Chapter 1: Introduction

Introduction to Virology
Learning Objectives:
Understand what a virus is. Summarize the history of virology. Describe techniques used to study viruses.

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Chapter 1: Introduction

Virus Diversity
There is much biological diversity between viruses. Viruses are successful parasites. Understanding diversity is the key to understanding viruses. At a molecular level:
protein-protein
protein-nucleic acid protein-lipid interactions
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Chapter 1: Introduction

Viruses are distinct from living organisms

Viruses are submicroscopic, obligate intracellular parasites. Rickettsiae and Chlamydiae are obligate intracellular parasitic bacteria. Therefore it is necessary to add further define what constitutes a virus.
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Chapter 1: Introduction

Virus Definition:
Virus particles are produced from the assembly of pre-formed components, other agents grow and reproduce by division.

Virus particles (virions) do not grow or undergo division

Viruses lack the genetic information necessary for the generation of energy or for protein synthesis (ribosomes)
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Viruses are energy parasites No known virus has the biochemical

or genetic potential to generate the energy necessary to drive biological processes. They are absolutely dependent on the host cell for energy.
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Are viruses are alive?

One view is that inside the host cell, viruses are alive, but outside it they are complex assemblages of inert chemicals. Chemical changes occur in extracellular virus particles, but these are not the 'growth' of a living organism.
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How big are viruses?

Viruses are always smaller than bacteria? Size alone does not distinguish them. The largest virus known (Mimivirus) is 400 nm in diameter. The smallest bacteria are only 200300 nm long.

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Chapter 1: Introduction

The History of Virology

First record of virus infection: poliovirus in ancient Egypt (3700 BC). Pharoh Ramses V died from smallpox in 1196 BC. Smallpox was endemic in China by 1000 BC. The Chinese invented variolation. Edward Jenner: Smallpox vaccination.
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Chapter 1: Introduction

The History of Virology

Antony van Leeuwenhoek (1632-1723) constructed the first microscopes and saw bacteria.

Robert Koch and Louis Pasteur (1880s) jointly

proposed the 'germ theory' of disease.

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Chapter 1: Introduction

Kochs Postulates
1. The agent must be present in every case of the disease. 2. The agent must be isolated from the host and grown in vitro. 3. The disease must be reproduced when a pure culture of the agent is inoculated into a healthy susceptible host.

4. The same agent must be recovered once again from the experimentally infected host.
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Chapter 1: Introduction

The History of Virology

Dimitri Iwanowski (1892). Martinus Beijerinick (1898). Freidrich Loeffler and Paul Frosch (1898).

Karl Landsteiner and Erwin Popper (1909).

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Discovery of bacteriophages
Frederick Twort (1915) and Felix d'Herelle (1917) were the first to recognize bacteriophages ("eaters of bacteria"). In the 1930s and subsequent decades, Salvador Luria, Max Delbruck and many others used these viruses as models. Important to understanding all types of virus. The history of virology is the development of experimental tools.
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Living Host Systems

1881: Louis Pasteur, attenuation of rabies. 1885, inoculation with the first artificially produced virus vaccine. Whole plants have been used to study plant viruses ever since Tobacco mosaic virus was discovered by Iwanowski.

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Living Host Systems

1900: Walter Reed demonstrated that yellow fever was caused by a virus and spread by mosquitoes. 1937: Max Theiler produced an yellow fever attenuated vaccine.

1930s-1950s: animal systems to identify

and propagate pathogenic viruses.
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Plaque Assays

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Tissue Culture
Eukaryotic cells grown in vitro ("tissue culture"). Embryonated hens eggs: Influenza virus, Vaccinia virus. Counting the 'pocks' on the chorioallantoic

membrane of eggs was the first

quantitative assay for a virus.
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Animal hosts still have their uses in virology:

For viruses which cannot be propagated in vitro Pathogenesis experiments Vaccine safety Nevertheless, they are increasingly being discarded: Expensive Complex and difficult to interpret

Host variation
Ethical? Overtaken by cell culture and molecular biology
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Cell Culture Methods

Began early in the twentieth century with whole-organ cultures, then progressed to methods involving individual cells, either: Primary cell cultures or: Immortalized cell lines
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Cell Culture Methods

1949: John Enders and colleagues propagate Poliovirus in primary human cell cultures. 1950s and 1960s: many viruses. 1952: Renato Dulbecco - plaque assay.
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Serological/Immunological Methods
1941: George Hirst - haemagglutination of red blood cells by influenza virus. An important tool for influenza and other viruses, for example, Rubella virus. Can measuring the titre (i.e. amount) of virus and determine the antigenic type.
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Serological/Immunological Methods
Complement fixation tests Radioimmunoassays Immunofluorescence (direct detection of virus antigens in infected cells or tissue) Enzyme-linked immunosorbent assays (ELISAs) Radioimmune precipitation Western blot assays
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Serological Methods

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Monoclonal Antibodies

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Ultrastructural Studies
Physical methods Chemical methods Electron microscopy

1930s: Physical measurements of virus

particles. 1960s: Sedimentation properties of viruses in ultracentrifuges.
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Differential Centrifugation

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Physical Methods

Spectroscopy
Electrophoresis X-ray diffraction by crystalline forms of purified virus
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Physical Methods
Complete structures of many viruses at a resolution of a few angstroms (). Not all viruses! Nuclear magnetic resonance (NMR). Only relatively small molecules can be analysed with NMR technology.
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Chemical Methods
Stepwise disruption of particles. Electrostatic interactions Non-ionic, hydrophobic interactions. Proteins which interact with lipids. Surface labelling. Cross-linking reagents.

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Denaturation of TMV

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Electron Microscopy
Electron microscopes. The first electron micrograph of a virus (TMV) was published in 1939.

Direct examination of viruses at magnifications of over 100,000 times.

Two types of electron microscope, the transmission electron microscope (TEM) and the scanning electron microscope (SEM).
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Electron Microscopy

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Molecular Biology
The terms 'molecular biology, 'genetic engineering' and 'genetic manipulation' have taken on the meaning of manipulating nucleic acids in vitro. Virus infection has long been used to probe the working of 'normal' (uninfected) cells. This new technology shifted the emphasis from proteins to nucleic acids.

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Hybridization Techniques
Nucleic acid-centred technology offers significant advances in detection of viruses and virus infection.

A labelled hybridization probe is allowed to react with a crude mixture of nucleic acids.
The specific interaction of the probe with complementary virus-encoded sequences reveals the presence of virus genetic material.
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Hybridization Techniques

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The Polymerase Chain Reaction (PCR)

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Bioinformatics
Computers are the ideal means of storing and processing nucleotide sequences. 'Bioinformatics' is a broad term used to describe any application of computers to biology. Specifically, the term applies to computer manipulation of biological sequence data.

Bioinformatics permits the inference of function from a linear sequence.

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Bioinformatics

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Bioinformatics
Computers are used to make predictions based on nucleotide sequences, including: open reading frames amino acid sequences of proteins control regions of genes secondary structure of proteins and nucleic acids molecular modelling Vast databases of nucleotide and protein sequence information.
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Three-dimensional structure of the DNA binding domain of SV40 T-antigen:

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Summary
Investigations of viruses - from particles via genomes back to proteins again - have have come full circle. The present pace of research in virology

shows that there is still far more we need to

know.
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