectron tomography. J Virol. 2008;82:10341-10348.

Marc Adrian (1945-2013)

Rift Valley fever virus, cryo-EM,

image reconstruction

In 1984, Marc Adrian, Jacques Dubochet, Jean

Lepault and Alasdair McDowall published the
first paper on cryo-electron microscopy, taking
advantage of the then surprising discovery
that liquid water can be cooled into a vitreous
(glasslike) solid, without crystallization. The
critical factor for successful vitrification
proved to be very small specimens that
could be plunged instantly into liquid ethane
(-188C), thereby avoiding crystallization. The
technology included specially adapted cryoelectron microscopes and computer image
reconstruction programs. Even in the first
papers, images of virions were prominent, and
soon the technology was adapted for cryoultramicrotomy, cryo-electron tomography,
cryo-negative staining and other methods.
Virion structure studies progressed to reveal
the intricacies of viral attachment to host cells,
entry, replication and exit. Such studies have
done as much as the original negative contrast
electron microscopic method and the rest of
the molecular biologic revolution to reveal the
viruses and viral infection processes as more
and more complex. The images underpinning
current understanding of this are incredibly
diverse and truly spectacular .

Adrian M, Dubochet J, Lepault J, McDowall

AW. Cryo-electron microscopy of viruses.
Nature 1984;308:32-36.

Adenovirus, cryo-EM

Dubochet J, Adrian M, Chang JJ, Homo JC,

Lepault J, McDowall AW, Schultz P. Cryoelectron microscopy of vitrified specimens. Q
Rev Biophys. 1988;21:129-228.

Schematic diagram of the image reconstruction process from digitization of the micrograph to the
dissemination of the structural results. Though some steps such as the boxing out of individual particle images
lend themselves to automation, many steps including the determination of particle origins and orientations
must be repeated and involve some trial-and-error decision making (e.g., to determine which data should or
should not be included). The scheme depicted here, which uses both cross-common lines and model-based
approaches to determine and refine particle origin and orientation parameters, is but one of many suitable
schemes.
Baker TS, Olson NH, Fuller SD. Adding the Third Dimension to Virus Life Cycles: Three-Dimensional
Reconstruction of Icosahedral Viruses from Cryo-Electron Micrographs. Microbiol Mol Biol Rev.
1999;63:862922.

1984 Marc Adrian, Jacques Dubochet, others development of cryo-electron microscopic analysis of virus structure

page 463

The concept underlying the polymerase chain reaction (PCR), which allows
the nearly limitless amplification of specific DNA sequences, was described
by Kjell Kleppe and Har Gobind Khorana in 1971. The major improvements
made by Kary Mullis allowed PCR to become a (perhaps the) central technique
in molecular biology, such that it was described by The New York Times as
virtually dividing biology into two epochs, before PCR and after. In 1983,
Mullis was working for the Cetus Corporation when he had the idea to use a
pair of primers to bracket a desired DNA sequence and to copy the sequence
over and over using DNA polymerase Mullis spent more than a year before
making his idea work, first in late 1983. Other Cetus scientists (Randall Saiki
and Henry Erlich) worked on developing AIDS and other tests utilizing PCR
Saiki was first author on the first paper (1983), while Mullis took longer to finish
the paper that described PCR itself (1984). A major shortcoming of the first PCR
was that the DNA polymerase was destroyed by the high heat used and had to
be replaced in each cycle. In 1986, Mullis started to use Thermophilus aquaticus
DNA polymerase (Taq polymerase) the enzyme from this bacterium, which
lives happily in Yellowstone hot springs, is heat resistant and needs to be
added only once, thus making the technique more affordable and amenable to
automation. In 1991, Cetus was sold to Chiron Corporation, its value largely
based upon its patent rights to PCR. PCR patent rights were then sold for $300
million to Hoffman-La Roche and its subsidiary, Roche Molecular Systems.

Kary B. Mullis and Sydney Brenner

at the Nobel Prize Celebration, Stockholm, 1993

Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N.
Enzymatic amplification of beta-globin genomic sequences and restriction site
analysis for diagnosis of sickle cell anemia. Science. 1985;20:230:1350-1354.
Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H. Specific enzymatic
amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb
Symp Quant Biol. 1986;51 Pt 1:263-273.

Scheme for the first three of usually 20-30 PCR cycles

1985 Kary Mullis, colleagues, Cetus, Perkin-Elmer invention of the polymerase chain reaction (PCR)
page 464

Kary
Mullis
first
thermocycler

Julio Isidro Maiztegui (1931-1993)

Julio Guido Barrera-Oro (1927-2013)

Candid #1 vaccine for Argentine hemorrhagic fever was developed

in 1985 by Julio Barrera Oro and Julio Maiztegui, in collaboration
with Gerald Eddy and George French of USAMRIID. The vaccine
was shown to be very effective in preventing disease in high risk farm
workers in the endemic region of Argentina. The vaccine was first
manufactured by the Salk Institute in the U.S. and became available in
Argentina in 1990. In 2006, the Instituto Nacional de Enfermedades
Virales Humanas Dr. Julio I. Maiztegui started production of the
vaccine in Argentina, with a goal of producing the 5 million doses to
vaccinate the entire population of the endemic area.
Sculpture, Instituto
Nacional de
Junin Virus Vaccine Development Team
Enfermedades
L-R: Francisco Pinheiro, C.J. Peters,
Virales Humanas
Gerald Eddy, Neal Halsey, Nick Tauraso,
Julio Maiztegui,
Julio Barrera Oro, George French, Julio
Pergamino, Argentina
Maiztegui, Alexis Shelokov, Kelly McKee

George French

1985 Julio Maiztegui, Julio Barrera-Oro, George French development of Argentine hemorrhagic fever vaccine

page 465

Abstracted from various reviews of Fields Virology over the years:

...Fields Virology was conceived by Bernard Fields, a distinguished
Harvard virologist of the 1980s and 1990s, as a means of bringing
fundamental and medical aspects of virology together in a more
integrated manner than previously available in contemporary
textbooks of virology. The project fulfilled the need and,
notwithstanding the early death of Bernard Fields, has progressed to
become one of the classic resources of virology.
Samuel Johnsons Dictionary defines Bible as the sacred volume
in which are contained the revelations of God. A current dictionary
has provided me with an additional definition: any book, reference
work, periodical, etc. accepted as authoritative, informative, or
reliable. When our virology graduate students were asked about their
use of Fields Virology, they said it was their virology Bible.
The Editors, many of them previous co-editors of Bernard Fields,
have taken particular care in entrusting the individual chapters to
internationally recognized experts in their fields; many of them have
contributed to previous editions, others have been recently recruited.
This is the most comprehensive book in virology, tying together
detailed accounts of the molecular biology, molecular pathology and
medical features of individual viruses in the broad context of general
virology. Not in vain is this opus regarded as the bible of virology
apart from all its other values, it is great fun to study.

David M. Knipe

Editors-in-Chief over the years: Bernard N. Fields,

David M. Knipe, Peter M. Howley

Bernard Nathan Fields (1938-1995)

Associate Editors over the years: Robert M. Chanock, Joseph

L. Melnick, Bernard Roizman, Robert E. Shope, Thomas P.
Monath, Stephen E. Strauss, Diane E. Griffin, Robert A. Lamb,
Malcolm A. Martin, Jeffrey I. Cohen and Vincent R. Racaniello

Peter M. Howley
Fields Virology:
1st ed. 1985
2nd ed. 1990
3rd ed. 1996
4th ed. 2001
5th ed. 2007
6th ed. 2013

1985 Bernard Fields, others publication of Fields Virology, now six editions

page 466

Acquired immunodeficiency syndrome (AIDS) first

appeared in 1981 and was first seen in hemophiliacs
and transfusion recipients in 1982. Subsequent studies
established the transmissibility of the causative agent
(HIV) by various routes, including blood transfusions,
untreated coagulation factor concentrates and
blood components needed by hemophiliacs. Once
transmissibility was established, some preventive
measures were put into place, although for some years
important aspects of the natural history of HIV infection
remained unknown. The first preventive measures
were to protect the blood supply. In 1983, a detailed
blood donor history questionnaire with deferral of
high-risk blood donors was implemented. Antibody
testing for HIV1 began in 1985, first for use in blood
banks, then for clinical diagnostics and epidemiologic
studies. The three pioneering firms that developed the
first commercially available enzyme immunoassays
(EIAs) were Abbott Laboratories, bioMerieux, Inc.
and Bio-Rad Genetic Systems. At first the antigen
used in these EIAs was a crude cell culture extract; in
recent years, synthetic peptides derived from highly
conserved, immunodominant regions of the HIV env
(envelope) and pol (polymerase) gene products have
been used. For clinical purposes, usually, one screening
test (often run more than once if the first test is positive
or indeterminate) have been employed, followed by a
confirmatory test where needed. EIA tests have been the
most widely used for screening, with the confirmatory
test now usually utilizing a western blot assay. The EIA
HIV tests have undergone many iterations that have
improved their sensitivity, specificity and reproducibility.
Additional methodologies have reduced the diagnostic
window, that is the period after infection but before
the appearance of detectable antibodies. PCR testing
for viral RNA or proviral DNA is now widely used
to confirm EIA tests its use has also narrowed the
diagnostic window considerably. Modern HIV serologic
and nucleic acid testing is extremely accurate. The
chance of a false-positive result in multi-step testing
protocols is estimated to be 0.0004% to 0.0007% in the
general U.S. population.

1985 bioMrieux, Bio-Rad Genetic Systems, Abbott development of first FDA approved HIV antibody tests (EIAs)

page 467

Norman L. Letvin (1949-2012)

Ronald C. Desrosiers

Phylogeny of the many SIVs,

each associated with a particular nonhuman primate species

Shortly after AIDS was recognized in humans a similar syndrome was observed in macaques housed at the New England and California National Primate Research Centers
(NENPRC and CNPRC). Although initially called simian AIDS, the syndrome was found in most instances to be caused by a type D retrovirus (now called simian retrovirus
1 (SRV1), related to Mason Pfizer monkey virus. Only after human AIDS emerged were transmission studies done; it was in these studies, done in 1983, that the first simian
immunodeficiency virus (SIV) was discovered at the NENPRC; this was the macaque SIV virus (SIVmac). Experimentally, this virus caused an AIDS-like disease that was fatal
in a rather short time (6 months to 2 years). SIVmac was shown to be closely related to HIV1 and virtually identical to HIV2 [which was even more closely related to the SIV
isolated from sooty mangabeys (SIVsmm)]. Many diverse SIV variants were then isolated from many species of seropositive, captive and wild African nonhuman primates,
including chimpanzees (SIVcpz, the variant most closely related to HIV1). Unlike HIV1 and HIV2 infections in humans, most SIV infections in their natural hosts appear to be
non-pathogenic, despite high levels of circulating virus the exceptions are important: SIVsmm infection in rhesus macaques causes simian AIDS (SAIDS) and SIVcpz causes
in chimpanzees an AIDS-like disease similar to HIV1 infection in humans. Indeed, further genomic/phylogenetic studies strongly suggest that HIV1 first infected humans via
zoonotic transmission of a SIVcpz-like virus and HIV2 of a SIVsmm-like virus. The presence of many of the variant SIVs in several of the National Primate Research Centers
was later traced back to animal transport in the 1960s and 1970s.
Daniel MD, Letvin NL, King NW, Kannagi M, Sehgal PK, Hunt RD, Kank PJ, Essex M, Desrosiers RC. Isolation of T-cell tropic HTLV-III-like retrovirus from macaques.
Science 1985;228:1201-1203.
Letvin NL, Daniel MD, Sehgal PK, Desrosiers RC, Hunt RD, Waldron LM, Mackey JJ, Schmidt DK, Chalifoux LV, King NW. Induction of AIDS-like disease in macaque
monkeys with T-cell tropic retrovirus STLV-III. Science 1985;230:71-73.
Gardner MB. Simian AIDS: an historical perspective. J Med Primatol. 2003; 32:180-186.

1985 Norman Letvin, Ronald Desrosiers, colleagues discovery of simian immunodeficiency viruses (SIVs)

page 468

Francis Barin

Max Essex

Phyllis Jean Kanki

Ronald Hunt

Although HIV1 and HIV2 are closely related, they maintain distinct epidemiologic and biologic
characteristics. HIV2 is largely confined to West Africa, while HIV1 infection is prevalent worldwide
and accounts for approximately 95% of all HIV infections globally. Importantly, disease progression to
AIDS occurs much more slowly in HIV2 in one way, HIV2 infection appears like an attenuated HIV
infection. The discovery of HIV2 in Senegal in the mid-1980s prompted numerous studies to determine
its geographic distribution and biologic significance. Through the use of type-specific serologic assays
it was found that HIV2 is present in most West African countries, but infection with HIV1 is more
prevalent than infection with HIV2, ranging from a 3- to 24-fold ratio (HIV1 versus HIV2). In fact, from
more recent serosurvey data it seems that HIV2 prevalence rates are diminishing and that HIV1 may
competitively displace HIV2 in the long term. Outside West Africa, sporadic cases of HIV2 infection
have been found, mostly related to human movement, with particular foci stemming from former
Portuguese colonies. It is now recognized that HIV2 and the SIV variant (SIVsmm) from the sooty
mangabey are so closely related that the human virus must have originated from zoonotic transmission
from the nonhuman primates of a SIVsmm-like virus to humans. It has been estimated that HIV1 and
HIV2/SIV viruses may have diverged from each other as recently as 50-60 years ago.
Barin F, MBoup S, Denis F, Kanki P, Allan JS, Lee TH, Essex M. Serological evidence for virus related to
simian T-lymphotropic retrovirus III in residents of west Africa. Lancet 1985;2:1387-1389.
Clavel F, Gutard D, Brun-Vzinet F, Chamaret S, Rey MA, Santos-Ferreira MO, Laurent AG, Dauguet
C, Katlama C, Rouzioux C, Klatzmann D, Champalimaud JL, Montagnier L. Isolation of a new human
retrovirus from West African patients with AIDS. Science 1986;233:343-346.

Phylogeny of HIV2 and related viruses

1985 Francis Barin, Phyllis Kanki, Max Essex, colleagues discovery of human immunodeficiency virus 2 (HIV2)

page 469

Alec J. Jeffreys

Bernard Roizman

HSV genomic DNAs digested with

SalI and electrophoresed in
agarose gel

DNA fingerprinting has had more than one definition seemingly, the first was the method of analyzing isolates of herpes simplex virus (HSV), by digestion of their DNA
with restriction endonucleases and visualization of fragments by gel electrophoresis, which originated in the laboratory of Bernard Roizman at the University of Chicago.
In 1978, he and his colleagues discriminated between HSV isolates in clusters of severe hospital infections, showing in one instance that there were two independent
introductions of HSV1 into a pediatric intensive care unit resulting in two clusters of epidemiologically related, but virologically independent chains of infection. The method
was subsequently used for distinguishing variants of other large DNA viruses and double-stranded RNA viruses (e.g., rotaviruses); the methodology was employed widely until
being replaced by various partial and complete viral genome sequencing methods. However, DNA fingerprinting is otherwise associated with Alec Jeffreys and his discovery in
1984 of a means of distinguishing the DNA of individuals. He had a eureka moment after looking at the X-ray film image of isotopically-labeled restriction enzyme fragments
of the DNA of different members of his technicians family. He realized that DNA fingerprinting could be used in forensics to identify individuals and also to resolve paternity
and immigration disputes. DNA fingerprinting was first used to identify the rapist and killer of two teenagers in 1983 and 1986. In 1985, DNA profiling, based on typing
individual highly variable minisatellites [also known as short tandem repeats (STRs)] in the human genome, was also developed by Jeffreys and his team by focusing on just
a few of these highly variable minisatellites, DNA profiling became sensitive, reproducible, and amenable to computer databasing; it has become the standard forensic DNA
system used worldwide. Jeffreys and his team also were also among the first to describe restriction fragment length polymorphism (RFLP) technology and the use of single
nucleotide polymorphisms (SNPs), which have had great value in viral pathogenesis research.
Linnemann CC, Light IJ, Buchman TG, Ballard JL, Roizman B. Transmission of herpes-simplex virus type 1 in a nursery for the newborn identification of viral isolates by DNA
fingerprinting. Lancet 1978;1:964-966.
Halperin SA, Hendley JO, Nosal C, B. DNA fingerprinting in investigation of apparent nosocomial acquisition of neonatal herpes simplex. J Pediatr. 1980;97:91-93.
Jeffreys AJ, Wilson V and Thein SL. Hypervariable minisatellite regions in human DNA. Nature 1985;314: 67-73.
Jeffreys AJ, Wilson V, Thein SL. Individual-specific fingerprints of human DNA. Nature 1985 316:76-79.

1985 Alec Jeffreys development of DNA fingerprinting (and detection of single nucleotide polymorphisms SNPs)

page 470

The first person to recognize that the sheep disease, scrapie, may have infected cows and caused bovine
spongiform encephalopathy (BSE) was Carol Richardson, a pathologist at the MAFF (Ministry of Agriculture,
Fisheries & Food, now DEFRA, the Department for Environment, Food and Rural Affairs) Central Veterinary
Laboratory (CVL) in the UK. In December 1984, a dairy cow was identified that had been behaving strangely,
showing tremors, and not responding to treatment. Soon other cows were identified with the same clinical signs;
tissues from the 10th cow were submitted to the CVL, where in September 1985 brain specimens were examined
by Richardson. She observed vacuolation in the neuriopil, similar to those she had seen many times before in
sheep affected with scrapie. Her colleagues agreed that this looked like a case of bovine scrapie, something
never seen before. But, the significance of the moment was lost it was only when more cases were referred to
the laboratory that it was realized that this was something new and significant. The first official MAFF record of
BSE in the UK was a memo from June 1987, although a confidential memo on the subject signed by the head of
the pathology department of the CVL circulated in MAFF in December 1986. It took until October 1987 before
Richardsons colleagues published the first description of the clinical syndrome and pathology of BSE in The
Veterinary Record. The epidemic surged even as control activities were initiated, reaching a peak in 1993. In a
2004 study, Roy Anderson and his epidemiology research group, first at Oxford University and then at Imperial
College London, estimated that 1.9 million cattle had been infected, 1.6 million of which had entered the food
chain. The overall cost of the epidemic to the British economy has been estimated at more than 4 billion.

Carol Richardson

Wells GA, Scott AC, Johnson CT, Gunning RF, Hancock RD, Jeffrey M, Dawson M, Bradley R. A novel
progressive spongiform encephalopathy in cattle. Vet Rec. 1987;121:419-420.
Left: MAFF Official data, BSE incidence,
UK, 1986 2009 (monthly plot)
(total of 181,123 confirmed cases on 36,191
farms, as of 2011) MAFF has always only
counted cases confirmed by histologic /
immuno-histochemical methods.
Right: The Economist, Aug 1996
[from: Anderson RM, Donnelly CA,
Ferguson NM, Woolhouse ME, et al.
Nature 1996;29;382:779-788.], showing that
1.9M cattle had been infected, not 181,000.

Carol Richardsons pathology case card, MAFF Central

Veterinary Laboratory, Weybridge, 19 September 1985

MAFF, Official first record of BSE in the UK,

MAFF, confidential memo of BSE in the UK,
17 June 1987 [but, November 1986 is the date given by the 19 December 1986, six months before public
UK government when BSE was first identified in UK]
announcement: advise keeping an open mind

1985 Carol Richardson discovery of bovine spongiform encephalopathy in the United Kingdom

page 471

Saul Kits thymidine kinase deletion mutant pseudorabies vaccine

The UL23 gene-encodes thymidine kinase, which phosphorylates

deoxythymidine into deoxythymidine-triphosphate. This enzyme is not
essential for virus growth in cell culture but is important for the replication
of the virus in vivo in differentiated cells

Saul Kit (1928-2008)

Herpesvirus infection, brain,

thin section electron microscopy

photo courtesy of Baylor College of Medicine Archives

Abstracted from the New Scientist and the New York Times, 1986: The first field test of a genetically altered virus vaccine took place in 1985 in the U.S. without the knowledge
of the public or a review by scientists appointed to approve such experiments. The Animal and Plant Health Inspection Service (APHIS), a division of the U.S. Department of
Agriculture, was accused of sidetracking the federal regulatory system in order to get a genetically altered virus vaccine onto the market quickly. When the permit for the first
release was suspended the controversy threatened to paralyze industrial biotechnology. The vaccine (Omnivac; TechAmerica, Omaha, Nebraska), for pseudorabies in swine,
was constructed by Saul Kit of Baylor College of Medicine as a thymidine kinase deletion mutant. The mutant virus replicates well in cell culture, but not in differentiated cells
in vivo. APHIS allowed the company to test the vaccine (which previously had only been tested in laboratory animals) in three states, but many scientists within the agency
were not aware of this, nor that the vaccine was approved for sale. The departments Agricultural Recombinant-DNA Research Committee, which was set up in 1976 to assess
proposals for biotechnology in agriculture, did not review the proposal prior to its release. The APHIS chief veterinarian stated that the proposal was mentioned informally
to the expert panel and there was no concern raised. He said that because only a single gene had been deleted, the virus was not considered worthy of special treatment. He
also argued that vaccination does not constitute release into the environment. Clearly, regulatory approval of novel vaccines, for animal or human use, has come a long way
since this episode. Even then, activist groups opposed to genetic engineering immediately filed law suits. In the end, years later, the vaccine was re-licensed.
Kit S, Kit M, Pirtle EC. Attenuated properties of thymidine kinase negative deletion mutants of pseudorabies virus. Am J Vet Res. 1985;46:1359-1367.

1985 Saul Kit, colleagues development of the first recombinant live-virus vaccine (pseudorabies)

page 472

The Development of Modern Viral Vaccinology Based Upon Innovative Technologies

Date

People

Virus

Technology

Attenuated Live-Virus Vaccines

1789

E Jenner

Variola virus

Vaccines produced from naturally occurring attenuated viruses

1935

M Theiler

Yellow fever virus

Vaccines produced by serial passage of viruses in heterologous host animal or embryonating eggs

1959

A Sabin, others

Polioviruses

Vaccines produced by serial passage of viruses in cultured cells and animals

1970s

H Maassab, others

Influenza viruses

Vaccines produced by selection of cold-adapted mutants and reassortant viruses

Non-Replicating Native Antigen Vaccines

1885

L Pasteur

Rabies virus

Vaccines produced from inactivated whole virions

1980s

G Laver, R Webster, others

Influenza viruses

Vaccines produced from purified native viral proteins

1981

M Hilleman

Hepatitis B virus

Vaccines produced from native viral subunits

Vaccines Produced by Recombinant-DNA and Other Innovative Technologies

1981

W Rutter, P Valenzuela,
M Hilleman, others

Hepatitis B virus

Vaccines produced by recombinant-DNA technology, expression in eukaryotic (yeast, mammalian, insect, plant) cells

1982

B Moss, E Paoletti

Vaccinia virus

Vaccines produced by recombinant-DNA technology, by the expression of viral antigens in viral vectors
(e.g., poxvirus vectors, rhabdovirus vectors, adenovirus vectors, picornavirus vectors, herpesvirus vectors)

1985

S Kit

Pseudorabies virus

Vaccines produced by recombinant-DNA technology, by the deletion of a gene(s) associated with pathogenicity

1990s

D Markowitz, I Verma,
A Stewart, M Hitt, others

Several viruses

Vaccines produced by recombinant-DNA technology, by the expression of viral antigens in defective viral vectors
(e.g., adenovirus vectors, AAV vectors, alphavirus vectors, flavivirus vectors, herpesvirus vectors)

1990s

A Charbit, M Hofnung,
R Curtiss, D OCallaghan

Several viruses

Vaccines produced by recombinant-DNA technology, by the expression of viral antigens in bacterial vectors

1990s

R Kennedy, H Kunkel, others

Hepatitis B virus

Vaccines produced by recombinant-DNA technology, by anti-idiotypic antibodies

1993

J Ulmer, others

Influenza viruses

Vaccines produced by recombinant-DNA technology, by the injection of viral DNA or plasmid DNA or replicons
containing viral gene(s)

2002

I Frazer, others

Human
papillomaviruses

Vaccines produced by recombinant-DNA technology, by viral proteins self-assembling in to virus-like particles (e.g.,
HBV, HPV, parvovirus B19, BTV) or pseudoparticles with/without heterologous epitopes

2002

T Monath, others

Yellow fever virus,

West Nile virus

Vaccines produced by recombinant-DNA technology, by the formation of viral chimeras (viruses with the replicative
machinery of one virus and the protective antigens of another)

This is not a place for coverage of the huge subject of modern, innovative vaccinology and the diverse methodologies involved in research, development and commercial
production of new vaccines, especially vaccines for those viral diseases where conventional vaccines have not succeeded (HIV/AIDS, hepatitis C virus, herpes simplex
virus, respiratory syncytial virus, Coxsackie A viruses, et al.). By the 1960s, there was a decline in the sort of innovation that had led to many successful vaccines, but after
the introduction of hepatitis B virus vaccine in the 1980s (recombinant HBsAg produced first in yeast and then in various hosts), a change for the better was seen, and now
at least in the areas of basic science and preclinical development, there is much ongoing innovation. It remains to be seen if the regulatory aspects and commercial (profitmaking) aspects of vaccinology will follow. Optimism stems from ongoing scientific, technological and manufacturing advances, most but not all grounded in molecular
and cell biology, molecular immunology, information technologies and the concept of platform technologies. In this context, the introduction of influenza vaccines made in
cell culture rather than in eggs, seemingly a prosaic change and not reflective of any really innovative technology, is nevertheless an important symbol. The table here might
have been placed in association with several key discoveries, but upon reflection it is the work of William Rutter, Pablo Valenzuela, Maurice Hilleman and their colleagues on
recombinant hepatitis B (HBsAg) vaccine in 1981 and of Saul Kit on thymidine kinase deletion mutant pseudorabies vaccine in 1985 that seem to stand as the benchmarks.

1981> The development of modern viral vaccinology based upon innovative technologies

page 473

Feline immunodeficiency virus (FIV) was first isolated from cats in

Petaluma, California in 1986 by Niels Pedersen, Janet Yamamoto
and their colleagues from the School of Veterinary Medicine at the
University of California Davis. The virus was shown to be a typical
lentivirus it replicates preferentially but not exclusively in feline
T-lymphoblastoid cells, where it causes a characteristic cytopathic
effect. Kittens experimentally infected with FIV manifest a transient
(several days to 2 weeks) fever and neutropenia beginning 4 to
8 weeks after inoculation. This is associated with a generalized
lymphadenopathy that persists for up to 9 months. Most cats
recover from this initial phase of the disease but complete recovery
is rare in nature or in the laboratory setting. The terminal AIDSlike phase of the illness has been seen mainly in naturally infected
cats. It appears a year or more following the initial infection in an
unknown proportion of infected animals. Similarities between FIV
infection of cats and HIV infection of humans are striking, making
FIV infection of cats a useful model for human AIDS. In spite of the
similarities between FIV and HIV, there is no evidence that FIV is a
health hazard to humans or other species of animals. FIV has been
identified in cats from all parts of the world. It is most prevalent in
high density populations of free roaming cats (feral and pet), with
prevalence rates of 1-12%.
Pedersen NC, Ho EW, Brown ML, Yamamoto JK. Isolation of a
T-lymphotropic virus from domestic cats with an immunodeficiencylike syndrome. Science. 1987;235:790-793.

Niels C. Pedersen

Janet K. Yamamoto

Yamamoto JK, Sparger E, Ho EW, Andersen PR, OConnor TP,

Mandell CP, Lowenstine L, Munn R, Pedersen NC. Pathogenesis of
experimentally induced feline immunodeficiency virus infection in
cats. Am J Vet Res. 1988;49:1246-1258.
Pedersen NC, Yamamoto JK, Ishida T, Hansen H. Feline
immunodeficiency virus infection. Vet Immunol Immunopathol.
1989;21:111-129.

A simplified phylogenetic tree

of the lentiviruses, based upon
multiple sources of genomic
data and analysis

1986 Niels Pedersen, Janet Yamamoto, colleagues discovery of feline immunodeficiency virus
page 474

Leroy E. Hood

Lloyd M. Smith

Automated DNA-sequencing output from a capillary

electrophoresis machine, where after size separation,
detection and recording of dye fluorescence is
visualized as shown, while the data are sent directly for
computer-based analysis and storage

Shortly after dideoxynucleotide chain-terminating DNA sequencing was introduced by Fredrick Sanger, Leroy Hood, Lloyd Smith, Tim and Mike Hunkapiller and their
colleagues, working in a Caltech basement, attempted to develop an automated sequencer it turned out to be a difficult job, taking several years. The difficulty was the
underlying chemistry and the means for detecting reaction products the chemistry for linking dyes to DNA did not exist and neither did a way to measure the different
colored fluorescent dyes (fluors). In a paper published in Nature in 1986, the group announced their prototype automated DNA sequencer, ushering in a new era in genomics
and in virology. The key to their success was fluorescence detection of enzymatically degraded DNA fragments using fluorophores (developed by DuPont), which were
covalently attached to oligonucleotide primers. A different colored fluorophore was used for each of the reactions specific for the four bases A, C, G and T. The reaction
mixtures were combined and co-electrophoresed down a single polyacrylamide gel tube, the separated fluorescent bands of DNA being detected near the bottom of the tube
as the electrophoresis continued and fragments ran out of the gel. The sequence information was fed directly into a computer. After the prototype was complete, Hood sold
the rights to Applied Biosystems, Inc., but he continued to work with the company to refine the original concept and machine. The invention became the engine of the global
effort to sequence the genomes of all prototypic viruses, as well as the engine of the Human Genome Project. Today, the electrophoresis is usually done in capillaries, thereby
allowing massively parallel runs there are also many different newer technologies in use today, supporting even more massive sequencing projects.
Smith LM, Fung S, Hunkapiller MW, Hunkapiller TJ, Hood LE. The synthesis of oligonucleotides containing an aliphatic amino group at the 5 terminus: synthesis of
fluorescent DNA primers for use in DNA sequence analysis. Nucleic Acids Res. 1985;13: 2399-2412.
Smith LM, Sanders JZ, Kaiser RJ, Hughes P, Dodd C, Connell CR, Heiner C, Kent SB, Hood LE. Fluorescence detection in automated DNA sequence analysis. Nature
1986;321:674-679.

1986 Leroy Hood, Lloyd Smith, Applied Biosystems, Inc. development of automated DNA sequencing

page 475

Frank Fenner (1914-2010)

Authors of Veterinary Virology, Third Edition

E. Paul J. Gibbs, Marian C. Horzinek, Michael J. Studdert, Frederick A. Murphy

In 1987, Frank Fenner, David White (1931-2004) and their

colleagues published a companion to the book, Medical Virology,
the first editions of which had been published in 1970 and 1976.
The first edition of Veterinary Virology was a success, so a second
edition was published in 1993, a third in 1999 and a fourth in
2010. Each edition followed Fenners unique style, and attempted,
in the words of M. Anthony (Tony) Epstein in his review in
Nature, to mimic Fenners seeming ease with which extremely
complicated topics are reduced to an orderly survey of the
basic facts involved, together with suitable explanations of their
significance. Fenners incomparable standard of scholarship, seen
in Veterinary Virology, was grounded in his memorable personal
qualities, his warm, generous, unpretentious character, matched
with his great fortitude, insight and understanding of his students.
Fenners vision was so broad as to extend from virologys historic
roots as a microbiological / pathological / clinical infectious
disease science, and later a molecular and cell biologic science, to
its present role as a major contributor to the overall advance of
our understanding and stewardship of life on earth.

Frank Fenner, Peter A. Bachmann, E. Paul J. Gibbs, Frederick A.

Murphy, Michael J. Studdert, David O. White, First Edition, 1987
Frank Fenner, E. Paul J. Gibbs, Frederick A. Murphy, Rudolf Rott,
Michael J. Studdert, David O. White, Second Edition, 1993
Frederick A. Murphy, E. Paul J. Gibbs, Marian C. Horzinek,
Michael J. Studdert, Third Edition, 1999
N. James Maclachlan, Edward J. Dubovi (Editors); Stephen W.
Barthold, Richard A. Bowen, Ronald P. Hedrick, Donald P.
Knowles, Michael D. Lairmore, Colin R. Parrish, Linda J. Saif,
David E. Swayne (Chapter Authors), Fourth Edition, 2010

1987> Frank Fenner, others publication of book, Veterinary Virology (currently four editions)
page 476

AZT - Zidovudine, structural model

Marty St. Clair

Samuel Broder

Azidothymidine (AZT) (Zidovudine, INN) (trade-names: Retrovir, Retrovis) was the first drug approved for the treatment of HIV infection /AIDS. It is an analog of thymidine.
Jerome Horwitz of Wayne State University School of Medicine first synthesized AZT in 1964; it was originally intended to treat cancer, but failed to show efficacy. In 1984,
shortly after HIV was shown to be the cause of AIDS, scientists at Burroughs Wellcome Co. (now GlaxoSmithKline) began searching for compounds to treat the disease.
Burroughs Wellcome had expertise in viral diseases, led by Gertrude Elion, George Hitchings David Barry, Phil Furman, Marty St. Clair, Janet Rideout, Sandi Lehman
and others. Their efforts focused on the viral reverse transcriptase; they identified and synthesized compounds and developed a screening test for activity against murine
retroviruses. One compound coded BW A509U showed potent activity against the murine viruses. At the same time, Samuel Broder, Hiroaki Mitsuya, and Robert Yarchoan
at the National Cancer Institute initiated a program to develop drugs active against HIV they soon developed a collaboration with Janet Rideout and others at Burroughs
Wellcome and confirmed that BW A509U had activity against HIV. A few months later, they started a phase 1 clinical trial of the drug, now named AZT, at the NCI and Duke
University. This trial showed that the drug could be safely administered to patients with HIV and that it could increase CD4 counts in AIDS patients. A placebo-controlled
randomized trial was subsequently conducted by Burroughs-Wellcome, in which it was shown that AZT could prolong the life of patients with AIDS. The FDA approved the
drug (via a then-new FDA accelerated approval system) in 1987. The time between the first demonstration that AZT was active against HIV in cell culture and its approval was
25 months, one of the shortest periods for drug development and licensing in history.
Mitsuya H, Weinhold K, Furman P, St Clair M, Lehrman S, Gallo R, Bolognesi D, Barry D, Broder S. 3-Azido-3-deoxythymidine (BW A509U): an antiviral agent that inhibits
the infectivity and cytopathic effect of human T-lymphotropic virus type III/lymphadenopathy-associated virus in vitro. Proc Natl Acad Sci USA. 1985;82: 7096-7100.
Broder S. The development of antiretroviral therapy and its impact on the HIV1/AIDS pandemic. Antiviral research 2009;85: 1-2.

1987 Marty St. Clair, Samuel Broder, others, Burroughs Wellcome/NCI first anti-HIV drug approved by FDA (AZT)

page 477

Mario R. Capecchi

Oliver Smithies

Mario Capecchi and Oliver Smithies were seeking ways of specifically altering the mammalian genome, Capecchi with a
view to inserting new genes into cells and Smithies in the hope of correcting genetic defects. They independently discovered
that they could use a natural mechanism, discovered decades before by Joshua Lederberg in bacteria, to introduce short
sequences of manipulated DNA into the chromosomes of cultured mammalian cells. Their techniques allowed them to
target individual genes, producing the genetic alterations they sought, but only at the cellular level. The embryonic stem cell
cultures that Martin Evans was then developing provided the necessary vehicle for taking such gene manipulations from cell
culture into whole animals. By modifying genes in embryonic stem cells and then injecting these cells into fertilized mouse
eggs, it became possible to rear mice with discrete inheritable genetic modifications knock-out mice.

Martin J. Evans

Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981;292:154-156.
Doetschman T, Maeda N, Smithies O. Targeted mutation of the hprt gene in mouse embryo stem cells Proc Natl Acad Sci
USA. 1988;85:8583-8587.
Mansour SL, Thomas KR, Capecchi MR. Disruption of the proto-oncogene Int-2 in mouse embryo-derived stem cells a
general strategy for targeting mutations to non-selectable genes. Nature 1988;336:348-352.
Evans MJ. Potential for genetic manipulation of mammals. Mol Biol Med. 1989;6:557-565.
Capeccci MR. Altering the genome by gene targeting. Trends Genet. 1989;5:70-76.

Mouse in which a gene affecting hair

growth has been knocked out

1988 Mario Capecchi, Oliver Smithies, Martin Evans knockout and other genetically manipulated mice
page 478

Exanthem subitum
(roseola infantum)
in a young child

Koichi Yamanishi

Takeshi Kurata

A virus called human herpesvirus 6 (HHV-6) was first isolated from adult patients
with lymphoproliferative disorders in 1986; however, soon it became evident that
there were two viruses, now named human herpesvirus 6A and 6B. The two viruses
are closely related, but show consistent differences in biological, immunological,
epidemiological and molecular properties. HHV-6B is the major causative agent
of exanthem subitum, but no clear association has been made between any clinical
disease and infection with HHV-6A. Exanthem subitum is also called roseola
infantum and sixth disease (as the sixth rash-causing childhood disease), but these
clinical terms are vague in that they encompass infections caused by other viruses
for example, HHV-7 can also cause the same clinical syndrome. HHV-6A, 6B
and HHV-7 are ubiquitous, with most infections occurring in infancy and with
more than 90% of adults having antibody to the three viruses. Typically, in infants
infected with HHV-6B there is sudden fever, which lasts for a few days, and a rash
which appears on the trunk and face and spreads to the lower extremities as the
fever subsides. More severe cases present diagnostic and treatment complexities
(thrombocytopenia, granulocytopenia, hepatitis, encephalopathy with seizures).
In adults, primary infections can cause a mononucleosis like disease. Since like
other herpesviruses, HHV-6B establishes latency following primary infection, it
is an important pathogen in immunocompromised hosts. Reactivation occurs in
patients after bone marrow transplantation, solid organ transplantation such as
liver, renal and heart transplantation, and AIDS.
Yamanishi K, Okuno T, Shiraki K, Takahashi M, Kondo T, Asano Y, Kurata T.
Identification of human herpesvirus-6 as a causal agent for exanthem subitum.
Lancet 1986;1:1065-1067.
Lopez C, Pellett P, Stewart J, Goldsmith C, Sanderlin K, Black J, Warfield D,
Feorino P. Characteristics of human herpesvirus-6. J Infect Dis. 1988;157:12711273.
Pellett PE, Lindquester GJ, Feorino P, Lopez C. Genomic heterogeneity of human
herpesvirus 6 isolates. Adv Exp Med Biol. 1990;278:9-18.
Yamanishi K. Human herpesvirus 6. Microbiol Immunol. 1992;36:551-561.

Philip E. Pellett

Carlos Lopez

Mori Y, Yamanishi K. HHV-6A, 6B, and 7: pathogenesis, host response, and clinical
disease. In: Arvin A, Campadelli-Fiume G, Mocarski E, Moore PS, Roizman B,
Whitley R, Yamanishi K, editors. Human herpesviruses: Biology, therapy, and
immunoprophylaxis. Cambridge: Cambridge University Press; 2007. Chapter 46.

1988 Koichi Yamanishi, Takeshi Kurata, Philip Pellett, Carlos Lopez human herpesvirus 6B and exanthem subitum

page 479

Internal ribosomal initiation sites (IRES) are

nucleotide sequences that allow for translation
initiation in the middle of a messenger RNA
(mRNA) sequence with consequent protein
synthesis. Usually, in eukaryotes, translation
can be initiated only at the 5 end of the mRNA
molecule, since 5 cap recognition is required.
IRES sequences were first discovered in 1988 in
encephalomyocarditis virus RNA and poliovirus
RNA in the labs of Eckard Wimmer and Nahum
Sonenberg, respectively. They were described
as distinct regions in mRNA molecules that
are able to attract the eukaryotic ribosome. It
was hypothesized and later proven that IRES
elements have a distinct secondary or even
tertiary structure within the mRNA, leading to
their correct spatial orientation with ribosomes.
IRES-ribosomal interactions also require the
recognition of viral mRNA by host factors.
Poliovirus IRES elements are the most complex
elements described to date, but they are very
efficient in driving internal translation initiation.
IRES elements have also been described in other
RNA viruses, including flaviviruses, retroviruses,
and several plant viruses. The IRES regions of
viral mRNAs also provide possible targets for
antiviral therapies.

Eckard Wimmer

Nahum Sonnenberg

Cartoon of poliovirus genome with its complex internal ribosomal entry site (IRES, blue)

Pelletier J, Sonenberg N. Internal initiation of

translation of eukaryotic mRNA directed by a
sequence derived from poliovirus RNA. Nature
1988;334:320-325.
Jang SK, Krusslich HG, Nicklin MJ, Duke GM,
Palmenberg AC, Wimmer E. A segment of the
5 nontranslated region of encephalomyocarditis
virus RNA directs internal entry of ribosomes
during in vitro translation. J Virol. 1988;62:26362643.
Fernndez-Miragalla O, Lpez de Quinto S,
Martnez-Salas E. Relevance of RNA structure
for the activity of picornavirus IRES elements.
Virus Res. 2009;139:172-182.

1988 Eckard Wimmer, Nahum Sonnenberg, colleagues internal ribosomal entry sites (IRES) in picornavirus mRNA
page 480

The National Center for Biotechnology Information (NCBI) is

part of the National Library of Medicine (NLM), a branch of
the National Institutes of Health (NIH). The NCBI was founded
in 1988 and houses several resources: (1) genome sequencing
data in GenBank; (2) an index of biomedical research articles
in PubMed; and (3) online databases through the Entrez search
engine. NCBI also houses a multi-disciplinary intramural
research program. Initially, the mission of the NCBI was to
aid in understanding the molecular mechanisms that affect
human health and disease. It was to create and maintain
public databases, develop software to analyze genomic data,
and to conduct research in computational biology. In time,
and through widespread use of the Internet, NCBI became
increasingly involved in supporting basic biological research,
that is molecular and cellular biology research. NCBI evolved
further to: (1) develop new methods to deal with the volume
and complexity of the data being accumulated faster and faster;
(2) develop methods to analyze the structure and function of
macromolecules; (3) develop computerized systems for storing
data in more accessible form; and (4) develop better means to
access analytic and computational tools by researchers and the
public. In the process of database development, NCBI formed
database standards such as the database nomenclature that is
also used by other databases. The NCBI database GenBank, the
nucleic acid sequence database, contains sequence information
from over 200,000 different organisms; it is the most popular
database in use today. The foundation of GenBank predates
the NCBI, having been formalized in 1982; it is linked to
the genomic database of the European Molecular Biology
Laboratory (EMBL) and the DNA Database of Japan (DDBJ), all
part of the International Nucleotide Sequence Database project.

PubMed:

Since 1997 free access to the entire

MEDLINE dataset (plus others)
20 million articles, 10.4 million
with abstracts
2 million articles with full text in
PubMed Central
21,000 journals
16 million requests per day

1988 National Library of Medicine National Center for Biotechnology Information (NCBI) and its public databases

page 481

H. Fred Clark (1937-2012) and Paul A. Offit

Pentvalent human-bovine reassortant rotavirus vaccine
(RotaTeq - Merck)

Albert Kapikian

Richard Ward

With colleagues. Human-bovine

With David Bernstein. Attenuated
reassortant rotavirus vaccine (several
human rotavirus vaccine (Rotarix international manufacturers)
GlaxoSmithKline)
In 1998, a rotavirus vaccine (RotaShield, Wyeth) containing an attenuated rhesus-human reassortant virus was licensed for use in the United States. Clinical trials in the
United States, Finland, and Venezuela had found it to be 80 to 100% effective at preventing severe diarrhea caused by the most common rotavirus variants, The vaccine,
however, was withdrawn from the market in 1999 after it was discovered that it may have contributed to a risk of intussusception in one of every 12,000 vaccinated infants.
Today, two newer vaccines (Rotarix, GlaxoSmithKline; RotaTeq, Merck) have been approved by FDA and another is in advanced development for use in developing countries
(Albert Kapikian, colleagues, NIH). Before the introduction of vaccine in the U.S. in 2006, rotavirus was the most common cause of severe gastroenteritis in children <5 years
of age. Although mortality is uncommon in the U.S., with an estimated 20 to 60 deaths annually, rotavirus gastroenteritis results in 55,000 to 70,000 hospitalizations, 200,000
emergency department visits, and 400,000 outpatient visits each year among children <5 years of age. Rotavirus disease in developing countries is much more significant:
worldwide, diarrhea is the second most common cause of fatal childhood disease, estimated to cause ~1.4 million deaths among children aged <5 years. Clinical trials in
developed countries have shown high efficacy for the presently licensed vaccines, reducing hospitalizations due to rotavirus gastroenteritis by 96% and office visits by 86% it
is anticipated that efficacy will be similar in developing countries as rotavirus vaccination programs expand globally.
Clark HF, Offit PA, Ellis RW, Eiden JJ, Krah D, Shaw AR, Pichichero M, Treanor JJ, Borian FE, Bell LM, Plotkin SA. The development of multivalent bovine rotavirus (strain
WC3) reassortant vaccine for infants. J Infect Dis. 1996;174(Suppl 1):S73-80.
Kapikian AZ, Simonsen L, Vesikari T, Hoshino Y, Morens DM, Chanock RM, La Montagne JR, Murphy BR. A hexavalent human rotavirus-bovine rotavirus (UK) reassortant
vaccine designed for use in developing countries and delivered in a schedule with the potential to eliminate the risk of intussusception. J Infect Dis. 2005;192(Suppl 1):S22-29.
Patel MM, Steele D, Gentsch JR, Wecker J, Glass RI, Parashar UD. Real-world impact of rotavirus vaccination. Pediatr Infect Dis J. 2011;30(Suppl):S1-5.

1988> Paul Offit, H. Fred Clark, Albert Kapikian, Richard Ward, others development of human rotavirus vaccines
page 482

Since 1988, morbilliviruses have been increasingly recognized as the

cause of mass mortality, first among harbor seals (Phoca vitulina)
and gray seals (Halichoerus grypus) (phocid distemper virus - PDV),
and then several other marine mammals: Baikal seals (Phoca sibirica)
(canine distemper viruses - CDV), bottlenose dolphins (Tursiops
truncatus) and striped dolphins (Stenella coeruleoalba) (both dolphin
morbillivirus - DMV), and harbor porpoises (Phocoena phocoena)
(porpoise morbillivirus - PMV). Morbillivirus antibody and isolates
have also been made and in some cases sequenced from: killer whales
(Orcinus orca), manatees (Trichechus manatus latirostris), polar
bears (Ursus maritimus), walruses (Odobenus rosmarus), long-finned
pilot whales (Globicephala melas) and fin whales (Balaenoptera
physalus). Clinical signs of morbillivirus infection in these animals
are rarely observed severe viral disease is likely to be incompatible
with life in the harsh environments in which these animals live;
however, infected animals have been found in poor condition and
in some instances with distemper-like lesions (oculonasal discharge,
conjunctivitis, keratitis, respiratory distress - pneumonia, diarrhea
and abortion).
Osterhaus AD, Groen J, Niesters H, van de Bildt M, Martina B,
Vedder L, Vos J, van Egmond H, Abou Sidi B, Ould Barham ME.
Morbillivirus in monk seal mass mortality. Nature 1997;388:838-839.
Osterhaus AD, Vedder EJ. Identification of virus causing recent seal
deaths. Nature 1988;335:20.

Albert D.M.E. Osterhaus

Morbillivirus, ruptured virion showing

herringbone nucleocapsid strands
negative contrast electron microscopy
Unrooted phylogenetic tree
(hemagglutinin gene) of
the morbilliviruses (marine
morbilliviruses in yellow)

Mahy BWJ, Barrett T, Evans S, Anderson EC, Bostock CJ.

Characterization of a seal morbillivirus. Nature 1988;336:115.
Kennedy S, Kuiken T, Jepson PD, Deaville R, Forsyth M, Barrett T,
van de Bildt MWG, Osterhaus AD, Eybatov T, Duck C, Kydyrmanov
A, Mitrofanov I, Wilson S. Mass die-off of Caspian seals caused by
canine distemper virus. Emerg Infect Dis. 2000;6:637-639.

An epizootic caused by dolphin

morbillivirus (DMV) killed
thousands of striped dolphins in the
Mediterranean Sea in 1990-1992.
The first victims were detected in
the Gulf of Valencia off the coast of
Spain; in the next two years, large
foci of infection were found further
east. Here, bottlenose dolphins
were also affected. By 1944, the
virus had moved into the Black
Sea, where it caused many deaths
among the subspecies of bottlenose
dolphin that inhabits that region.

1988> Albert Osterhaus, colleagues discovery of phocine distemper virus, marine mammal morbilliviruses

page 483

Manfred Eigen

Juan de la Torre

Esteban Domingo

Peter Schuster

John Holland

Eugene Koonin

Adrian Gibbs

Luis Villarreal

1988 Manfred Eigen, others development of modern concepts of the origin of viruses, virus evolution, quasispecies
page 484

Development of modern concepts of the origin of viruses

From the earliest International Congresses for Virology, sessions on the origin and
evolution of the viruses were very popular, but most of the content was entirely
speculative and ungrounded in observational or experimental proofs lots of fun, but
not too serious. In the past decade or so this has changed: only data-based concepts
now have credibility and these are largely grounded in genomics and the rise of
evolutionary biology, overall. The subject has gotten bigger entire conferences are
devoted to it, proceedings are published and biologists from outside the world of
virology contribute. Nevertheless, data-based or not, the subject is full of strongly
held opinions. A decade ago, students were taught that there were three hypotheses
to explain the origin of viruses: (1) the progressive, or escape, hypothesis stated that
viruses arose from genetic elements (DNA, RNA) that escaped from cellular control
and gained the ability to move between cells; (2) the regressive, or reductionistic,
hypothesis asserted that viruses are remnants of cellular organisms or organelles;
and (3) the virus-first hypothesis stated that viruses originated in a pre-cellular
world and predate or coevolved with their current hosts. The first hypothesis was
usually given most credibility, based on the notion that bacteriophages originated
from bacterial genomes and eukaryotic viruses from eukaryotic genomes, etc. The
discovery that virus genes came from diverse sources, as well as unique sources,
and the discovery of the archaea and viruses of archaea confounded this, but better
notions arose only after further progress was made in discerning very distant
viral genomic relationships and in discerning molecular structure/function
relationships among viral and cellular proteins. Slowly, it came to be realized
that viruses form a world of their own, and that their molecular features
strongly suggest that they are ancient, predating the Last Universal Cellular
Ancestor (LUCA). The main debate has shifted to those who suggest a long
period of acellular evolution (up to the actual emergence of the archaea and
bacteria), versus those who favor an early appearance of cells (or something
very similar to cells). Those who suggest the former have revived the virusfirst hypothesis. That is, viruses emerged from an assemblage of selfreplicating elements thriving in special environments (say, hydrothermal
vents or such), using inorganic compartments as their hosts. For those who
favor an early emergence of cellular organisms, viruses are usually considered to have originated in cells in which RNAs played the part of enzymes
(ribozymes) and were also the stuff of inheritance. This has been called the
RNA world. The literature on the evolution of life (whether viruses are alive
or not is another subject!) from the point of self-replication is large and
diverse the literature on the initial steps transforming inorganic molecules
(the primordial soup) into the constituents of living organisms is small, not
having moved much from the famous experiments of Stanly Miller and
Harold Urey in 1952, in which they simulated hypothetical conditions
thought to be present on the early Earth and synthesized organic compounds
from inorganic precursors. Once amino acids were formed, the rest was easy
but hardly explained. At another level of evolution, that of one virus

evolving into another via Darwinian mutation/selection processes, it is widely

presumed, on the basis of all evidence, that most of the families of viruses originated
and evolved separately that the families of viruses that relate to the archaea, to the
bacteria and to eukaryotic organisms originated and evolved separately, as suggested
in the diagram below, where a pre-virus appears from the ancient mist and enters one
of the three virospheres, coincident with the formation of the three domains of life.
Eugene Koonin and his colleagues have envisioned the emergence of the eukaryotic
cell as another melting pot of virus evolution, from which the major groups of
eukaryotic viruses originated as a result of extensive recombination of genes from
various bacteriophages, archaeal viruses, plasmids, and bits of evolving eukaryotic
genomes. As remarkable as this early end of the evolutionary spectrum is, it is
equaled at the other end of the spectrum, where today we are still concerned with the
evolution of new viral genotypes and consequent phenotypes (pathotypes, topotypes,
etc.), which explain, say, the emergence of variant viruses with importantly altered
transmission and/or pathogenicity characteristics.
Miller SL, Urey HC. Organic compound synthesis on the primitive Earth. Science
1959;130: 245-251; and Origin of Life. Science 1959;130:1622-1624.
Koonin EV, Senkevich TG, Dolja VV. The ancient virus world and evolution of cells.
Biol Direct. 2006;1:29.
Prangishvili D, Forterre P, Garrett RA. Viruses of the archaea: a unifying view. Nat Rev
Microbiol. 2006;4:837-848.

1988 Manfred Eigen, others development of modern concepts of the origin of viruses, virus evolution, quasispecies

page 485

Development of the concept of viral quasispecies

Every virus species, as defined by conventional phenotypic properties, exists as a genetically

dynamic, diverse population of virions in which individual genotypes have only a fleeting
existence. Most individual viral genomes differ in one or more nucleotides from the consensus
or average sequence of the population and over relatively short times genotypic drift occurs as
particular variants gain advantage. Genotypic drift over longer times leads to the evolution of
substantially different viruses. Manfred Eigen, John Holland and their colleagues introduced the
term quasispecies to describe such diverse, rapidly evolving and competing viral populations. If
viral nucleic acid replication were without error all progeny would be the same and there would
be no evolution of phenotypes. If the error rate was too high, mutants of all sorts would appear
and the viral population would lose its integrity. However, at an intermediate error rate, such as
is seen with most or all RNA viruses, the viral population becomes a coherent, self-sustaining
entity that resembles a metaphorical cloud of variants centered around a consensus sequence, but
capable of continuous expansion and contraction in different directions as new mutants continue
to emerge and others disappear within the population. Darwinian selection limits the survival of
the most extreme mutants extreme outliers do not survive and favors variants near the center
of the cloud since these best achieve fit in the environmental niche. Just as the center of a cloud
is unclear, so the consensus sequence at the heart of the quasispecies is considered inscrutable.
Any published viral genomic nucleotide sequence reflects a random choice of starting material,
one biological clone among many, more or less representative of the consensus sequence of the
genome of the population as a whole. Using new technologies, such as deep sequencing, it is
hoped that the kinds of population structures built around consensus sequences will become
discernable. Do population structures radiate outward from consensus in a stochastic manner,
creating a symmetrical cloud focused around one single master genome? Or do they radiate out
from a multiple number of dominant genotypes. How do these populations differ in different hosts
or different cell types or tissues? How do small adaptive changes, mutations that become fixed
in the consensus sequence, alter the surrounding mutant cloud? The evolution of quasispecies
would be expected to be most conspicuous in viruses with large RNA genomes, where nonlethal changes might accumulate rapidly. Indeed, for example, the genomes of coronaviruses,
with the largest RNA viral genomes known, are fraught with genetic defects. At the mutation
rate commonly seen in single-stranded RNA viruses, that is 10-3 per nucleotide per replication
cycle (i.e., 1 out of every 1,000 nucleotides in every coronavirus genome in every round of
replication), would be changed. Since coronavirus genomes contain about 30,000 nucleotides,
every genome must differ from the next by at least one nucleotide. From this, one might wonder
how coronaviruses or other RNA viruses can maintain their identities as pathogens over any
evolutionarily significant period of time; why have these viruses not mutated out of existence?
The answer lies in the quasispecies concept, with all its stabilizing influences.
Eigen M. Viral quasispecies. Sci Am. 1993;269:42-49.
Prangishvili D, Forterre P, Garrett RA. Viruses of the archaea: a unifying view. Nat Rev Microbiol.
2006;4:837-848.
Vignuzzi M, Andino R. Picornaviruses fidelity mutants and biological implications. In: Domingo
E, Ehrenfeld E, Roos R, editors. Picornaviruses: molecular biology, evolution and pathogenesis.
Washington DC. ASM Press; 2010.

Depiction of the quasispecies concept of Manfred Eigen

The box represents sequence space, that is the confines of all possible
genetic variants that might occur when a virus replicates through
many infection cycles. The central spot represents the lack of variance
that would follow if the replication process was perfectly accurate and
environmental selective pressures were constant. The cloud represents
the viral population diversity that actually follows upon an intermediate
error rate in replication. The population, overall, becomes a coherent,
self-sustaining entity that metaphorically resembles a cloud in that its
center, the original consensus sequence, is inscrutable, while its edges
represent probes pushing into the environment seeking better-and-better
fit. It has been found that the evolution of many viruses operates at the
level of the quasispecies, not at the level of individual virion genotypes,
and the result is the continuing emergence of new viral phenotypes,
some of which cause new and/or more severe disease.

1988 Manfred Eigen, others development of modern concepts of the origin of viruses, virus evolution, quasispecies

page 486

Since the discovery in children with acute

gastroenteritis of picobirnaviruses by Helio Pereira,
Thomas Flewett and their colleagues in the late 1980s,
many efforts have been made to prove their etiologic
role in diarrheal disease. However, quite often the
viruses have been detected in healthy children and
adults, in some instances in an unexpected percentage
of fecal samples tested, and in some instances for
prolonged periods. There are some suggestions
that they may be the cause of persistent diarrhea in
immunocompromised patients, patients with advanced
AIDS, et al. Further, after the viruses were found in
black-footed pigmy rice rats, distinct genotypes were
also found worldwide in pigs, calves, foals, lambs,
rabbits, guinea pigs, birds and snakes. Still, their
zoonotic potential is unproven. At present, there
is no cell culture system or animal model for these
viruses RT-PCR assays are used for epidemiologic
and natural history studies and virions purified from
feces are used for molecular biologic and structural
studies. The picobirnaviruses (family Picobirnaviridae)
are nonenveloped, double-stranded RNA viruses with a
bisegmented genome.
Pereira HG, Fialho AM, Flewett TH, Teixeira JM,
Andrade ZP. Novel viruses in human faeces. Lancet
1988;2:103-4.

Helio Gelli Pereira (1918-1994)

Thomas Henry Flewett (1922-2006)

Pereira HG, Flewett TH, Candeias JA, Barth OM,

1988b. A virus with a bisegmented doublestranded RNA
genome in rat (Oryzomys nigripes) intestines. J Gen
Virol 1988;69:2749-2754.
Ganesh B, Bnyai K, Martella V, Jakab F, Masachessi G,
Kobayashi N. Picobirnavirus infections: viral persistence
and zoonotic potential. Rev Med Virol. 2012;22:245256.
Smits SL, van Leeuwen M, Schapendonk CM, Schrch
AC, Bodewes R, Haagmans BL, Osterhaus AD (2012)
Picobirnaviruses in the human respiratory tract. Emerg
Infect Dis 18(9):153940

Picobirnavirus
model from
cryo-electron microscopic
image reconstruction

1988 Helio Pereira, Thomas Flewett, colleagues discovery of picobirnaviruses

page 487

Thomas D. Brock, Yellowstone National Park

In the early 1980s, Kary Mullis at Cetus Corporation

invented the polymerase chain reaction (PCR). In its
first iteration, a new aliquot of the thermolabile DNA
polymerase from E. coli had to be added after each
cycle, because the enzyme was denatured by the >90C
temperature used to dissociate the strands of the newly
formed DNA hence, PCR was slow and laborious.
Switching to the thermostable DNA polymerase, Taq
polymerase, was key to the evolution of PCR into its
present form, using fully automated thermal cyclers
and other streamlining instruments. Because of these
improvements over the initial invention, PCR became the
most widely applicable technique in molecular biology.
Taq polymerase was isolated from the thermophilic
bacterium, Thermus aquaticus, which was discovered
in 1966 in Mushroom Spring in the Lower Geyser Basin
of Yellowstone National Park by Thomas Brock and
Hudson Freeze, then of Indiana University. The optimum
temperature for Taq polymerase activity is 75-80C
and it can survive 97C for some time; it can replicate a
1,000 base pair strand of DNA in less than 10 seconds
at 72C. At Cetus, purifying the Taq polymerase was
achieved by David Gelfand and Susanna Stoffel and proof
of the fidelity of its enzymatic activity was done by Ray
Saiki. Gelfand and Stoffel then cloned and successfully
over-expressed the enzyme in E. coli so that large
amounts of extremely pure product became available.
The refined PCR technique was seen to be so important
that Hoffmann-LaRoche paid more than $300 million
to acquire rights to it. Essentially, the company was
acquiring the rights to two patents, that of Gelfand, et al.
(1989) for purified Taq polymerase and that of Mullis, et
al. (1990) for the PCR technique itself. In 1989, Science
magazine established a new award called The Molecule
of the Year. Taq polymerase was the first awardee.
Brock TD, Freeze H. Thermus aquaticus, a
nonsporulating extreme thermophile. J Bact. 1969;98:289297.

Thermus aquaticus, scanning electron microscopy

1988 Thomas Brock discovery of Taq polymerase

page 488

The Taq polymerase molecule

is shaped like a hand. The DNA
fits into the palm (between
the green and magenta residues)
when the enzyme is active

Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi

R, Horn GT, Mullis KB, Erlich HA. Primer-directed
enzymatic amplification of DNA with a thermostable
DNA polymerase. Science. 1988;239:487-491.
Mullis KB. The unusual origin of the polymerase chain
reaction. Sci Am. 1990;262:56-65.

Choo QL, Kuo G, Weiner AJ, Overby

LR, Bradley DW, Houghton M.
Isolation of a cDNA clone derived
from a blood-borne non-A, non-B
viral hepatitis genome. Science
1989;244:359-362.
Kuo G, Choo QL, Alter HJ, Gitnick
GL, Redeker AG, Purcell RH,
Miyamura T, Dienstag JL, Alter MJ,
Stevens CE, Tegtmeier GE, Bonino
F, Colombo M, Lee W-S, Kuo C,
Berger K, Shuster JR, Overby LR,
Bradley DW, Houghton M. An
assay for circulating antibodies to
a major etiologic virus of human
non-A, non-B hepatitis. Science.
1989;244:362-364.
Houghton M. The long and winding
road leading to the identification
of the hepatitis C virus. J Hepatol.
2009;51:939-948.

George Kuo, Qui-Lim Choo, Michael Houghton

Daniel W. Bradley

The effort to discover the etiologic agent of non-A, non-B hepatitis, that is hepatitis C virus
(HCV), which started in 1982, took more than five years and was based in techniques that were
novel and unproven at the time. Daniel Bradley, at the Centers for Disease Control and Prevention,
showed that the agent could be transmitted to chimpanzees and that plasma from these animals
was infectious. However, despite heroic efforts the agent could not be grown in cell cultures. Then,
Michael Houghton and his colleagues George Kuo and Qui-Lim Choo at Chiron Corporation (now
part of Novartis International AG), entered into collaboration with Bradley; in 1985, they cloned
all the DNA and RNA they could find in samples of infected chimpanzee plasma. They carried out
large-scale immunoscreening of bacterial cDNA libraries derived from the chimpanzee plasma (this
was prior to the development of PCR) using sera from patients presumed to have post-infection
antibodies. Eventually, this approach led to the isolation of a single small cDNA clone that was proven
to be derived from the HCV genome by Harvey Alter at NIH. The clone was expressed and used to
develop an initial serologic test; Alter then ran this antigen against a coded reference set of human
sera and proved that the virus had been found. The clone also provided a handle allowing the entire
genome to be isolated and sequenced. Next, a blood test was developed and by the early 1990s all
blood donors were tested in a successful program to eliminate the virus from the blood supply the
yearly rate of new infections in the U.S. dropped from ~250,000 to ~25,000. The development of a
HCV vaccine has proven to be far more difficult the high mutation rate of the virus, the production
of quasispecies variation, and the frequent establishment of chronic infection and long-term shedding
has confounded vaccine development, which is just now showing promise of success.

The approach that led to the

isolation and identification of HCV

1989 Michael Houghton, Qui-Lim Choo, George Kuo, Daniel Bradley discovery of hepatitis C virus

page 489

Stephen P. A. Fodor

Patrick O. Brown

Fodor SPA, Read JL, Pirrung MC, Stryer L, Lu AT, Solas D. Light-directed, spatially addressable
parallel chemical synthesis. Science 1991;251:767-773.
Schena M, Shalon D, Davis RW, Brown PO. Quantitative monitoring of gene expression patterns
with a complementary DNA microarray. Science 1995;270:467-470.
Lenoir T, Giannella E. The emergence and diffusion of DNA microarray technology. J Biomed Disc
Collab. 2006;1:11.

Several methods for generating large peptide and oligonucleotide

libraries through what was called combinatorial chemistry came on
the scene in the mid-1980s. The field got its start in 1963, when R. Bruce
Merrifield introduced solid phase synthesis, whereby polypeptide or
oligonucleotide chains could be made in assembly-line fashion using
automated synthesizers. Microarrays and gene chips then grew out of
efforts by a team of scientists concerned with developing novel chemical
approaches to automate drug discovery. This team, at the time a unique
collaboration between academic and industrial scientists, was assembled
by Alex Zaffaroni in 1988 and had members from the company
Affymax (Stephen Fodor and his colleagues), Stanford University
(Patrick Brown and his colleagues), and others. In 1989, Affymax staff
members Leighton Read and Michael Pirrung invented VLSIPS (Very
Large Scale Immobilized Polymer Synthesis); this was based on the
analogy of semiconductor chip manufacture by photolithography it
was called light-directed spatially addressable chemical synthesis
and involved depositing onto a glass microscope slide peptides that
could be photoactivated and photoprotected in a complex system using
fluorescent markers to control the composition of the forming peptide
chain. Stephen Fodor became the overall project leader and advanced
the construction of high-density arrays of peptides and oligonucleotides
substantially he also invented a scanner employing fluorescent labels
and confocal laser microscopy to measure each individual binding event
on the surface of the chip with extraordinary sensitivity and precision. At
this time, 1993, Fodor co-founded Affymetrix, Inc., first as a division of
Affymax, then as an independent entity. The first commercial microarray
system, the Affymetrix GeneChip, was developed in 1994, and by 2000
there was an explosion of technological advances and biomedical
applications, mostly following upon hundreds of millions of dollars of
investment by biotechnology and pharmaceutical companies. The initial
microarray consisted of 1,024 peptides in a 1.6 cm2 area generated in
a ten-step process [today, resolution is so good that >250,000 sites per
square centimeter is possible]. The Affymetrix groups 1991 paper in
Science became a citation classic and received the Newcomb-Cleveland
Award as the outstanding paper published in Science that year. About
the same time the Affymetrix group was developing the GeneChip,
several academic teams were developing alternative microarray systems;
of particular importance has been spotted microarray technology
developed at Stanford by Patrick Brown, Dari Shalon, Stephen J.
Smith, Mark Schena and Ron Davis. In this system, the probes are
oligonucleotides; i.e., cDNA or PCR products that correspond to
mRNAs. The probes are synthesized beforehand and then spotted
onto glass slides. [By convention the term probe is the ligand bound to
the solid substrate, that is the glass slide, and the target is the sample of
interest in the liquid phase.]

1989 Stephen Fodor, Patrick Brown, others development of microarray technology

page 490

van Regenmortel MHV. Virus species, a much overlooked

but essential concept in virus classification. Intervirology
1990;31:241-254.
van Regenmortel MHV. Viruses are real, virus species
are man-made taxonomic constructions. Arch Virol.
2003;148:2481-2488.
Gibbs A. Virus nomenclature descending into chaos. Arch
Virol. 2000;145:1505-1507.
Condit RC. Principles of virology. In: Knipe DM, Howley
PM, editors. Fields virology, fifth edition. Philadelphia:
Lippincott, Williams & Wilkins; 2007. p. 25-27.

Marc H. V. van Regenmortel

The science of virology had grown so much and so many viruses were recognized by the early 1960s that Andr Lwoff led the first comprehensive efforts to build a taxonomy
(and consistent nomenclature); this evolved by 1966 into the International Committee on Nomenclature of Viruses (soon changed to the International Committee on
Taxonomy of Viruses, ICTV). The ICTV wrestled with the fundamental problem of developing a taxonomic system that would accommodate the unique properties of viruses
and which could also anticipate advancements in the identification and characterization of viruses. One critical issue was whether the system should consider virus properties
in a monothetical, hierarchical fashion (a system based on a single characteristic, modeled after the Linnean system), or a polythetic, hierarchical fashion (a system based on
more than one shared common characteristic, without any one characteristic being essential for membership in the group). The taxonomy system chosen was monothetical
at the family and genus levels, but for many years the species level was left in a rather informal, vernacular status. One reality behind this was that virologists dealing with the
various virus families and genera, that is the ICTV Study Groups, used different characteristics in distinguishing viruses and groups of viruses below the genus level. From the
beginning, the system differed from that used by any other taxonomists (e.g., for bacteria or any higher organisms), but it was useful, it was used to the exclusion of competing
systems, and it stayed close to the interests of those virologists more interested in the product than the process (e.g., diagnostic virology, pathology). By 1970, the ICTV had
established two virus families each containing two genera, 24 floating genera, and 16 plant groups. It was inevitable that the species level should be addressed and in 1989
Marc van Regenmortel proposed that a virus species be defined as a polythetic class of viruses that constitutes a replicating lineage and occupies a particular ecological niche.
This definition was accepted by the ICTV in 1991 and work began to apply it to the entire system. By 2010, 6 orders, 87 families, 19 subfamilies, 348 genera and 2,285 species
of viruses had been formally recognized. Even though there had hardly ever been any disagreement over the use of the system at the order, family or genus level, there has
been considerable confusion at the species level, partly based in misunderstanding over the difference between the man-made taxonomic construction, the species, and the
real entity, the virus. This was exacerbated by controversial efforts to change the formal nomenclature for virus species, and by disagreements between the lumpers-and-thesplitters, especially when important pathogenic viruses were reduced to sub-species levels in the hierarchy based solely on genomic sequence relationships. van Regenmortel
and a few colleagues have spent much effort trying to educate the community and resolve the differences of opinion and the widespread indifference that still leaves many
venues of virologic communication using various vernaculars that stand in the way of the kind of precision that the universal taxonomy system was meant to fix.

1989 Marc van Regenmortel definition of virus species

page 491

ivind Bergh

Curtis A. Suttle

When the first marine virus was described in 1955, it was largely ignored; however,
when in 1989 ivind Bergh and his colleagues recognized that viruses were
abundant in marine environments others entered the field and made astounding
discoveries. An amazing diversity of virion morphologies were discovered, but
more importantly, marine viruses have been recognized as having an important
role in ocean ecology and energy and nutrient cycles. These viruses infect
bacterioplankton, phytoplankton, microzooplankton and other unicellular
organisms, which constitute 90% of the living biomass of the oceans and produce
50% of the Earths oxygen it is estimated that viruses kill approximately 20%
of this biomass per day. This viral shunt moves organic matter from living
organisms into the particulate and dissolved pools of organic matter, where
some of it is converted into CO2 by degradative processes, but the rest is reused
in new organismal growth without being recycled to the bottom of the ocean
where reuse cycles are much slower. It is estimated that as much as one-quarter
of the primary organic synthesis in the ocean ultimately flows through the viral
shunt. The level of viral infection in the oceans is not at a steady state, so it has an
influence on temporal changes in CO2 levels in surface water and air. The mineral
elements that are liberated during viral lysis (iron, nitrogen, phosphorous) are
rapidly re-assimilated, also greatly influencing the abundance of microorganismal
communities. Each recent estimate of the abundance of marine viruses has been
higher than the preceding one (each being based upon newer methods): surface
seawater abundances routinely are found to be greater than 106 virus particles per
ml. In marine sediments, abundances are even higher, with 108-109 particles per
ml typical in nearshore sediments. Electron microscopic studies had revealed a
plethora of virion morphologies, many absolutely bizarre, but genomic sequencing
of isolates and metagenomic sequencing of marine viral communities has revealed
an even more amazing diversity of species. Among marine virus genomes,
typically 60-80% of open reading frames show no similarity to any sequences in
GenBank. Furthermore, 65-95% of marine viral metagenomic sequences are not
similar to previously described sequences, suggesting that we have only begun to
scratch the surface of the true diversity of this econiche.
Bergh , Brsheim KY, Bratbak G, Heldal M. High abundance of viruses found in
aquatic environments. Nature 1989;340: 467-468.
Suttle CA, Chan AM, Cottrell MT. Infection of phytoplankton by viruses and
reduction of primary productivity. Nature 1990;347;467-469.
Fuhrman JA. Marine viruses and their biogeochemical and ecological effects.
Nature 1999;399:541-548.

J. Craig Venter

Venters Sorcerer II Global Ocean

Sampling Expedition: relative
proportion of viral sequences
by virus type

Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA, Smith
HO, et al. Environmental genome shotgun sequencing of the Sargasso Sea. Science
2004;304:66-74.
Suttle C. The virosphere: the greatest biological diversity on Earth and driver of
global processes. Environ Microbiol. 2005;7:481-482.

1989 ivind Bergh, Curtis Suttle, Craig Venter, others high concentrations of diverse viruses in the ocean
page 492

In 1989, W. Ian Lipkin and his colleagues were the first to identify a virus, Borna disease virus, in clinical
specimens using purely molecular tools. Borna disease virus has been known for many years as the cause of a
meningoencephalitis of horses and sheep in central Europe. The introduction of sensitive molecular and serologic
assays enabled surveys that indicated a wider geographic and species distribution, and experimental infections in a
wide variety of vertebrates, including chickens, quail, parrots, rats, rabbits, cats, shrews, and nonhuman primates,
indicated a broader host range. The methods have changed over the years, lately adapting probes and microarrays
and deep sequencing. Lipkins work not only characterized this phenotypically unusual virus, but placed it in
its own, new taxon, a new family, Bornaviridae. In 1996, the first evidence that Borna disease virus can infect
humans was reported initial suspicion that it was associated with mental disorders (memory loss, depression,
schizophrenia) have not been substantiated.
Lipkin WI, Travis GH, Carbone KM, Wilson MC. Isolation and characterization of Borna disease agent cDNA
clones. Proc Natl Acad Sci USA. 1990;87:4184-4188.

W. Ian Lipkin

Borna disease virus

thin section electron
microscopy

1989 W. Ian Lipkin, colleagues first identification of a virus in clinical specimens using purely molecular tools

page 493

Primary Human Genome Project Sequencing Sites

(Members of the International Human Genome Sequencing Consortium):

U.S.DOE Joint Genome Institute, Walnut Creek, California, integrating three

genome centers at Department of Energy national laboratories.
Baylor College of Medicine Human Genome Sequencing Center,
Department of Molecular and Human Genetics, Houston, Texas
The Wellcome Trust Sanger Institute, The Wellcome Trust Genome Campus,
Hinxton, Cambridgeshire, United Kingdom
Washington University School of Medicine Genome Sequencing Center,
St. Louis, Missouri

Automated Sample Preparation, Whitehead Institute

Whitehead Institute/MIT Center for Genome Research,

Cambridge, Massachusetts

1990 International Human Genome Sequencing Consortium launching of the Human Genome Project
page 494

Nitza Frenkel

Herpesvirus
scanning electron Human herpesvirus 7 (HHV7) was identified serendipitously in the cells of
microscopy a healthy adult in 1990 by Nitza Frenkel and her colleagues. In infants and

Herpesvirus capsid model

Case of exanthem subitum

caused by human herpesvirus 7

children the virus often acts together with HHV6B causing exanthem subitum
(roseola), but it is the latter virus that is the primary cause of this childrens
disease. Like HHV6B, HHV7 infection is nearly universal antibody prevalence
for HHV7 reaches 75% in 3- to 6-year-old children and 98% in adults. In adults,
clinical manifestations of HHV7 infection have been primarily associated
with reactivation in immunocompromised persons the virus becomes latent
in peripheral blood mononuclear cells, particularly mature CD4+ T cells.
Although HHV7 antigen expressing cells are detectable in a wide number
of other sites, including skin, lungs, salivary glands, mammary glands, liver
and kidney, it is not know if all these tissues are involved in reactivation.
HHV7 DNA has been found in 93% of lesions from pityriasis rosea patients,
although often diagnosis in this adult disease is confounded by the close
genetic relationship between HHV6B and 7. In immunocompromised hosts,
particularly transplant recipients (and in particular bone marrow transplant
recipients), there is increased risk for symptomatic primary or reactivation
disease associated with HHV6B as well as HHV7 infection interstitial
pneumonitis and encephalitis has been recognized, but are not common.
Frenkel N, Schirmer E, Wyatt L, Katsafanas G, Roffman E, Danovich R, June C.
Isolation of a new herpesvirus from human CD4+ T cells. Proc Natl Acad Sci
USA. 1990;87:748-752.
BlackJ, PellettP. Human herpesvirus 7. Rev Med Virol. 1999;9:245-262.

1990 Nitza Frenkel, colleagues discovery of human herpesvirus 7 (HHV7)

page 495

PRRS virus

thin section electron microscopy

Cathrinus Terpstra

James E. Collins

David A. Benfield

Veterinary clinicians in the U.S. in the late 1980s noted the appearance in swine herds of a previously unrecognized disease with a clinical presentation of severe reproductive
losses in late gestation, increases in the number of weak live-born piglets, severe pneumonia in neonatal and nursery pigs, and poor growth and increased mortality (porcine
reproductive and respiratory syndrome, PRRS). A similar clinical syndrome was initially described in Germany in 1990 and spread to other countries in Europe over the
following years. The causative agent remained unknown, leading to the name mystery swine disease in the U.S. and several other names in Europe. Gert Wensvoort,
Cathrinus Terpstra and their colleagues at the Central Veterinary Institute (Lelystad), The Netherlands, were the first to describe a small, enveloped RNA virus as the causative
agent. A similar virus was isolated in the U.S. by James Collins, David Benfield and their colleagues in South Dakota and Minnesota. Comparison of the genomic sequence
of the isolates from Europe and the U.S. indicated that they belonged to the family Arteriviridae, genus Arterivirus, but differed genomically by ~40%. Since then, PRRS
has emerged as the most important disease of swine in the world; in the U.S. annual losses are estimated at $560 million. The virus continues to evolve, producing varying
pathotypes with varying mortality rates. For example, in recent years quite virulent strains have become a major problem in China.
Wensvoort G, Terpstra C, Pol JMA, Lask EA, Bloemraad M, de Kluyver EP, Kragten C, van Butten L, den Besten A, Wagenaar F, Broekhuijsen JM, Moonen PJM, Zetstra T, de
Boer EA, Tibben AhJ, de Jong MF, vanr Veld P, Groenland GJR, van Gennep JA, Voets MTh, Verheijden JHM, Braamkamp J. Mystery swine disease in the Netherlands: the
isolation of Lelystad virus Vet Q. 1991;13:121-130.
Terpstra C, Wensvoort G, Pol JM. Experimental reproduction of porcine epidemic abortion and respiratory syndrome (mystery swine disease) by infection with Lelystad virus:
Kochs postulates fulfilled. Vet Q. 1991;13:131-136.
Benfield DA, Nelson E, Collins JE, Harris L, Goyal SM, Robison D, Christianson WT, Morrison RB, Goryca D, Chladek D. Isolation of swine infertility and respiratory
syndrome virus (isolate ATCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs. J Vet Diagn Invest. 1992;4:117-126.
Benfield DA, Nelson E, Collins JE, Harris L, Goyal SM, Robison D, Christianson WT, Morrison RB, Goryca D, Chladek D. Characteristics of swine infertility and respiratory
syndrome (SIRS) virus (isolate ATCC VR-2332) J Vet Diagn Invest. 1992;4:127-133.

1990 Cathrinus Terpstra, others discovery of porcine reproductive and respiratory syndrome virus (PRRS virus)

page 496

The foundations of systems biology (by any name) goes back before the 19th century and the development of
organismal biology disciplines, but in virology the subject is usually considered to stem from the expansion of
molecular virology and functional genomics in the 1980s and 1990s. That expansion also has roots in the Human
Genome Project, with its massive advance in computational science. Its emergence constitutes a true paradigm
shift, a shift to more holistic thinking; this may seem ironic in that the rise of molecular virology is usually seen to
have led to more and more reductionistic thinking. Systems Biology is a huge subject, not adequately introduced
here, but it is documented by many excellent reviews.
Various authors have defined Systems Biology / Systems Virology so differently that the central theme is diffcult
to isolate: it is an inter-disciplinary subject that focuses on complex physiological / pathological host-virusenvironment interactions, and is usually grounded in high-throughput molecular profiling of pathogen effects
on its host and host response to the pathogen (most often using microarray technology). The subject is rooted in
advances in massive data collection, data storage and computational analyses. It usually includes experimental
perturbation (i.e., data from clinical medicine, or more often from experimental infection in animal models), and
analyses of changes from normal homeostatic life processes. It is usually rather translational in promise, offering opportunities for better diagnostics, therapeutics and vaccines and even personalized medicine. The promise
is to analyze and integrate data from the full hierarchical sequence of biological / physiological systems , from
molecule to environment: DNA a mRNA a protein a
protein interactions a informational pathways a informational networks a cells a tissues, organs, networks of cells a organisms a populations a ecologies, enviThe systems virology paradigm: experimental models and
ronments. The promise is to analyze all the elements of these compartments while they are functioningdata are
technologies are used to generate multidimensional data (e.g.,
to be integrated, graphically displayed, and ultimately subjected to computational meta-analyses. One product
virology, pathology, immunology, ecology data). Analyses of
is seen as very large databases, such as in ViPR and IRD. In this, some authorities see biology / pathology as a
such data, in combination with mathematical modeling, are used
straightforward information science, quite different from hypothesis-driven classical sciences. For example, proto generate comprehensive, integrated, predictive models of
ponents of Systems Biology argue, The reductionist approach has successfully identified most of the components
biological systems and virus-host interactions. Resulting predictions and hypotheses guide subsequent experimental perturbaand many of the interactions among life processes but, unfortunately, offers no convincing concepts or methods to
tions, leading in turn to a deeper understanding of the complex
understand how system properties emerge... the pluralism of causes and effects in biological networks is better adbiological processes involved. These findings are then used by the
dressed by observing, through quantitative measures, multiple components simultaneously (genomics, phenomics,
scientific community to guide development of various disease
epigenomics, transcriptomics, proteomics; metabolomics; etc.), and by rigorous data integration with mathematiprevention and control strategies. From Law GL, et al.
cal models. An integration of the two sciences may take the edge off arguments among various proponents.
Ideker T, Galitski T, Hood L. A new approach to decoding life: systems biology. Annu Rev Genet Hum Genet.
2001;2:343-372.
Trewavas A. A brief history of systems biology. Plant Cell 2006;18:2420-2430.
Aderem A, Adkins JN, Ansong C, Galagan J, Kaiser S, Korth MJ, Law GL, McDermott JG, Proll SC, Rosenberger C,
Schoolnik G, Katze MG. A systems biology approach to infectious disease research: innovating the pathogen-host
research paradigm. mBio 2011;2:e00325-10.
Law GL, Korth MJ, Benecke AG, Katze MG. Systems virology: host-directed approaches to viral pathogenesis and
drug targeting. Nat Rev Microbiol. 2013;11:455-466.
The dissemination of -omics data sets to the community is an important element of systems virology. It is envisioned that
complex virus-host interactions will be unraveled through meta-analyses that rely on advanced computational and mathematical approaches and the integration of large multi-dimensional data sets. Two US NIAID-sponsored Bioinformatics
Resource Centers, ViPR (Virus Pathogen Resource) and IRD (Influenza Research Database), are charged with capturing,
sharing, storing, integrating and visualizing systems-wide high-throughput -omics data sets that detail host responses to
viral infections. Data sets include genomic, transcriptomic, proteomic, metabolomic, and other sets, as well as metadata
that provide users with experimental details needed for data interpretation. Linking metadata to several forms of -omics
data (raw, analysed, modeled) enables ViPR and IRD to support diverse end-user needs. From Law GL, et al.

~1990s> The development of Systems Biology / Systems Virology as a paradigm shift in viral disease research

page 497

In 1977, Carl Woese established the overall phylogenetic structure of cellular life: his graphic product was the now ubiquitous
tripartite tree based upon comparative DNA sequencing of the gene encoding the small subunit of ribosomal RNA (ssrRNA).
This had a monumental effect on evolutionary thought, as well as on our understanding of the emergence of life and the evolutionary processes that followed. Molecular phylogenetics changed the way evolutionary relationships were viewed, ending years
of rather subjective, opinion-based discourse. New focus on the bacteria (and archaea) followed, but soon much was also written
about the viruses and whether or not to place them in the organismal Tree of Life. Meanwhile, separate phylogenies of the various
families of viruses, based solely on genomic sequencing and derived trees (mostly via various Bayesian phylogenetic methods),
were constructed, became pervasive, and settled as part of the basic fabric of virology.
Most viruses, especially RNA viruses, rapidly accumulate genetic variation because of their short generation time, high mutation rate and absence of an error-correction mechanism. The phylogenetic patterns resulting vary greatly with varying transmission routes and dynamics. The patterns are also affected by other pathogenetic, epidemiologic, immunologic, and evolutionary
processes, especially those linked to virulence, tissue tropism, innate and acquired immunity and immune escape. Then, investigators turned to underlying evolutionary drivers. Edward Holmes and his colleagues combined concepts lodged in (1) the
epidemiological concept of infectious disease population dynamics (especially epidemic potentialR0>>1, when more and more
secondary cases follow upon the introduction of a pathogen into a new population); (2) the epidemiological concept of critical
community size, that is the number of individuals in a population required for a pathogen to persist; and (3) the population genetics concept referred to as coalescence theory. The latter seeks key evolutionary processes grounded in changing demographics
over time and place. The goal has been to link viral genome sequence variance (phylogenetic trees) and the epidemiology of the
disease caused by the virus. Of particular interest has been new viral species jumping events (cross-species transmission, zoonotic transmission, host-range extension) and codivergence events (patterns resulting from very long-term association between
virus and host). Understanding the evolution of species jumping transmission is fundamental to understanding the nature of new,
emerging and reemerging viral diseases. Eugene Koonin and his colleagues have carried similar concepts back to the dawn of life
on Earth and the evolutionary role of viruses in organismal evolution. The ultimate origins of the RNA viruses are the subject of
much debate based on a growing mass of comparative genomic sequence data and metadata; in any case, the great diversity and
wide distribution of the RNA viruses indicate that they have existed for millennia, whether tied to animal and human evolution
or not. Our general assumption is that infection, morbidity and mortality due to RNA viruses has been one of the greatest challenges facing vertebrate host species survival and consequent evolution, with many host defense systems having evolved just to
deal with invading microorganisms and viruses. The coevolution of humans and human pathogens continues: Holmes cites four
major transitions in human evolution affecting Darwinian natural selection and requiring new evolutionary adaptations: (a) the
establishment of farming; (b) the development of urbanization; (c) the rise of global travel; and (d) the modern human world,
characterized by new societal dynamics, major changes in land use (e.g., deforestation), climate change, increased immunodeficiency states, et al. Phylogenetic, phylogeographic and phylodynamic studies are providing a very long-term view of the past and,
hopefully, a clearer and clearer view of the future.
Woese CR, Fox GE. Phylogenetic structure of the prokaryotic domain: the primary kingdoms. Proc Natl Acad Sci USA.
1977;74:5088-5090.
Grenfell BT, Pybus OG, Gog JR, Wood JL, Daly JM, Mumford JA, Holmes EC. Unifying the epidemiological and evolutionary
dynamics of pathogens. Science 2004;303:327-332.
Holmes EC. The phylogeography of human viruses. Mol. Ecol. 2004;13:745-756.
Koonin EV, Senkevich TG, Dolja VV. The ancient virus world and evolution of cells. Biol. Direct.2006;1:29-56.
Holmes EC. Evolutionary history and phylogeography of human viruses. Ann Rev Microbiol. 2008;62:307-328.
Koonin EV. The logic of chance: the nature and origin of biological evolution. Upper Saddle River (NJ): FT Press; 2011.
Khnert D, Wu CH, Drummond AJ. Phylogenetic and epidemic modeling of rapidly evolving infectious diseases. Infect Genet Evol.
2011;11:1825-1841.
Poon AF, Walker LW, Murray H, McCloskey RM, Harrigan PR, Liang RH. Mapping the shapes of phylogenetic trees from human
and zoonotic RNA viruses. PLoS One. 2013;8:e78122.

1990s> Development of viral phylogenetics, phylogeography, phylodynamics

page 498

Diversity in RNA virus phylogenetic tree shapesinfluenza A (HA), HIV-1, dengue 1 and Coxsackie A24
viruses, each shaped differently by phylodynamic
processes. The influenza A HA pattern is driven by
antigenic drift (few lineages persist between epidemics). In contrast, the life-long nature of HIV infection
enables rapidly diverging lineages to persist over time.
From Poon AF, et al. Mapping the shapes of phylogenetic trees from human and zoonotic RNA viruses.
PLoS One. 2013;8:e78122.

Phylogenetic / phylogeographic patterns, human

viruses, shaped by: (a) Spatial dynamics; viruses
from different regions, separate clades. (b) Temporal dynamics; viruses from different times, separate
clades. (c) Congruence of host and virus phylogenies,
codivergence. (d) Incongruence of host and virus
phylogenies in place and time, revealing the different time-scales of evolution of host and virus. From
Holmes EC. The phylogeography of human viruses.
Mol. Ecol. 2004;13:745-756.

The World Wide Web (WWW, the Web) is a system of interlinked hypertext documents accessed via the
Internet. The term is often mistakenly used as a synonym for the Internet itself, but the Web is a service
that operates over the Internet, as e-mail does. Web pages may contain text, images, videos, or multimedia;
navigation between pages is via hyperlinks. In 1984 at CERN (the European Organization for Nuclear
Research), engineer and computer scientist Tim Berners-Lee considered the problem of information sharing:
to link and access information of various kinds as a web of nodes in which the user can browse at will. He
was concerned that physicists from around the world who needed to share data had no common hardware or
software with which to do so. He wrote a proposal in March 1989 for a large hypertext database with typed
links, but it generated little interest. He then collaborated with Robert Cailliau, who rewrote the proposal in
a more persuasive style and sought resources within CERN to implement it. It worked. By 1990, Berners-Lee
had built all the tools necessary for a working Web: the HyperText Transfer Protocol (HTTP), the HyperText
Markup Language (HTML), the first Web browser (named WorldWideWeb, which was also a Web editor), the
first HTTP server software (CERN httpd), the first web server (http://info.cern.ch), and the first Web pages that
described the project itself and accessed the CERN telephone directory. However, the system could run only on
NeXT computers; Nicola Pellow created a simple text browser that could run on almost any computer. With all
this at hand, CERN made major investments in the expansion and refinement of the Web and it took off as the
Internet expanded. Berners-Lee is now Director of the World Wide Web Consortium.
Berners-Lee T, Fischetti M. Weaving the web: the original design and ultimate destiny of the World Wide Web
by its inventor. SanFrancisco: Harper; 1999.

Tim Berners-Lee

Visualization of various routes through a portion of the Internet

1991 Tim Berners-Lee development of the World Wide Web (WWW)

page 499

J. Craig Venter

Hamilton Smith

Sequencing Lab, J. Craig Venter Institute

Since the chain termination method of DNA sequencing only yields

short products, longer sequences are obtained by fragmentation,
sequencing and re-assembly of fragment sequences to give the overall
sequence. One way this is done is by shotgun sequencing, which is fast
but requires multiple overlapping reads of the target DNA, obtained
by performing several rounds of fragmentation and sequencing.
Complex computer programs are then used to assemble the overlapping
ends of the reads forming the continuous sequence. This process
uses enormous amounts of computing power to deal with errors and
repetitive sequences; for example, to complete the Human Genome
Project, most of the human genome was sequenced at 12X or greater
coverage. In 1995, shotgun cloning/sequencing was adapted to larger
targets and was used by the Institute for Genomic Research (TIGR) to
sequence the genome of the bacterium Haemophilus influenzae and
then in 2000 by Celera Genomics to sequence the genome of Drosophila
melanogaster. It was then used in the Human Genome Project.
Although shotgun sequencing was the most advanced technique for
sequencing genomes from about 1995-2005, other technologies have
surfaced since then, called next-generation sequencing. These are very
fast but use even more computer power to assemble fragments.

1991 Craig Venter, Hamilton Smith (TIGR), others invention of shotgun cloning/sequencing methods
page 500

SISPA

Gregory Reyes

RDA

Michael H. Wigler

Virus discovery has been aided greatly by the development of sequence independent methodologies for the generation of genomic data and, indeed, quite a few important
viruses have been discovered in recent years solely by the use of such methods (table). The most prominent of these methodologies include representational difference analysis
(RDA) and sequence-independent, single-primer amplification (SISPA), each with several variations. The SISPA method, first developed by Gregory Reyes and Jungsuh Kim in
1991, entails the directional ligation of an asymmetric primer at either end of a DNA molecule. Following several cycles of denaturation, annealing and amplification, minute
amounts of the initial DNA are enriched and then cloned, sequenced, PCR amplified and analyzed. SISPA is especially useful when no nucleotide sequence data are available
and the nucleic acid of interest is present in only very small amounts these are the conditions present in the initial isolation and cloning of previously uncharacterized viral
genomes. When first developed, SISPA was incredibly tedious and took weeks to run, but, again, advances made it much more practicable. The same was the case for RDA
when first developed by Michael Wigler and his colleagues, but again technical improvements over the years have made it more practicable. This technique is based upon
amplifying sequences that are different in two genomic or cDNA samples. Genomes or cDNA sequences from two samples (i.e., a known and unknown virus or an unknown
virus in a clinical specimen) are PCR amplified and differences
Some Examples of Viral Genomes Initially Discovered or Characterized by Molecular Methods
analyzed using subtractive DNA hybridization. Both methods also
employ high-throughput equipment, virus concentration methods
Virus
Disease
Know* Genome
Method
Date
such as ultracentrifugation, and modern assays for detection and
Parvovirus B19
Fifth disease
Yes ssDNA
Molecular cloning and DNA hybridization
1984
C virus
Non-A, non-B hepatitis No ssRNA
Transmission in primates; molecular cloning and immunoscreening
1989
further characterization of products of interest. Both methods also Hepatitis
Hepatitis E virus
Non-A, non-B hepatitis No ssRNA
Transmission in primates; molecular cloning and sequence similarity
1990
Retroviruses
Various
Yes ssRNA/dsDNA Degenerate or consensus primer PCR and sequencing
1990>
are tied to downstream production of large amounts of such
Rotavirus
Gastroenteritis
Yes Seg dsRNA
SISPA
1992
products for diagnostic antigens, nucleic acid probes, etc.
Astrovirus
Gastroenteritis
Yes ssRNA
SISPA and immunoscreening
1993
Reyes GR, Kim JP. Sequence-independent, single-primer
amplification (SISPA) of complex DNA populations.
Mol Cell Probes 1991;5:473-481.
Lisitsyn N, Lisitsyn N, Wigler M. Cloning the differences
between two complex genomes. Science 1993;259: 946-951.

HHV-8, KSHV
GB viruses A and B
GB virus C
TTV (Torque teno)
Bovine parvovirus
Coronavirus V-NL63
Bocavirus
Parvovirus 4

Kaposis sarcoma
Not known
Not known
Not known
Not known
Respiratory disease
Respiratory disease?
Not known

No dsDNA
No ssRNA
Yes ssRNA
No ssDNA
No ssDNA
No ssRNA
No ssDNA
No ssDNA

RDA
Transmission in primates and RDA
SISPA, cloning and sequencing
RDA
SISPA, cloning and sequencing
Cell culture and VIDISCA
Random PCR, cloning, sequencing
SISPA, cloning and sequencing

1994
1995
1996
1997
2001
2004
2005
2005

*Were the investigators searching for a specific type of virus genome?

SISPA, Sequence Independent Single Primer Amplification; VIDISCA, Virus Discovery cDNA AFLP (a SISPA variant);
RDA, Representational difference analysis

1991> Gregory Reyes, Michael Wigler, others development of novel techniques to find uncultivable viruses

page 501

Rebecca Rico-Hesse

Robert Tesh, Rosalba Salas

Robert Ellis Shope (1929-2004)

Short-tailed cane mouse

(Zygodontomys brevicauda)

In 1989, physicians in the state of Portuguesa, Venezuela, became aware of an outbreak of a severe febrile disease, mainly among rural inhabitants of the southern part of
the state, which was characterized by fever, headache, myalgia, sore throat, weakness, anorexia, nausea, vomiting and occasionally convulsions. Many of the patients were
hospitalized because of unremitting fever, weakness, dehydration and hemorrhagic manifestations (epistaxis, bleeding gums, hematemesis, melena and menorrhagia). Between
1989 and 1997, 220 cases were reported with a fatality rate of 33%. The disease is now called Venezuelan hemorrhagic fever (VHF). All age groups and both sexes are affected,
but the highest incidence is in males 15-44 years of age mostly agricultural workers. A new arenavirus, Guanarito virus, was identified as the etiologic agent of the disease.
This was done by Rosalba Salas and her colleagues at the Venezuelan National Institute of Hygiene, working with international colleagues. Its natural reservoirs are the rodent
Zygodontomys brevicauda (cane mouse) and the cotton rat Sigmodon alstoni.
Salas RA, de Manzione N, Tesh RB, Ricco-Hesse R, Shope RE, Betancourt A, Godoy O, Bruzual R, Pacheco ME, Ramos B, Taibo ME, Tamayo JG, Jaimes E, Vasquez C, Araoz F,
Querales J. Venezuelan hemorrhagic fever. Lancet 1991;338:1033-1036.
de Manzione N, Salas RA, Paredes H, Godoy O, Rojas L, Araoz F, Fulhorst CF, Ksiazek TG, Mills JN, Ellis BA, Peters CJ, Tesh RB. Venezuelan hemorrhagic fever: clinical and
epidemiological studies of 165 cases. Clin Infect Dis. 1998;26: 308-313.

1991 Rosalba Salas, Robert Tesh, others discovery of Guanarito virus, the cause of Venezuelan hemorrhagic fever
page 502

Rabies Virus Variants in Terrestrial Reservoir Host Species, United States

Jean S. Smith

Phylogenetic relationship of
rabies virus variants in U.S.
(N gene, 1350 bp)

Rabies virus (genotype 1) is distributed worldwide and is endemic throughout the tropical, subtropical, and temperate regions
of Africa, North and South America, Asia, Europe, and Australia. Within each region virus genotypic variants exhibit different
degrees of host specialization and geographic compartmentalization. North American virus variants in terrestrial carnivores
show significant species-specific geographic distributions (map). The restriction of distinct variants of the virus in single or a few
related mammalian reservoir host species has been only recently appreciated it was made possible through the work of Jean
Smith and her collegues at the U.S. Centers for Disease Control and Prevention (CDC) and Charles Rupprecht and his colleagues,
first at the Wistar Institute and then with Smith at CDC, first by the use of monoclonal antibodies and then more recently by
genomic sequencing of large numbers of virus isolates. Genomic sequence analysis suggests that geographic variants of major
terrestrial carnivore hosts cluster phylogenetically within specific host lineages (skunk-bites-skunk-bites-skunk), without any
regard to the phylogenetic relatedness of the hosts, per se. This has made genotyping and the mapping of geographic spread and
temporal distribution of virus variants valuable in support of wildlife rabies control by bait-delivered vaccination. Genotyping and
the mapping of variants may have started with the U.S. and Canada, but today it is being done all around the world, again yielding
valuable information for prioritizing control programs. It is being done with bat rabies isolates as well, where control may be far off
but understanding the complex natural history in so many bat species is also proving valuable.
Rupprecht CE, Glickman LT, Spencer PA, Wiktor TJ. Epidemiology of rabies virus variants. Differentiation using monoclonal
antibodies and discriminant analysis. Am J Epidemiol. 1987;126:298-309.
Smith JS, Orciari LA, Yager PA, Seidel HD, Warner CK. Epidemiologic and historical relationships among 87 rabies virus isolates as
determined by limited sequence analysis. J Infect Dis. 1992;166:296-307.

Charles E. Rupprecht

Real LA, Childs JE. Spatial-temporal dynamics of rabies in ecological communities. In: Collinge SK, Ray C, editors. Disease ecology
- community structure and pathogen dynamics. Oxford; Oxford University Press; 2006. p. 170-187.

1992 Jean Smith, Charles Rupprecht, others rabies virus genotyping and mapping of wildlife species variants

page 503

Joshua Lederberg

Stephen Morse

Robert Shope

(1925-2008)
(1929-2004)
It was the perfect storm a tempest that may happen only once in a century a noreaster created by so
rare a combination of factors that it could not possibly have been worse... (The Perfect Storm: A True Story
of Men Against the Sea, Sebastian Junger) A transcendent moment nears upon the world for a microbial
perfect storm. Unlike the meteorological perfect storm happening just once in a century the microbial
perfect storm will be a recurrent event. The two events share a common feature; a combination of factors is
the driving force behind each (Microbial Threats to Health, Emergence, Detection and Response, 2003)

Margaret Hamburg

Mark Smolinski

The Convergence Model

1992

1996

2003

Convergence of factors leading to the emergence of an infectious disease.

Microbial Threats to Health, Emergence, Detection and Response.
Washington DC: National Academies Press; 2003.

1992> Joshua Lederberg, Stephen Morse, others the concept of New, Emerging and Re-emerging Infectious Diseases

page 504

Development of the Concept of New, Emerging and Re-emerging Infectious Diseases

The future of humanity and microbes likely will unfold as episodes of a suspense thriller
that could be titled Our Wits Versus Their Genes.
Joshua Lederberg (2000)
With the success of sanitary improvements, antibiotics, vaccines and other drugs in
the twentieth century, public awareness in developed countries drifted away from the
infectious diseases, with attention directed more toward cancer, heart disease and
other chronic diseases. In 1962 McFarland Burnett stated, By the end of the Second
World War it was possible to say that almost all of the major practical problems of
dealing with infectious disease had been solved. An urban legend attributed to U.S.
Surgeon General, William H. Stewart (1921-2008), in 1967 has been misquoted ever
since: It is time to close the book on infectious diseases, and declare the war against
pestilence won. When the HIV/AIDS epidemic emerged in 1981, research funding escalated but was appropriately focused and did not greatly affect interest in other infectious diseases. Then, in 1989, Stephen S. Morse organized a conference in Washington, DC, co-sponsored by the Rockefeller University, the National Institute of Allergy
and Infectious Diseases and the Fogarty International Center: Emerging Viruses: The
Evolution of Viruses and Viral Diseases. There were 200 participants and extensive
press coverage. Concerns were expressed over the apparent complacency of the scientific and medical communities and political leaders toward the threat of epidemics.
Recognizing these concerns, in 1991 the U.S. Institute of Medicine (IOM) convened
a 19-member committee, the Committee on Emerging Microbial Threats to Health,
with Josua Lederberg (1925-2008) and Robert E. Shope (1929-2004) as co-chairs
and Stanley C. Oaks, Jr. as editor. In 1992, the committee issued its report, Emerging
Infections -- Microbial Threats to Health in the United States. A second committee,
co-chaired by Josua Lederberg and Margaret Hamburg, with Mark Smolinski as editor, issued a report in 2003, Microbial Threats to Health Emergence, Detection and
Response. These have been landmark documents, affecting government policy and
public interest, and especially after the terrorism events of 2001 (9/11 and the anthrax
letters) driving large amounts of new research funding. Thus, these two documents
remarkably revitalized the infectious disease sciences, especially medical and comparative virology. At present, some leaders are saying that this emerging disease and
biodefense flag under which infectious disease research has marched for all these
years is aging, losing its luster, while others say it is still valuable, dynamically flexible
as new diseases take center stage in the public eye. Time will tell, as extraordinary
special funding for HIV/AIDS, tuberculosis and malaria presents a counterbalance.
Since the 2003 Microbial Threats report, the IOM has issued more than a dozen
other reports on the subject, most narrowly focused on specific disease threats or
intervention strategies. As with the original two reports, all of these contain formal
recommendations for action. In response, federal agencies have issued research and
response plans and have developed new infrastructure to assure action. There are
several repeating themes in all these driving documents:
1. Raising public and political awareness of new and emerging disease threats, most

clearly by listing recent outbreaks/epidemics having potential for substantial impact. There are long and short lists: most start with HIV/AIDS and include Ebola
hemorrhagic fever, hantavirus pulmonary syndrome, severe acute respiratory
syndrome-SARS, West Nile viral encephalitis, dengue, variant Creutzfeldt-Jakob
disease/bovine spongiform encephalopathy, influenza caused by unusual viral variants, several bacterial diseases, and diseases stemming from biowarfare and bioterrorism. The morbidity and mortality represented in such lists is impressive, indeed;
certainly anchoring public attention.
2. Raising awareness of the diverse factors favoring new disease emergence in this rapidly changing world. There were six factors listed in 1992: Microbial adaptation and
change; Economic development and land use; Human demographics and behavior;
International travel and commerce; Technology and industry; and Breakdown of
public health measures. In 2003 seven more were added: Human susceptibility to
infection; Climate and weather; Changing ecosystems; Poverty and social inequality; War and famine; Lack of political will; and Intent to harm. Various authors have
had their own lists and terms, but they are all quite similar in substance.
3. Raising awareness of the central role of zoonoses as the cause of most emerging
human diseases. Some have raised a new flag, that of One Medicine / One Health,
to emphasize the complexity of viral host range extensions and changing human
disease potential of infectious agents with reservoirs in animals.
4. Raising awareness of the necessity for the complete chain of research and development, from the most basic molecular biology to the most applied clinical / epidemiological sciences. The analysis of the details of past disease emergence episodes,
it is hoped to be able to identify risks of future events and, thereby, better target
surveillance, early detection and public health response actions.
5. Raising awareness of how interrelated all factors favoring disease emergence are.
The best illustration of this is the IOMs Convergence Model (previous page). To
understand a disease emergence event one must assess the impact of all factors
acting in concert. The Black Box at the center of the graphic connotes the complexity and difficulty of understanding the true nature of an infection, an outbreak, an
epidemic. Useful as it is, the graphic suggests nothing about how disease outbreaks
progress, in time and place, and nothing about the timeline and logistical resources
needed for prevention and control actions, locally, nationally and internationally.
6. Raising awareness of the need for better global disease surveillance, global laboratory-based etiologic investigation, global communication systems, and global infectious disease professional community development.
Lederberg J, Shope RE, Oaks SC, editors, and Committee on Emerging Microbial Threats
to Health. Emerging infections: microbial threats to health in the United States. Washington,
DC: National Academy Press; 1992. Smolinski M, Hamburg MA, Lederberg J, editors, and
Committee on Emerging Microbial Threats to Health in the 21st Century. Microbial threats to
health emergence, detection and response. Washington, DC: National Academy Press; 2003.

1992> Joshua Lederberg, Stephen Morse, others the concept of New, Emerging and Re-emerging Infectious Diseases

page 505

The online Program to Monitor Emerging Diseases (ProMED and ProMED-Mail, the name first suggested by Robert Shope)
was established in 1994 under the auspices of the Federation of American Scientists. It was launched largely by the initiative
of Stephen S. Morse, following meetings with an advisory group. Early on, Morse was joined by Jack Woodall and Barbara
Hatch Rosenberg, the three becoming ProMEDs steering committee. Its principal intent was to assist local, national, and
international organizations in disseminating, as rapidly as possible, reports of outbreaks of infectious diseases wherever
they occur; these reports are taken from sources such as media reports, online summaries, local observers, official reports,
and others. ProMED circulates reports on animal, plant and human diseases and toxins from all corners of the globe.
This is presently done through 26 expert moderators, who add guidance and context to the reports. As volunteers, the
moderators are free to act independently, enabling ProMED to play a silent but important role in furthering more government
transparency and even gently coercing governments and international organizations into action. With minimum funding,
the program started with 40 subscribers in 1994. Challenges that confronted the system early on, such as e-mail access in
remote locations, have proven less daunting than anticipated. That seems clear: the service now has over 50,000 subscribers
in more than 180 countries. Since 1999, the service has been housed at the International Society for Infectious Diseases, at
the Harvard School of Public Health. The online system is run by Oracle. In 2009, collaboration with HealthMap.org added
automated map-based geographic information to online reports. Further upgrades in the technology have been underwritten
by Google.org.
Morse SS, Rosenberg BH, Woodall J. Global monitoring of emerging diseases: design for a demonstration program. Health
Policy 1996;38:135-153.
Morse, SS. Global infectious disease surveillance and early warning systems: ProMED nd ProMED-Mail. In: Global infectious
disease surveillance and detection: assessing the challengesfinding solutions, workshop summary, Institute of Medicine,
Forum on Microbial Threats. Washington DC: National Academies Press; 2007.

Stephen S. Morse

ProMEDs
original
Steering
Committee,
1994 (L-to-R):
Stephen Morse,
Jack Woodall,
Barbara Rosenberg

1993> Stephen Morse, others founding of the Program for Monitoring Emerging Diseases (ProMED)
page 506

Stuart T. Nichol

C.J. Peters

Thomas G. Ksiazek,
Cynthia S. Goldsmith

Pierre E. Rollin

In 1993, a new hantavirus disease, an acute respiratory distress syndrome, was recognized in the southwestern region of the USA. Clinical signs included hypoxia, shock and,
in many cases, death. The story of how the etiology of the disease was determined and the virus characterized presents a lesson in modern virologic and epidemiologic science:
as the first patients were identified by clinicians and pathologists in New Mexico, specimens were sent to the U.S. Centers for Disease Control and Prevention. All test results
were negative except for low level serologic signals for the hantavirus, Puumala virus a most unexpected result, given that Puumala virus, from Finland, had never been
associated with such a disease. PCR primers were synthesized to Puumala virus genomic sequences and used as probes on tissues from patients results were positive and new
primers were made specifically to the virus present in the tissues. With new probes, tissues from 10 of 10 patients were positive. As more and more viral genomic sequence
was determined, a bacterial expression system was used to synthesize viral proteins, which were used as antigens for testing humans and animals for the presence of antibody.
Without a virus isolate, within 30 days of the first reported death, the virus had been identified as a new hantavirus, 42 cases had been confirmed, the principal virus reservoir
host had been identified (the deer mouse, Peromyscus maniculatus) and the public was informed on how to minimize risk. Months
later, the virus, named Sin Nombre virus, was isolated from deer mouse tissues by tedious, patient cell culture efforts. In 1996, it
was recognized that many other hantaviruses are also present in rodent populations throughout the western hemisphere, some
proven to be human pathogens, others not.
Nichol ST, Spiropoulou CF, Morzunov S, Rollin PE, Ksiazek TG, Feldmann H, Sanchez A, Childs J, Zaki S, Peters CJ. Genetic
identification of a hantavirus associated with an outbreak of acute respiratory illness. Science. 1993;262:914-917.
Childs JE, Ksiazek TG, Spiropoulou CF, Krebs JW, Morzunov S, Maupin GO, Gage KL, Rollin PE, Sarisky J, Enscore RE, Frey JK,
Peters CJ, Nichol ST. Serologic and genetic identification of Peromyscus maniculatus as the primary rodent reservoir for a new
hantavirus in the southwestern United States. J Infect Dis. 1994;169:1271-1280.
Elliott LH, Ksiazek TG, Rollin PE, Spiropoulou CF, Morzunov S, Monroe M, Goldsmith CS, Humphrey CD, Zaki SR, Krebs JW,
Maupin G, Gage K, Childs JE, Nichol ST, Peters CJ. Isolation of the causative agent of hantavirus pulmonary syndrome. Am J Trop
Sin nombre virus by Cynthia Goldsmith
Med Hyg. 1994;51:102-108.

1993 Stuart Nichol, C.J. Peters, Thomas Ksiazek, others Sin Nombre virus and hantavirus pulmonary syndrome

page 507

Encyclopedia of Virology, Third Edition, five volumes, 2008, 3,234 pages, list price US$1,600.00.
The largest single reference work in virology, organized mini-review articles, covering all
biological, molecular, and medical topics concerning viruses of animals, plants, bacteria
and insects. [This work has also been issued by the publisher as four separate volumes: Desk
Encyclopedia of General Virology, Desk Encyclopedia of Human and Medical Virology, Desk
Encyclopedia Animal and Bacterial Virology, and Desk Encyclopedia of Plant and Fungal Virology,
Mahy BWJ, van Regenmortel MHV, editors, 2009.]

Robert G. Webster

Allan Granoff (1923-2012)

Brian W. J. Mahy

Marc van Regenmortel

Paraphrased from reviews: ...The encyclopedia has the intention of being all-encompassing at a
high level of quality. This is reflected in a large number of specialists (over 640; almost a Whos
Who of virology), coordinated by the two Editors-in-Chief and 12 Associate Editors, who have
all contributed in their areas of special expertise. ...It is amazing that viruses are found as obligate
parasites in cells of practically all branches of the tree of life (Eubacteria, Archaea and Eukarya). The
Encyclopedia presents an enormous body of knowledge in this respect in a very comprehensive way.
...The chapters are alphabetically ordered, frequently according to the names of individual viruses or
virus species, genera, families or orders, interspersed with articles on particular diseases or on general
virology topics. Classification issues have received close attention, following the ICTV 8th Report
on Virus Taxonomy (2005). Glossaries preceding some of the chapters are useful in explaining
terms of specific significance for the chapter. ... This magnum opus represents a tremendous effort in
providing a synopsis of present knowledge in all branches of virology.

Granoff photo courtesy St. Jude Childrens Hospital

1994> Robert Webster, Alan Granoff, Brian Mahy, Marc van Regenmortel Encyclopedia of Virology (three editions)
page 508

Yuan Chang

Patrick S. Moore

With the onset of the AIDS epidemic in the early 1980s,

there was a sudden epidemic resurgence of Kaposi
sarcoma (KS) affecting primarily gay AIDS patients, with
up to 50% of AIDS patients having this tumor. Analysis of
epidemiologic data by Valerie Beral, Thomas Peterman,
Ruth Berkelman and Harold Jaffe led them to propose
that KS was caused by an unknown sexually transmitted
virus that only causes tumors when the person becomes
immunosuppressed, as in AIDS. As early as 1984, it was
reported that by electron microscopy herpesvirus-like
structures were evident in KS tumors from this clue,
more than 20 agents were subsequently described as the
cause of KS, including cytomegalovirus and HIV itself.
The etiologic agent of KS, human herpesvirus 8 (HHV8),
was discovered by Yuan Chang, Patrick Moore, a wife
and husband team then at Columbia University, and their
team, in 1994. They did this by isolating fragments of the
genomic DNA of the virus from a KS tumor in an AIDS
patient. They used representational difference analysis
(RDA), a method based upon comparing DNA fragments
in the KS tumor tissue to unaffected normal tissue from
the same patient. In their initial RDA experiment, they
isolated two small DNA fragments that represented
less than 1% of the viral genome. These fragments
were similar to but distinct from known herpesvirus
genomic sequences, indicating the presence of a new
virus. Starting from these fragments, and working for
two years, they sequenced the entire genome of the virus
and using expression systems made immunoreagents.
Since this discovery, HHV8 has also been associated
with other diseases: primary effusion lymphoma,
multicentric Castlemans disease, angioendotheliomatosis,
angiosarcomas and dermatofibroma.
Beral V, Peterman TA, Berkelman RL, Jaffe HW.
Kaposis sarcoma among persons with AIDS: a sexually
transmitted infection?. Lancet 1990;335: 123-128.
Chang Y, Cesarman E, Pessin MS, Lee F, Culpepper J,
Knowles DM, Moore PS. Identification of herpesvirus-like
DNA sequences in AIDS-associated Kaposis sarcoma.
Science. 1994;266:1865-1869.

Moore PS, Chang Y. Detection of herpesvirus-like DNA

sequences in Kaposis sarcoma in patients with and
Kaposi sarcoma in an AIDS patient; tissue stained with H&E (left) and with a monoclonal antibody directed without HIV infection. N Engl J Med. 1995;332:11811185.
at latent nuclear antigen, a protein consistently expressed in HHV8 infected cells (right)

1994 Yuan Chang, Patrick Moore, colleagues discovery of human herpesvirus 8 Kaposi sarcoma herpesvirus

page 509

Directed genetic manipulation of RNA virus genomes depends on the ability to produce recombinant RNAs which
are accepted as templates by particular viral RNA-dependent RNA polymerases. Transcripts generated by the DNAdependent RNA polymerases most commonly used (e.g., phage T7 RNA polymerase and cellular RNA polymerase II)
are recognized by the polymerases of many positive-strand RNA viruses. This has allowed the recovery of infectious
viruses or replicons from cells transfected with cDNA transcripts this, in turn, has allowed the application of this kind
of recombinant DNA technology to the analysis of virus genomes, the production of mutants for pathogenesis studies,
the use of these viruses as as vectors for expressing foreign proteins, etc. However, the templates of the polymerases
of negative-strand RNA viruses are exclusively viral ribonucleoprotein complexes (RNPs), consisting of the genomic
RNA tightly encapsidated (within N or NP) and associated with a phosphoprotein (P). The RNPs apparently never
disassemble and RNA synthesis does not change the structure of the RNA-nucleoprotein template. That is, the RNP
proteins are actively required for viral gene expression and can be regarded as part of the template. Therefore, since the
genomic RNAs of these viruses do not function as mRNAs all the proteins involved in replication and transcription
have to be provided by other means. This hindered the application of recombinant DNA technology for study of these
viruses and the infections they cause. Success in developing reverse genetics of negative-strand RNA viruses with large,
non-segmented genomes (e.g., filo-, paramyxo- and rhabdoviruses) started with rabies virus and the work of Karl-Klaus
Conzelmann and his colleagues. Their approach was soon extended to many other nonsegmented negative-strand RNA
viruses and to the segmented negative-strand viruses as well. The key to recovering recombinant viruses was the use
of plasmids directing transcription of antigenome (positive-strand, messenger-sense) RNAs for each gene, rather than
genome RNAs. The use of positive-sense RNAs turned out to be successful for recovering infectious cDNA from every
rhabdovirus and paramyxovirus tried.
Schnell M.J., Mebatsion T., and Conzelmann K.K. Infectious rabies viruses from cloned cDNA. EMBO J. 1994;13:41954203.
Conzelmann K.K. Nonsegmented negative-strand RNA viruses: genetics and manipulation of viral genomes. Annu Rev
Genet. 1998;32:123-162.

Karl-Klaus Conzelmann
Some Negative-Strand RNA Viruses
Recovered from cDNA:

Gene order of nonsegmented negative-strand RNA viruses; comparison of representative virus genomes.
Equivalents of the five basic genes are drawn as filled boxes: N (NP in paramyxoviruses; nucleoprotein),
P (phosphoprotein), M (matrix protein), and L (Large; catalytic subunit of the RNA polymerase).
Rabies virus (red) was the first negative-strand RNA virus expressed from cDNA

Rabies virus
Vesicular stomatitis viruses
Measles virus
Rinderpest virus
Human respiratory syncytial virus
Sendai virus (PIV1)
Human parainfluenza virus 3 (HPIV3)
SV5 virus
Bunyamwera virus

1994 Karl-Klaus Conzelmann, colleagues development of reverse genetics for negative-strand RNA viruses
page 510

Only one known case of naturally

contracted Sabi virus infection
has been documented, yet
the virus remains important
due to at least two laboratory
infections that have occurred.
The original natural case of Sabi
virus infection occurred in a
woman in the village of Sabi,
outside of Sao Paulo Brazil, in
1990. In this instance, severe
liver damage led physicians to
an initial diagnosis of yellow
fever. Following the patients
death, the virus was identified as
a then unknown arenavirus. The
viriologist who was responsible
for this identification, however,
contracted the disease; he,
fortunately, survived. Four years
later, at the Yale Arbovirus
Research Unit a visiting research
scientist was exposed to the virus
when a centrifuge tube broke
while he was purifying virus from
cell culture fluid. He became very
ill, but recovered.

The team from the Instituto Adolfo Lutz in So Paulo, Brazil, that discovered Sabi virus
From left to right: Front - Luiza T. M. de Souza, Danya Moyses Fialho, Terezinha Lisieux Moraes Coimbra, Iray Maria Rocco;
Back - Luiz Eloy Pereira, Ivani Bisordi Ferreira, Elza da Silva Nassar, Esther L. Boccato Chamelet, Raimundo Nonato

Coimbra TLM, Nassar ES,

Burattini MN, de Souza LT,
Ferreira I, Rocco IM, da Rosa
AP, Vasconcelos PF, Pinheiro
FP, LeDuc JW, Rico-Hesse R,
Gonzalez J-P, Tesh RB, Jahrling
PB. New arenavirus [Sabi]
isolated in Brazil. Lancet
1994;343:391-392.

Left to right:
Amelia Travassos da Rosa
Francisco Pinheiro
Rebeca Rico-Hesse
James LeDuc
Peter Jahrling
Robert Tesh

1994 Terezinha Lisieux Moraes Coimbra, others Sabi virus and Brazilian hemorrhagic fever

page 511

Keith Murray

director, Australian Animal Health

Laboratory, 1994, with Fred Murphy

Investigating a Hendra virus outbreak

Queensland, Australia

Alex Hyatt

Peter Hooper

Hendra virus

negative contrast electron microscopy

Hendra virus (HeV) was first identified during the first recorded outbreak of the disease that took place in the
Brisbane suburb of Hendra, Australia, in 1994. The index case, a mare, housed with 23 other horses, died after
two days of illness. Subsequently, 19 of the remaining horses became ill and 13 died. Both the trainer and a
stable hand who were involved in nursing the index case fell ill with an influenza-like illness within one week
of the horses death. The stable hand recovered, but the trainer died of respiratory and renal failure. A total of
13 outbreaks of Hendra virus infection have occurred since 1994, all confined to the east coast of Australia,
all involving infection of horses. The case fatality rate in horses has been approximately 75%. Four of these
outbreaks have spread to humans as a result of direct contact with infected horses. Horses have been identified
as intermediate hosts, transmitting infection from reservoir host fruit bats (flying foxes - family Pteropodidae,
genus Pteropus) to humans through close contact during care or necropsy of ill or dead horses. Symptoms of
HeV infection in humans range from mild influenza-like illness to fatal respiratory and neurological disease.
Neurologic disease has been associated with late sequelae (as long as 14 months after initial infection); this has
included persistent convulsions, personality changes and eventually death this resembles subacute sclerosing
panencephalitis caused by persistent measles virus infection.
Murray, K, Selleck P, Hyatt A, Gould A, Gleeson L, Westbury W, Hiley L, Selvey L, Rodwell B, Ketterer P.
A morbillivirus that caused fatal disease in horses and humans. Science 1995;268:94-97.
Murray K, Rogers R, Selvey L, Selleck P, Hyatt A, Gould A, Gleeson L, Hooper P, Westbury H. A novel
morbillivirus pneumonia of horses and its transmission to humans. Emerg Infect Dis. 1995;1:31-33.

1995 Keith Murray, Peter Hooper, Alex Hyatt, colleagues discovery of Hendra virus and its reservoir host fruit bats
page 512

Richard Preston

Richard Preston with

projected image of
Ebola virus in background

Abridged from book reviews: Marburg virus first showed

up in 1967 in a vaccine factory in Germany, and was traced
to cells from African green monkeys. Seven people died, a
quarter of those infected. The first known Ebola outbreak
was in Sudan in 1976. The virus spread rapidly from
village to village, killing half of its victims. Two months
later, an even deadlier strain of Ebola hit Zaire, erupting
simultaneously in some 50 villages, killing nine out of ten
people it infected. Zaires president, Mobutu Sese Seko,
called out his army to seal off the entire zone of infected
villages, with orders to shoot anyone trying to come out.
Prestons account makes these events and the people
who investigated them unforgettable, tracing events back
to individuals with names and faces and stories, not only
the victims but the doctors and scientists willing to risk
their own lives to treat and investigate these mysterious
outbreaks. Centered on the events in Zaire, Preston opens
with a disturbingly graphic description of the meltdown
of a human body invaded by Ebola virus. He describes
the infection as essentially liquifying the body. The story
switches to the United States: in the fall of 1989, a monkeyimporting company with a primate quarantine unit in
Reston, VA (about ten miles from Washington, D.C.),
noted that monkeys were dying at an alarming rate and
with suspicious symptoms. When the cause was found to
be Ebola virus all hell broke loose. The Army quickly took
over, and a team was sent in to halt the spread of the virus.
Random House, 1994
The complicated and hazardous job of entering the monkey
house (the hot zone), killing each monkey, and retrieving
tissue samples is dramatically described. This operation had
to be conducted in secret to avoid public panic. That the
virus, now known as Ebola Reston, turned out not to affect
humans is small comfort: viruses mutate rapidly, and the
rain forests are only a plane ride away. ...A totally convincing
page turner, proving that truth is scarier than fiction.
Preston casts the story as a scientific thriller, which it is.
And he writes in the manner of such popular novelists as
Michael Crichton and Robin Cook, who have made the
strange virus outbreak into a literary convention of hightech, neo-Gothic horror. As a result, this book is hard to put
down, very scary, crammed with the detail that can make
fiction seem real or reality read like fiction. The genre
New York Times #1 nonfiction
Preston has inherited from the fiction writers draws you in
bestseller for more than a year,
by amassing small, even trivial details, and he is a master at
published in more than 30 languages,
this.
sold more then 2.5 million copies

1995 Richard Preston publication of the influential book, The Hot Zone

page 513

Robert Will

James Ironside

Variant Creutzfeldt-Jakob disease, human, left-to-right:

Spongiform change with single and multiloculated vacuoles

varying in size from one to 50 in diameter in the neuropil
of cortical gray matter. H&E.
Florid plaques in the neuropil of cortical gray matter. H&E.
Immunohistochemical staining for PrP; heavy staining of
focus in a lymphoid follicle.
The crystallographic structure of the prion protein (PrP).

John Collinge

Geographical distribution of patients

at onset of symptoms of vCJD, United
Kingdom, through 2009 (n=172)

March 20, 1996: announcement from the UK Spongiform Encephalopathy Advisory Committee
(SEAC) that 10 people may have become infected with the BSE agent through exposure to beef: ...a
previously unrecognized and consistent disease pattern... ...although there is no direct evidence of a
link, on current data and in the absence of any credible alternative, the most likely explanation is that
these cases are linked to exposure to BSE before the bovine offal ban in 1989. This is a cause for great
concern.
Nature, 1996: John Collinge and his colleagues report that PrPsc from patients with variant CJD
has strain characteristics distinct from other types of CJD, but similar to PrPSc from mice, cats and
macaques infected with BSE. ...this is consistent with BSE being the source of this new disease [vCJD].

1996 Robert Will, James Ironside, John Collinge BSE prion, the cause of human variant Creutzfeldt-Jakob disease
page 514

Molecular Guidelines for Establishing

Microbial Disease Causation
A nucleic acid sequence belonging to a
putative pathogen should be present in most
cases of an infectious disease. Microbial
nucleic acids should be found preferentially
in those organs or gross anatomic sites known
to be diseased (i.e., with anatomic, histologic,
chemical, or clinical evidence of pathology)
and not in those organs that lack pathology.
Fewer, or no, copy numbers of pathogenassociated nucleic acid sequences should
occur in hosts or tissues without disease.
With resolution of disease (for example,
with clinically effective treatment), the copy
number of pathogen-associated nucleic
acid sequences should decrease or become
undetectable. With clinical relapse, the
opposite should occur.
When sequence detection predates disease, or
sequence copy number correlates with severity
of disease or pathology, the sequence-disease
association is more likely to be a causal
relationship.

David N. Fredricks

The nature of the microorganism inferred

from the available sequence should be
consistent with the known biological
characteristics of that group of organisms.
When phenotypes (e.g., pathology, microbial
morphology, and clinical features) are
predicted by sequence-based phylogenetic
relationships, the meaningfulness of the
sequence is enhanced.

Flow diagram demonstrating various approaches for the

detection and identification of viruses in clinical samples
using sequence-based technologies

Tissue-sequence correlates should be sought

at the cellular level: efforts should be made
to demonstrate specific in situ hybridization
of microbial sequence to areas of tissue
pathology and to visible microorganisms or to
areas where microorganisms are presumed to
be located.
These sequence-based forms of evidence for
microbial causation should be reproducible.

David A. Relman

Fredricks DN, Relman DA. Sequence-based

identification of microbial pathogens: A
reconsideration of Kochs postulates. Clin
Microbiol Rev 1996; 9:18-33.

1996 David Fredricks, David Relman sequence-based criteria for proof of causation of viral diseases

page 515

Martin S. Hirsch

David Ho

Thomas C. Merigan

The concept and basic studies supporting combination antiretroviral therapy for HIV infection were developed in the laboratory of Martin Hirsch during the late 1980s, and
brought into clinical trials in the 1990s. Drug therapy has evolved ever since, embracing the concept of highly active antiretroviral therapy (HAART) and more recent iterations
of the concept. HAART has been defined in various ways, but it is commonly considered to consist of therapy using at least three anti-retroviral drugs, typically two nucleoside
or nucleotide reverse transcriptase inhibitors (NRTIs) plus a non-nucleoside reverse transcriptase inhibitor (NNRTI) or a protease inhibitor (PI) or a second NRTI. HAART
affords a potent way of suppressing viral replication while attempting to prevent the virus from developing resistance to individual drugs. HAART has been clearly shown to
delay progression to AIDS and prolong life. Presently, there are ~25 drugs and several fixed-dose drug combinations approved for use in HIV/AIDS patients: individual drugs
target many of the critical steps in the HIV replication cycle entry, reverse transcription, integration, and proteolytic processing. Choosing which drugs to use in which
clinical circumstance is complex and depends upon viral load, intercurrent infections, the practicality of each drugs use, etc. In the years following the introduction of HAART
therapy, the death rate from AIDS was reduced by 50 to 80% and changed from a nearly universally fatal illness to what is now often a manageable chronic illness.
Hammer SM, Katzenstein DA, Hughes MD, Gundacker H, Schooley RT, Haubrich RH, Henry WK, Lederman MM, Phair JP, Niu M, Hirsch MS, Merigan TC. A trial
comparing nucleoside monotherapy with combination therapy in HIV-infected adults with CD4 cell counts from 200 to 500 per cubic millimeter. AIDS Clinical Trials Group
Study 175 Study Team. N Engl J Med. 1996;335:1081-1090.

1996 Martin Hirsch, David Ho, Thomas Merigan, others development of HAART therapy for AIDS

page 516

In 1996, Jules Hoffmann and his colleagues discovered the main mechanism
by which fruit flies (Drosophila spp) combat infections. They used flies with
mutations in several different genes, including Toll genes, which encode
seven distinct families of inducible antimicrobial peptides. When they
infected flies with bacteria or fungi, they discovered that Toll mutants died.
They found that the product of the Toll genes were involved in sensing
pathogenic microorganisms and Toll activation was needed for successful
defense against invading pathogens. All were found to act via transmembrane
receptors, called Toll receptors. The discovery that Toll receptors are
essential for innate resistance to infection in flies led to the discovery of the
innate immune sensing functions of the mammalian Toll-like receptors, and
provoked a revolution in immunology. The mammalian Toll family, a dozen
invariant receptors, recognizes evolutionarily conserved microbial/viral
molecules, which activate expression of many different immune-response
genes, many of which regulate T cell responses. [Hoffmann is now studying
antiviral defenses in Drosophila. His findings point to RNA interference as a
potent defense mechanism, along with a STAT-JAK pathway (regarded as an
analogue of the mammalian interferon system)].

Jules A. Hoffmann

Bruce A. Beutler

Diagram from InvivoGen, 2011

In 1998, Bruce Beutler and his colleagues were searching for a receptor that
could bind the bacterial product, lipopolysaccharide (LPS), which can cause
septic shock by overstimulating the immune system. They discovered that
mice resistant to LPS had a mutation in a gene that was quite similar to the
Toll genes of the fruit fly. This Toll-like receptor (TLR) turned out to be the
elusive LPS receptor, which activates a cascade of mediators of inflammation.
These findings showed that mammals and fruit flies use similar molecules
to activate innate immunity. Beutler went on to discover the structure and
function of many of the Toll-like receptors, each detecting different signature
molecules that herald infection. These receptors also mediate severe illnesses,
including shock and systemic inflammation as it occurs in the course of septic
infection. They are also central to the pathogenesis of sterile inflammatory
and autoimmune diseases such as systemic lupus erythematosus.
Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA. The
dorsoventral regulatory gene cassette sptzle/Toll/cactus controls the potent
antifungal response in drosophila adults. Cell 1996;86:973-983.
Beutler B, Eidenschenk C, Crozat K, Imler JL, Takeuchi O, Hoffmann JA,
Akira S. Genetic analysis of resistance to viral infection. Nature Reviews of
Immunology 2007;7:753-766.

Toll-Like Receptors (TLRs) recognize pathogen-associated microbial patterns (PAMPs),

which when stimulated initiate complex signaling cascades that lead to the activation of
transcription factors, such as AP-1, NF-B and IRFs, which in turn induce the secretion of
pro-inflammatory cytokines that direct the adaptive immune response.

Mueller S, Gausson V, Vodovar N, Deddouche S, Troxler L, Perot J, Pfeffer S,

Hoffmann JA, Saleh MC, Imler JL. RNAi-mediated immunity provides strong
protection against the negative-strand RNA vesicular stomatitis virus in
Drosophila. Proc Natl Acad Sci USA. 2010;107:19390-19305.

1996> Jules Hoffmann, Bruce Beutler, others discovery of Toll genes / Toll-like receptors, keys to innate immunity

page 517

The first indication that viruses could be used as transneuronal

markers came in the studies of Albert Sabin in 1938. He
studied the different routes of entry into the CNS of mice
following intranasal instillation by various neurotropic
viruses. Each virus studied produced a unique pattern: e.g.,
pseudorabies virus produced transneuronal infection in
multiple systems (sympathetic, parasympathetic, and sensory
systems); However, it was not until 1983 that Xavier Martin
and Michel Dolivo used pseudorabies virus to map trigeminal
nerve pathways. The current explosion of research in neuronal
pathway tracing started with the work of Henricus (Hans)
Kuypers (1925-1989)and Gabriella Ugolini, who in 1990
showed that herpes simplex virus could be used as a selfamplifying transneuronal tracer. Since then, each neurotropic
virus used has had particular advantages: rabies virus has been
particularly valuable because after its injection into muscles or
nerves it is propagated exclusively by retrograde transneuronal
transfer without infection of unconnected neurons or
supporting glial cells, but it crosses many synapses enroute
to target sites in the brain. Recently, rabies virus mutants
Tracing monosynaptic inputs to single neurons: A
have been used; e.g., a glycoprotein gene deletion mutant has
single neuron in a slice of the cortex of a mouse was
been used which is only capable of infecting a single cell and
electroporated with plasmids encoding fluorophores
jumping across one synapse (first-order synaptic partner); this
and rabies glycoprotein (green). In a complex
Transneuronal tracers: (A) wheat germ
has allowed investigation of local connectivity of neurons.
protocol, after six days the electroporated neuron
agglutinin-horseradish peroxidase (WGAOther neurotropic viruses such vesicular stomatitis and mouse
expressed markers indicating successful transfection
HRP); (B) herpes simplex virus 1 (HSV 1) and hepatitis viruses have been used in mouse and rat models.
and virus infection. Thirty-six neurons were infected
pseudorabies virus (PrV); and (C) rabies virus. The advance of this technology to answer neuro-anatomic,
trans-synaptically by the virus (red). From the work of
With conventional tracers (A), only a small
-physiologic and -pathologic questions has required incredible
James H. Marshel, Takuma Mori1, Kristina J. Nielsen
amount of the marker is transferred from
attention to the details of virus strain and genotype/phenotype,
and Edward M. Callaway: Targeting single neuronal
directly labeled first-order neurons (1) to
amount of virus used, timing, anatomic localization of entry
networks for gene expression and cell labeling in vivo.
second and third-order neurons (2, 3), thereby (in some instances via microinjection) and all specifics of the
Neuron 2010;67:562-574.
limiting usefulness. Viruses function as selfanimal model employed. In the case of rabies, much is being
amplifying markers: however, herpesviruses
learned about the neuropathogenesis of the infection as well,
(B) induce neuronal necrosis (X) and can also the infection that drives the host to the fury that leads to bite
propagate via cell-to-cell spread to local glial transmission.
cells and non-contacted neurons. In contrast,
rabies virus (C) propagates exclusively via
retrograde transneuronal transfer, regardless of Kuypers HG, Ugolini G. Viruses as transneuronal tracers. Trends Neurosci. 1990;13:71-75.
the dose and postinoculation time, and thereby Song CK, Enquist LW, Bartness TJ. New developments in tracing neural circuits with herpesviruses. Virus Res.
is particularly useful in tracing pathways.
2005;111:235-249.
Luo L, Callaway EM, Svoboda K. Genetic dissection of neural circuits. Neuron 2008;57:634-660.
Curanovic D, Enquist L. Directional transneuronal spread of alpha-herpesvirus infection. Future Virol. 2009;4:591-603.
Ugolini G. Rabies virus as a transneuronal tracer of neuronal connections. Adv Virus Res. 2011;79:165-202.

1996 Hans Kuypers, Gabriella Ugolini, others viruses as neuronal pathway tracers in viral pathogenesis research
page 518

PubMed was first released in 1996 as an

experimental database under the Entrez retrieval
system with full access to MEDLINE. The term
experimental was removed the next year. There
were approximately two million PubMed searches
per month by June 1997 current usage typically
exceeds three million searches per day. By 1998, the
PubMed database was redesigned to change the way
information is stored and retrieved, dramatically
increasing the speed of the system. Internet Grateful
Med started using the PubMed system to search
MEDLINE and MedlinePlus began linking to
PubMed for Health Topics. In 2000, PubMed began
linking to PubMed Centrals full-text articles and
the Bookshelf s initial book, Molecular Biology of
the Cell. PubMed continued to grow PubMed
daily statistics for 2014: (a) 3.5 million Web searches
(730,000 users); (b) 5.5 million abstract views (1.7
million users); (c) 5.0 million E-utilities (API format
converter) searches; (d) 75,000 Mobile searches
(18,000 users); (e) > 23 million records with about
500,000 new records are added each year. Of these,
13.1 million of PubMeds records are listed with
their abstracts, and 14.2 million articles have links
to full-text (of which 3.8 million articles are available
full-text for free for any user).

Inauguration of PubMed, 1996

National Center for Biotechnology
Information director David Lipman
(left) coaches Vice President Al Gore
(seated) as he searches PubMed. NIH
director Harold Varmus (center) and
National Library of Medicine director
Donald Lindberg (right) look on. Gore
inaugurated the PubMed search system
at a Capitol Hill press conference.
With helpful prompts from Lipman,
Gore demonstrated the usefulness of
PubMed by performing the first three
searches. At one point, Gore typed in
Should I get a flu shot?

1996 National Center for Biotechnology Information development of PubMed

page 519

Robert G. (Rob) Webster

Kennedy F. Shortridge

A strain of H5N1 influenza virus (highly pathogenic avian influenza virus, HPAI A/H5N1) was
the causative agent of the epidemic that started in China and in 1997 spread to chicken farms
in the New Territories of Hong Kong. Surveillance of retail and wholesale poultry markets in
Hong Kong in late 1997 revealed that almost 20% of chickens and a small percentage of ducks
and geese were infected with this virus. The virus killed tens of millions of birds in China
and Hong Kong and hundreds of millions of others were culled to stem the spread of the
virus. It was considered to be an incipient pandemic situation, the chicken being the source
of virus for humans it was the first instance where a pandemic may have been averted. This virus was exceptional in that it infected humans directly; by 2010 there had been
510 human cases and 303 deaths. The case fatality rate was ~60%, but the bigger worry has been that this virus will continue to mutate and acquire increased transmissibility
characteristics. Indeed, over the period since 1997 the virus has continued to mutate, branching into five clades as it has moved around the world, but its capacity to infect
humans and cause severe disease and death has not changed. There is much speculation, but no consensus, about future virus evolution and its public health consequences.
Shortridge KF, Zhou NN, Guan Y, Gao P, Ito T, Kawaoka Y, Kodihalli S, Krauss S, Markwell D, Murti KG, Norwood M, Senne D, Sims L, Takada A, Webster RG.
Characterization of avian H5N1 influenza viruses from poultry in Hong Kong. Virology. 1998;252:331-342.
Zhou NN, Shortridge KF, Claas EC, Krauss SL, Webster RG. Rapid evolution of H5N1 influenza viruses in chickens in Hong Kong. J Virol. 1999;73:3366-3374.
Li KS, Guan Y, Wang J, Smith GJ, Xu KM, Duan L, Rahardjo AP, Puthavathana P, Buranathai C, Nguyen TD, Estoepangestie AT, Chaisingh A, Auewarakul P, Long HT, Hanh
NT, Webby RJ, Poon LL, Chen H, Shortridge KF, Yuen KY, Webster RG, Peiris JS. Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern
Asia. Nature 2004;430: 209-213.

1997 Robert Webster, others highly pathogenic H5N1 influenza virus and lethal disease in humans in Hong Kong
page 520

Tsutomu Nishizawa

Hiroaki Okamoto

Torque teno virus 1 (TTV1, transfusion transmitted

virus) was discovered in 1997 in the plasma of a
Japanese patient suffering from posttransfusion
hepatitis by Tsutomu Nishizawa, Hiroaki Okamoto
and their colleagues. The virus was initially
discovered by means of representational difference
analysis (RDA) this technology allowed the
development of nucleic acid probes and expressed
proteins for use as immunological reagents.
Remarkably, given the notion that most human
viruses had been discovered by the 1990s, the virus
was found to be extremely common, reaching 100%
prevalence in some countries, and present in ~10%
of blood donors in the U.S. and U.K. Still more
remarkably, the virus was found to be the first of a
very large number of related viruses found in large
numbers in many animal species: chimpanzees,
several species of African monkeys, tamarins, cows,
pigs, cats, dogs, and tree shrews. These viruses
(there are at least 103 of them) have been classified
as a new family, the Anelloviridae, comprising
nine genera. The virions are 19-27nm in diameter,
non-enveloped, with icosahedral symmetry, and
have genomes that are circular, non-segmented,
negative-sense, single-stranded DNA, ~3,0004,000 nucleotides in size. The viruses or genomic
DNA have been found in commercial vaccines for
swine, enzyme preparations such as trypsin, cell
cultures and human drugs containing components
of porcine origin, so their significance as pathogens
is a matter of much ongoing research.

Nishizawa T, Okamoto H, Konishi K, Yoshizawa H, Miyakawa Y, Mayumi

M. A novel DNA virus (TTV) associated with elevated transaminase
levels in posttransfusion hepatitis of unknown etiology. Biochem Biophys
Res Commun. 1997;241:92-97.
Okamoto H, Nishizawa T, Ukita M. A novel unenveloped DNA virus
(TT virus) associated with acute and chronic non-A to G hepatitis.
Intervirology. 1999;42:196-204.

Torque teno virus 1

negative contrast electron microscopy

Okamoto H, Takahashi M, Nishizawa T, Tawara A, Fukai K, Muramatsu

U, Naito Y, Yoshikawa A. Genomic characterization of TT viruses (TTVs)
in pigs, cats and dogs and their relatedness with species-specific TTVs in
primates and tupaias. J Gen Virol. 2002;83:1291-1297.

1997 Tsutomu Nishizawa, Hiroaki Okamoto, colleagues torque teno virus, anelloviruses, new family, Anelloviridae

page 521

Some commercially
available analytes:

An example of microbead-based virus detection technology:

James Malony, Sylvia Chong and their colleagues developed a multiplexed
PCR assay for the detection of 20 different respiratory viruses (types/
subtypes) in a single test. The assay employed 14 virus-specific primer
pairs, followed by a multiplexed Target-Specific Primer Extension (TSPE)
reaction using 21 primers for specific respiratory virus types and subtypes.
TSPE products were sorted and identified by using a fluid microspherebased Luminex array technology. The assay detected influenza A and B
viruses; influenza A virus subtypes H1, H3, and H5 (including subtype
H5N1, Asian lineage); parainfluenza virus types 1, 2, 3, and 4; respiratory
syncytial virus types A and B; adenoviruses; metapneumovirus;
rhinoviruses; enteroviruses; and coronaviruses OC43, 229E, and SARS
coronavirus. They tested their assay using large numbers of diagnostic
nasopharyngeal swab specimens, which were also tested by conventional
methods, including culture they obtained very good sensitivity and
specificity.
Mahony J, Chong S, Merante F, Yaghoubian S, Sinha T, Lisle C, Janeczko
R. Development of a respiratory virus panel test for detection of twenty
human respiratory viruses by use of multiplex PCR and a fluid microbeadbased assay. J Clin Microbiol. 2007;45:2965-2970.

Adenovirus / Human
Cytomegalovirus / Human
Epstein-Barr virus EA / Human
Epstein-Barr virus NA / Human
Epstein-Barr virus VCA / Human
HBV virus Core / Human
HBV virus Envelope / Human
HBV virus Surface (Ad) / Human
HBV virus Surface (Ay) / Human
HCV virus Core / Human
HCV virus NS3 / Human
HCV virus NS4 / Human
HCV virus NS5 / Human
Hepatitis A virus / Human
Hepatitis D virus / Human
Hepatitis E virus orf2 3KD / Human
Hepatitis E virus orf2 6KD / Human
Hepatitis E virus orf3 3KD / Human
Herpes simplex virus 1 gD / Human
Herpes simplex virus 1&2 / Human
Herpes simplex virus 2 gG / Human
HIV1 gp120 / Human
HIV1 gp41 / Human
HIV1 p24 / Human
HTLV-1&2 / Human
Human papillomavirus / Human
Influenza A H3N2 viruses / Human
Influenza A viruses / Human
Influenza B viruses / Human
Measles virus / Human
Mumps virus / Human
Parainfluenza 1 virus / Human
Parainfluenza 2 virus / Human
Parainfluenza 3 virus / Human
Polioviruses / Human
Respiratory syncytial virus / Human
Rubella virus / Human
Varicella zoster virus / Human

Detection and identification of TSPE reaction

products captured onto beads employs proprietary
anti-tag oligonucleotides that hybridize to
complementary tag oligonucleotides. The
microbeads are sorted with a Luminex flow cell
instrument, which identifies spectrophotometrically
colored beads with one laser and a different colored
signal on the beads with a second laser.

1997> Luminex Corporation, others development of the first high throughput viral diagnostics instruments

page 522

Mechanism of RNA interference:

A. On entering the cell, long dsRNA
acts as a trigger of the RNAi
process.
B. It is first processed by the RNase
III enzyme Dicer.
C. Dicer processes long dsRNA
into 21-23 nt siRNA with 2-nt 3
overhangs.
D. The siRNAs are incorporated into
the RISC RNAi effector complex
(Argonaute protein, which cleaves
and discards the sense strand
leading to an active RISC.
E and F. The remaining antisense
strand of the siRNA duplex serves
as the guide strand and guides the
RISC to its homologous mRNA,
resulting in the endonucleolytic
cleavage of the target mRNA.

Andrew Z. Fire and Craig C. Mello

In 1998, Andrew Fire and Craig Mello discovered a mechanism that can degrade mRNA
from a specific gene; this mechanism, RNA interference (RNAi), is activated when doublestranded RNA molecules occur in the cell. Double-stranded RNA activates the RNAi
mechanism which then degrades mRNA molecules with the same sequence as the doublestranded RNA. When the mRNA molecules disappear, the corresponding gene is silenced
and no protein is made. RNAi is of great importance for the regulation of gene expression,
keeps jumping genes under control and participates in defense against viral infections.
RNAi is already being widely used in basic science as a method to study the function of
genes and it is hoped that it will lead to novel therapies in the future. In a series of simple
but elegant experiments, Fire and Mello deduced that RNAi is sequence-specific, that
RNAi can spread between cells and can even be inherited. It worked with tiny amounts of
injected double-stranded RNA, causing Fire and Mello to proposed that RNAi is a catalytic
process. The components of the RNAi machinery were identified during the following
years. Double-stranded RNA binds to a protein complex, Dicer, which cleaves it into
fragments. Another protein complex, RISC, binds these fragments. One of the RNA strands
is eliminated but the other remains bound to the RISC complex and serves as a probe to
detect mRNA molecules. When an mRNA molecule can pair with the RNA fragment
on RISC, it is bound to the RISC complex, cleaved, degraded and silenced. In lower
organisms, such as arthropods, that have no immune system, it seems that RNAi plays a
particularly important role in defense against microorganisms and viruses.
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic
interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998;391:806-811.

1998 Andrew Fire, Craig Mello discovery of RNA interference (RNAi, RNA silencing by dsRNA)

page 523

Sergey Brin and Larry Page

In 1995, Larry Page and Sergey Brin met at Stanford University as graduate
students in computer science. By January of 1996, they began collaborating on
writing a program for a search engine to do back-link analysis. Fueled by rave
reviews for the search engine, they began working on an improvement, named
Google. Operating out of their dorm rooms, the pair built a server network
using cheap, used, and borrowed PCs. They tried to license their search engine
technology for what now seems a pittance, however, they failed to find a buyer
and decided to keep Google and seek financing to improve the product and take
it to the public directly. Co-founder of Sun Microsystems, Andy Bechtolsheim,
after a demo wrote a check for $100,000 and within a few weeks another $900,000
was obtained. In 1998, Google Inc. opened with a beta search engine quickly it
was answering 10,000 search queries per day. By 2010, worldwide, there are over
2 billion searches on Google per day. In addition to running the worlds most
popular search service, Google also runs a number of vertical search services
that are proving to be of great use to virologists, including: Google Book Search
(started in 2004); Google Scholar (2004); Google Maps (2004); Google Trends
(2004); Google Analytics (2007); and Google Universal Search (2007). The last
four of these have contributed to the entry of the company into public health
data analysis. For example, the Global Trends program led to Google Flu Trends,
which uses aggregated search data to estimate current influenza activity around
the world in near real-time. In a project done in collaboration with the Centers
for Disease Control and Prevention, certain search terms were found to be good
indicators of influenza activity as millions of users around the world search for
health information online. A close relationship was found between how many
people search for influenza-related topics and how many people actually have
influenza-like symptoms; results compared closely with traditional flu surveillance
systems and identified hot spots with regional and state-level specificity, all with
significantly less delay than with other systems. This project is being extended to
other infectious diseases, such as West Nile virus infection, respiratory syncytial
virus infection, and avian influenza (internationally).
Ginsberg J, Mohebbi MH, Patel RS, Brammer L, Smolinski MS, Brilliant
L. Detecting influenza epidemics using search engine query data. Nature
2009;457:1012-1014.
Carneiro HA, Mylonakis E. Google trends: a web-based tool for real-time
surveillance of disease outbreaks. Clin Infect Dis. 2009;49:1557-1564.

the flu
Correlation between Google Flu Trends (blue)
and CDC surveillance data (red) for the U.S.
Mid-Atlantic Region from 2004 through 2008.

1998 Larry Page, Sergey Brin development of Google and web-based surveillance tools
page 524

Nancy Cox

Yoshihiro Kawaoka

Robert G. Webster

The molecular determinants that make certain influenza viruses highly pathogenic for mammalian species, including humans, remained poorly understood until the advent
of reverse genetic technology, wherein virulence characteristics of each viral gene from virulent and avirulent isolates could be assessed. The more that was learned, the more
complex the subject became. Influenza virus virulence in mammalian species is a polygenic trait, requiring, as first pointed out by Rudolf Rott and Christoph Scholtissek in the
1960s, a constellation of genes they also realized that viral virulence, host range and transmissibility are distinct genetic characteristics with different genetic linkages. In
1997, when highly pathogenic avian influenza (HPAI - H5N1) virus infected chickens and humans in Hong Kong with lethal effects, efforts were made in many labs, particularly
those of Yoshihiro Kawaoka, Hans-Dieter Klenk, Nancy Cox, Robert Webster and their colleagues, to determine the specific molecular determinants responsible. Determinants
responsible for: (1) receptor-binding and interspecies transmission (per hemagglutinin protein HA), (2) for host range restriction (per RNA polymerase PB2 protein); and (3)
interferon antagonistic activity (per nonstructural protein NS1) were found to correlate with virulence in complex ways. The best known virulence mechanism, discovered
earlier, is that based in the variable cleavability of the HA protein into its two active forms (HA1, HA2). The HA protein is synthesized as a precursor protein and its cleavage
is a prerequisite for fusion of viral and endosomal membranes, a key to viral infectivity in target tissues. The HAs of low pathogenicity viruses have amino acid sequences at
the cleavage site that are recognized by extracellular proteases that are secreted only by cells in the respiratory and intestinal tracts and consequently limit infections to these
organs. By contrast, highly pathogenic viruses have polybasic sequences at the cleavage site that are recognized by ubiquitous proteases that thus can trigger systemic infection.
The acquisition of a highly cleavable HA had been shown to be the key to the conversion of avirulent strains to virulent strains in several avian epidemics and this correlation
was then extended to humans (for example, all the avian viruses that killed people in Hong Kong in 1997 possessed a highly cleavable HA). Specific mutations in the PB2 and
NS1 proteins have also been specifically linked to changes in virus phenotype. More recently, mutations in several other genetic loci have been implicted in increased lethality.
Bender C, Hall H, Huang J, Klimov A, Cox N, Hay A, Gregory V, Cameron K, Lim W, Subbarao K. Characterization of the surface proteins of influenza A (H5N1) viruses
isolated from humans in 1997-1998. Virology 1999,254:115-23.
Suarez DL, Perdue ML, Cox N, Rowe T, Bender C, Huang J, Swayne DE. Comparisons of highly virulent H5N1 influenza A viruses isolated from humans and chickens from
Hong Kong. J Virol. 1998;72:6678-6688.
Hatta M, Gao P, Halfmann P, Kawaoka Y. Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses. Science 2001;293:1840-1842.
Bogs J, Veits J, Gohrbandt S, Hundt J, Stech O, Breithaupt A, Teifke JP, Mettenleiter TC, Stech J. Highly pathogenic H5N1 influenza viruses carry virulence determinants
beyond the polybasic hemagglutinin cleavage site. PLoS One 2010;5:e11826.

1998> Nancy Cox, Yoshihiro Kawaoka, Robert Webster, others molecular basis for influenza virus virulence

page 525

Kaw Bing Chua

Thomas G. Ksiazek

Kenneth Lam Sai Kit

Bryan Eaton

William J. Bellini

Nipah, first episodes

In 1998, an outbreak of acute febrile encephalitis associated with high

mortality was reported in one district of Malaysia, among pig-farmers; this was
followed by outbreaks in three other districts and in Singapore, all traceable
to the movement of pigs. A total of 265 cases of encephalitis with 105 deaths
occurred in Malaysia; this first episode was controlled through the culling of
>1 million pigs. At first Japanese encephalitis was suspected, so strongly that
alternatives were ignored. Then, Kaw Bing Chua, working in the Department
of Medical Microbiology, Faculty of Medicine, University of Malaya, isolated a
virus from serum and cerebrospinal fluid of patients. The virus rapidly caused
syncytia in infected Vero cell cultures. Chua used the infected cells as antigen
on spot slides and did indirect immunofluorescent antibody tests using patient
sera as the source of antibody (along with appropriate controls). In classical
fashion, Chua said, when I looked at the reaction, I could feel a chill going
down my spine. All the patients sera and cerebrospinal fluids had antibodies
that reacted with the infected Vero cells. The infected syncytial cells lighted
up as like green fluorescence lanterns. I immediately called up the Head of
Department I told him that it was most likely a new virus and we needed
international assistance urgently to identify the virus Chua was sent to the
Centers for Disease Control and Prevention (CDC), in Fort Collins, Colorado
(not Atlanta, since the outbreak was still thought to be caused by an arbovirus).
At CDC, Nick Karabatsos ran immunofluorescence assays using the virus and
a panel of typing antibodies against known arboviruses all were negative.
Bruce Cropp examined by electron microscopy thin sections of virus-infected
Vero cells that Chua has brought from Malaysia but had not been able to
examine earlier they saw what appeared to be paramyxovirus nucleocapsids.
Everything was packed up and Chua traveled to CDC in Atlanta, where he
worked with a team led by Brian Mahy, William Bellini and Thomas Ksiazek.
The virus was rapidly identified as a novel paramyxovirus, which reacted
heterologously with antibodies to Hendra virus from Australia. This prompted
a search for the virus among fruit bats, the natural host and reservoir of
Hendra virus. Indeed, the virus was isolated from the urine and saliva of fruit
bats (Pteropus hypomelanus) in Malayia. In following years, outbreaks of
encephalitis occurred in India and Bangladesh and there have been further
outbreaks in Malaysia.
Chua KB, Goh KJ, Wong KT, Kamarulzaman A, Tan PS, Ksiazek TG, Zaki SR,
Paul G, Lam SK, Tan CT. Fatal encephalitis due to Nipah virus among pigfarmers in Malaysia. Lancet. 1999;354:1257-1259.
Chua KB, Bellini WJ, Rota PA, Harcourt BH, Tamin A, Lam SK, Ksiazek TG,
Rollin PE, Zaki SR, Shieh W, Goldsmith CS, Gubler DJ, Roehrig JT, Eaton
B, Gould AR, Olson J, Field H, Daniels P, Ling AE, Peters CJ, Anderson LJ,
Mahy BW. Nipah virus: a recently emergent deadly paramyxovirus. Science.
2000;288:1432-1435.

Nipah virus encephalitis, human, H&E and immunohistochemistry

Chua KB. The discovery of Nipah virus: A personal account. Neurology Asia
2004;9: 59-63.

1999 Kaw Bing Chua, Kenneth Lam, William Bellini, Thomas Ksiazek, others discovery of Nipah virus
page 526

New York
City, 1999

Marcelle Layton

W. Ian Lipkin

Robert S. Lanciotti

In the summer of 1999, an outbreak of encephalitis was detected in New York City, which as it progressed over the following years became more and more significant. One
way to relate the story is with a timeline. August 23-28: Deborah Asnis, chief of infectious diseases at Flushing Hospital in Queens County, New York, recognized neurological
disease in several elderly patients. She notified Marcelle Layton of the New York City Department of Health and sent samples of blood and spinal fluid to the NY State
Department of Health and to the Centers for Disease Control and Prevention (CDC). August 31: CDC sent epidemiologists to New York City to investigate what was then
being called encephalitis of unknown etiology. September 3: CDC completed tests on specimens from the patients; all were serologically positive for St. Louis encephalitis
virus, and CDC announced this as the cause of the outbreak. New York Citys mayor announced a $6 million campaign to wipe out the citys mosquitoes and aerial spraying
started. September 21: based on partial genomic sequencing of a virus isolated from a patient, W. Ian Lipkin and his colleagues at the University of California, Irvine, surmised
that the virus was not St. Louis encephalitis virus but a related virus, either Kunjin or West Nile virus. September 24: Lipkins information was communicated to the New
York State and City Departments of Health and CDC. September 25: CDC scientists reported that the mosquito-borne illness might not be St. Louis encephalitis but West
Nile virus. This was a startling finding as West Nile virus had never before been found in the Western Hemisphere. Further sequencing done by Lipkin and his colleagues and
Jonathan Smith and his colleagues at the U.S. Army Medical Research Institute for Infectious Diseases (USAMRIID) (the latter using virus from bird tissues from the Bronx
Zoo submitted by Tracey McNamara) and by Robert S. Lanciotti and his colleagues at the CDC determined that the disease was caused by a particular genetic lineage of West
Nile virus, one that had likely originated in Israel. Before the first frost and the end of the mosquito transmission season, there had been ~8,200 people infected (~2.6% of the
risk population), with ~1,700 cases of febrile illness, 62 cases of encephalitis, and 7 deaths. There were 25 cases of encephalitis in horses, with ~10 deaths. Concurrently, large
numbers of crows died in the greater New York metropolitan area. In succeeding years, the virus marched across the U.S., ultimately infecting humans, horses and wild birds
in 48 states (not Hawaii or Alaska), and Canada. There were an estimated ~16,000 cases of neuroinvasive disease by 2010. The virus also moved south, spreading across Mexico
and Central America.
Asnis DS, Conetta R, Teixeira AA, Walman G, Sampson BA. The West Nile virus outbreak of 1999 in New York: Flushing Hospital experience. Clin Infect Dis. 2000;30:413-418.
Nash D, Mostashari F, Fine A, Miller J, OLeary D, Murray K, Huang A, Rosenberg A, Greenberg A, Sherman M, Wong S, Layton M. 1999 West Nile Outbreak response
working group. The outbreak of West Nile virus infection in the New York City area in 1999. N Engl J Med. 2001;344:1807-1814.
Briese T, Jia XY, Huang C, Grady LJ, Lipkin WI. Identification of a Kunjin/West Nile-like flavivirus in brains of patients with New York encephalitis. Lancet 1999;354:12611262.
Lanciotti RS, Roehrig JT, Deubel V, Smith J, Parker M, Steele K, Crise B, Volpe KE, Crabtree MB, Scherret JH, Hall RA, MacKenzie JS, Cropp CB, Panigrahy B, Ostlund
E, Schmitt B, Malkinson M, Banet C, Weissman J, Komar N, Savage HM, Stone W, McNamara T, Gubler DJ. Origin of the West Nile virus responsible for an outbreak of
encephalitis in the northeastern United States. Science 1999;286:2333-2337.

1999 Deborah Asnis, Marcelle Layton, W. Ian Lipkin, Robert Lanciotti, others West Nile virus in the United States

page 527

Albert D.M.E. Osterhaus

In 2001, Albert Osterhaus, Bernadette van den Hoogen and their

colleagues in the Department of Virology of the Erasmus Medical
Center, Rotterdam, reported the discovery of a novel virus from
children with respiratory tract illness in The Netherlands. The virus
was detected in respiratory secretions of 28 children, but its genetic
characterization remained a mystery until use of a technique known
as randomly primed PCR (RAP-PCR), which allows identification of
unknown viruses grown in cultured cells. Based on sequence data,
the virus appeared to be closely related to avian pneumovirus and
so was named human metapneumovirus (hMPV). Several lines of
evidence suggested that hMPV was a common human respiratory
pathogen, causing upper respiratory tract infections, bronchiolitis,
and pneumonia; by the age of 5 nearly all individuals had evidence
of hMPV infection. It accounts for approximately 10% of respiratory
tract infections in children and may be the second most common
cause (after respiratory syncytial virus) of lower respiratory
infection in young children. The virus is distributed worldwide and
has a seasonal distribution comparable to that of influenza viruses
during winter. Reinfections with hMPV occur throughout adult life;
infections are generally subclinical or mild in adults but may be more
serious in the elderly, adults with underlying pulmonary disease, and
those who are immunocompromised. Phylogenetic analysis of strains
of hMPV has revealed that the epidemiology of hMPV is complex and
dynamic hundreds of variants have been identified. Unlike influenza
virus, where two or three strains spread around the world each year,
outbreaks of hMPV appear to be a local phenomenon. Strains of
hMPV differ from community to community, and strains identified
in one location may be quite similar to strains identified in other
locations in different years.

Immunohistochemistry of experimental
human metapneumovirus (hMPV)
infection in the cynomolgus macaque.
Immunoperoxidase staining of hMPV
antigens in bronchial epithelium,
especially in the cilia and apical areas of
ciliated epithelial cells.

Human metapneumovirus

negative contrast electron microscopy

(inset) ribonucleoprotein
van den Hoogen BG, de Jong JC, Groen J,
Kuiken T, de Groot R, Fouchier RA, Osterhaus
AD. A newly discovered human pneumovirus
isolated from young children with respiratory
tract disease. Nat Med. 2001;7:719-724.
Kuiken T, van den Hoogen BG, van Riel DA,
Laman JD, van Amerongen G, Sprong L,
Fouchier RA, Osterhaus AD. Experimental
human metapneumovirus infection of
cynomolgus macaques (Macaca fascicularis)
results in virus replication in ciliated epithelial
cells and pneumocytes with associated lesions
throughout the respiratory tract. Am J Pathol.
2004;164:1893-1900.

2001 Albert Osterhaus, Bernadette van den Hoogen, colleagues discovery of human metapneumovirus
page 528

The 2001 anthrax attacks in the United States occurred in two waves over the course of several weeks, beginning one week after the September 11th attacks. Letters containing
anthrax spores were mailed from Princeton, New Jersey, to several news media offices and to two U.S. Senators. There were eleven cases of inhalational anthrax, with five
deaths, and eleven cases of cutaneous anthrax, with no deaths. Seven letters are believed to have been mailed (three of these were inferred from the location of cases),
containing powders of two different qualities, one a crude brownish powder, the other a highly refined powder consisting of nearly pure Bacillus anthracis spores. The ensuing
investigation became one of the largest and most complex in history over the succeeding years FBI investigators interviewed 9,000 people, conducted 67 searches and issued
over 6,000 subpoenas. It is estimated that the cleanup and investigation cost >$1 billion. The analysis of the bacterium isolated from several of the envelopes indicated that
it was the Ames strain, a strain that had been distributed to sixteen labs within the U.S. (and to labs in Canada, Sweden and the United Kingdom). This strain was used most
prominently by the United States Army Medical Research Institute for Infectious Diseases (USAMRIID), Fort Detrick, Maryland. In 2008, the Department of Justice and the
FBI announced that charges were about to be brought against one individual, an employee of USAMRIID, but that he had taken his own life as he was about to be arrested.
In 2010, the government formally concluded the investigation and issued a final report, but there was so much criticism that the National Academy of Sciences was asked to
review the FBIs science and conclusions. In 2011, a report was issued that stated that there was insufficient evidence to support a definitive conclusion about the origin of the
letters mailed in 2011. There has been little progress reported since then. The anthrax attacks, in concert with the September 11th attacks, spurred significant increases in U.S.
government funding for biodefense research. For example, biodefense-related funding at the National Institute of Allergy and Infectious Diseases (NIAID) increased by $1.5
billion in 2003, and in 2004 Project Bioshield was funded, providing $5.6 billion over ten years for stockpiling vaccines and drugs against infectious agents considered most
likely to be used by terrorists. Other civilian federal agencies added considerably to the total expenditure, which reached ~$5 billion per year throughout the decade.
U.S. Department of Justice. Amerithrax investigative summary. 2010. Available from: http://www.justice.gov/amerithrax/docs/amx-investigative-summary.pdf.
Committee on review of the scientific approaches used during the FBIs investigation of the 2001 Bacillus Anthracis mailings. Review of the scientific approaches used during
the FBIs investigation of the 2001 anthrax letters. Washington DC: National Academies Press; 2011.

2001> U.S. Government anthrax bioterrorism events, start of large biodefense research and response programs

page 529

Foot-and-mouth disease epidemic, United Kingdom, 2001

The 2001 epidemic of foot-and-mouth disease (FMD) in the United Kingdom was the most extensive ever recorded
in a developed country and instructive of how the virus can cause incredible economic loss. The 2001 epidemic
started silently on a pig farm illegally feeding unprocessed garbage (food scraps, probably from an imported meat
product); soon 90% of the herd exhibited clinical signs. The disease spread to a slaughterhouse servicing this farm,
and to a nearby sheep farm (the disease is difficult to recognize clinically in sheep), and from there through sheep
markets across the country. Only after the widespread movement of infected sheep was a diagnosis made and
control efforts initiated; by this time, the virus had spread throughout England, Scotland and Wales. Although a
strategic stockpile of vaccine was available, the control campaign relied upon the traditional policy of stamping
out, that is slaughtering all animals on farms where infected animals were identified, and also all animals on farms
where there was suspicion of exposure (called dangerous contact farms), and also on farms that were considered contiguous premises.
In one area, this extended to all animals on premises within a 3 km zone around an infected premises. Stop movement orders also led to
the slaughter of many animals on welfare grounds when feedstuff ran short. The economic consequences of infection within the United
Kingdom were severe, partly grounded in the dilemma between the government wish (a) to achieve disease-free status as quickly as
possible, thereby allowing exports to resume, and (b) to achieve disease control with the minimum impact on the livestock industries and
communities. Clearly, these two ideals came into conflict, with great impact across the country. Since 2001, the British government and
the European Commission have issued new plans to deal with future epidemics, but they are remarkably similar to those in place for many,
many years: they are based on stamping-out of infected and in-contact herds, and on regional restrictions on the movement of susceptible
animals and their products. Provisions are made for the use of emergency vaccination. [abstracted from Gibbs P. The foot-and-mouth

disease epidemic of 2001 in the UK: implications for the USA and the war on terror. J Vet Med Educ. 2003;30:121-132.]

Impact of foot-and-mouth
disease, United Kingdom, 2001:
10 million animals killed
(1-in-8 of all farm animals)
cost 8-13 billion in 2001
(0.8% of GDP)(tourism 3.2
billion, agriculture 2.4
billion, plus 1.5 billion in
2002 and substantial
continuing costs)
10,157 farms, 25% of all farms
adversely affected
7,800 farmers and farm
workers lost their jobs
Estimates at the national level
conceal the extent of the
impact in the hardest-hit
rural areas
[in some instances official data
are are at odds with later studies]

2001 Foot-and-mouth disease epidemic in United Kingdom, destruction of very large numbers of livestock
page 530

Genomic structure of poliovirus 1 (PV1,

strain Mahoney) and strategy for the synthesis
of its full-length cDNA. (A) The positivestranded RNA of poliovirus is shown with
VPg at the 5 end of the non-translated
region (NTR). In the cDNA, VPg is replaced
by the T7 RNA polymerase promoter. The
polyprotein contains one structural (P1) and
two nonstructural (P2 and P3) domains. The 3
NTR contains a heteropolymeric region and is
polyadenylated (shown as AAAn). (B) PV1
cDNA carrying a T7 RNA polymerase
promoter at the 5 NTR end was subdivided
into three large fragments for the synthesis of
full-length cDNA. The sizes of the fragments
are shown. The genome sequence encoded by
each fragment was described separately. (C)
The three DNA fragments were synthesized
and assembled stepwise via common unique
restriction endonuclease cleavage sites to
yield full-length cDNA (F1-2-3 pBR322). The
sequence of the cDNA was confirmed.
Cello J, Paul AV, Wimmer E. Chemical
synthesis of poliovirus cDNA: generation
of infectious virus in the absence of natural
template. Science 2002;297:1016-1018.

Eckard Wimmer
In 2001, Eckard Wimmer and his colleagues, at the State University of New York at Stony Brook, stirred world-wide controversy when they announced that they had made
synthetic poliovirus in a test tube without a natural DNA or RNA template and without the use of living cells. Starting with the genomic sequence that is publicly available
from GenBank, they purchased from a commercial supplier oligonucleotides (averaging 69 nucleotides each) representing the cDNA for the entire virus genome. Given the
state of the art in 2001, the process of ligating the fragments was time consuming, but straight-forward. Once they had assembled the full-length cDNA, as shown above,
they transcribed it with RNA polymerase to make the single-stranded RNA genome of poliovirus. Next, they made infectious virus by incubating the RNA with a cell-free
extract from Hela cells. When injected into mice, the animals developed a neurological disease indistinguishable from that caused by native virus. This result for the first
time demonstrated the feasibility of biochemically synthesizing an infectious agent in the absence of a template. Two bases for controversy emerged: first, as the WHO Polio
Eradication Programme moved forward and it was presumed that one day polio vaccination would cease, the possibility that terrorists might easily synthesize a startup stock
of virus raised the question if vaccination could ever be stopped. Second, was the more theoretical question of whether other pathogens might someday also be synthesized
variola virus, et al. This, in turn, raised the question of whether genomic sequences of pathogens should be published or entered into public databases. The controversy
heated up further when the poliovirus synthesis which took two years of work was followed by Craig Venters announcement that the synthesis of the slightly smaller X174
bacteriophage from its genomic sequence had taken only a matter of weeks. Finally, the NIH, through its Recombinant DNA Advisory Committee (RAC), was forced to add a
new mandate for Institutional Biosafey Committees, that is a manditory review of biosafety considerations before approval of any recombinant DNA experiments.

2002 Eckard Wimmer, colleagues synthesis of poliovirus complementary DNA by assembling oligonucleotides

page 531

Joseph L. DeRisi
(Virochip, 2002)

W. Ian Lipkin

(GreeneChip, 2005)

Until 2002, most molecular methods useful for virus discovery

were dependent upon precise complementarity between the
probes used and their viral targets such methods were not
easily extendable to the discovery of viruses that were not
represented in databases and probe or primer sets, although
some success was had using degenerate primers, etc. From 2002
onward, in the laboratories of Joseph DeRisi at the University
of California San Francisco and Ian Lipkin at Columbia
University, microarray technology employing longer probes
(e.g., >60 nt), which are more tolerant of sequence mismatches
and may detect agents that have only modest similarity to
those already known, evolved into platforms named Virochip
by DeRisi and GreeneChip by Lipkin. Although they differ in
design, both employ random amplification strategies to allow
an unbiased detection of viral and microbial targets. One key
to the success of these arrays was methodology for dealing
with host DNA and RNA sequences which are amplified along
with the viral or microbial sequences in the sample. Still, these
arrays have been most successful with acellular specimens,
such as virus cell culture supernatants, serum, plasma,
cerebrospinal fluid, urine, etc. Usually, hybridization of viral
or microbial sequences in specimens to probes representing
pathogen targets is detected by binding of a fluorescent label;
other readout methods are in development. Virochip and
GreeneChip arrays have had important successes: in a Marburg
hemorrhagic fever outbreak in Africa; in an Ebola virus (variant
Reston) outbreak in pigs in the Philippines; and in the discovery
that the etiologic agent of severe acute respiratory syndrome
(SARS) is a new coronavirus. The DeRisi and Lipkin labs have
established collaborative service systems to help others with
virus and microbial discovery.
Wang, D., L. Coscoy, M. Zylberberg, P. C. Avila, H. A. Boushey,
D. Ganem, and J. L. DeRisi. Microarray-based detection
and genotyping of viral pathogens. Proc Natl Acad Sci USA.
2002;99:15687-15692.
Palacios, G., P. L. Quan, O. J. Jabado, S. Conlan, D. L.
Hirschberg, Y. Liu, J. Zhai, N. Renwick, J. Hui, H. Hegyi, A.
Grolla, J. E. Strong, J. S. Towner, T. W. Geisbert, P. B. Jahrling,
C. Buchen-Osmond, H. Ellerbrok, M. P. Sanchez-Seco, Y.
Lussier, P. Formenty, M. S. Nichol, H. Feldmann, T. Briese, and
W. I. Lipkin. Panmicrobial oligonucleotide array for diagnosis
of infectious diseases. Emerg Infect Dis. 2007;13:73-81.
Lipkin WI. Microbe hunting. Microbiol Mol Biol Rev.
2010;74:363-377.

2002> Joseph DeRisi, W. Ian Lipkin, others application of microarray technology for identification of viruses

page 532

Ian Frazer vaccinating a young woman with HPV vaccine

Human papillomavirus virus-like particles
(VLPs), the basis for HPV vaccine

More than 99% of cervical cancers contain one or more of the

approximately 15 human papillomavirus (HPV) genotypes that
have been associated with the development of cervical cancer.
Approximately 50-60% of these cancers contain HPV16, and
another 10-20% contain HPV18 DNA; hence, these two HPV
genotypes were the focus of vaccine development efforts. For
many years, it was difficult to develop practical papillomavirus
vaccines because these viruses do not grow efficiently in
cultured cells. Even if they did, live attenuated vaccines would
contain viral oncogenes, which would prohibit their use. Ian
Frazer and Jian Zhou started work on a human papillomavirus
vaccine in 1990, using molecular methods to synthesize
virus-like particles (VLPs) that could mimic the virus as an
immunogen. In 1991 Zhous wife, Xiao-Yi Sun, combined two
expressed viral proteins into the first VLPs. Frazer and Zhou
filed for a patent in 1991 and sold partial rights to Merck,
where further development went forward. GlaxoSmithKline
independently used the same VLP-approach to develop a
similar vaccine under licensing of Frazers intellectual property.
It was found that a vaccine based solely on the L1 major capsid
protein was feasible the L1 protein alone folded correctly
and self-assembled into virus-like particles (VLPs) when
expressed in eukaryotic cells. These VLPs not only closely
resemble native virions morphologically, but also induce high
titers of protective antibodies. Since L1 is the only viral protein
required for the self-assembly of VLPs, in 2002 they began
producing it in recombinant yeast and baculovirus systems.
In 2008-2009, the FDA issued licenses to Merck and Glaxo
SmithKline and a drive to vaccinate young women began.
Zhou J, Sun XY, Stenzel DJ, Frazer IH. Expression of vaccinia
recombinant HPV 16 L1 and L2 ORF proteins in epithelial cells
is sufficient for assembly of HPV virion-like particles. Virology
1991;185:251-257.
Kirnbauer R, Booy F, Cheng N, Lowy DR, Schiller JT.
Papillomavirus L1 major capsid protein self-assembles into
virus-like particles that are highly immunogenic. Proc Natl
Acad Sci USA. 1992;89:12180-12184.
Koutsky LA, Ault KA, Wheeler CM, Brown DR, Barr E,
Alvarez FB, Chiacchierini LM, Jansen KU. A controlled trial
of a human papillomavirus type 16 vaccine. N Engl J Med.
2002;347:1645-1651.
Schiller JT, Davies P. Delivering on the promise: HPV vaccines
and cervical cancer. Nat Rev Microbiol. 2004;2:343-347.

2002 Ian Frazer, Sanofi Pasteur, Merck Sharp & Dohme, GlaxoSmithKline development of papillomavirus vaccine

page 533

Max Theiler, 1951

Thomas Weller, Frederick

Robbins, John Enders, 1954

Peter Doherty, Rolf Zinkernagel, 1996

Stanley Prusiner, 1997

Erling Carl Jacob Norrby

Norrby E. A century of Nobel Prizes. Proc Am Philos Soc.

2002;146, No. 4, 2002.
Norrby E. Yellow fever and Max Theiler: the only Nobel
Prize for a virus vaccine. J Exp Med. 2007;204:2779-2784.
Norrby E, Prusiner SB. Polio and Nobel prizes: looking back
50 years. Ann Neurol. 2007;61:385-395.
Norrby E. Nobel Prizes and the emerging virus concept.
Arch Virol. 2008;153:1109-1123.
Norrby E. Nobel prizes and life sciences. Singapore: World
Scientific; 2010.
Norrby E. Nobel prizes and natures surprises. Singapore:
World Scientific; 2013.

Since 2002, Erling Norrby has published a series of papers, and then a book, on the Nobel Laureates whose
discoveries in the field of virology have been seminal in the advance of the science. He has used his experiences
as Permanent Secretary of the Royal Swedish Academy of Sciences from 1997 to 2003, as well as those as a
distinguished virologist and professor and chair of the Department of Virology of the Karolinska Institute, as
sources of information and perspective. The proceedings leading to the award of the Nobel Prizes are secret, but the
archives are opened 50 years after each award it is from these archives that much of the content of these papers
and book have come. Such interesting insight into the views and decisions of those involved! As more and more
Nobel Prizes in virology and related fields reach this 50 year anniversary, it must be hoped that Norrby will continue
with this project.

2002> Erling Norrby papers and books describing the awarding of the Nobel Prizes pertinent to virology

page 534

Eric Leroy and Jean-Paul Gonzalez

Pierre Formenty

Robert Swanepoel

Peter D. Walsh

Walsh PD, Abernethy KA, Bermejo M, Beyers R, De Wachter P, Akou ME, Huijbregts B, Mambounga DI, Toham AK, Kilbourn AM,
Lahm SA, Latour S, Maisels F, Mbina C, Mihindou Y, Obiang SN, Effa EN, Starkey MP, Telfer P, Thibault M, Tutin CE, White LJ,
Wilkie DS. Catastrophic ape decline in western equatorial Africa. Nature 2003;422:611-614.
Leroy EM, Rouquet P, Formenty P, Souquire S, Kilbourne A, Froment JM, Bermejo M, Smit S, Karesh W, Swanepoel R, Zaki SR,
Rollin PE. Multiple Ebola virus transmission events and rapid decline of central African wildlife. Science 2004;303:387-390.
Rouquet P, Froment JM, Bermejo M, Yaba P, Dlicat A, Rollin PE, Leroy EM. Wild animal mortality monitoring and human Ebola
outbreaks, Gabon and Republic of Congo, 2001-2003. Emerg Infect Dis. 2005;11:283-290.

Pierre Rollin

Since the 1990s Ebola virus (strains

Zaire and Cte dIvoire) has been
spreading across the habitat of the
western lowland gorilla, decimating
the highly endangered species, even
possibly threatening its extinction. For
example, in the Democratic Republic of
Congo an epidemic that started in 2002
killed from 3,500 to more than 6,000
individuals, and it continues to spread
through the largest reserves in the
region. In the same region, in the same
epidemic, the disease also killed ~83%
of chimpanzees. As these episodes
continue, preliminary plans have been
made to test and use one or more of the
Ebola virus vaccines currently under
development for human use, but the
feasibility (vaccination via darts or
oral baiting with heat-stable vaccine),
approvals and funding for this pose
substantial challenges.

Bermejo M, Rodrguez-Teijeiro JD, Illera G, Barroso A, Vil C, Walsh PD. Ebola outbreak killed 5000 gorillas. Science. 2006;314:1564.

2003 Peter Walsh, Eric Leroy, others epidemics of Ebola disease in endangered great apes threaten species survival

page 535

Scenes from sequencing centers of the Human Genome Project

The Human Genome Project (HGP) was the largest international collaboration ever undertaken in
biology. The HGP consortium included thousands of scientists worldwide who undertook the immense
task of sequencing the 3 billion bases of the euchromatic human genome, arrayed in its 24 chromosomes.
There were 20 sequencing centers in the United States, Great Britain, France, Germany, Japan, and
China. The five institutions that generated the most sequence were Baylor College of Medicine, Houston;
Washington University School of Medicine, St. Louis; Whitehead Institute/MIT Center for Genome
Research, Cambridge; Department of Energys Joint Genome Institute, Walnut Creek; and The Wellcome
Trust Sanger Institute, Cambridge, England. Between 2000, when the draft sequence was reported,
and 2003, the finished sequence was completed (with an accuracy of 99.99%), with highly contiguous
long runs (with the only remaining gaps corresponding to regions whose sequence cannot be reliably
resolved with current technology centromere, telomere regions, etc.). The estimated number of human
genes dropped below 25,000, according to the analysis of the finished human genome sequence. This
is a third fewer genes than was reported for the draft genome sequence, mostly because of improved
Improvements in the speed of DNA sequencing over the past
discrimination between genes and pseudogenes. The workhorse of the HGP was the automated capillary
30 years and into the future
sequencer, which at the time was the state-of-the-art for throughput and accuracy. Even so, it took
over a year to read a DNA fragment one gigabase (one billion bases, or a third of the human genome) in size at a cost of ~$0.10 per 1000 bases. [By 2008, the worlds largest
sequencing center, the Wellcome Trust Sanger Institute, sequenced the equivalent of 300 human genomes in 6 months, and since then throughput has increased again using
machines that have reduced the cost per base down to ~$0.001 per 1000 bases.] In the HGP, the genome was first broken into conveniently sized chunks, fragments of about
150 kilobases. Each fragment was inserted into a bacterial artificial chromosome (BAC) cloning vector and the DNA fragments were produced in large amounts. The BACs
were then mapped, making re-assembly of the sequenced fragments easier and more accurate. Each of the large clones was then shotgunned broken into pieces of ~1,500
base pairs, either enzymatically or by physical shearing and the fragments were sequenced separately. Computers then analyzed the sequences of the small fragments and
assembled the original sequence of the clone. Finally, the whole genome was assembled and published in open databases.
International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature 2001;409:860-921.
International Human Genome Sequencing Consortium. Finishing the euchromatic sequence of the human genome. Nature. 2004;431:931-945.

2003 International Human Genome Project completion of consensus sequence of the euchromatic human genome
page 536

Carlo Urbani (1956-2003)

Leo Poon

Thomas Ksiazek

Albert Osterhaus

Malik Peiris

Severe acute respiratory syndrome (SARS) emerged in 2003 in Guangdong, China,

and quickly spread to Hong Kong, and from there to 37 countries. By the time the
epidemic ended there had been 8,422 cases and 916 deaths (a case-fatality rate of 10.9%).
Carlo Urbani, the WHO staffer in Hong Kong, became infected in the course of his
epidemiologic investigation and died. Malik Peiris and his colleagues at the University
of Hong Kong discovered a new coronavirus as the cause of the disease, first by growing
it in cell culture, then by electron microscopy. Albert Osterhaus and his colleagues in
Rotterdam proved the etiologic association of the virus and disease using a cynomolgus
macaque (Macaca fascicularis) model. Tissues of palm civets (Paguma spp.) sold as food
in local markets were found to be silently infected with the virus, but later the reservoir
host was found to be bats (Rhinolophus spp.), which also were infected silently.
Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S, Tong S, Urbani C,
Comer JA, Lim W, Rollin PE, Dowell SF, Ling AE, Humphrey CD, Shieh WJ, Guarner J,
Paddock CD, Rota P, Fields B, DeRisi J, Yang JY, Cox
N, Hughes JM, LeDuc JW, Bellini WJ, Anderson LJ.
A novel coronavirus associated with severe acute
respiratory syndrome. N Engl J Med. 2003;348:19531966.
Drosten C, Gnther S, Preiser W, van der Werf
S, Brodt HR, Becker S, Rabenau H, Panning M,
Kolesnikova L, Fouchier RA, Berger A, Burguire
AM, Cinatl J, Eickmann M, Escriou N, Grywna
K, Kramme S, Manuguerra JC, Mller S, Rickerts
V, Strmer M, Vieth S, Klenk HD, Osterhaus AD,
Schmitz H, Doerr HW. Identification of a novel
coronavirus in patients with severe acute respiratory
syndrome. N Engl J Med. 2003;348:1967-1976.

2003 Carlo Urbani, Malik Peiris, Leo Poon, others discovery of SARS coronavirus and the global epidemic

page 537

Bernard B. La Scola

Didier Raoult

Acanthamoeba polyphaga mimivirus

thin section electron microscopy

During routine tests for Legionella bacteria in water from

a cooling tower in Bradford, UK in 1992, a strange new
type of virus was unknowingly isolated by a team led
by Thomas Rowbotham. At first, due to its size, it was
thought to be just another bacterium and was put in the
freezer. Subsequent investigations led to the discovery
of the remarkable Acanthamoeba polyphaga mimivirus
(mimivirus; mimi from mimicking microbe), initially
described by Bernard La Scola, Didier Raoult, JeanMichel Claverie and their colleagues at the Universit
de la Mditerrane, Facult de Mdecine, in 2003. The
virus was shown to infect the protozoan Acanthamoeba
polyphaga. At the time, mimivirus had the largest virion
size and largest genome of any virus known larger
than some bacteria. The genome is 1.18 Mbp in size,
and encodes ~1,262 genes, only ~10% of which encode
proteins of known function. The mimivirus icosahedral
capsid is about 500nm in size. From the capsid, there is
a dense fringe of 125nm long fibers, so the virion has a
total diameter of ~750nm (0.75 m; when stained the
virus is visible in the light microscope). The interior of the
virion appears quite complex, with two lipid membranes
and a double-teardrop-shaped core. Recently, La Scola
and his colleagues discovered of an even larger relative
of mimivirus in a cooling tower in Paris; they named it
mamavirus. This virus was shown to infect the protozoan
Acanthamoeba castellanii. Mimivirus was shown to be
related to viruses infecting certain phytoplankton found
in marine environments the first of these organisms
shown to harbor a virus has been a marine microflagellate
called Cafeteria roenbergensis and the virus cafeteria
roenbergensis virus; it was discovered by Curtis Suttle and
his colleagues. The size and complexity of the mimivirus
genome challenge the established dividing line between
viruses and parasitic cellular organisms, suggesting that
ancient DNA viruses could have given rise to eukaryotic
cells as found in the plant and animal kingdoms. Mimivirus
has also been shown to be a human pathogen, associated
with community and hospital-acquired pneumonia.
La Scola B, Audic S, Robert C, Jungang L, de Lamballerie
X, Drancourt M, Binges R, Claverie JM, Raoult D. A giant
virus in amoebae. Science 2003;299:2033.
La Scola B, Marrie TJ, Auffray JP, Raoult D. Mimivirus in
pneumonia patients. Emerg Infect Dis. 2005;11:449-452.

2003 Bernard La Scola, Didier Raoult, others discovery of mimivirus, at the time the largest virus known
page 538

APDS (Autonomous Pathogen Detection System)

Instrument next to lamppost in Times Square, New York City
Even before the 9/11 and anthrax terrorist attacks in 2001, a team at Lawrence Livermore National Laboratory (LLNL) had been developing what became the Autonomous
Pathogen Detection System (APDS) to provide early warning of aerosol-delivered biothreats. After 2001, its ownership fell to the U.S. Department of Homeland Securitys
BioWatch Program and was linked to the Centers for Disease Control and Preventions Laboratory Response Network. [The manual instrumentation developed before the
automted, autonomous system was called BASIS (Biological Aerosol and Sentry Information System)]. The analytical core of the series of instruments that were built over
the decade was a Luminex assay platform and flow cytometer, eventually to allow detection of many pathogen signatures at once. The program included instrumentation for
continuous air sampling and collection of micro-particulates, biological reagent (signature) development, technology for sample preparation, a system for result analysis, proof
testing, field packaging, communications, maintenance support, and remote monitoring. A requirement was that the system needed to continuously sample the air at a location
of interest over many months and produce regular timely reports on whether a biological threat agent was present. Other operational systems with similar missions were the
U.S. Postal Services Biohazard Detection System and the Department of Defenses Joint Biological Point Detection System (JBPDS). By 2002, units were developed that ran
multiplexed assays unattended for at least a week at a time, with results transmitted via a radio link. By 2004, the APDS was deployed to dozens of classified locations around
the country, and to two acknowledged locations: a Washington, DC metro station and Times Square in New York City. By 2006, in addition to multiplexed immunoassays
the APDS incorporated nucleic acid detection (using a flow-through PCR module and multiple primers developed for each pathogen signature of interest, running through a
Luminex platform). Criticisms have been that only few outdoor and indoor sites are covered, and that by focusing on air sampling other threats are left uncovered (e.g., water,
food, agricultural threat sites). The worst criticisms have concerned the endless jurisdictional turf wars between federal and other government agencies involved.

2003> Lawrence Livermore National Laboratory Autonomous Pathogen Detection System for biothreat agents

page 539

Top: Pierre Formenty,

Tai Forest, Gabon, 1997
Middle: Egyptian
fruit bats, Rousettus
aegyptiacus
Bottom: Marburg
virus reservoir host
investigation, Kitaka
mine and cave, 2007
Right: Robert
Swanepoel, Rousettus
aegyptiacus
From the time of the first known outbreak of Ebola hemorrhagic fever, in Sudan in 1976, bats were
suspected of playing a role in the natural history of the filoviruses. Bats were found under the roof of
the Nzara Cotton Factory where the index case and two other primary cases worked. The index case
of a 1979 outbreak also worked at the Nzara Cotton Factory. In the years following, some effort was
made to study the role of bats as reservoir hosts, usually with negative results, but in 1996 Robert
Swanepoel and his colleagues succeeded in finding evidence of infection in bats after inoculation of
Ebola virus. Then, between 2001 and 2005 Ebola virus was detected by Eric Leroy and his colleagues
at the Centre International de Recherches Mdicales de Franceville, Gabon, in three different bat
species (Hypsignathus monstrosus, Epomops franqueti and Myonycteris torquata) in Gabon and the
Republic of the Congo of 679 bats studied, specific Ebola antibody was found in 16 and viral RNA in
13. In the same region, in the wake of a large outbreak of Ebola hemorrhagic fever, massive migrations
of fruit bats were observed, with native people hunting them as bushmeat, and with index cases found
to have purchased freshly killed bats. In 2007, Marburg virus was detected in fruit bats in Gabon
and Republic of Congo in a study in which liver and spleen samples of 1,138 bats were analyzed
using a Marburg virus-specific real-time RT-PCR assay, four bats, all the Egyptian fruit bat Rousettus
aegyptiacus, were positive.
Leroy EM, Kumulungui B, Pourrut X, Rouquet P, Hassanin A, Yaba P, Dlicat A, Paweska JT, Gonzalez
JP, Swanepoel R. Fruit bats as reservoirs of Ebola virus. Nature 2005;438:575-576.
Towner JS, Pourrut X, Albario CG, Nkogue CN, Bird BH, Grard G, Ksiazek TG, Gonzalez JP, Nichol
ST, Leroy EM. Marburg virus infection detected in a common African bat. PLoS One. 2007;2:e764.
Leroy EM, Epelboin A, Mondonge V, Pourrut X, Gonzalez JP, Muyembe-Tamfum JJ, Formenty P.
Human Ebola outbreak resulting from direct exposure to fruit bats in Luebo, Democratic Republic of
Congo, 2007. Vector Borne Zoonotic Dis. 2009;9:723-728.
Pourrut X, Souris M, Towner JS, Rollin PE, Nichol ST, Gonzalez JP, Leroy E. Large serological
survey showing cocirculation of Ebola and Marburg viruses in Gabonese bat populations, and a high
seroprevalence of both viruses in Rousettus aegyptiacus. BMC Infect Dis. 2009;9:159.

2005> Eric Leroy, Jonathan Towner, others finding that reservoir hosts of Ebola and Marburg viruses are fruit bats
page 540

Jeffery K. Taubenberger

Terrence M. Tumpey

Peter Palese

Beginning in 1995, Jeffery Taubenberger and his colleagues

at the Armed Forces Institute of Pathology obtained gene
sequences of the 1918 influenza virus from formalin-fixed
lung autopsy materials and from frozen lung tissues from
an Alaskan influenza victim buried in permafrost in 1918.
Painstakingly, over ten years the eight viral RNA genome
segments were reconstructed by plasmid-based reverse
genetics; that is, viral RNA was isolated (from fragments
under 200 nucleotides in length), then each fragment was
reverse transcribed into cDNA and amplified by RT-PCR.
The cDNAs were inserted into plasmids and amplified in
bacteria and then sequenced and assembled for infection
of cells in culture. When the reconstructed virus obtained
from the cell cultures was analyzed, it was found that all
eight genome segments were of avian derivation and all
differed in important ways from all other human viruses.
When the reconstructed virus was used to infect mice it was
extremely virulent it replicated to very high titer and was
much more lethal than contemporary viruses. Reassortant
viruses containing mixtures of genes from the 1918 virus
and other virus strains indicated that virulence was largely
based in the hemagglutinin gene, but was polygenic. Some
researchers had predicted that the 1918 virus would not be
more pathogenic than other human influenza viruses, that
its massive human toll in 1918 was the result of secondary
bacterial pneumonias and the absence of antibiotics, perhaps
along with the stress from World War I. The death of mice by
the reconstructed 1918 virus makes that scenario less likely.
Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning
TG. Initial genetic characterization of the 1918 Spanish
influenza virus. Science 1997;275:1793-1796.
Reid AH, Taubenberger JK, Fanning TG. Evidence of an
absence: the genetic origins of the 1918 pandemic influenza
virus. Nat Rev Microbiol. 2004;2:909-914.

Adolfo Garcia-Sastre
Reconstructed 1918 influenza
virus, negative contrast
electron microscopy
Cynthia Goldsmith, CDC

The nucleotide sequence of

the eight RNA segments was
obtained using random primers,
reverse transcriptase and PCR.
cDNAs were constructed from
synthetic oligonucleotides and
the cDNAs cloned in plasmids.
Cells were transfected with
the plasmids plus four helper
plasmids and influenza virus was
rescued and used to infect mice.

Tumpey TM, Garca-Sastre A, Taubenberger JK, Palese P,

Swayne DE, Basler CF. Pathogenicity and immunogenicity of
influenza viruses with genes from the 1918 pandemic virus.
Proc Natl Acad Sci USA. 2004;101:3166-3171.
Tumpey TM, Basler CF, Aguilar PV, Zeng H, Solrzano A,
Swayne DE, Cox NJ, Katz JM, Taubenberger JK, Palese P,
Garca-Sastre A. Characterization of the reconstructed 1918
Spanish influenza pandemic virus. Science 2005;310:77-80.

2005 Jeffery Taubenberger, Terrence Tumpey, others 1918 influenza virus sequenced and virus reconstructed

page 541

Renaud Mahieux

Antoine Gessain

Phylogenetic
relationships of
HTLV1/STLV1,
HTLV2/STLV2,
STLV3/HTLV-3
per 6812 bp gagpol-env-tax of
HTLV3
(PTLV=primate
T-lymphotropic virus)

The discovery of close homologies between human T-lymphotropic virus 1 (HTLV1) and simian T-lymphotropic virus 1 (STLV1) subtypes (in a quite complex phylogeny),
led to the demonstration that most HTLV1 subtypes arose from interspecies transmission between monkeys and humans. Since a great number of STLV viruses had been
discovered in many species of monkeys in several countries in East, Central, and West Africa, in very diverse habitats, that it seemed likely that at least some of these viruses
would also have been transmitted to humans. Antoine Gessain, Renaud Mahieux and their colleagues of the Institut Pasteur in Paris investigated whether there might be
other human T-lymphotropic retroviruses and whether they might be pathogenic. This was technically difficult, since there could be no specific molecular probes, primers or
immune-reagents to search for unknown viruses. In 2005, they detected two new viruses in clinically normal people from Cameroon and named them HTLV3 and 4. [These
names are confusing: HTLV-III had been used for HIV early on, and HTLV-IV had been used to describe HIV2.] Sequencing showed that HTLV3 is a close homolog of STLV3,
but HTLV4 is not related to any known STLV. As part of their approach, they used a series of PCR assays that were designed to amplify all known HTLVs and STLVs. They also
used HTLV1 and 2 serologic tests (ELISA and western blot) results suggested that the new viruses are widespread but their prevalence is low. There was a substantial number
of indeterminate western blot test results, suggesting that there are more HTLVs yet to be discovered, of course with undetermined human pathogenicity.
Mahieux R, Gessain A. Les rtrovirus humains HTLV-3 et HTLV-4: nouveaux membres de la famille des HTLV [New human retroviruses: HTLV-3 and HTLV-4]. Med Trop
(Mars). 2005;65:525-528.
Calattini S, Chevalier SA, Duprez R, Bassot S, Froment A, Mahieux R, Gessain A. Discovery of a new human T-cell lymphotropic virus (HTLV-3) in Central Africa.
Retrovirology 2005;2:30.
Mahieux R, Gessain A. The human HTLV-3 and HTLV-4 retroviruses: new members of the HTLV family. Pathol Biol (Paris). 2009;57:161-166.

2005 Renaud Mahieux, Antoine Gessain discovery of human T lymphotropic viruses 3 and 4
page 542

Hepatitis C virus infection of a hepatoma cell line; immunofluorescence

Takaji Wakita

Charles M. Rice

After the discovery of hepatitis C virus (HCV) in 1988, and the rapid development of
nucleic acid-based diagnostic tests which allowed exclusion of the virus from the blood
supply, further progress was greatly inhibited by the inability to grow the virus in cell
culture. Then, in 2005 there were great breakthroughs in the labs of Frank Chisari at the
Scripps Research Institute and Charles Rice at Rockefeller University. The breakthroughs
stemmed from the use of an unusual isolate of HCV that was discovered in 2001 by
Takaji Wakita and his colleagues at the Tokyo Metropolitan Institute for Neuroscience
a replicon made from the nonstructural regions of this virus, when transfected into
hepatoma cells, was found to support virus replication; higher titers were obtained by
further optimizing hepatoma cell lines to be maximally compatible with the replicon. Full
length infectious clones were produced and drug targets identified, but since all this was
done with only one of the six major HCV genotypes, chimeras had to be produced to
capture the specificities of the other genotypes. Based on these advances, an HCV vaccine
now for the first time seems feasible.
Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M, Zhao Z, Murthy K, Habermann
A, Krusslich H-G, Mizokami M, Bartenschlager R, Liang TJ. Production of infectious
hepatitis C virus in tissue culture from a cloned viral genome. Nat Med. 2005;11:791-796.
Lindenbach BD, Evans MJ, Syder AJ, Wlk B, Tellinghuisen TL, Liu CC, Maruyama T,
Hynes RO, Burton DR, McKeating JA, Rice CM. Complete replication of hepatitis C virus
in cell culture. Science 2005;309:623-626.
Zhong J, Gastaminza P, Cheng G, Kapadia S, Kato T, Burton DR, Wieland SF, Uprichard S,
Wakita T, Chisari FV. Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci USA.
2005;102:9294-9299.
Moradpour D, Penin F, Rice CM. Replication of hepatitis C virus. Nat Rev Microbiol.
2007;5:453-463.

2005 Takaji Wakita, Charles Rice, others in vitro replication of hepatitis C virus by manipulation of virus and cells

page 543

Roche/454 workstation the bright

dots on the screen are individual
sequencing reactions occurring in the
wells in a PicoTiter plate. If the entire
plate were full, there would be about
1.2 million bright dots.

Jonathan M. Rothberg

Jonathan Rothberg is the inventor of massively parallel

DNA sequencing; he founded 454 Life Sciences
Corporation, later acquired by Roche Diagnostics, and
other companies, that have commercialized technologies
for DNA sequencing that have significantly reduced
the cost and increased throughput. The 454 technology
has been considered one of the great breakthroughs in
molecular biology. He has also invented other futuristic
technologies: an instrument for sequencing on a chip with
genomic information fed directly to digital information
analysis software; the first non-bacterial cloning system;
etc.

Margulies M, Egholm M, Altman WE,

Attiya S, Bader JS, Bemben LA, Berka
J, Braverman MS, Chen YJ, Chen Z,
Dewell SB, Du L, Fierro JM, Gomes
XV, Godwin BC, He W, Helgesen S,
Ho CH, Irzyk GP, Jando SC, Alenquer
ML, Jarvie TP, Jirage KB, Kim JB,
Knight JR, Lanza JR, Leamon JH,
Lefkowitz SM, Lei M, Li J, Lohman
KL, Lu H, Makhijani VB, McDade KE,
McKenna MP, Myers EW, Nickerson
E, Nobile JR, Plant R, Puc BP, Ronan
MT, Roth GT, Sarkis GJ, Simons JF,
Simpson JW, Srinivasan M, Tartaro
KR, Tomasz A, Vogt KA, Volkmer GA,
Wang SH, Wang Y, Weiner MP, Yu
P, Begley RF, Rothberg JM. Genome
sequencing in microfabricated highdensity picolitre reactors. Nature.
2005;437:376-380.
Rothberg JM, Leamon JH. The
development and impact of 454
sequencing. Nat Biotechnol.
2008;26:1117-1124.

Sample Input and Fragmentation

A variety of starting materials including
genomic DNA, PCR products, BACs and cDNA
may be used. Large DNAs are fractionated into
small, 300- to 800-basepair fragments.
Library Preparation
Short adaptors specific for both the 3 and 5
ends are added to each fragment. The adaptors
are used for purification, amplification, and
sequencing steps. Single-stranded fragments
with A and B adaptors compose the sample
library.
Bead Technology
The single-stranded DNA library is
immobilized onto DNA capture beads. Each
bead carries a unique single-stranded DNA
library fragment. The beads are emulsified (the
water phase contains PCR reaction ingredients)
resulting in isolation of each reaction bead in its
own lipid micelle, thereby excluding competing
or contaminating sequences.
Emulsion PCR Amplification
Each DNA fragment is amplified within its own
lipid micelle bead microreactor resulting in a
copy number of several million per bead. This is
done in a massively parallel instrument. Then,
the emulsion is removed and the amplified
fragments remain bound to their specific beads.
Sequencing
The clonally amplified DNA fragments are
enriched and loaded into a liquid-handling
workstation (e.g., PicoTiterPlate, which
has wells of a diameter that allow only one
bead per well). The sequencing reactions
(pyrosequencing by primer extension) are read
in the hundreds of thousands of wells using
chemiluminescent signals recorded by a CCD
camera-microscope.
Data Analysis
The positional information generated across
the plate device allows software to determine
the sequence of ~1,200,000 individual reads
every 10-hour run. Sequencing data analysis
and other bioinformatics tools support de novo
assembly of up to 400 megabases.

2005 Jonathan Rothberg, 454 Life Sciences (Roche) development of 454 pyrosequencing
page 544

Tomteboda Home of European Centre for Disease Prevention and Control

(ECDC), Stockholm, Sweden

The European Centre for Disease Prevention and Control (ECDC) was established in 2005. It is an EU agency aimed at strengthening Europes defenses against infectious
diseases. It is seated in Stockholm, Sweden. Its mission is to identify, assess and communicate current and emerging threats to human health posed by infectious diseases.
ECDC works in partnership with national health protection bodies across Europe to strengthen and develop continent-wide disease surveillance and early warning systems. By
working with experts throughout Europe, ECDC pools Europes health knowledge to develop authoritative scientific opinions about the risks posed by current and emerging
infectious diseases. The Centre collects, collates, evaluates and disseminates relevant scientific and technical data; provides scientific opinions and scientific and technical
assistance including training; provides timely information to the European Commission, the Member States, Community agencies and international organizations active
within the field of public health; coordinates European networking of organizations operating in the public health; and exchanges information, expertise and best practices, and
facilitates the development and implementation of joint actions. What is does not do directly is laboratory work and research; instead it relies on each member country to carry
out these activities. [This is quite different from the work of the U.S. Centers for Disease Control and Prevention, where research is an integral part of all activities.]

2005 Founding of the European Centre for Disease Prevention and Control (ECDC)

page 545

Tobias Allander

Human bocavirus:
(top) negative contrast electron
microscopy, colorized;
(bottom) model reconstruction
of virion from cryo-electron
microscopic images

Human bocavirus (HBoV) was discovered in 2005 by

Tobias Allander and his colleagues at the Karolinska
University Hospital in Stockholm. Since the first report,
the virus has been shown to occur worldwide. The
virus was the first to be identified by a procedure based
on DNase treatment of clinical specimens, random
PCR amplification and cloning, followed by large scale
sequencing and bioinformatic analyses the virus has
not been grown in cell culture or experimental animals
it is only known from its DNA sequence. By its genomic
organization and sequence homology, the virus has
been classified in the family Parvoviridae, subfamily
Parvovirinae, the first member of the genus Bocavirus.
There are now four genotypes of the virus, types 1 to 4.
The fact that HBoV was first detected in nasopharyngeal
aspirates of children with respiratory tract infections
suggested that the virus may be etiologically associated
with such infections. In the first study this hypothesis
was supported, but proving it has been difficult. Most
evidence indicates that the virus is associated with
clinical signs of acute respiratory tract infection, but
because the virus is only detected by PCR and because
the virus persists after primary infection for a much
longer period than other respiratory viruses (in some
cases for months), it cannot be certain from prevalence
data alone just when the virus is the cause of the acute
infection of interest and when it is just a confounding
finding. This is also making it difficult to prove an
etiologic association of the virus with gastroenteritis
and lower respiratory infections. This problem is being
resolved with serologic studies using as antigen viruslike particles (VLPs) derived from expressed major virus
capsid protein cloned into a baculovirus vector and
transfected into insect cells.
Allander T, Tammi MT, Eriksson M, Bjerkner A,
Tiveljung-lindell A, Andersson B. Cloning of a human
parvovirus by molecular screening of respiratory tract
samples. Proc Natl Acad Sci USA. 2005;102:12891-12896.
Allander T, Jartti T, Gupta S, Niesters HG, Lehtinen
P, Osterback R, Vuorinen T, Waris M, Bjerkner A,
Tiveljung-Lindell A, van den Hoogen BG, Hyypi T,
Ruuskanen O. Human bocavirus and acute wheezing in
children. Clin Infect Dis. 2007;44:904-910.
Allander T. Human bocavirus. J Clin Virol. 2008;41:29-33.

2005 Tobias Allander, colleagues discovery of human bocavirus (parvovirus)

page 546

Laura H. Kahn

Bruce Kaplan

Thomas P. Monath

The One Health Initiative is a movement to forge collaborations between physicians, veterinarians and other professionals, all of whom recognize the merit of collaboration
at the interface of medicine, veterinary medicine, public health and related fields, all with the purpose of advancing the understanding, prevention and control of disease. The
concept, first called One Medicine, originated in the writings of Rudolf Virchow and William Osler in the mid-1800s, and was resurrected by Calvin Schwabe in the 1960s,
only to lose visibility by the 1990s. The concept, now called One Health, was again resurrected, this time in 2006 by Laura Kahn, Bruce Kaplan, Thomas Monath and Jack
Woodall. The Initiative now has a website, newsletter, publications, educational activities, and the endorsement of many organizations, institutions and individuals.
Schwabe CW. Veterinary medicine and human health. Baltimore: Williams & Wilkins; 1964.
Kahn LH, Kaplan B, Steele JH. Confronting zoonoses through closer collaboration between medicine and veterinary medicine (as one medicine). Vet Ital. 2007;43:5-19.
Kahn LH, Kaplan B, Monath TP, Steele JH. Teaching one medicine, one health. Am J Med. 2008;121:169-170.

2006 Laura Kahn, Bruce Kaplan, Thomas Monath, others development of the One Health / One Medicine Initiative

page 547

Attendees at an Extended Evolutionary Synthesis Workshop, 2008: From left to right: Sergey Gavrilets, Stuart Newman, David Sloan
Wilson, John Beatty, John Odling-Smee, Michael Purugganan, Greg Wray, David Jablonski, Marc Kirschner, Ers Szathmary, Gnter
Wagner, Werner Callebaut, Eva Jablonka, Gerd Mller, Massimo Pigliucci, Alan Love.
The modern evolutionary synthesis (yellow oval) was a union of ideas from several biological fields which provided a widely accepted account of evolution; the concept
evolved throughout the 1930s-1940s, and added to the concepts originated by Charles Darwin (e.g., common descent, natural selection cyan oval). In this era before the
dawn of molecular biology, it added ideas from population genetics, experimental Mendelian genetics, paleontology, botany and ecology the term was coined by Julian
Huxley in his book, Evolution: The Modern Synthesis (1942). Other major figures in the modern synthesis included Theodosius Dobzhansky, R. A. Fisher, J.B.S. Haldane,
Sewall Wright, E.B. Ford, Ernst Mayr, Bernhard Rensch, Sergei Chetverikov, George Gaylord Simpson, and G. Ledyard Stebbins. The extended evolutionary synthesis
(magenta oval) was elaborated in ~2007 to reflect more recent research, especially derived from the molecular biology revolution (genomics and other -omics), genetic study
of natural populations, phylogenetics, species-level stasis and punctuated evolution, and developmental biology (evo-devo evolution via genetic changes in developmental
processes). It is largely additive, not undoing any of Darwins central concepts or any concepts from the earlier modern
evolutionary synthesis. The new concepts include evolvability, developmental plasticity, phenotypic and
genetic accommodation, punctuated equilibrium, phenotypic innovation, facilitated variation, epigenetic
inheritance, and multi-level selection. The new concept has paid little attention to the viruses, but
phenomena studied widely in virology are part of its substance: recombination, horizontal gene
transfer, genetic drift and shift, etc. Horizontal gene transfer (the non-genealogical transmission
of genetic material from one organism to another) is now known to be an important force
driving the evolution of bacteria and archaea, as well as that of unicellular eukaryotes again,
many evolutionary scientists seem to ignore the viruses and their great role in horizontal
gene transfer (not so much their central role as vectors of gene transfer, but targets of gene
transfer). Overall, the extended evolutionary synthesis has been quite controversial, especially
in trying to integrate its elements into the Darwinian foundation for example, how important
is horizontal gene transfer or recombination in the evolution of vertebrates? Does horizontal
gene transfer challenge the construction of phylogenetic trees of higher organisms? How
does horizontal gene transfer challenge the traditional view of evolution as a gradual process of
variation?

Pigliucci M, Mller GB, editors. Evolutionthe extended synthesis. 2nd ed. Cambridge: MIT Press; 2010.

2007 Massimo Pigliucci, Gerd Mller, others development of the Extended Evolutionary Synthesis
page 548

Two human polyomaviruses, BK virus and JC virus, were discovered

and associated with serious diseases, including polyomavirus
nephropathy and progressive multifocal leukoencephalopathy, more
than four decades ago. Only recently has the list been expanded by the
identification of three new viruses (and a fourth, not yet associated
with disease). Viral genomes cloned from DNA sequences have been
designated KI polyomavirus in work from the lab of Tobias Allander
at the Karolinska University Hospital in Stockholm; WU polyomavirus
in work from the lab of David Wang at Washington University, St.
Louis; and Merkel cell (MC) polyomavirus in work from the lab of
Patrick Moore and Yuan Chang at the University of Pittsburgh School
of Medicine. KI and WU viruses were identified from large-scale
high-throughput screens of respiratory secretions from patients with
respiratory tract infections and MC virus was identified in Merkel cell
carcinomas (MCC) using digital transcriptome subtraction. MC virus
has been implicated in the etiology of Merkel cell carcinoma, a rare
but aggressive form of skin cancer that is increasing in incidence.
Allander T, Andreasson K, Gupta S, Bjerkner A, Bogdanovic G,
Persson MA, Dalianis T, Ramqvist T, Andersson B. Identification of a
third human polyomavirus [KI polyomavirus]. J Virol. 2007;81:41304136.

Tobias Allander

David Wang

Gaynor AM, Nissen MD, Whiley DM, Mackay IM, Lambert SB, Wu G,
Brennan DC, Storch GA, Sloots TP, Wang D. Identification of a novel
polyomavirus from patients with acute respiratory tract infections
[WU polyomavirus]. PLoS Pathog. 2007;3:e64.
Feng H, Shuda M, Chang Y, Moore PS. Clonal integration of a
polyomavirus in human Merkel cell carcinoma [MC polyomavirus].
Science 2008;319:1096-1100.

Masahiro Shuda, Huichen Feng, Yuan Chang, Patrick Moore

Merkel cell carcinoma infiltrating skin tissue,

stained for MC polyomavirus large T protein

2007 Tobias Allander, David Wang, Yuan Chang, others human polyomaviruses KI, WU, MC (Merkel carcinoma)

page 549

Bernard La Scola

Didier Raoult

After the discovery of the giant mimivirus in 2007, Bernard La Scola, Didier Raoult and their
colleagues, at the Universit Aix-Marseille, in 2008, discovered an even larger virus infecting the
amoeba Acanthamoeba castellanii, and named it mamavirus (Acanthamoeba polyphaga mimivirus,
APMV). Most remarkably, mamavirus was found to be infected by a smaller virus, which they
named Sputnik by analogy to the term bacteriophage, they coined the term virophage for viruses
infecting viruses. Sputnik virophage virions are icosahedral, 50nm in diameter, and have a circular,
double-stranded DNA genome, 18.3 kb in size, which encodes 21 ORFs, some of which are
derived from the mamavirus, some from the amoeba and many of unknown origin and function.
Sputnik virophage replication occurs only in amoeba co-infected with mamavirus its presence
is detrimental to the replication of mamavirus, causing abortive or abnormal capsid assembly, and
greatly lowered yield of infectious virions. Sputnik has no known homologues with other viruses
and will be the founding member of a new family. In 2011, two more virophages were discovered:
one called mavirus (short for maverick virus), which infects the giant cafeteria roenbergensis virus
of amoeba; the other called Organic Lake virophage, which was found infecting a phycodnavirus in a
phototrophic algae in Organic Lake, a hypersaline lake in Antarctica.
La Scola B, Desnues C, Pagnier I, Robert C, Barrassi L, Fournous G, Merchat M, Suzan-Monti M,
Forterre P, Koonin E, Raoult D. The virophage as a unique parasite of the giant mimivirus [Sputnik].
Nature. 2008;455:100-104.
Fischer MG, Suttle CA. A virophage at the origin of large DNA transposons [mavirus]. Science.
2011;332:231-234.

Top: Sputnik virophage virions and two mamavirus virions at the

edge of a cytoplasmic factory where replication/assembly occur.
Bottom: Sputnik virophage virions within a mamavirus virion.
Thin section electron microscopy. Cryo-electron microscopy
reconstruction of Sputnik virophage.

2008 Bernard La Scola, Didier Raoult, others discovery of the first virophage Sputnik, infecting mamavirus
page 550

Yoshihiro Ozawa

Peter Roeder

William Taylor

Ricky Ireri

Rinderpest, a disease that had devastated cattle herds and their

human keepers across Eurasia and Africa for millennia, was
eradicated globally by a vaccination campaign organized principally
by the U.N. Food and Agriculture Organization (FAO) it joins
smallpox as the only infectious diseases to have been eradicated.
Following World War II, precarious food supplies were threatened
by rinderpest epidemics, leading the newly created FAO to launch a
regional vaccination campaign in Africa. One key to the feasibility
of this was the development in the 1960s by Walter Plowright of a
safe, efficacious attenuated live-virus vaccine. Campaign followed
campaign, each failing as disease incidence declined locally, only
to be followed by a resurgence of disease as interest waned. One
campaign initiated in 1962 came close to freeing Africa of the virus
but, again, as incidence declined countries terminated vaccination
and surveillance programs and by the late 1970s the disease fanned
out from two lingering foci of infection in Mali and Sudan; the same
thing happened in Asia, from Turkey to Bangladesh. One estimate
was that 100 million cattle died during this resurgence. By the
1980s, it was realized that reintroduction of the virus from Asia into
Africa via the live cattle trade was such that only a global campaign
could succeed from this the FAO Global Rinderpest Eradication
Programme (GREP) was initiated in 1993. In 1990, Jeffrey Mariner,
at Tufts University School of Veterinary Medicine, developed a
heat-stable variation of the Plowright vaccine that allowed vaccine to
be backpacked into the remote areas where the virus lingered. The
program met its goal of eradicating the disease by 2004 and from
then until 2010 intensive surveillance was done to assure that there
were no lingering pockets of contagion. The virus was last detected
in 2001 in wild buffalo in Meru National Park in Kenya. Many people
worked in Africa and Asia for all the years of the GREP campaign,
particularly an international group of veterinarians (e.g., Peter
Roeder, William Taylor, Yoshihiro Ozawa, many others) and as time
went along many African and Asian veterinarians and technicians
played central roles. Over time, surveillance and epidemiological
investigations were augmented by new molecular and serological
tools unavailable in previous campaigns. Molecular analyses
allowed the tracking of outbreak viruses to their source reservoirs.
Sensitive tests for antibodies helped monitor the effectiveness of
vaccination and were crucial for checking for pockets of infection
once vaccination was stopped. The FAO announced in 2010 that
the global eradication effort had succeeded and that all follow-up
fieldwork had ended. Curiously, this grand achievement has received
far less public notice than might be expected; this, in turn, may
dampen chances of further global eradication campaigns against
animal or human viral diseases.

2010 FAO Global Rinderpest Eradication Programme global eradication of rinderpest

page 551

Berndt Hoffmann

Martin Beer

Thomas Mettenleiter

Schmallenberg virus
thin section electron microscopy

In 2011, a novel virus was detected in plasma samples from cattle with fever, diarrhea and
reduced milk yield in the German town of Schmallenberg. Disease was also seen in sheep
and goats. The virus, Schmallenberg virus (SBV), was identified at the Friedrich-LoefflerInstitut using next-gen sequencing; the virus is a member of the Simbu serogroup, genus
Orthobunyavirus, family Bunyaviridae. As with the related virus, Akabane virus, transplacental
infection results in the birth of malformed calves, lambs and kids (arthrogryposis, torticollis,
hydranencephaly, porencephaly, etc.). This Culicoides-borne virus has spread widely across,
presenting an economic threat of unknown scale. If Schmallenberg virus behaves like its relative,
Akabane virus, which is found in tropical and sub-tropical regions of Africa, the Middle East,
south-east Asia and Australia, it may have little long-term impact. In such regions animals
are exposed to Akabane virus early in life and develop long-lasting immunity, so they are not
susceptible during pregnancy. However, high levels of virus transmission are needed to ensure
early-life infection and immunity, and in Europe Culicoides spp. density is erratic, heavy in some
years, light in othersthis may not favor the development of herd immunity in cattle, sheep,
goats and wildlife species at risk.
Hoffmann B, Scheuch M, Hper D, Jungblut R, Holsteg M, Schirrmeier H, Eschbaumer
M, Goller KV, Wernike K, Fischer M, Breithaupt A, Mettenleiter TC, Beer M. Novel
Orthobunyavirus in Cattle, Europe, 2011. Emerg Infect Dis. 2012;18:469-472.
Beer M, Conraths FJ, Van Der Poel WHM. Schmallenberg virusa novel orthobunyavirus
emerging in Europe. Epidemiol Infect. 2012;10:1-8.

Schmallenberg virus infection, cattle, sheep, Europe, 2012

2011 Berndt Hoffmann, Martin Beer, Thomas Mettenleiter, colleagues discovery of Schmallenberg virus
page 552

Middle East respiratory syndrome coronavirus (MERS-CoV)

was described in a ProMED post in 2012 by Ali Mohamed
Zaki in Jeddah, Saudi Arabia. He isolated this previously
unknown coronavirus from the lungs of a 60-year-old patient
with pneumonia and renal failure. Using a pan-coronavirus
primer in RT-PCR for preliminary genomic sequencing,
Zaki found the new virus was related to bat coronaviruses.
The virus was sent by Zaki to Ron Fouchier of the Erasmus
Medical Center in Rotterdam, from where the full genomic
sequence was published and submitted to GenBank. Saudi
officials had not given permission to Zaki to release the virus
and were angered when Fouchier tried to patent the genomic
sequence. In turn, Zaki was fired.

As of March 2014, there were 206 laboratory confirmed

cases, with 86 deaths. Most patients have developed a severe
acute respiratory distress syndrome, with fever, cough,
and shortness of breath. So far, all the cases have been
linked to six countries in or near the Arabian Peninsula.
The virus has spread from ill people to others through close
contact; however, the virus has not been shown to spread
in a sustained way in communities other than in health care
settings.

Ali Mohamed Zaki

Ron Fouchier

The suspicion that camels may serve as a reservoir host for

MERS-CoV, and the source of virus infecting humans, has
led to several serologic and PCR-based studies. Antibody
and PCR-signal have been found in camels, with patterns
suggesting repeated introductions into human populations
from camels. It is still not clear how the virus is transmitted
from camel-to-camel and then indirectly to humans.
Present evidence suggests, but does not prove, that camels
are a significant reservoir species, but there is no evidence
confirming the continuous transmission of the virus in this
species.
Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus
AD, Fouchier RA. Isolation of a novel coronavirus from
a man with pneumonia in Saudi Arabia. N Engl J Med.
2012;367:1814-1820.

MERS-CoV, cell culture

Thin section electron microscopy
Maureen Metcalfe & Azaibi Tamin, CDC

Alagaili AN, Briese T, Mishra N, Kapoor V, Sameroff SC,

de Wit E, Munster VJ, Hensley LE, Zalmout IS, Kapoor A,
Epstein JH, Karesh WB, Daszak P, Mohammed OB, Lipkin
WI. 2014. Middle East respiratory syndrome coronavirus
infection in dromedary camels in Saudi Arabia. mBio
5(2):e00884-14.

2012 Ali Mohamed Zaki, Ron Fouchier, W. Ian Lipkin Middle East respiratory syndrome coronavirus (MERS-CoV)

page 553

Pandoraviruses and pithovirus sibericum, the largest viruses known

Chantal Abergel

Jean-Michel Claverie

After discovery of mimivirus (capsid diameter 400 nm; genome size ~1.1 Mb) in 1992, and
recognition that it is a virus and that infects amoeba, the search for other giant viruses was
expanded, especially in econiches where host unicellular eukaryotes might be present. Megavirus
was discovered in 2011 in seawater off the coast of Chile, and other viruses of similar virion and
genome size soon followed. In 2013, Jean-Michel Claverie, Chantal Abergel and their colleagues
reported the discovery of two much larger viruses infecting amoebas, which they named
pandoraviruses: pandoravirus salinus, found in seawater off the coast of Chile, and pandoravirus
dulcis, found in a freshwater pond in Melbourne, Australia, With their discovery in 2014 of an even
larger virus, pithovirus sibericum (isolated from a >30,000-year-old radiocarbon-dated Siberian
permafrost soil sample, propagated in acanthamoeba culture), there now seems to be at least three
distinct clades of giant viruses, viruses that are large enough to be visible under a light microscope:
1. Viruses brought together under the proposed family name Megaviridae. Virions have a distinctive
structure: a unique external fiber layer enclosing a pseudo-icosahedral capsid about 0.5 m in
diameter, containing lipid membranes surrounding an electron-dense nucleocapsid. They have
a linear DNA genome up to 1.26 Mb in size, encoding up to 1,120 proteins. They encode a full
transcription apparatus allowing them to replicate in the hosts cytoplasm. The viruses: Megavirus,
Mimivirus (both infecting acanthamoeba), cafeteria roenbergensis, infecting the marine
microflagellate grazer cafeteria roenbergensis and phaeocystis globosa virus.
2. Pandoraviruses. Virions are amphora-shaped, 1-1.2 m in their longest dimension, have a linear
DNA genome up to 2.8 Mb in size, encoding up to 2,500 proteins. Virions do not incorporate
transcription machinery that would allow them to entirely replicate in the hosts cytoplasm. They
do not appear to be genetically related to the member viruses of the family Megaviridae.
3. Pithovirus. Virions are pandoravirus-like, amphora-shaped, but larger than pandoraviruses, 1.5
m in their longest dimension. Virions have a unique apex aperture (cork), and a thick electrondense tegument lined by a lipid membrane enclosing a rather unorganized internal compartment.
Surprisingly, they have a smaller linear DNA genome, about 600 kb in size, predicted to encode
a mere 467 proteins. They encode a full transcription apparatus allowing them to replicate in the
hosts cytoplasm.
Perhaps most remarkable is the finding that the genome of pandoravirus salinus contains 2,556
putative protein-coding sequences, of which only 6% have recognizable relationships with genes
from known viruses, microorganisms or eukaryotes. Phylogenetic analysis has suggested to some
investigators that these viruses form a previously unknown domain, a fourth branch of the tree of
lifeothers have said that it is too early to draw such a conclusion. In any case, the virus world is
getting more and more interesting.
Philippe N, Legendre M, Doutre G, Cout Y, Poirot O, Lescot M, Arslan D, Seltzer V, Bertaux L,
Bruley C, Garin J, Claverie JM, Abergel C. Pandoraviruses: amoeba viruses with genomes up to 2.5
Mb reaching that of parasitic eukaryotes. Science 2013;341:281-286.
Claverie JM, Abergel C. Open questions about giant viruses. Adv Virus Res. 2013;85:25-56.

Left: pandoravirus salinus [particle length ~1 m (1,000nm),

genome size ~2.5 Mb]. Right: pithovirus sibericum
[particle length ~1.5 m, genome size ~ 600 kb]

Legendre M1, Bartoli J, Shmakova L, Jeudy S, Labadie K, Adrait A, Lescot M, Poirot O, Bertaux L,
Bruley C, Cout Y, Rivkina E, Abergel C, Claverie JM. Thirty-thousand-year-old distant relative of
giant icosahedral DNA viruses with a pandoravirus morphology. Proc Natl Acad Sci U S A. 2014
[Epub ahead of print]

2013-2014 Chantal Abergel, Jean-Michel Claverie, colleagues discovery of pandoraviruses and pithovirus sibericum
page 554

The WHO global Polio Eradication Initiative, begun in 1988 and led by the World Health Organization,
UNICEF and the Rotary and Gates Foundations, has reduced the number of cases from the hundreds
of thousands per year to <1,000 (223 cases in 2012). Despite great difficulties in the end-game, it is
hoped that the disease will be eradicated by 2018 it will be the third disease globally eradicated, after
smallpox and rinderpest. The most important step in polio eradication is interruption of endemic
transmission by universal infant vaccination using oral vaccine (OPV; often via national immunization
days), supplementary IPV vaccination campaigns where needed, intensive surveillance of cases of flaccid
paralysis, and in some places detection of virus in sewage. The poorest countries of the world are anxious
to complete the eradication program and then eventually to stop vaccination, thereby avoiding continuing
costs this will prove to be a very difficult decision with global political ramifications.

~2018 WHO Global Polio Eradication Initiative planned global eradication of poliomyelitis

page 555

Further Reading
In a volume on history, it is obvious that a Further Reading list will include some
papers and books that are out-of-date. Such is true here; the following list includes
contemporary works along with timeless classics that were highly influential in their
day. The latter may create a sense of the context of the time, a sense of what it was like
to be around when great discoveries were made by the scientists who populate this
book:

Reed W, Carroll J, Agramonte A, Lazear JW. The etiology of yellow fever - a preliminary note. Public Health
Pap Rep. 1900;26:37-53.
Rivers TM. Filterable viruses: A critical review. J Bacteriol. 1927;14:217-258.
Rivers TM, editor. Filterable viruses. Baltimore: Williams and Wilkins; 1928.
Rivers TM. Some general aspects of pathological conditions caused by filterable viruses. Am J Pathol.
1928;4:91-124.
Woodruff AM, Goodpasture EW. The susceptibility of the chorio-allantoic membrane of chick embryos to
infection with the fowl-pox virus. Am J Pathol. 1931;7:209-222.
McKinley EB. A concept of the ultramicroscopic virus diseases and a classification. Science 1932;76:449454.
Theiler M, Smith HH. The use of yellow fever virus modified by in vitro cultivation for human
immunization. J Exp Med. 1937;65:787-800.
Ellis EL, Delbrck M. The growth of bacteriophage. J Gen Physiol. 1939;22:365-384.
Williams G. Virus Hunters. New York: Knopf; several editions, 1941-1959.
Fenner F. The pathogenesis of the acute exanthems; an interpretation based on experimental investigations
with mousepox (infectious ectromelia of mice). Lancet 1948;2:915-920.
Watson JD, Crick FH. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature
1953;171:737-738.
Watson JD, Crick FH. Genetical implications of the structure of deoxyribonucleic acid. Nature
1953;171:964-967.
Lumsden LL. St. Louis encephalitis in 1933; observations on epidemiological features. Public Health Rep.
1958;73:340-353.
Meselson M, Stahl FW. The replication of DNA in Escherichia coli. Proc Natl Acad Sci USA.
1958;44:671-682.
Casals J. Procedures for identification of arthropod-borne viruses. Bull World Health Organ.
1961;24:723-734.
Coons AH. The beginnings of immunofluorescence. J Immunol. 1961;87:499-503.
Hirst GK. Development of virology as an independent science. Br Med J. 1962;1:1431-1437.
Kissling RE, Murphy FA, Henderson BE. Marburg virus. Ann N Y Acad Sci. 1970;174:932-945.
Kalter SS, Heberling RL. Comparative virology of primates. Bacteriol Rev. 1971;35:310-364.
Andrewes C. Fifty years with viruses (Christopher H. Andrewes). Annu Rev Microbiol. 1973;27:1-11.
Wilkinson L. The development of the virus concept as reflected in corpora of studies on individual
pathogens. 1. Beginnings at the turn of the century. Med Hist. 1974;18:211-221.
Stuart-Harris C. The contribution of virology to contemporary medicine. Br J Prev Med. 1975;29:1-17
Murphy FA. Arenavirus taxonomy: a review. Bull World Health Organ. 1975;52:389-391.
Wilkinson L, Waterson AP. The development of the virus concept as reflected in corpora of studies on
individual pathogens. 2. The agent of fowl plague a model virus. Med Hist. 1975;19:52-72.
Wilkinson L. The development of the virus concept as reflected in corpora of studies on individual
pathogens lessons of the plant viruses. 3. Tobacco mosaic virus. Med Hist. 1976;20:111-134.
Johnson KM, Lange JV, Webb PA, Murphy FA. Isolation and partial characterisation of a new virus causing
acute haemorrhagic fever in Zaire [Ebola virus]. Lancet 1977;1:569-571.
Wilkinson L. The development of the virus concept as reflected in corpora of studies on individual
pathogens. 4. Rabies two millennia of ideas and conjecture on the aetiology of a virus disease. Med
Hist. 1977;21:15-31.
Waterson AP, Wilkinson L. An introduction to the history of virology. Cambridge, United Kingdom:
Cambridge University Press; 1978.

Andrewes C. The growth of virus research 1928-1978. Postgrad Med J. 1979;55:73-77.

Wilkinson L. The development of the virus concept as reflected in corpora of studies on individual
pathogens. 5. Smallpox and the evolution of ideas on acute (viral) infections. Med Hist. 1979;23:1-28.
Witkowski JA. Alexis Carrel and the mysticism of tissue culture. Med Hist. 1979;23:279-296.
Enders JF, Robbins FC, Weller TH. Classics in infectious diseases. The cultivation of the poliomyelitis
viruses in tissue culture. Rev Infect Dis. 1980;2:493-504.
Wilkinson L. Rinderpest and mainstream infectious disease concepts in the eighteenth century. Med Hist.
1984;28:129-150.
Murphy FA. The epidemiology of infectious diseases of livestock. Onderstepoort J Vet Res. 1985;52:195200.
Fenner F, Gibbs A. Portraits of viruses: a history of virology. Basel, Switzerland: Karger; 1988.
Hsiung GD. The impact of cell culture sensitivity on rapid viral diagnosis: a historical perspective. Yale J Biol
Med. 1989;62:79-88.
Grafe A. A history of experimental virology. Vienna, Austria: Springer-Verlag; 1991.
Monath TP. Yellow fever: Victor, Victoria? Conqueror, conquest? Epidemics and research in the last forty
years and prospects for the future. Am J Trop Med Hyg. 1991;45:1-43.
Kevles DJ. Renato Dulbecco and the new animal virology: medicine, methods, and molecules. J Hist Biol.
1993;26:409-442.
Koprowski H. Yesterday, today and tomorrow in virology. Intervirology 1993;35:9-15.
Mahy BWJ, Lvov D, editors. Concepts in virologyFrom Ivanovsky to the present. Chur, Switzerland:
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Johnson RT. The Soriano Award Lecture. Emerging infections of the nervous system. J Neurol Sci.
1994;124:3-14.
Shope RE. The discovery of arbovirus diseases. Ann N Y Acad Sci. 1994;740:138-145.
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Microbiol. 1995;46:79-90.
Fredericks DN, Relman DA. Sequence-based identification of microbial pathogens: a reconsideration of
Kochs postulates. Clin Microbiol Rev. 1996;9:18-33.
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1996;22:179-187.
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del Regato JA. James Carroll: a biography. Ann Diagn Pathol. 1998;2:335-349.
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1998;10:97-101.
Barrett T, Rossiter PB. Rinderpest: the disease and its impact on humans and animals. Adv Virus Res.
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Brock T. Milestones in microbiology. Classic papers translated and edited by Thomas Brock. Chapter:
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Gottesman M. Bacteriophage lambda: the untold story. J Mol Biol. 1999;293:177-180.

Gubler DJ, Meltzer M. Impact of dengue/dengue hemorrhagic fever on the developing world. Adv Virus
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2000;18:1436-1447.
Kapikian AZ. The discovery of the 27-nm Norwalk virus: an historic perspective. J Infect Dis. 2000;181
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2000;355:1713-1717.
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2001;356:413-420.
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2001;11:59-70.
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genetic data in order to explain the origin and virulence of the 1918 Spanish influenza virus. Philos
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Americas. Clin Lab Med. 2002;22:981-1020.
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Some Major Image Libraries

Of course, an eBook like this would have been impossible to write and illustrate without Google Images (http://www.google.com/imghp) and the main Google search engine
(http://www.google.com/). The work has also been helped very much by the resources of PubMED (http://www.ncbi.nlm.nih.gov/pubmed/), Wikipedia (http://www.wikipedia.
org/) and especially by the staff of the University of Texas Medical Branch Moody Medical Library. Since this is primarily a picture book, public and private image libraries,
archives and databases have been explored exhausively, not only to find images with suitable content, but to find images of the highest quality available without resorting to
expensive stock photo agencies.
U.S National Library of Medicine (NLM). History of Medicine Division (HMD). Images from the History of Medicine (IHM) (access to 70,000 images).
http://www.nlm.nih.gov/hmd/ihm/.
National Library of Medicine. Images from the History of the Public Health Service. Disease Control and Prevention.
http://www.nlm.nih.gov/exhibition/phs_history/ind.html.
Library of Congress (LOC). Prints & Photographs Online Catalog.
http://www.loc.gov/pictures/.
Smithsonian Libraries. Digital Collection (20 libraries).
http://siris-libraries.si.edu/.
Smithsonian Libraries. Dibner Library of the History of Science and Technology.
http://www.sil.si.edu/DigitalLibrary.cfm.
Wellcome Collection. Wellcome Library. History of Medicine Collection. Wellcome Images.
http://images.wellcome.ac.uk/.
Institut Pasteur. Scientific Image Bank.
http://www.pasteur.fr/infosci/biblio/english/ressources/imagebank/.
Rockefeller Archive Center. Rockefeller Foundation Archives.
http://www.rockarch.org/collections/rf/
University of Texas Medical Branch, Moody Medical Library, Truman G. Blocker Jr. History of Medicine Collection. Most of image collection not digitized.
http://ar.utmb.edu/ar/Library/BlockerHistoryofMedicineCollection/tabid/183/Default.aspx.
University of Virginia. Claude Moore Health Sciences Library. Philip S. Hench Walter Reed Yellow Fever Collection.
http://yellowfever.lib.virginia.edu/reed/.
U.S. National Academy of Sciences. Biographical Memoirs.
http://www.nasonline.org/site/PageServer?pagename=Memoirs_A.
National Institutes of Health. Office of History.
http://history.nih.gov/.
The Royal Society. Biographical Memoirs of the Fellows of the Royal Society.
http://rsbm.royalsocietypublishing.org/.
U.S. Centers for Disease Control and Prevention. Public Health Image Library (PHIL).
http://phil.cdc.gov/phil/home.asp.
The Nobel Foundation.
http://nobelprize.org/nobel_prizes/.
National Museum of Health and Medicine (formerly part of the Armed Forces Institute of Pathology, now in transition to new facilities, many digital images still available)
http://nmhm.washingtondc.museum/news/exhibits_closing.html.
University of Wisconsin. Bock Laboratories. Institute for Molecular Virology. VirusWorld. Jean-Yves Sgro.
http://www.virology.wisc.edu/virusworld/viruslist.php.
The Garry Lab. David. M. Sander, Department of Microbiology and Immunology, School of Medicine, Tulane University. The Big Picture book of Viruses and All the Virology on the WWW.
http://www.virology.net/big_virology/BVHomePage.html.

Index
3 poly(A) tail on many mRNAs, discovery of 383
5 cap on messenger RNAs, discovery of 413
5-hydroxymethylcytosine in some phage DNA, discovery
of 258
5-iodo-2-deoxyuridine, the first antiviral drug 309
454 pyrosequencing, development of 544
Abbe, Ernst Karl 26
Abelson, Herbert T. 248
Abergel, Chantal 554
Acanthamoeba polyphaga mimivirus 538
Achong, Bert Geoffrey 345, 394
achromatic lenses, invention of 26
acquired immunodeficiency syndrome (AIDS) 445, 446
acyclovir, antiviral drug, development of 388
adeno-associated viruses, discovery of 351
adenoviruses, discovery of human 259
adenovirus structural / functional elements, discovery
of 352
Adil Bey, Mustafa 91
adjuvants for viral vaccines 134
Adrian, Marc 463
Aedes aegypti 53
Aedes aegypti, disease control 203
African horse sickness viruses, discovery of 84
African swine fever virus, discovery of 135
agarose gel electrophoresis, development of 402
Agramonte, Aristides 77, 81
Ahmed, Rafi 316
AIDS 445
Aitken, Thomas H. G. 342
Aleutian disease of mink virus, discovery of 284
Allander, Tobias 546, 549
Almeida, June 350
Alpers, Michael 359
Alter, Harvey J. 349
Altman, Sidney 443
American Committee on Arthropod-Borne Viruses,
founding of 310
American Society for Virology, founding of 451, 452
Amici, Giovanni Battista 26
Anderson, Charles R. 342
Anderson, John F. 113
Anderson, Norman G. 335
Andrewes, Christopher Howard 162, 170
anelloviruses, discovery of 521
An Introduction to the History of Virology, publication
of book 431
Anslow, Ralph Owen 314
anthrax 44, 49
anthrax bioterrorism events 529, 539
antibody molecules, discovery of structure 305
antigen presentation, discovery of dendritic cells 403
Appel, Max J. G. 429
Arber, Werner 373
arboviruses, antigenic classification of 327
Archaea, discovery of viruses of 408
Archiv fr die gesamte Virusforschung (now Archives of
Virology), first international virology journal 200

Argentine hemorrhagic fever vaccine, development of

465
Arita, Isao 423
Armstrong, Charles 157, 158, 174, 187, 201, 202
arthropod transmission of disease, first proof of 67
Arvin, Ann 341
Ashburn, Percy Moreau 106
Asibi, yellow fever patient 183
Asnis, Deborah 527
association of a specific pathogen with a specific disease
44
strm, Karl-Erik 298
astroviruses, discovery of 412
Atchison, Wayne 351
atomic structure of viruses, visualization of 432
Aujeszky, Aladr 89
Australia antigen, hepatitis B virus, discovery of 349
autoclave, invention of 59
Autonomous Pathogen Detection System 539
Avery, Oswald Theodore 223
avian infectious bronchitis virus, discovery of 191
avian leukemia viruses, discovery of 108
AZT, first anti-HIV drug, development of 477
Babesia bigemina 67
Bacon, Francis 10
bacteria, discovery of 12
bacteriophages, discovery of 126
bacteriophage, toxins 144
bacteriophage , discovery of 249
Baer, George Martin 395
Balayan, Mikhail Surenovich 458
Baltimore, David 251, 380, 381, 390, 447
Bang, Frederik B. 262
Bang, Oluf 108
Barin, Francis 469
Barr-Sinoussi, Francoise 459
Barr, Yvonne 345
Bauer, Johannes 151
Bawden, Frederick Charles 185
Bazin, Herve 56
Beard, Joseph Willis 115, 171, 188
Beards, Graham 457
Beasley, R. Palmer 434
Beaudette, Fred Robert 191
Beckman, Arnold O. 179, 235
Beer, Martin 552
Beijerinck, Martinus Willem 70
Bellini, William J. 526
Benfield, David A. 496
Benzer, Seymour 275, 399
Bergh, ivind 492
Berg, Paul 275, 399
Bernal, John Desmond 192
Bernard, Claude 46
Berners-Lee, Tim 499
Bernhard, Wilhelm 265
Berson, Solomon A. 307
Beutler, Bruce A. 517
Bhatt, Pravin N. 288
BHK-21 cell line, development of 322
Biggs, Hermann Michael 29, 65

Biggs, Peter M. 365

Billingham, Rupert E. 264
Binnig, Gerd 448
biodefense research and response programs 529
Biohazards in Biological Research, Asilomar Conference
399
biomedical illustration 35
Bishop, David H. L. 451
Bishop, J. Michael 417
Bishop, Ruth F. 401
Bittner, John Joseph 168
BK virus (kidney, bladder disease), discovery of 392
Blanden, Robert 316
bluetongue viruses, discovery of 92
Blumberg, Baruch 349, 434
Boophilus annulatus 67
Bordet, Jules 54
Borna disease virus, discovery of 154
Borrel, Amde 90
Borst, Piet 402
Boshell-Manrique, Jorge 176
Bouteille, Michel 348
bovine rotavirus, discovery of 376
bovine spongiform encephalopathy (BSE), discovery in
United Kingdom 471
bovine torovirus, discovery of 457
bovine viral diarrhea virus, discovery of 290
Boyer, Herbert W. 398, 439
Boyle, Robert 10
Bradley, Daniel W. 489
Brakke, Myron Kendall 252
Breese Hall, Caroline 241
Breinl, Anton 132
Brenner, Sydney 302, 320, 323, 399
Brent, Leslie Baruch 264
Brin, Sergey 524
Brock, Thomas D. 488
Broder, Samuel 477
Broderson, Roger 329
Brown, J. Howard 144
Brown, Patrick O. 490
Bryans, John T. 289
Buchmeier, Michael 316
Buckley, Sonja 375
budding, release of virus at the cell surface, discovery
of 262
Buist, John Brown 61
Bunyamwera virus, discovery of 232
Burke, Alexander 205
Burkitt, Denis Parsons 297
Burkitts lymphoma, description of 297
Burmester, Ben Roy 365
Burnet, Frank Macfarlane 164, 234, 285
Butel, Janet S. 387
Buynak, Eugene 360
B virus, discovery of 169
Cairns, John 324
Calisher, Charles 327
Callaway, Edward M. 518
Camargo, Lus Patio 176
Campbell, Carlos (Kent) 375

canine distemper virus, discovery of 103

canine parvovirus, discovery and characterization of 429
Capecchi, Mario R. 478
carbolic acid, disinfection 32
Carey, Donald E. 393
Carmichael, Leland E. (Skip) 429
Carr, Henri 99, 103
Carrel, Alexis 118, 119
Carroll, James 77, 78, 80
Carruthers, Marvin 460
Carter, Henry Rose 76
Casals-Ariet, Jordi 327, 375
Caspar, Donald 332
catalytic properties of RNA, discovery of ribozymes 443
categorization of viruses based on replicative path to
mRNA 390
Cech, Thomas R. 443
cell as unit of structure of all organisms 28
cell culture, development of 118, 119, 120
cell culture, first cultivation of virus in 121
cell culture methodology for polio, measles viruses,
development of 243
cellular immune system recognition of virus-infected
cells 406
cellular (microscopic) pathology, father of 39
Centanni, Eugenio 88
Centers for Disease Control and Prevention 325, 363
cervical carcinoma, papillomaviruses, discovery of
association 409
Chaille, Stanford Emerson 51
Chalfie, Martin 338
Chamberland, Charles 55, 57, 59
Chang, Yuan 509, 549
Chanock, Robert Merritt 277, 282
Chargaff, Erwin 245
Chargaffs Rules, A+T=C+G 245
Chase, Martha Cowles 253
Chelle, Paul-Louis 186
chemotherapy, founding of 54
Chermann, Jean-Claude 459
Chevalier, Charles 27
chikungunya virus, discovery of 281
childbed fever 32
Childs, Jamie E. 507
cholera epidemic, London 29
Choo, Qui-Lim 489
Chow, Marie 432
Christofferoni, J. 153
chromosome, nature and role of 112
chronic wasting disease of deer and elk, discovery as
prion disease 442
Chua, Kaw Bing 526
Chumakov, Mikhail Petrovich 194, 228
Churchill, Anthony 365
circoviruses, discovery of 410
Ciuffo, Giuseppe 107
Clark, H. Fred 378, 482
classical swine fever virus, discovery of 93
classification of organisms, system for 14
classification of viruses based upon virion characteristics
331

Claverie, Jean-Michel 538, 554

Cleland, John Burton 132
clonal selection as the central mechanism in immunity,
discovery of 285
Cohen, Seymour Stanley 258
Cohen, Stanley Norman 398
Cohn, Zanvil A. 403
Coimbra, Terezinha Lisieux Moraes 511
Cold Spring Harbor Symposium - Basic Mechanisms in
Animal Virus Biology 330
Cole, Gerald 316
Collinge, John 514
Collins, James E. 496
Colorado tick fever virus, discovery of 233
common descent, concept of 40
Compans, Richard 433
comparative pathology, father of 39
compound microscope, invention of 9
confocal microscope, invention of 286
contagium vivum fluidum 70
Convergence Model, factors in viral emergence 504
Conzelmann, Karl-Klaus 510
Corey, Lawrence 388
Cosgrove, Albert S. 334
Cosgrove, B. P. 412
Cossart, Yvonne Edna 411
Coto, Celia 296
Cotton, William 142
Courtois, Ghislain F. G. J. 228
Cowdry, Edmund Vincent 62
cowpox virus 17, 18, 19
Cox, Herald Rea 164, 300
Cox, Nancy 525
Coxsackieviruses, discovery of 239
Craig, Charles Franklin 106
Creutzfeldt, Hans Gerhard 136
Creutzfeldt-Jakob disease, description of 136
Crick, Francis 260, 323
Crimean-Congo hemorrhagic fever virus, discovery of
228
cryo-electron microscopy 463
Cuill, Jean 186
Curran, James 445
Curtice, Frederick Cooper 67, 68
cytology, viral disease 62
Dalldorf, Gilbert 239
Dalton, Albert J. 265
Danna, Kathleen J. 373, 405
Darnell, James E. Jr. 383
Darwin, Charles Robert 40, 41
Daubney, Robert 165
Davaine, Casimir 44
Davenport, Frederick 227
Davidson, Geoffrey 401
DDT, discovery of the insecticidal qualities and use for
mosquito control 203
Death in a Sailors Suit, Yellow fever in New York 183
De Cock, Kevin 445
de Hevesy, George 116
de Kruif, Paul Henry 141

de la Rivire, Ren Dujarric 130

de la Torre, Juan 484
Delbrck, Max Ludwig Henning 204, 226, 231
dendritic cells, discovery of role in immunity 403
dengue hemorrhagic fever and shock syndrome 426
dengue viruses, discovery of 106
Department of Filterable Viruses, Johns Hopkins
University 147
DeRisi, Joseph L. 532
de Schweinitz, Emil Alexander 93
Desrosiers, Ronald C. 468
de Vries, Hugo Marie 85
dHerelle, Flix 126
Diener, Theodore Otto 391
Dimitrov, Dimiter S. 404
Dimock, William W. 177
Dinter, Zvonimir 276
disinfection, methods of 32
di Vestea, Alfonso 96, 97
Dixon, Frank 284, 315
DNA cloning in phage 435
DNA-dependent RNA polymerase, discovery in virus
364
DNA fingerprinting, development of 470
DNA, identified as the material of inheritance 223, 253
DNA of T2 phage, discovery of its nature 324
DNA sequencing, development of automated 475
DNA structure, discovery of 260, 261
DNA synthesis technology, development of commercial
460
Doane, Frances 241
Dobzhansky, Theodosius 190
Dochez, Alphonse Raymond 131
Doermann, Augustus 195
Doerr, Robert 111, 198, 200
Doherty, Peter C. 406
Doherty, Ralph L. 343
Doll, Elvis Roger 177, 289
Dollond, John 26
Domingo, Esteban 484
Donn, Alfred 34
Dorset, Marion 93
double-stranded viral RNA, discovery of 339
Dowdle, Walter Reed 336, 441
Downs, Wilbur George 310, 342
Doyle, Thomas Michael 143
dsRNA-dependent RNA polymerase, discovery of 368
Dubochet, Jacques 463
Dubovi, Edward J. 476
Dulbecco, Renato 257, 267
Dunkin, George 103
Eagle, Harry 120
Earle, Wilton R. 208
eastern equine encephalitis virus, discovery of 180
Eaton, Bryan 526
Ebola and Marburg viruses, reservoir host fruit bats 540
Ebola disease in endangered great apes 535
Ebola virus, discovery of 415
eclipse period in virus replication, discovery of 195
Eddy, Bernice 294

Eddy, Gerald 465

Edelman, Gerald Maurice 305
Ehrlich, Paul 54
Eigen, Manfred 484
electronic pH meter, invention of 179
electron micrographs of viruses, first 199
electron microscope, invention of 172, 173
electrophoresis, development of 98
Eletr, Sam H. 460
Elford, William Joseph 162
Elion, Gertrude Belle 388
Ellerman, Vilhelm 108
Elliott, Luanne H. 507
Ellis, Emory Leon 204
embryonating hens eggs, development as host for viruses
163, 164
Encyclopedia of Virology, publication of book 508
Enders, John Franklin 120, 243, 340
Engvall, Eva 389
Enquist, Lynn W. 435, 518
enzyme-linked immunoassays (EIAs, ELISAs),
development of 389
epidemiology, founding of 29
Epstein-Barr virus, association with Burkitts lymphoma
345
Epstein-Barr virus, association with infectious
mononucleosis 369
Epstein, Michael Anthony 345, 394
equine abortion virus, discovery of 177
equine arteritis virus, discovery of 289
equine herpesviruses, discovery of 177
equine infectious anemia virus, discovery of 99
equine rhinopneumonitis virus, discovery of 177
erythema infectiosum, fifth disease 411
Essex, Max 468, 469
estimating 50% endpoints, development of methods for
196
Eugene Koonin 498
European Centre for Disease Prevention and Control
(ECDC) 545
Evans, Alfred S. 419
Evans, Martin J. 478
evolutionary science, father of 40
experimental pathology, founding of 20
Extended Evolutionary Synthesis, development of 548
extrinsic incubation period, mosquito 76
Farr, William 29
Far Side 444
Feinstone, Stephen M. 400
Feldman, Joseph 315
feline immunodeficiency virus, discovery of 474
feline leukemia virus, discovery of 346
feline panleukopenia virus, discovery of 153
Feng, Huichen 549
Fenner, Frank 237, 357, 379, 423, 476
Field, Anne 392, 411
Fields, Bernard Nathan 316, 466
Fields Virology, publication of book 466
Fiers, Walter 421
fifth disease, erythema infectiosum 411

filariasis 50
Filterable Viruses, first major virology book 147
Finlay, Carlos Juan 53, 77
Fire, Andrew Z. 523
Fite, George L. 175
Flewett, Thomas Henry 401, 487
Flexner, Simon 87
Florio, Lloyd 233
focus assay, Rous sarcoma virus 293
Fodor, Stephen P. A. 490
Folks, Thomas 394
foot-and-mouth disease epidemic, United Kingdom 530
foot-and-mouth disease vaccines, development of 140
foot-and-mouth disease virus, discovery of 71, 73
formaldehyde to inactivate viruses for vaccines 134
Formenty, Pierre 535, 540
Forterre, Patrick 408
Foucault, Lon 34, 35
Fouchier, Ron 553
fowl plague virus (avian influenza virus), discovery of 88
fowl plague virus is an influenza virus, discovery that 276
Fracastoro, Girolamo 8
Fraenkel-Conrat, Heinz 274, 279
Francis, Donald 445
Francis, Thomas Jr. 227, 268
Franois Jacob 320
Franco, Roberto 176
Franklin, Rosalind Elise 192, 261
Frazer, Ian 533
Fredricks, David N. 515
French, Eric Lancelot 132
French, George 465
Frenkel, Herman 140
Frenkel, Nitza 495
Friend, Charlotte 248
Frobisher Jr., Martin 144
Frosch, Paul 71
Gajdusek, D. Carleton 359
Gallo, Robert C. 440
Galloway, Ian A. 154
Galtier, Pierre-Victor 20
Garcia-Sastre, Adolfo 541
Gardner, Phillip 241
Gardner, Sylvia D. 392
Garnham, Percy Cyril 165
Gay, Frederick Parker 169
Gayle, Helene 445
Gelderblom, Hans 410
GenBank, development of 455
gene, definition of 275
Genentech, Inc. 439
genetically manipulated (knockout) mice, development
of 478
genetic code, deciphering of 326
genetic control of enzyme and virus synthesis, discovery
of mechanisms 318, 319
genetic engineering, development of 398
genetic recombination in bacteria, discovery of 229
genetic recombination in viruses, discovery of 231
Genetics and the Origin of Species, publication of book 190

genetics, father of 45
genetics, rediscovery of Mendels work 85
Gerin, John L. 425
germ theory, father of 8, 30
Gershon, Anne 341
Gessain, Antoine 542
Gey, George Otto 247
Gibbs, Adrian 484
Gibbs, Clarence Joseph 359
Gibbs, E. Paul J. 438, 476
Gierer, Alfred 279
Gilbert, Walter 356, 424
Gillespie, James R. 290
Ginsberg, Harry S. 259, 451
global eradication of smallpox 423
Goad, Walter 455
Goldberger, Joseph 113, 114
Golde, David 440
Goldsmith, Cynthia S. 507
Goldwasser, Robert 295
Gomatos, Peter J. 339
Gonzalez, Jean-Paul 535
Gonzlez-Scarano, Francisco 316
Goodpasture, Ernest W. 163, 178
Google, development of 524
Gorgas, William Crawford 100
Gorham, John R. 284
Gottschalk, Alfred 234
Gowans, James Learmonth 299
graded collodion membranes, to determine virion size
162
Granoff, Allan 508
Grassi, Giovanni Battista 50
Graunt, John 29
green fluorescent protein, discovery and development
of 338
Green, Robert Gladding 155
Griffin, Diane 316
Griffith, Fred 149
Gross, Ludwik 248
Guanarito virus (Venezuelan hemorrhagic fever),
discovery of 502
HAART (highly active antiretroviral therapy),
development of 516
Haase, Ashley 316
Haddow, Alexander John 232
Hadlow, William J. 359
Hallauer, Curt 198, 200
Halonen, Pekka Eljas 241, 385
Halstead, Scott B. 426
Hamburg, Margaret 504
Hammon, William McDowall 106, 214
Handbuch der Virusforschung, publication of book 198
Hantaan virus, discovery of 428
Harde, Edna Steinhardt 123
Haring, Clarence Melvin 159
Harpers Weekly 52
Harrison, Ross Granville 105
Harrison, Steven C. 432
Haselkorn, Robert 451
Hayashi, Michitomo 181

Hayflick, Leonard 355

HBsAg produced in yeast, first recombinant human
vaccine 453
Heberling, Richard L. 386
HeLa cell line, establishment of 247
Helenius, Ari 10, 404
Hellman, Alfred 399
hematology, founding of 54
hemorrhagic fever with renal syndrome 428
Henderson, Donald A. 423
Henderson, William 31
Hendra virus, discovery of 512
Henle, Friedrich Gustav Jakob 30
Henle, Gertrude S. 369
Henle-Koch postulates revisited 193
Henle-Koch postulates revisited again 419
Henle-Loeffler-Koch Postulates 30
Henle, Werner 369
Henson, James B. 284
hepatitis A and B, separation of two diseases 225
hepatitis A virus, discovery of 400
hepatitis B virus, discovery of 349
hepatitis B virus infection, association with hepatocellular
carcinoma 434
hepatitis C virus, discovery of 489
hepatitis C virus, in vitro replication of 543
hepatitis delta virus, discovery of 425
hepatitis E virus, discovery of 458
hepatocellular carcinoma, hepatitis B virus infection 434
herpes simplex virus 1, discovery of 133
herpes simplex viruses, differentiation of HSV 1 and 2
336
herpes simplex virus, molecular biology and replication,
discovery of 353
Hershey, Alfred Day 231, 253
Hess, Alfred Fabian 124
Hierholzer, John C. 329
high-containment virology laboratories, development
of 363
highly active antiretroviral therapy (HAART),
development of 516
Hilleman, Maurice 312, 341, 360, 453
Hippocrates 6
Hiro, Yoshibumi 124
Hirsch, Martin S. 516
Hirst, George K. 227, 272
His, Wilhelm 27
Hitchings, George H. 388
HIV/AIDS Epidemic 446
HIV antibody tests (EIAs), development of 467
Ho, David 516
Hodgkin, Dorothy Crowfoot 192
Hoerlein, A. B. 290
Hoffmann, Berndt 552
Hoffmann, Jules A. 517
hog cholera virus, discovery of 93
Hogle, James M. 432
Holden, Margaret 169
Holland, John 484
Holley, Robert W. 326
Holloway, Ann 340

Holmes, Edward 498

Holmes, Ian H. 401
Holmes, Kathryn 316
Holmes, Sr., Oliver Wendell 32
Hood, Leroy E. 475
Hooke, Robert 11
Hooper, Peter 512
Horne, Robert W. 302, 303, 331
Horsfall, Frank Lappin Jr. 148, 240
Horstman, Dorothy 451
Horzinek, Marian C. 457, 476
Hotta, Susumu 106
Houghton, Michael 489
Howitt, Beatrice 159
Howley, Peter M. 466
h-ras, cloning of first tumor-derived oncogene 450
Hsiung, Gueh-djen (Edith) 241
Huang, Alice S. 251, 380
Hudson, Charles B. 191
Hudson, John Richard 165
Hudson, Noel 151
Huebner, Robert J. 259, 374
Hughes, James M. 441
Hughes, Thomas 205
human adenoviruses, discovery of 259
human astroviruses, discovery of 412
human bocavirus, discovery of 546
human coronaviruses, discovery of 350
human cytomegalovirus, discovery of 283
human diploid cell strains (WI-1 through WI-44),
establishment of 355
Human Genome Project 494, 536
human herpesvirus 6B (exanthem subitum), discovery
of 479
human herpesvirus 7 (exanthem subitum), discovery of
495
human herpesvirus 8 (Kaposi sarcoma herpesvirus),
discovery of 509
human immunodeficiency virus 1 (HIV1), discovery of
459
human immunodeficiency virus 2 (HIV2), discovery of
469
human metapneumovirus, discovery of 528
human papillomaviruses and cervical carcinoma,
discovery of 409
human papillomaviruses, discovery of 107
human parainfluenza viruses, discovery of 282
human polyomaviruses KI, WU, MC, discovery of 549
human rhinoviruses, discovery of 278
human rotaviruses, discovery of 401
human rotavirus vaccines, development of 482
human T-lymphotropic viruses 1 & 2, discovery of 440
human T lymphotropic viruses 3 and 4, discovery of 542
Hunter, John 20
Hunt, Ronald 469
Hurlbut, Herbert 273
Hwang, Lu-yu 434
Hyatt, Alex 512
hygiene, development of methods 32
icosahedral structure of viruses, discovery of principles
of 332

IL-2, discovery/characterization of first cytokine 354

immune complexes in viral diseases, discovery of 315
immune enhancement, discovery of 426
immunofluorescence in viral pathogenesis research,
development of 246
immunological tolerance, discovery of 264
immunology, founding of 54
immunopathology in viral diseases, discovery of 315
inclusion bodies 31, 61, 62
infectious bovine rhinotracheitis virus, discovery of 280
infectious bursal disease virus, discovery of 334
infectious canine hepatitis virus, discovery of 155
infectious disease sciences, father of 37
influenza pandemic of 1918-1919 128, 129
influenza virus, 1918 virus sequenced / reconstructed
541
influenza virus, discovery of 130, 131
influenza viruses, discovery of the link between avian and
human viruses 337
influenza virus hemagglutinin, determination of atomic
structure of 449
influenza virus, highly pathogenic H5N1 variant,
discovery of 520
influenza virus, molecular basis for virulence 525
influenza virus, proof of viral etiology 170
innate immunity 517
Institute of Medicine of the U.S. National Academy of
Sciences, founding of 377
Institut Pasteur, founding of 64
interferons, discovery of 287
internal ribosomal entry sites (IRES) in virus mRNA,
discovery of 480
International Committee on Taxonomy of Viruses,
founding of 357, 358
International Congresses for Virology 370, 371, 372
International Human Genome Project 536
International Human Genome Sequencing Consortium
494
Introduction to the Study of Experimental Medicine 46
Ireri, Ricky 551
Ironside, James 514
Isaacs, Alick 287
Ishida, Nakao 263
isopycnic density gradient ultracentrifugation, invention
of 291
Israeli, Clara 121
Ivanovsky, Dmitry 69
Jackson, Dale A. 397
Jackson Memorial Laboratory 168
Jacob, Franois 318, 320
Jaffe, Harold 445
Jakob, Alfons Maria 136
Jansen, Sacharias 9
Japanese encephalitis virus, discovery of 181
Jarrett, William F. H. 346
JC virus, progressive multifocal leukoencephalopathy,
discovery of 298
Jeffreys, Alec J. 470
Jenner, Edward 17, 18, 19
Jerne, Niels Kaj 285
Jesty, Benjamin 16

Johnson, Claud 178

Johnson, Karl M. 347, 363, 415, 428
Johnson, Richard T. 316
Joklik, Wolfgang Karl 451
Jorge Boshell-Manrique 176
Journal of General Virology, publication of journal 272
Journal of Virology, publication of journal 272
Jubil de Pasteur a la Sorbonne 38
Jung, Rudolf 27
Junin virus (Argentine hemorrhagic fever), discovery of
296
Kahn, Laura H. 547
Kalfayan, Bernard 314
Kalter, Seymour S. (Sy) 386
Kalyanaraman, Vaniambadi S. 440
Kanki, Phyllis Jean 469
Kapikian, Albert Z. 396, 400, 401, 482
Kaplan, Bruce 547
Kaposi sarcoma 445
Kaposi sarcoma herpesvirus (HHV-8), discovery of 509
Krber, G. 196
Kasahara, Shiro 181
Kasakura, Shinpei 354
Kates, Joseph R. 364, 383
Katz, Samuel 340
Kawakita, Yosio 328
Kawaoka, Yoshihiro 525
Kelly, Thomas 373
Kemp, Graham E. 378
Kendrew, John Cowdery 250
Kerr, Jerome Austin 176
Khorana, Har Gobind 326
Kilbourne, Edwin 227
Kilham, Lawrence 301
Kim, Jungsuh 501
Kinyoun, Joseph J. 63
Kissling, Robert E. 362
Kit, Saul 472
Kjeldgaard, Niels 244
Kleinschmidt, Albrecht Karl 304
Klenk, Ernst 234
Klug, Aaron 332
Knipe, David M. 466
knockout (genetically manipulated) mice, development
of 478
Knoll, Max 172, 184
Koch, Heinrich Hermann Robert 49, 72
Kochs Postulates 30, 49
Kochs Postulates, revisited 193
Kochs Postulates, revisited again 419
Kochs Postulates, revisted yet again (sequence-based
criteria) 515
Khler, Georges 407
Koonin, Eugene 484
Koprowski, Hilary 300
Kornberg, Arthur 308
Kossel, Ludwig Karl Martin Leonhard Albrecht 47
Kozloff, Lloyd 272
Kraft, Lizbeth M. 329
Kraneveld, Frederik Cornelis 143

Krugman, Saul 225

Ksiazek, Thomas G. 507, 526, 537
Kubes, Vladimir 197
Kuo, George 489
Kurata, Takeshi 479
kuru prion, transmission to non-human primates 359
Kuypers, Hans H. G. 11, 518
Kyasanur Forest disease virus, discovery of 288
Lacaillade, Charles Jr. 180
lac operon of E. coli 356
La Crosse virus, discovery of 314
lactate dehydrogenase-elevating virus, discovery of 313
Laemmli, Ulrich K. 366
Lagos bat virus 378
Laidlaw, Patrick Playfair 103
Laigret, Jean 152
lambda bacteriophage, discovery of 249
Lambert, Robert A. 121
Lam Sai Kit, Kenneth 526
Lancefield, Rebecca 150
Lanciotti, Robert S. 527
Landsteiner, Karl 109, 110
Lane, David P. 436
Lange, James V. 415
Larson, Gary 444
La Scola, Bernard 538, 550
Lasher, Hiram N. 334
Lassa virus (Lassa fever), discovery of 375
latent period, discovery of 267
Laveran, Charles Louis 50
Laver, William Graeme 337
Layton, Marcelle 527
Lazear, Jesse William 77, 81
L cell line, L929 cloned cell line, development of 208
Leader, Robert 284
Lebailly, Charles 130
Lederberg, Esther Miriam Zimmer 249
Lederberg, Joshua 229, 255, 504, 505
Lee, Ho Wang 428
Lee, Kyu M. 290
Lee, Pyung Woo 428
Lennette, Edwin H. 150
Lpine, Pierre 268
Lerner, Richard 315
Leroy, Eric M. 535, 540
Leslies Illustrated Newspaper 52, 183
Letvin, Norman L. 468
Levaditi, Constantin 110
Levine, Arnold J. 436
Levkovich, Elizabeth N. 194
Levy, Jay 445
Lewis, Sinclair 141
Lincoln, Abraham 43
Lindbergh, Charles 119
Lindenmann, Jean 287
Linnaeus, Carolus 14
Lipkin, W. Ian 493, 527, 532, 553
Lipman, David 519
Lister, Joseph 32
Lister, Joseph Jackson 26, 32

Little, Clarence Cook 168

Loeffler, Friedrich A. J. 71, 72, 73, 74
Loeffler-Koch Postulates 30
Lopez, Carlos 479
Lwenstein, A. 8, 133
Luciw, Paul 394
Lumsden, William H.R. 281
Luria, Salvador Edward 226
Lwoff, Andr 244, 318, 331
lymphocyte recirculation, discovery of 299
lymphocytic choriomeningitis (LCM) virus, discovery
of 187
lymphocytic choriomeningitis (LCM) virus
immunopathology 315
lysogeny, induction, discovery of 244
lyssaviruses, discovery of 378
Maalin, Ali Maow 423
Maass, Clara Louise 83
MacCallum, Fred O. 225
Machupo virus (Bolivian hemorrhagic fever), discovery
of 347
MacKenzie, Ronald 347
Maclachlan, N. James 476
MacLeod, Colin Munro 223
Macpherson, Ian 322
Madeley, C. Richard (Dick) 412
Madin, Stewart Harvey 280
Mahaffy, Alexander Francis 232
Mahieux, Renaud 542
Mahy, Brian W. J. 508
Maitland, Hugh Bethune 120
Maitland, Mary Cowan 120
Maizel, Jacob V. Jr. 366
Maiztegui, Julio I. 465
malaria, etiologic agent 50
Manson, Patrick 50
Maramorosch, Karl 256
Marburg virus, discovery of 362
Mareks disease virus, discovery of 365
Marine Hospital Service 24
marine mammal morbilliviruses, discovery of 483
marine viral metagenomics 492
Marion, Andre F. 460
Marshall, Ian 238
Marshel, James H. 518
Martini, Gustav 362
Materia Medica 7
Mathis, Constant 152
Matsumoto, Seiichi 344
Matteucci, Mark 460
Matthaei, J. Heinrich 326
Matthew Meselson 320
Matthews, Richard Ellis Ford 357
Maxam, Allan 424
Mayer, Adolf Eduard 60
Mayr, Ernst Walter 206
McAuslan, Brian R. 364
McCarthy, Kevin 340
McCarty, Maclyn 223
McClintock, Barbara 207

McCordock, Howard A. 175

McFadyean, John 84
McIntosh, Kenneth 241
McKercher, Delbert Grant 280
McNamara, Tracy 527
measles, Faroe Islands 36
measles vaccine, development of 340
measles virus, discovery of 113
Mebus, Charles A. (Chuck) 376
Medawar, Peter Brian 264, 299
Medical Virology, publication of book 379
medicine, father of modern 6, 48
Meiklejohn, Gordon 227
Meister, Joseph 56
Mello, Craig C. 523
Melnick, Joseph L. 351, 370, 372
Mendel, Gregor Johann 45
Meredith, Courtney D. 378
Merigan, Thomas C. 516
Merkel cell carcinoma (MC polyomavirus), discovery
of 549
Merrifield, Robert Bruce 317
Merrill, Malcolm H. 180
Meselson, Matthew 291, 292
Mesopotamian medicine 7
messenger RNA 320
Metchnikoff, Elie 54
Mettenleiter, Thomas 552
Mettler Instruments AG, single-pan analytical balance,
development of 236
Meyer, Harry Jr. 341
Meyer, Karl Friedrich 159, 160
microarray technology, development of 490
microarray technology for identification of viruses 532
microbead-based virus detection technology,
development of 522
microbiology, co-founder of 49
microbiology, father of 37
Micrographia, publication of book 11
microorganism, first depiction of 11
microscope, simple (single lens), invention of 12
microscopes, improvements in design 26
microtiter system for virologic and serologic assays,
invention of 271
microtome, invention of 27
Middle East respiratory syndrome coronavirus (MERSCoV) 553
Miescher, Johan-Friedrich 47
Miller, Jacques 321
Miller, Mabel 233
Milovanovic, Milan 340
Milstein, Csar 407
mimivirus, discovery of 538
Mims, Cedric 246, 316
Minot, Charles Sedgwick 27
Minsky, Marvin Lee 286
Mitus, Anna 340
Miyamoto, Kaneatsu 344
Mizutani, Satoshi 381
MMWR 325, 445

modern evolutionary synthesis, developmnet of 206, 548

Mogabgab, William J. 278
molecular virus identification 493
molluscum contagiosum virus 31
Moloney, John B. 248
Monath, Thomas P. 375, 547
monoclonal antibodies, development of 407
Monod, Jacques 318
Montagnier, Luc 459
Montagu, Lady Mary Wortley 13
Montgomery, Robert Eustace 127, 135
Moore, Patrick S. 509, 549
Morbidity and Mortality Weekly Report, publication of
325
Morens, David 129
Morgan, Councilman 10, 404
Morgan, Thomas Hunt 112
Morris, J. Anthony 277
Morse, Stephen S. 504, 506
Moss, Bernard 413, 462
mouse, development for virus research 157, 158
mouse mammary tumor virus, discovery of 168
Muckenfuss, Ralph S. 174
Mudd, Stuart 58
Muench, Hugo 196
Mugrage, Edward 233
Mller, Gerd B. 548
Mller, Paul Hermann 203
Mullis, Kary B. 464
mumps vaccine 360
mumps virus, discovery of 178
murine cytomegalovirus, discovery of 269
murine leukemia and lymphoma viruses, discovery of
248
murine parvoviruses, discovery of 301
murine polyoma virus, discovery of 294
Murphy, Frederick A. 329, 362, 378, 415, 416, 441, 451,
476
Murphy, James Bumgardner 262
Murray, Andrew W. 461
Murray, Keith 512
Murray Valley encephalitis virus, discovery of 132
myxoma virus 75, 164
Nahmias, Andr J. 336
Nairobi sheep disease virus, discovery of 127
Nathans, Daniel 373, 405
Nathanson, Neal 316
National Center for Biotechnology Information (NCBI)
and its public databases, development of 481
National Center for Infectious Diseases (CDC), founding
of 441
National Institute of Allergy and Infectious Diseases,
founding of 63
National Institutes of Health, founding of 63
natural history of arboviruses in vectors 214
natural selection, concept of 40
negative contrast electron microscopy, invention of 302
Negri, Adelchi 94, 104
Negri body 94
Negri body, discovery of nature of 344

neuronal pathway tracers, viruses as 11, 518

Newcastle disease virus, discovery of 143
New, Emerging and Re-emerging Infectious Diseases,
development of concept of 504, 505
Newhouse, Verne 375
New York City Department of Health, founding of 25
New York City Department of Health microbiology
laboratory, founding of 65
Nichol, Stuart T. 507, 540
Nicolau, Stefan S. 154
Nicolle, Charles 130
Nicolle, Maurice 91, 104
Nipah virus, discovery of 526
Nirenberg, Marshall W. 326
Nishizawa, Tsutomu 521
Nisonoff, Alfred 305
Nobel Prizes pertinent to virology, publication of papers
and book 534
nonhuman primate virology, development of 386
noroviruses, discovery of 396
Norrby, Erling Carl Jacob 352, 534
Norwalk virus, discovery of 396
Nothing in Biology Makes Sense Except in the Light of
Evolution 190
Nowell, Peter 354
nucleic acids, discovery of 47
nucleoprotein, RNA, tobacco mosaic virus, discovery
of 185
Oatley, Charles 184
Ochoa de Albornoz, Severo 308
Offit, Paul A. 482
Okamoto, Hiroaki 521
Oker-Blom, Nils 370
Oldstone, Michael B. A. 315, 456
Olitsky, Peter Kosciusko 131, 142
One Health / One Medicine Initiative 547
One Medicine, concept of 48
one-step growth experiment 204
On the Origin of Species 41
optimum constellation of genes determines virulence,
development of concept 418
Orenstein, Walter A. 341
origin of viruses, development of modern concepts of
484, 485
orthoreoviruses, discovery of 266
Osler, William 48
Osterhaus, Albert D.M.E. 483, 528, 537
Oxman, Michael N. 399
Ozawa, Yoshihiro 551
p53 tumor suppressor protein in SV40 cells, discovery
of 436
Padgett, Billie L. 298
Page, Irvine 377
Page, Larry 524
Palese, Peter 541
Panama Canal 100, 101, 102
pandoraviruses, discovery of 554
Panum, Peter Ludwig 29, 36
Paoletti, Enzo 462
papillomavirus vaccines, development of 533

Parkman, Paul 341

Parodi, Armando S. 296
Parrish, Colin R. 429
parvovirus B-19 (fifth disease), discovery of 411
Pasteur, Louis 37, 55, 64
Patrick Playfair Laidlaw 170
Pauling, Linus Carl 250
Paul, John Rodman 205
Payne, James 365
Pedersen, Niels C. 474
Peebles, Thomas Chalmers 340
Peiris, Malik 537
Pellett, Philip E. 479
Pelon, William 278
Penhoet, Edward E. 453
Pereira, Helio Gelli 352, 487
Perlmann, Peter 389
Perutz, Max Ferdinand 250
peste des petits ruminants virus 438
Peters, C.J. 507
Phage Course at Cold Spring Harbor Laboratory,
founding of 226
pH meter, invention of 179
phocine distemper virus, discovery of 483
photomicroscopy 34
phylodynamics 498
phylogenetics 498
phylogeny based on mRNA gene sequences 427
phylogeography 498
Pickels, Edward 242
picobirnaviruses, discovery of 487
Pigliucci, Massimo 548
Pintard, John 25
Pirie, Norman Wingate 185
Pirosky, Ignacio 296
pithovirus sibericum 554
plaque assay, virus quantification, development of 257
Plotkin, Stanley A. 341, 361
Plowright, Walter 311, 384, 551
Pneumocystis carinii pneumonia 445
Poiesz, Bernard J. 440
Polio Hall of Fame, Warm Springs, Georgia 202
polio, planned global eradication of 555
polio vaccine, attenuated live-virus, development of 300
polio vaccine, inactivated virus, development of 268
poliovirus, adaptation to cotton rats and mice 201
poliovirus, complementary DNA synthesized in vitro 531
poliovirus, complete genomic sequence of 447
polioviruses, discovery of 109
poliovirus, infectious clone, development of 447
Pollack, Robert 399
polyacrylamide gel electrophoresis, development of 366
polymerase chain reaction (PCR), invention of 464
Poon, Leo 537
Popper, Erwin 109
porcelain ultrafilter, development of 57, 58
porcine circovirus, discovery of 410
porcine reproductive and respiratory syndrome virus,
discovery of 496
Porterfield, James 327

Porter, Rodney R. 305

Prangishvili, David 408
Preston, Richard 513
Price, Winston H. 278
prion disease 136
prions, etiologic agents of the spongiform
encephalopathies 454
Program to Monitor Emerging Diseases (ProMED),
development of 506
projection microscope 35
protein structure, X-ray crystallography, discovery of 250
Pruden, Mitchell 29
Prusiner, Stanley B. 454
Prusoff, William H. 309
pseudorabies vaccine, development of 472
pseudorabies virus 89
Ptashne, Mark 356
PubMed (NCBI), development of 519
puerperal fever 32
Purcell, Robert H. 400
quasispecies, development of concept of 484, 486
rabbit (Shope) papillomavirus, discovery of 171
rabbit (Shope) papillomavirus, progression to carcinoma
188
rabies 7, 94
rabies immunofluorescence diagnostic method,
development of 295
rabies-like viruses, discovery of 378
rabies, oral vaccination of wildlife 395
rabies vaccine, development of 55, 56, 117
rabies virus, discovery of 97
rabies virus, genotyping / mapping of wildlife variants
503
rabies virus, morphology/morphogenesis of 344
Racaniello, Vincent 447
radioimmunoassays, invention of 307
radioisotopic labeling, development of 116
Ramon, Gaston 134
Raoult, Didier 538, 550
rapid sequencing of DNA, development of the technology
for 424
rapid virus diagnostics linked to clinical care 385
Ratcliffe, Francis 238
rate-zonal ultracentrifugation, invention of 335
rational antiviral drug design 388
Rauscher, Frank J. Jr. 248
Rayer, Pierre-Franois-Olive 44
recombinant DNA technology, application of 398
recombinant-DNA technology, development of 397
recombinant live-virus vaccine, development of first 472
Reed, Lowell 196
Reed, Walter 77, 78, 82, 83
Reeves, William Carlisle 132, 214, 310
Relman, David A. 515
Remlinger, Paul Antoine 96, 104
reoviruses, discovery of 266
replication of DNA and RNA, discovery of mechanisms
308
representational difference analysis (RDA) 501
repressor genes, discovery of 356

respiratory syncytial virus, discovery of 277

restriction endonucleases, discovery of 373
restriction enzyme map of SV40 virus DNA 405
retroviral oncogenes, discovery of cellular origin of 417
retroviruses, morphology/morphogenesis/classification
of 265
reverse genetics, development of 430
reverse genetics for negative-strand RNA viruses 510
reverse transcriptase in retroviruses, discovery of 381
Reyes, Gregory 501
rhinoviruses, discovery of 278
ribavirin, broad spectrum antiviral drug 382
ribozymes, catalytic RNAs 443
Rice, Charles M. 543
Richardson, Carol 471
Rico-Hesse, Rebecca 502
Rifat Bay 96
Rift Valley fever virus, discovery of 165, 166, 167
Riley, Vernon Todd 313
rinderpest 49
rinderpest, global eradication of 551
rinderpest vaccine, attenuated-live virus, development
of 311
rinderpest virus, discovery of 91
Rios, Francisco A. 197
Rivers, Thomas Milton 58, 74, 145, 146, 147, 148, 187,
193, 240
Rivers, Thomas Milton, criteria for proof of viral disease
causation 193
Rizzetto, Mario 425
RNA-dependent DNA polymerase in retroviruses,
discovery of 381
RNA-dependent RNA polymerase in VSV, discovery of
380
RNA interference (RNAi), discovery of 523
RNA splicing and split genes, discovery of 422
Robbins, Frederick Chapman 120, 243
Roberts, Richard J. 422
Robinson, Harriet 316
Robinson, Marion 281
Robinson, Roslyn Q. 362
Robins, Roland K. 382
Rockefeller Foundation Virus Laboratory, founding of
148
Rockefeller Institute for Medical Research, founding of
86
Rockefeller, John D. Jr. 86
Rockefeller, John D. Sr. 86
Rodriguez-Boulan, Enrique 433
Rodriguez, F.R. 288
Roeder, Peter 551
Rohrer, Heinrich 448
Roizman, Bernard 353, 470
role of monocytes in infections 138
Rollin, Pierre E. 507, 535, 540
Rosenberg, Barbara 506
Rossman, Michael G. 432
Ross River virus 343
Ross, Ronald 50
Ross, R.William 281

rotaviruses, discovery of human 376

Rothberg, Jonathan M. 544
Rott, Rudolf 418
Rous, Francis Peyton 115, 171, 188
Rous sarcoma virus, discovery of 115
Roux, Pierre-Paul-Emile 55
Rowe, Wallace P. 259, 283, 351
Rowson, Kenneth Edmund 313
Royal Society 10
rubella vaccine 361
rubella virus, discovery of 124
Rubin, Harry 293
Ruck, Brian 401
Ruckle, Gisele 340
Rueckert, Roland R. 432
Rupprecht, Charles E. 503
Rush, Benjamin 22, 23
Ruska, Ernst 172, 199
Ruska, Helmut 139, 199
Russian Spring-Summer encephalitis virus, discovery
of 194
Rutter, William J. 453
Sabatini, David 433
Sabattini, Marta 296
Sabi virus (Brazilian hemorrhagic fever), discovery of
511
Sabin, Albert Bruce 106, 169, 266, 300
Sabin, Florence Rena 138
Salas, Rosalba A. 502
Salk, Jonas 268
Salzman, Norman 272
Sambrook, Joseph 402
Sanarelli, Giuseppe 75
sandfly (phlebotomus) fever viruses, discovery of 111
Sanford, Katherine K. 208
Sanger, Frederick 421, 424
SARS coronavirus, discovery of 537
Sawyer, Wilbur Augustus 148, 151
scanning electron microscope, invention of 184
scanning tunneling electron microscope, invention of
448
Schaeffer, Priscilla 451
Schfer, Werner 276
Scherer, William 310
Schleiden, Matthias 28
Schmaljohn, Connie S. 393
Schmallenberg virus, discovery of 552
Schmidt, Nathalie J. 150
Schneweis, Karl-Eduard 336
Schoening, Harry 142
Scholtissek, Christoph 418
Schramm, Gerhard 279
Schrdinger, Erwin 224
Schuster, Peter 484
Schuurs, Anton 389
Schwabe, Calvin W. 547
Schwann, Theodor 28
scrapie prion, discovery of transmissibility of 186
SDS polyacrylamide gel electrophoresis, development
of 366

Sellards, Andrew 152

semi-conservative mode of replication of DNA, discovery
of 292
Semmelweis, Ignaz Philipp 32
Semple, David 117
Sencer, David J. 416, 420
Sendai virus (murine parainfluenza virus 1), discovery
of 263
sequence-independent, single-primer amplification
(SISPA) 501
Sever, John L. 271, 348
Shah, Keerti 387
Sharp, Phillip A. 402, 422
Shatkin, Aaron J. 368, 413
sheeppox virus 90
Shelokov, Alexis 310
Shimomura, Osamu 338
Shope, Richard Edwin 148, 161, 171
Shope, Robert Ellis 327, 343, 378, 393, 502, 504
Shortridge, Kennedy F. 520
shotgun cloning/sequencing, invention of 500
Shuda, Masahiro 549
Sickles, Grace 239
Siegert, Rudolf 362
Sigurdsson, Bjrn 270
simian foamy virus, infection in humans 394
simian immunodeficiency viruses (SIVs), discovery of
468
Siminovitch, Louis 244
Simon, Charles Edmund 147
Simpson, David I. 378
Sindbis virus, discovery of 273
Singer, Beatrice A. 279
Singer, Maxine 275, 399
single burst experiment 204
single cell burst size, discovery of 267
single nucleotide polymorphisms (SNPs), detection of
470
single stranded DNA viral genome, discovery of 306
Sin Nombre virus (hantavirus pulmonary syndrome),
discovery of 507
Sinsheimer, Robert L. 306
Sipe, Jean D. 368
Skehel, John J. 449
Slenczka, Werner 362
slow viruses, development of the concept of 270
smallpox 16, 17, 18, 19
smallpox, global eradication of 423
Smithburn, Kenneth C. 205, 232
Smith, Geoffrey 316
Smith, Hamilton 373, 500
Smith, Hugh Hollingsworth 183
Smithies, Oliver 478
Smith, Jean S. 503
Smith, Kendall 354
Smith, Lloyd M. 475
Smith, Margaret Gladys 269, 283
Smith, Theobald 67
Smith, Wilson 170
Smolinski, Mark 504

Snow, John 29
solid phase synthesis technology, development of 317
Song, Jin-Won 393
Sonnenberg, Nahum 480
Soper, Fred L. 176
southern blotting, development of 414
Southern, Edwin Mellor 414
Spallanzani, Lazzaro 15
specificity of disease causation 37
Spence, Leslie 342
Spinco Model E analytical ultracentrifuge, development
of 235
Spinco Model L preparative ultracentrifuge, development
of 242
spontaneous generation, refutation of concept 15
Sputnik, virophage, discovery of 550
Stahl, Frank W. 291, 292
Stanley, Wendell Meredith 182, 254
Stark, George R. 437
St. Clair, Marty 477
Steck, Franz 395
Steinhardt, Edna Harde
Harde, Edna Steinhardt 121
Steinman, Ralph A. 403
Stent, Gunther 226
Sternberg, George Miller 66
Stewart, Sarah Elizabeth 294
St. Louis encephalitis virus, discovery of 174
Stoker, Michael George Parke 322
Stokes, Adrian 151
Studdert, Michael J. 177, 476
subacute sclerosing panencephalitis, discovery of etiology
348
Summers, Max D. 451
Suttle, Curtis 492, 538, 550
SV40 virus as cause of human tumors, controversy 387
SV40 virus (simian virus 40), discovery of 312
Svedberg, Theodor 137
Swanepoel, Robert 535, 540
Swanson, Robert A. 439
Sweet, Benjamin 312
swine flu vaccination episode of 1976 420
swine influenza virus, discovery of 161
Symons, Robert H. 397
Systems Biology / Systems Virology 497
Szmuness, Wolf 434
Szostack, Jack W. 461
Tacaribe virus, discovery of 342
Takahashi, Michiaki 341
Takatsuki, Kiyoshi 440
Taktsy, Gyula 271
Talmage, David 285
Tamm, Igor 240, 339
Taq polymerase 464, 488
Tasaka, S. 124
Tatum, Edward Lawrie 229
Taubenberger, Jeffery K. 129, 541
Taylor, Grant 333
Taylor, Richard Moreland 273, 310, 327
Taylor, William P. 438, 551

Temin, Howard Martin 293, 381

Ten Broeck, Carl 180
ter Meulen, Volker 316
Terpstra, Cathrinus 496
Tesh, Robert B. 384, 502
Texas fever 67
The Hot Zone, publication of book 513
Theiler, Arnold 92
Theiler, Max 148, 157, 183
Thompson, Wayne H. 314
Thottapalayam virus, discovery of 393
Thuillier, Louis 55
thymus, role in cellular immunity, discovery of 321
tick-borne encephalitis virus, discovery of 194
Tignor, Gregory H. 378
Tischer, Ilse 410
Tiselius, Arne Wilhelm Kaurin 98
tobacco mosaic disease 60
tobacco mosaic virus, crystallization of 182
tobacco mosaic virus, discovery of 69, 70
tobacco mosaic virus, infectivity of RNA, discovery of
279
tobacco mosaic virus, purification and crystallization
of 182
tobacco mosaic virus, reconstitution from protein and
RNA 274
tobacco mosaic virus, structure 192
Todaro, George 374
Toll-like receptors 517
toroviruses, discovery of 457
torque teno virus, discovery of 521
Torsvik, Vigdis 408
Tournier, Paul 331
Towner, Jonathan S. 540
transduction: DNA transfer by bacteriophage, discovery
of 255
transformation in bacteria, discovery of 149
transmissible spongiform encephalopathy, scrapie 186
transmission electron microscope, invention of 172, 173
transovarial transmission of viruses by arthropods 384
transposable elements, transposons, development of the
concept of 207
Traub, Erich 187
Traum, Jacob 142, 189
Tree of Life, based on mRNA gene sequences 427
Trentin, John J. 333
triplet coding strategy of DNA, discovery of 323
trypsinization for dispersal of cells in culture 125
Tsien, Roger 338
tuberculosis 49
tumors in hamsters induced by human adenoviruses,
discovery of 333
Tumpey, Terrence M. 541
Twort, Frederick William 126
Tyrrell, David Arthur John 350
Uganda Virus Research Institute 232
Ugolini, Gabriella 11, 518
ultracentrifuge, development of 137
ultrafilterable virus, concept of 60
ultrafiltration 58
uncultivable viruses, development of techniques to find

501
Urbani, Carlo 537
U.S. Army Yellow Fever Commission 82
U.S. Centers for Disease Control and Prevention 218,
325, 441
U.S. Communicable Disease Center 325, 363
U.S. National Academy of Sciences, founding of 43
U.S. National Center for Biotechnology Information
(NCBI), development of PubMed 519
U.S. National Library of Medicine 481
U.S. Public Health Service, founding of 24
vaccination 16, 17, 18, 19
vaccinia virus 121
vaccinia virus, discovery of 61, 104
vaccinia virus recombinants, for vectored viral vaccines
462
Valentine, Robin C. 352
Valenzuela, Pablo 453
Valle, Henri 99, 140
van den Hoogen, Bernadette 528
van der Groen, Guido 415
van der Westhuizen, Barnard 367
van Leeuwenhoek, Anton 12
van Regenmortel, Marc H. V. 72, 491, 508
van Weemen, Bauke 389
variant Creutzfeldt-Jakob disease (vCJD), BSE prion 514
varicella-zoster virus, discovery of 139
variolation 13
variola virus, discovery of 61, 104
Varmus, Harold E. 417
Venezuelan equine encephalitis virus, discovery of 197
Venter, J. Craig 492, 500
Verge, Jean-Louis-Armand 153
Vero cell line, establishment of 328
vesicular exanthema virus of swine, discovery of 189
vesicular stomatitis viruses, discovery of 142
Veterinary Virology, publication of book 476
Villarreal, Luis 484
Villerm, Louis-Ren 29
Vinograd, Jerome 235, 291
Viral and Rickettsial Infections of Man, publication of
book 240
viral damage to cellular homeostasis, development of
concept 456
viral diagnostic instruments, development of high
throughput 522
viral diagnostics linked to clinical care 385
viral disease diagnostics, broad application of methods
241
viral disease diagnostics, founding of 150
viral disease pathogenesis research, founding of 237
viral genomes, sequencing of MS2 and 174 421
viral oncogene hypothesis, development of 374
viral pathogenesis research, founding of the modern era
316
viral receptors on target cells, discovery of 234
viral vaccines based upon innovative technologies 473
viral vaccines, development, promulgation and
commercialization of 341
Virchow, Rudolf Ludwig Karl 39
viroids, discovery of 391

virology, establishment as a distinct field of science 145

virology, father of 37
Virology, publication of journal 272
virophage, Sputnik, discovery of 550
Virosphere 427
virus entry mechanisms 404
virus entry mechanisms, discovery of 10, 404
viruses as neuronal pathway tracers 11, 518
viruses in the ocean 492
viruses of Archaea, discovery of 408
viruses replicating in both plants and insects, discovery
of 256
virus evolution, development of modern concepts 484
virus, first visualization 61
Virus Laboratory, University of California Berkeley,
founding of 254
virus-like particles 533
virus neutralization, discovery of 66
virus quantification by plaque assay, development of 257
virus species, definition of 491
visualization of viral DNA by electron microscopy 304
Vogt, Marguerite 257, 267
Volberding, Paul 445
von Ardenne, Manfred 184
von Behring, Emil Adolf 54
von Borries, Bodo 199
von Magnus, Preben & Herdis 251
von Prowazek, Stanislaus 104
Wagner, Robert 272
Wakita, Takaji 543
Waldmann, Otto 140
Walker, Duard Lee 298
Walsh, Peter 535
Wandeler, Alexander 395
Wang, David 549
Ward, Richard 482
Waring blender experiment 253
Waterson, Anthony Peter 431
Watson, James Dewey 260
Watts, Douglas M. 384
Weaver, Scott 484
web-based surveillance tools (Google), development of
524
Webb, Patricia A. 347, 415
Webster, Leslie Tillotson 157, 174
Webster, Robert G. 337, 508, 520, 525
Weigle, William 315
Weinberg, Robert A. 450
Weissenbacher, Mercedes 296
Weissmann, Charles 430
Weller, Thomas Huckle 120, 139, 243, 283
Wensvoort, Gert 496
western blotting, invention of 437
western equine encephalitis virus, discovery of 159
West Nile virus, discovery of 205
West Nile virus in the United States 527
What is Life?, publication of book 224
White, David Ogilvie 379
Whitfield, Sylvia G. 415
Whitley, Richard J. 388
WHO Global Polio Eradication Initiative 555

Wigler, Michael H. 450, 501

Wildy, Peter 272, 357, 370
Wiley, Don C. 449
Wilkins, Maurice Hugh 260
Williams, Elizabeth S. 442
Williams, Robley Cook 274
Will, Robert 514
Wilson, Ian A. 449
Wimmer, Eckard 447, 480, 531
Witkowski, Jan A. 105, 118, 119
Witkowski, Joseph 382
Woese, Carl Richard 427
Wollman, lie Leo 195
Wollman, Elisabeth 195
Wollman, Eugne 195
Woodall, John P. 228, 506
Woode, Gerald N. 457
Woodruff, Alice Miles 163
Woodruff, Gwen 238
Work, Telford Hindley 273, 288, 310
World Wide Web, development of 499
Wright, Arthur 169
Wyckoff, Ralph Walter 197
X-ray crystallography 192
Yabe, Yoshiro 333
Yalow, Rosalyn Sussman 307
Yamamoto, Janet K. 474
Yamanishi, Koichi 479
Yamanouchi, T. 130
Yanagihara, Richard 393
Yasumura, Yosihiro 328
yeast artificial chromosomes, cloning of large DNA
fragments 461
yellow fever 51, 53, 76, 100, 101, 102
yellow fever epidemic, Philadelphia 22
yellow fever epidemics in U.S. 52
yellow fever vaccine, development of 183
yellow fever virus, discovery of 77, 78, 79, 80, 81
yellow fever virus, transmission to rhesus macaques 151,
152
York, Charles J. 280
Yoshida, Mitsuaki 440
Youngner, Julius 268
Young, Stuart 442
Zaitlin, Milton 451
Zaki, Ali Mohamed 553
Zaki, Sherif 166
Zeiss, Carl 27
Zhdanov, Victor 370
Zhou, Jian 533
zidovudine, first anti-HIV drug 477
Zilber, Lev Alexandrovich 194
Zillig, Wolfram 408
Zinder, Norton 255
Zinke, Georg Gottfried 20
Zinkernagel, Rolf M. 406
zur Hausen, Harald 409
Zu Rhein, Gabriele M. 298
Zworykin, Vladimir 184

The Author

Frederick A. Murphy

Frederick A. Murphy is a professor in the Department of

Pathology, University of Texas Medical Branch (UTMB),
Galveston, Texas. He holds a BS and DVM from Cornell
University and a PhD from the University of California, Davis.
He has served as Dean and Distinguished Professor, School of
Veterinary Medicine, and Distinguished Professor, School of
Medicine, University of California, Davis. Before that he served
as Director of the Division of Viral and Rickettsial Diseases and
then Director of the National Center for Infectious Diseases,
Centers for Disease Control, Atlanta. He is a member of the
Institute of Medicine of the U.S. National Academy of Sciences
(where he has served on 10 committees), the Deutschlands
Nationale Akademie der Wissenschaften (German National
Academy of Sciences - Leopoldina) and the Acadmie Royale
de Mdecine de Belgique (Belgian Royal Academy of Medicine).
His honors include an honorary Doctor of Medicine and
Surgery from the University of Turku, Turku, Finland; an
honorary Doctor of Science from the University of Guelph,
Ontario, Canada; an honorary Doctor of Veterinary Medicine
from the University of London, United Kingdom; an honorary
Doctor of Science from University College Dublin, Ireland; the
Presidential Rank Award of the U.S. Government; the PennVet
World Leadership Award from the University of Pennsylvania;
the Distinguished Microbiologist Award from the American

College of Veterinary Microbiologists; the Richard Moreland

Taylor Award from the American Committee on ArthropodBorne Viruses, American Society of Tropical Medicine and
Hygiene; the University of California Davis Medal; the K.F.
Meyer Gold Headed Cane from the American Veterinary
Epidemiology Society and American Veterinary Medical
Association; and an Honorary Fellowship from the John Curtin
School of Medical Research, the Australian National University.
At UTMB he is a member of the Institute for Human Infections
and Immunity, the Center for Biodefense and Emerging
Infectious Diseases, the Galveston National Laboratory, and
the McLaughlin Endowment for Infection and Immunity. His
professional interests, in association with many colleagues,
include the pathology and epidemiology of highly pathogenic
viruses/viral diseases: (1) Rabies: long running studies leading
to the identification of more than 25 viruses as members of the
virus family Rhabdoviridae, identification and characterization
of the first rabies-like viruses, and major studies of rabies
pathogenesis in experimental animals, including the initial
descriptions of infection events in salivary glands and in
muscle. (2) Arboviruses: long running studies of alphaviruses,
flaviviruses and bunyaviruses with the initial proposal for the
establishment and naming of the virus family Bunyaviridae,
and characterization of reo-like viruses culminating in the
establishment and naming of the virus genus Orbivirus. (3) Viral
hemorrhagic fevers: long running studies leading to the initial
discovery of Marburg and Ebola viruses, and characterization
of several other hemorrhagic fever viruses, culminating in the
establishment and naming of the virus families Arenaviridae
(e.g., Lassa and Machupo viruses) and Filoviridae (Marburg
and Ebola viruses), and elucidation of the pathology and
pathogenesis of the diseases in humans and experimental
animals caused by these exceptionally virulent agents. (4)
Viral encephalitides: long running studies of the pathogenesis
of neurotropic viruses in experimental animals, including
alphaviruses, flaviviruses, bunyaviruses, enteroviruses,
paramyxoviruses, herpesviruses, and others. He has been
a leader in advancing the concept of new and emerging
infectious diseases and new and emerging zoonoses, the
concept that has reenergized the infectious disease research
sciences. Most recently his interests have included the threat
posed by bioterrorism and national efforts in prevention of this
threat.

1998: John, Terence, Irene,

Fred, Rick and Tim Murphy