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VETERINARY VIROLOGY

VMP-5355

JOSEPH E. GYIMAH, BS, BVM, MS, PhD School of Veterinary Medicine Ross University, St. Kitts, West Indies

TABLE OF CONTENTS

PRINCIPLES OF VIROLOGY 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Nature and Structure of Viruses ..1 Classification and Nomenclature of Viruses...9 Virus Cultivation and Purification ...21 Virus Quantitation and Replication...................27 Effects of Viruses on Host Cells 41 Pathogenesis of Viral Infections 52 Epidemiology of Viral Diseases .67 Viral Chemotherapy .76 Host Response to Viral Infections .79 Laboratory Diagnosis of Viral Diseases 84

VETERINARY AND ZOONOTIC VIRUSES 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Poxviridae ..91 Circoviridae ..101 Herpesviridae ..104 Asfarviridae ..125 Parvoviridae .128 Papillomaviridae ..136 Adenoviridae ...141 Retroviridae ..147 Rhabdoviridae ..164 Picornaviridae ...171

21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 33. 34.

Paramyxoviridae .178 Orthomyxoviridae 189 Reoviridae 197 Coronaviridae ..204 Togaviridae ..215 Flaviviridae ...220 Arteriviridae . 230 Bunyaviridae 235 Caliciviridae . 239 Birnaviridae . 243 Bornaviridae ... 245 Astroviridae ..... 247 PRION AND PRION DISEASES . 248 Introduction to Serology .... 255 Antigen and Antibody Immunoassays . 259

NATURE AND STRUCTURE OF VIRUSES


Some Landmarks In Virology Tobacco mosaic virus. First virus to be recognized as filterable by Dmitri Iwanoski in Russia in1892.
In an attempt to isolate the cause of tobacco mosaic disease, Dmitri Iwanoski filtered the sap of diseased tobacco plants through a porcelain filter that was designed to retain bacteria. He expected to find the microbe trapped in the filter; instead, he found that the infectious agent had passed through the minute pores of the filter. When he injected healthy plants with the filtered fluid, they contracted tobacco mosaic disease. Iwanoski still believed the infectious agent to be a bacterium that was small enough to pass through the filter. In 1899, Martinus Beijerinck and his coworkers in Holland observed that the behavior of the agent was different from that of bacteria. In 1935, an American chemist, Wendell M. Stanley, isolated the tobacco mosaic virus, making it possible for the first time to carry out chemical and structural studies on a purified virus.

Foot-and-mouth disease virus. First filterable animal virus demonstrated by Friedrich August Lffler and Paul Frosch in 1898. Yellow fever virus. First human virus demonstrated by Walter Reed et al. in 1901. Adelchi Negri. Discovered the inclusion bodies of rabies virus in 1903. Peyton Rous. First demonstration of a solid tumor virus, Rous sarcoma virus, in 1911. Bacteriophages. Discovered by the British bacteriologist Frederick Twort and the microbiologist Flix dHrelle in France in 1916-17. Ernest Goodpasture and Alice Woodruff. Reported on the use of fertile hens egg as a medium for cultivating viruses in 1931. John F. Enders et al. Reported in 1949 that nonneural tissue supports poliovirus replication in culture. Max Knoll and Ernst Ruska. Invented the electron microscope in 1932. Microbial World Microorganisms such as protozoa, fungi, and bacteria are unicellular microorganisms. They possess the biologic equipment necessary for the production of meta-

2 bolic energy and macromolecules [nucleic acids, proteins, polysaccharides, and lipids]. Viruses, however, are not cells; they are totally dependent on their cellular hosts for these necessary functions. Outside a susceptible cell, the virus particle is metabolically inert.
A Comparison of Viruses and Bacteria Conventional Rickettsia Bacteria Chlamydophila + + + + + + + + + + + + + + -

Property > 300 nm diameter

Viruses + +

Obligate intracellular parasite Plasma membrane Binary fission Possess both DNA and RNA Functional ribosomes ATP-generating metabolism Sensitive to antibiotics Sensitive to interferon

The size of a virus in comparison to a bacterial and animal cell.

3 Definition of a Virus Viruses are submicroscopic particles whose genomes are elements of nucleic acid that replicate inside living cells using the cellular synthetic machinery for the production of progeny virions.

Virus is the Latin word for fluid poison. Originally used to describe infectious agents that passed through bacteriologic filters, ie, filterable viruses. Terms and Definitions in Virology

Host range: Range of animal species and tissue cells that the virus can infect. The host range for a given virus may be broad or extremely limited. Viruses infect invertebrates, vertebrates, plants, fungi, bacteria, etc. Structural protein [Protomer]: Protein subunit which may be assembled into capsomeres. Maybe one type of protein subunit or diverse protein subunits. Capsomeres: Morphological subunits from which the virus capsid is built. Capsid: The protein shell or coat that encloses the nucleic acid genome. Envelope: A lipid-containing membrane that surrounds some viruses. Nucleocapsid: The capsid together with the enclosed nucleic acid. Virion: The complete infective virus particle. Viral attachment proteins [VAPs]. Capsid and envelope proteins that mediate the attachment of viruses to specific host cell receptors. Incomplete virion: Virion without nucleic acid [empty capsid]. Defective virus: A virus that cannot replicate because it lacks a full complement of viral genes; results from mutations or errors in the production and assembly of virions. Replication occurs only in mixed infections with a helper virus. Pseudotype: When related viruses infect the same cell, the genome of one virus may be enclosed in the heterologous capsid of the second virus. Pseudovirion: During viral replication, the capsid sometimes encloses host nucleic acid rather than viral nucleic acid. Such particles look like ordinary virus particles when observed by electron microscope, but do not replicate. Episome: Autonomous extra-chromosomal genetic element [which may later become integrated into chromosomal DNA or may remain separate].

4 Provirus: Viral DNA that is integrated into a host cell chromosome in a latent state and must be activated before it is transcribed, leading to production of progeny virions. It is transmissible from a parent cell to its daughter cells. Structure of Viruses Knowledge of virus structure enhances our understanding of the mechanism of certain processes such as the interaction of virus particles with cell surface receptors and neutralizing antibodies. Structurally, a virus consists of nucleic acid, surrounded by a protein coat, the capsid. In some viruses the capsid is enveloped, in others it is naked or nonenveloped.

Schematic diagram of the structure of a virus

Capsid (Capsa-Latin-box) Capsids are made up of capsomeres held together by noncovalent bonds. Most viruses have 1 capsid except the reoviruses that have an outer, middle, and inner capsids. Viral architecture can be grouped into three types based on the arrangement of the morphologic subunits of the capsid: Cubic symmetry [Icosahedron]: Cubic symmetry is of the icosahedron pattern, the most efficient arrangement for subunits in a closed shell. An icosahedron is a solid with 12 corners [vertices], 20 equilateral triangular faces, and 30 edges.

The capsomeres are arranged in a regular pattern on an icosahedron and their numbers vary in different viruses. They are solid in most viruses, but hollow in herpesviruses and reoviruses. Pentons: Capsomeres located at the vertices of icosahedral virions; each is surrounded by five neighbors. Both DNA and RNA viral groups contain viruses with cubic symmetry.

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Naked icosahedral Enveloped icosahedral

Helical symmetry The capsomeres and nucleic acid molecule(s) self-assemble as a helix. Hence, because of the interaction between capsid protein and nucleic acid, it is not possible for empty helical particles [incomplete virions] to form.

In all animal viruses, helical nucleocapsids are wound into a coil and enclosed within a lipoprotein envelope.
Naked helical Enveloped helical

Complex viruses. Some virus particles do not exhibit simple cubic or helical symmetry but are more complicated in structure.

Poxviruses are brick-shaped with ridges on the external surface and a core and lateral bodies inside.

6 Functions of a capsid

Responsible for the structural symmetry of the virus particle. Encases and protects the viral nucleic acid from nucleases in biologic fluids. Facilitates the attachment of virus to susceptible cells. Some viral capsids contain enzymes which play roles in the infection process, eg, many viruses contain their own nucleic acid polymerases which transcribe the viral nucleic acid into mRNA. Determines the antigenic characteristics of the virus [the hosts protective immune response is directed against antigenic determinants exposed on the surface of the virus capsid]. Envelope

Enveloped virions acquire their envelopes when the nucleocapsid buds from cellular membranes, eg, cytoplasmic membrane, nuclear membrane, or Golgi complex. Budding occurs only at sites where virus-specific proteins have been inserted into the host cell membrane. The envelope consists of: Virus-specified proteins. Associated with a number of crucial activities:

Receptor binding: Associated with glycoprotein peplomeres, observed as spikes in electron micrographs. The polysaccharides are derived from the host cell. Membrane fusion: Associated with fusion proteins. Fusion proteins are associated with peplomeres and are involved in key steps in viral entry into and viral release from cells. Matrix protein: Found as a layer in the inside of some envelopes. It serves as a recognition site for the nucleocapsid at the plasma membrane and also provides added rigidity to the envelope.

Lipid bilayer. Derived entirely from host cell membrane.

The integrity of the envelope is maintained only in aqueous or moist environments, hence it is readily disrupted by drying, acidic conditions, etc. Solvents such as ether or chloroform or detergents such as sodium deoxycholate are used to disrupt the integrity of the envelope. Such disruption results in loss of infectivity, except in some poxviruses [capsids and envelopes both have viral attachment proteins]. Chemical Composition of Virions Viral Proteins

Proteins constitute up to 50-70% of the virion. The proteins include structural proteins, enzymes, regulatory proteins, apoptosis inhibitors, etc.

Virusencoded enzymes include: [1] enzymes that transcribe viral genome into mRNAs, [2] enzymes that copy the nucleic acid genome, [3] enzyme that copies virion RNA into DNA (RNA-dependent DNA polymerase [reverse transcriptase]; carried by retroviruses and hepadnaviruses). Viral Nucleic Acids

Encode the genetic information necessary for replication of the virus. Using the synthetic machinery of the host cell, the genome directs the synthesis of many copies of all the viral components.

All viral genomes are haploid, ie, they contain only one copy of each gene. The genome may be monopartite [all viral genes contained in a single chromosome] or multipartite [segmented, ie, viral genes distributed among several chromosomes that constitute the viral genome].

DNA genome. The genome of all DNA viruses of vertebrates is monopartite. Double-stranded or single-stranded [Parvoviruses and Circoviruses]; circular or linear.

RNA genome Single-stranded or double-stranded [Reoviruses and Birnaviruses]. Monopartite [most RNA viruses] or multipartite. Positive-sense RNA genome. Can function as mRNA in the infected cell; thus, naked RNA extracted from a [+] sense RNA virus is infectious when injected into host cell.

8 By convention, since mRNA can be directly translated into polypeptides, it is designated plus [+] mRNA.

Negative-sense RNA genome. Cannot function as mRNA, therefore virion carries own transcriptase enzymes; injected naked RNA is not infectious.

Viral Lipids Found only in the envelope. It is a typical lipid bilayer with virus-coded glycolprotein peplomeres and, in some cases, other viral proteins, embedded in it. Viral Glycoproteins Most occur as membrane-anchored spikes which extend outward from the envelope. Stability of Viruses Like all infectious agents, viruses face harsh environmental conditions both in the body and on the outside. These include pH changes, bile salts, proteases, temperature changes, osmotic changes, sunlight, dessication, humidity, etc. To maintain transmissibility and, hence infection in a susceptible population, a virus must overcome some of these conditions.

In general, naked viruses survive well in the body and on the outside. On the other hand, enveloped viruses are more susceptible to environmental factors such as gastric acidity, drying and bile salts. Such differences in stability influence the ways in which naked and enveloped viruses can be transmitted.

Temperature. Viral surface proteins are denatured within a few minutes at temperatures of 55 to 60oC, making the virion incapable of normal cellular attachment, penetration, and/or uncoating. pH. Acidic conditions can lead to reversible or irreversible disassembly of the viral capsid. Most viruses survive best at physiologic pH, however, some viruses can tolerate a wide pH range.

CLASSIFICATION AND NOMENCLATURE OF VIRUSES


The main criteria for the classification of viruses established by the International Committee on Taxonomy of Viruses [ICTV] are: (1) the type and character of the viral genome, (2) the strategy of viral replication, and (3) the morphology of the virion. Family: Viruses are divided into families based on the following:

Size, structure and symmetry of the virus particle [icosahedral, helical or complex]; type of nucleic acid genome; number of nucleic acid strands and their polarity; and replication strategy. Families end with the suffix viridae, eg, Herpesviridae.

Subfamily: Deals with complex interrelationships among member viruses in a family. Subfamilies end with the suffix virinae, eg, chordopoxvirinae. Genera and Species: The classification of genera and species within a family are based on criteria which include host species, pathogenesis, nucleic acid homology and antigenic differences. Viral genera end with the suffix virus, eg, herpesvirus.
Currently, there are approximately 200 viral species in some 22 viral families known to infect the eight main domestic animal species [cattle, sheep, goats, pigs, dogs, cats, horses, and poultry].

VIRAL PROPERTIES THAT DISTINGUISH VIRUS FAMILIES Properties of the Virions of DNA Virus Families
Virion Diameter (nm) Envelope 17-22 25 42 55 45 80-100 150 + 175-215 + 130-300 + 250x200x200 Genome Size Nature (kb, kbp) ss, circular 1.7-2.3 ss, linear 5 ds, circular 3.2 ds, circular 8 ds, circular 5 ds, linear 36-44 ds, linear 125-235 ds, linear 170-190 ds, linear 150-350 ds, linear 170-250

Family Circoviridae Parvoviridae Hepadnaviridae Papillomaviridae Polyomaviridae Adenoviridae Herpesviridae Asfarviridae Iridoviridae Poxviridae

Capsid Symmetry Icosahedral Icosahedral Icosahedral Icosahedral Icosahedral Icosahedral Icosahedral Icosahedral Icosahedral Complex

10 Pictorial Representation of DNA Virus Families

OVERVIEW OF DNA VIRUS FAMILIES Family Parvoviridae (parvus, small) Parvoviruses are very small viruses. The family consists of two subfamilies: the subfamily Parvovirinae [contains viruses of vertebrates] and the subfamily Densovirinae [contains viruses of insects].

Replication occurs only in actively dividing cells; replication and assembly take place in the nucleus. Parvoviruses form acidophilic intranuclear inclusion bodies. The virions are remarkably resistant, remaining viable in the environment for long periods. Some Common Pathogens

Feline parvovirus [feline panleukopenia]; Canine parvovirus 1, 2a, 2b and 2c. Family Papillomaviridae

Papillomaviruses are small viruses. They cause chronic, latent, and transforming infections in their natural hosts.

Viral genome may be episomal or integrated into host cell DNA. Papillomaviruses replicate within the nucleus.

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Papillomaviruses encode proteins that promote cell growth. The viruses are stable in the environment. Some Common Pathogens

Papillomaviruses. Cause warts in domestic animals, eg, bovine papillomaviruses, equine papillomaviruses, and canine papillomavirus. Human papillomaviruses [causes warts; some are associated with cancer of the pharynx, cervix, and anus]. Family Adenoviridae (adenos, gland)

Adenoviruses are medium-sized viruses that possess filaments/fibers which project from vertex capsomers.

The fiber contains the viral attachment proteins. The penton base and fiber are toxic to cells. Adenoviruses replicate in the nucleus of host cells, producing basophilic intranuclear inclusion bodies. The viruses cause latent infections; may be reactivated by immunosuppression. Some adenoviruses are oncogenic. Some Common Pathogens

Canine adenovirus 1 [infectious canine hepatitis]; canine adenovirus 2 [kennel cough]; avian; equine, bovine, and ovine adenoviruses. Family Asfarviridae (African Swine Fever And Related Viruses)

Asfarviruses are large virions containing many proteins and several enzymes.

Virus replication occurs in the cytoplasm of host cells and in soft ticks of the Ornithodorus species. Produce intracytoplasmic inclusion bodies. The only pathogen is Asfivirus [African swine fever]. Family Herpesviridae (Greek herpein, to creep)

The family consists of three subfamilies: the subfamily Alphaherpesvirinae, the subfamily Betaherpesvirinae, and the subfamily Gammaherpesvirinae.

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The viruses replicate in the nucleus and mature by budding through the nuclear membrane. Produce acidophilic intranuclear inclusion bodies. Members of the family tend to be fragile and therefore require close contact for transmission. Some herpesviruses induce cytomegaly and/or syncytia, or are oncogenic. A feature of all herpesvirus infections is lifelong persistent infection, usually in latent form. Excretion of virus may occur continuously or intermittently, with or without episodes of recurrent clinical signs. Some Common Pathogens

Porcine herpesvirus 1 [pseudorabies]; avian herpesvirus 2 [Mareks disease]; feline herpesvirus 1; canine herpesvirus 1 [fading puppy syndrome]; equine herpesviruses 1-5; bovine herpesviruses 1-5. Family Poxviridae (poc, pustule)

The family Poxviridae consists of two subfamilies: the subfamily Chordopoxvirinae [contains poxviruses that infect vertebrates] and subfamily Entomopoxvirinae [invertebrate poxviruses].

Largest of all the viruses that infect domestic animals. They are brick-shaped or ovoid and complex in structure. Replication occurs entirely within the cell cytoplasm and virions are released by budding [enveloped virions] or by cell lysis [nonenveloped virions]. Both enveloped and nonenveloped poxviruses are infectious. Virions produce eosinophilic intracytoplasmic inclusion bodies. All poxviruses produce skin lesions. Some are oncogenic. Poxviruses can survive for many months or years in dried scabs. Some Common Pathogens

Variola virus [smallpox]; cowpox virus; horsepox virus; sheeppox virus; and contagious pustular dermatitis virus. Family Hepadnaviridae (hepar, liver)

The genome of hepadnaviruses consists of partially double-stranded and partially single-stranded DNA.

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The virions replicate in the nucleus of hepatocytes. Replication involves an RNA intermediate and requires a virus-coded reverse transcriptase [RNAdependent DNA polymerase]. Hepadnaviruses cause acute and chronic hepatitis which may progress to cirrhosis and primary hepatocellular carcinoma. Some Common Pathogens

Hepatitis B virus [humans]; woodchuck hepatitis B virus; ground squirrel hepatitis B virus; and duck hepatitis B virus. Family Circoviridae (circo, circular)

Circoviruses are the smallest known viruses of vertebrates and plants. They possess single-stranded DNA genome.

Replication occurs in the nucleus of cells in the S phase of the cell cycle. They produce large intranuclear or intracytoplasmic inclusion bodies. Circoviruses are stable in the environment. Some Common Pathogens

Chicken anemia virus, psittaccine beak and feather disease virus and porcine circovirus type 2 (postweaning multisystemic wasting syndrome [PMWS]). Family Polyomaviridae

Polyomaviruses are small viruses. The viruses are associated with inapparent infections in most hosts. They are highly host-specific.

The virions replicate in host cell nucleus; genome may be integrated with host cell DNA. Avian polyomaviruses have been associated with budgerigar fledgling disease and French molt.

14 Properties of the Virions of RNA Virus Families


Virion Family Picornaviridae Caliciviridae Arteriviridae Astroviridae Togaviridae Flaviviridae Bornaviridae Retroviridae Reoviridae Birnaviridae Bunyaviridae Arenaviridae
Orthomyxoviridae

Diameter (nm) 28-30 30-38 50-70 28-30 70 45-60 50-60 80-100 60-80 60 80-120 100-300 80-120

Envelope + + + + + + + +

Capsid Symmetry Icosahedral Icosahedral Icosahedral Icosahedral Icosahedral Icosahedral Icosahedral Icosahedral Icosahedral Icosahedral Helical Helical Helical

Genome Size Nature (kb, kbp) ss, (+), linear 7.2-8.4 ss, (+), linear 7.4-7.7 ss, (+), linear 15 ss, (+), linear 7.2-7.9 ss, (+), linear 9.7-11.8 ss, (+), linear 9.5-12.5 ss, ( -), linear 8.9 ss, (+), linear 7-11 ds, linear, 16-27 10-12 segments ds, linear, 7 2 segments ss, ( -), linear, 11-21 3 segments ss, ( -), linear, 10-14 2 segments ss, ( -), linear, 10-13.6 6-8 segments ss, (+), linear ss, ( -), linear ss, ( -), linear ss, ( -), linear 20-32 19.1 13-16 15-16

Coronaviridae Filoviridae Rhabdoviridae


Paramyxoviridae

80-220 790-970x80 180x75 150-300

+ + + +

Helical Helical Helical Helical

Pictorial Representation of RNA Virus Families

15 OVERVIEW OF RNA VIRUS FAMILIES Family Picornaviridae (pico, micro-micro) The family is made up of small, ether-resistant viruses. The RNA genome is single-stranded and positive sense, ie, it can also serve as mRNA.

Replication and assembly take place in the cell cytoplasm and the virions are released via cell lysis. The viruses are rapidly cytolytic, thus, infection is generally acute; persistent infections may occur with some picornaviruses. Multiple serotypes may produce same disease syndrome, with very little or no cross-protection between serotypes. Some Common Pathogens

Aphthovirus [foot-and-mouth disease]; rhinoviruses [common cold]; porcine enteroviruses 1-8; avian enterovirus [epidemic tremor]; polioviruses 1, 2, and 3; and hepatitis A virus [humans]. Family Caliciviridae (calyx, cup or goblet)

Caliciviruses are similar to picornaviruses but are slightly larger. Viral particles have cup-shaped depressions on the surface.

[+] sense RNA genome. Replication and assembly take place in the cell cytoplasm and the virions are released via cell lysis. Some Common Pathogens

Feline calicivirus; hepatitis E virus [humans]; lagovirus [rabbit hemorrhagic disease]. Family Togaviridae (toga, cloak)

Togaviruses possess a [+] sense RNA genome and an envelope which is tightly applied to an icosahedral nucleocapsid.

Replication takes place in the cytoplasm, and assembly involves budding through host cell membranes. Infection of host cells is acute and cytolytic.

16 Some Common Pathogens

Equine alphaviruses [eastern equine encephalomyelitis; etc]; rubivirus [rubella in humans]. Family Arteriviridae (from arteritis)

Virions possess [+] sense RNA genome and a closely adherent envelope.

Replication takes place in the cytoplasm, and assembly involves budding through host cell membranes. Primary host cells are macrophages. Persistent infections are frequently established. Some Common Pathogens

Equine arterivirus [equine viral arteritis] and Lelystad virus [porcine reproductive and respiratory syndrome].

Family Flaviviridae (flavus, yellow) Virions are enveloped and the genome consists of a [+] sense RNA genome.

Replication occurs in the cytoplasm, and assembly involves envelopment in the endoplasmic reticulum [true budding is not seen]. Infection of vertebrate cells is cytolytic. Labile viruses. Some Common Pathogens

Bovine pestivirus [bovine viral diarrhea-mucosal disease]; porcine pestivirus [hog cholera]; human flavivirus [yellow fever]; West Nile meningoencephalitis.

Family Reoviridae (sigla, respiratory enteric orphan) Medium-sized virions possessing two- or three-layered capsids and segmented, double-stranded RNA genome.

Segmentation results in genetic reassortment in dual infections. Replication and assembly take place in the cytoplasm. Virions produce acidophilic, intracytoplasmic, perinuclear inclusion bodies.

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Member viruses are moderate to stable in the environment. Some Common Pathogens

Ovine orbivirus [bluetongue]; equine orbivirus [African horse sickness]; rotaviruses [gastroenteritis in all domestic animals]. Family Birnaviridae (sigla, bi-rna, two segments of RNA)

Medium-sized virions possessing two molecules of double-stranded RNA genome.

Virions assemble and accumulate in the cell cytoplasm and are released by cell lysis. Birnaviruses are stable in the environment. Some Common Pathogens

Avibirnavirus [infectious bursal disease, Gumboro disease], and aquabirnavirus [infectious pancreatic necrosis of salmonid fish]. Family Coronaviridae (corona, crown)

Medium-sized virions possessing an envelope with club-shaped surface peplomers arranged in a fringe with a solar corona-like appearance.

The genome consists of a single molecule of [+] sense RNA. Replication occurs in the cell cytoplasm and virions mature by budding through the endoplasmic reticulum and Golgi membranes. The viruses have a narrow host range and readily establish persistent infections. Some Common Pathogens

Porcine coronavirus [transmissible gastroenteritis]; feline coronavirus [feline infectious peritonitis]; bovine coronavirus [gastroenteritis]; avian coronavirus [infectious bronchitis]; human coronaviruses [common cold]. Family Paramyxoviridae

Large virion with a helical nucleocapsid surrounded by a pleomorphic envelope [spherical to filamentous].

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The genome consists of a single, [-] sense RNA. Virions penetrate host cell by fusion with the plasma membrane. Replication occurs in the cell cytoplasm and assembly occurs via budding from the plasma membrane. All members produce intracytoplasmic inclusion bodies; in addition, some members produce intranuclear inclusion bodies [morbilliviruses]. Viruses induce cell-cell fusion, producing syncytia or multinucleated giant cells. Some member viruses cause persistent infections. Labile virions. Some Common Pathogens

Canine morbillivirus [canine distemper]; human morbillivirus [measles]; bovine morbillivirus [rinderpest]; and avian paramyxovirus 1 [Newcastle disease].

Family Orthomyxoviridae (orthos, straight; myxa, mucus) Medium-sized, enveloped, pleomorphic virions [spherical or tubular]. Envelope contains surface projections with hemagglutinin or neuraminidase activity.

The genome is segmented, [-] sense, single-stranded RNA. The segmented genome permits ready genetic reassortment when two influenza viruses infect the same cell. Replication takes place in the nucleus and cytoplasm, and assembly occurs via budding from plasma membranes. Orthomyxoviruses are labile in the environment. Some Common Pathogens

Influenza A viruses of humans, equine, swine, and avian. Family Rhabdoviridae (rhabdos, rod)

Enveloped virions resembling a bullet, flat at one end and round at the other.

The genome is single-stranded, [-] sense RNA. Replication occurs in the cytoplasm and assembly occurs via budding from plasma [vesiculoviruses] or intracytoplasmic [lyssaviruses] membranes.

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Rabies virus produces prominent cytoplasmic inclusion bodies [Negri bodies]. The viruses have broad host ranges. Virions are rapidly inactivated by UV light. Some Common Pathogens

Lyssavirus [rabies]; vesiculovirus [vesicular stomatitis]; ephemerovirus [bovine ephemeral fever]. Family Retroviridae (retro, backwards)

Spherical enveloped viruses with a diploid genome consisting of two identical copies [unique among viruses] of [+] sense, single-stranded RNA.

Replication is unique: the virion contains a reverse transcriptase enzyme [RNA-dependent DNA polymerase] that produces a DNA copy of the RNA genome in the cytoplasm. Integration of proviral DNA into host chromosomal DNA is brought about by viral integrase present in the nucleocapsid. Regions in the viral integrase determine the site of binding and integration of provirus into the host cell DNA; this can influence host cell function.

The virus is then replicated from the integrated provirus DNA copy. Virion assembly occurs by budding from plasma membrane. Hosts remain chronically infected. All oncogenic RNA viruses belong to the family. Labile viruses. Some Common Pathogens

Feline lentivirus [feline immunodeficiency disease]; human lentivirus [HIV]; and feline gammaretrovirus [feline leukemia]. Family Bunyaviridae (Bunyamwera = a town in Uganda, East Africa)

Spherical or pleomorphic enveloped virions. The genome is made up of triplesegmented, single-stranded, [-] sense RNA.

Due to their segmented genomes, closely related viruses can undergo genetic reassortment in mixed infections. Replication occurs in the cytoplasm, and viruses bud from Golgi membranes. All members of the family are transmitted by various arthropods [arboviruses] with the exception of the hantaviruses. Virions are labile in the environment.

20 Some Common Pathogens

Hantavirus, Akabane virus, and Phlebovirus [Rift Valley fever]. Family Bornaviridae (Borna: a town in Germany)

Spherical enveloped virions with [-] sense, single-stranded genome.

Virus replication occurs in the host cell nucleus. Viruses produce intranuclear inclusion bodies. Virion has a particular affinity for nervous tissues. Labile virion. Some Common Pathogens

Bornavirus [meningoencephalomyelitis of horses, sheep, and humans]. Family Astroviridae (astron, star)

Small, nonenveloped virions with [+] sense ssRNA genome. Some astroviruses have characteristic five- or six-pointed stars on their surface.

Replicate in the cytoplasm. No inclusion bodies. Moderately stable. Some Common Pathogens

Bovine astrovirus, feline astrovirus, etc. Gastroenteritis. Family Arenaviridae (arena, sand) Virions are pleomorphic and enveloped [envelope possesses large club-shaped peplomers]. Host cell ribosomes resembling grains of sand occur within virions.

The genome consists of two molecules of single-stranded [-] sense RNA. Replication occurs in the cytoplasm. Arenaviruses cause chronic, often lifelong inapparent infections in specific rodent reservoir hosts. Some Common Pathogens

Lymphocytic choriomeningitis virus of mice and humans; Lassa virus [Lassa fever, humans] and Machupo virus [Bolivian hemorrhagic fever, humans].

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VIRUS CULTIVATION AND PURIFICATION


Viruses are obligate intracellular parasites, hence can replicate only in living cells. Most viruses are grown in cultured cells, embryonated hens egg, or laboratory animals. A few viruses are yet to be grown under laboratory conditions. Each system used has advantages and disadvantages. The system[s] employed will depend on availability, laboratory facilities, personnel, and viral agent being studied. Collectively, they are used under the following circumstances:

Isolation and identification of viruses Characterization of viruses Pathologic examination Serologic tests Vaccine production CELL CULTURE (TISSUE CULTURE)

Cell culture involves the growth of dispersed animal cells in vitro, either as cells in suspension or as a monolayer [lawn of cells] on a solid surface such as the inside surface of a polystyrene culture flask.

In 1949, Enders, Weller, and Robbins reported that poliovirus could be grown in cultured nonneural cells with the production of recognizable cytopathic changes. Since then hundreds of viruses have been isolated and identified in cell cultures. Types of Monolayer Cell Cultures

Primary cell cultures. These are cultures established from tissue taken directly from humans or animals. The cells have the same chromosomes and chromosome number as the original tissue. Primary cultures contain several differentiated cell types, hence they are the best cell culture systems for isolation and propagation of viruses.

Primary cells can be detached from the cell culture flask using either trypsin or the chelating agent EDTA, diluted and used to initiate new cell cultures. Such subcultures are called secondary [transfer] cultures. However, primary cell cultures have limited life span [capable of only 5 to 20 subcultures]. Primary cell cultures are used in producing viral vaccines.

Serially propagated cell cultures [Cell lines]

Diploid cell lines. Consist of a homogenous population of a single cell type usually derived from human embryonic tissue [eg, WI-38; Wistar Institute; derived from human embryonic lung] or subcultures of a primary culture.

22 Diploid cell lines can be subcultured up to 100 times before growth stops. In spite of the numerous subcultures, the cells retain their original morphology and the diploid chromosome number. They may be used in producing some viral vaccines, eg, rabies; poliomyelitis vaccines, etc.

Continuous [heteroploid; immortal] cell lines. These are cells of a single type that are capable of indefinite propagation in vitro. Such immortal cell lines are derived from tumor cells [eg, HeLa cells] or by treating a primary cell culture or diploid cell line with a mutagenic chemical. The cells often no longer bear close resemblance to their cell of origin because of mutations and are often abnormal in chromosome morphology and number. FDA regulations prohibit their use in vaccine production. Preparation of Monolayer Cell Culture

Growth medium. This is a chemically defined medium containing all the nutrients required for cell growth, including amino acids, vitamins, inorganic salts, and other growth factors such as glucose.

Media may be purchased in liquid or powdered form, eg, Eagles minimum essential medium [MEM], RPMI 1640, and Leibovitzs [L-15] medium. Serum. 5-10% sheep, horse, or fetal calf serum is added to provide additional growth factors and ensure continued viability of the cultured cells. Serum must be free of antibodies to the virus[es] under study. pH indicator. Phenol red [turns medium light orange or light pink]. Most media are buffered to pH 7.4. Antimicrobial agents. Penicillin, streptomycin, gentamicin, mycostatin, and kanamycin [against Mycoplasma spp.].

Maintenance medium. Growth medium without added serum or 1-2% of serum may be added. Replaces growth medium when cell growth is complete and the cells are to be infected with virus. Trypsinization. Minced organ or tissue is treated with a protease, usually 0.25% trypsin warmed at 37oC or collagenase, to separate the cells. Preparation of cell suspension. The cells are washed, counted, diluted in a growth medium, and allowed to settle on the flat surface of a glass or polystyrene container (the cells produce glycoprotein-like materials that permit them to adhere to the containers; such attachment to a rigid support is essential for the growth of most normal cells, ie, anchorage dependence).

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Most types of cells adhere quickly and under optimal conditions they divide about once a day until the surface is covered with a confluent monolayer.

Incubation. Cell cultures are either incubated in tightly sealed containers or in open containers, eg, Petri dish, under 5% CO2. The incubation temperature is usually about 37oC.

Rhesus monkey kidney [RhMK] cell culture.

Rapid Cell Culture Systems Shell vial [1-dram] cell culture. A shell vial is a small borosilicate glass vial containing a coverslip. The monolayer is grown on the coverslip. It is ideal for centrifuge-enhanced virus inoculation of the monolayer.

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Centrifuge-enhanced inoculation. Growth medium is decanted from the vial and the processed clinical sample is placed directly on the monolayer. The inoculated vial is spun in a centrifuge at low speed [700 x g] for an hour, fresh culture medium is added, and the vial is incubated at 35-37oC. At a designated time interval, pre-CPE [see Cytopathic Effect, page 41] detection of virus can be carried out by staining the infected monolayer with virus-specific horseradish peroxidase [HRP]- or FITC-labeled monoclonal antibodies [MAbs] to detect viral antigen or, later, the monolayer can be examined microscopically for CPE with an inverted microscope.

Co-cultivated cells. This technique involves combinations of different cell types grown together as a single monolayer. It is ideal for clinical samples that may contain multiple viruses. Various MAbs, each labeled with a different fluorochrome, are used to detect different viruses growing in the same vial. EMBRYONATED EGGS The technique of growing viruses in the embryonated hens egg [developing chick embryo] was devised by Goodpasture in 1930. The eggs are obtained from a disease-free flock (specific pathogen-free [SPF] eggs). Embryonated eggs containing antibodies to a virus are not suitable for the isolation of the virus.

The embryos are used when they are between 5 and 13 days old, depending on the virus and route of inoculation. Routes of Inoculation

Following candling of the egg, a hole is drilled in the eggshell and a viral suspension [0.1-0.2 ml] or suspected virus-containing tissue is injected into the fluid of the egg. There are several membranes in an egg, and the virus is injected near the one most appropriate for its growth. The hole is sealed using melted wax.

Chorioallantoic membrane inoculation

Yolk sac. 5 to 7-day-old embryos. 22-gauge, 1-in length needle. Can be used for togaviruses, avian enterovirus [encephalomyelitis], etc.

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Chorioallantoic membrane [CAM]. 9 to 12-day-old embryos. 26- or 28gauge needle. Poxviruses [except parapoxviruses], some herpesviruses, etc. Amniotic route. 7 to 13-day-old embryos. Influenza A viruses, etc. Allantoic route: 7 to 13-day-old embryos. Influenza A viruses, Newcastle disease virus, infectious bronchitis virus, etc. Intravenous route. 10 to 12-day-old embryos. Ovine orbivirus [bluetongue]. Incubation Conditions

Incubator must have good air circulation to ensure a balanced temperature and also automatic turning racks to rotate the eggs.

Temperature: 32oC to 37oC, depending on the virus. Relative humidity: 62%. Signs of Virus Growth

Embryos can respond to infection in a number of ways. Some responses which may be noticed following inoculation of the embryonating egg are:

stunted growth [dwarfing of embryo] pocks [necrotic lesions] on chorioallantoic membrane urate deposits in mesonephros hemorrhage and congestion encephalitis [sluggish movements] death of embryo lesions of the extracellular membranes, eg, edema presence of hemagglutinins in embryonic fluids [avian influenza, Newcastle disease]. LABORATORY ANIMALS

The animal[s] selected should be free of antibodies. Commonly used laboratory animals include mice, hamsters and other rodents, rabbits, guinea pigs, monkeys, chickens, etc.

Used for infectious agents that cannot yet be grown in cell culture. Studying pathogenic mechanisms and the immune response. eg, hamsters are widely used in tumor virology because they are highly susceptible to tumor production by a number of oncogenic viruses.

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Serology. Rabbits are extensively used for producing antisera, whereas mice are commonly used for intraperitoneal implantation of hybridomas which secrete monoclonal antibody as an ascitic fluid. CONCENTRATION AND PURIFICATION OF VIRUSES

To study the physicochemical characteristics, infectivity, antigenic properties, etc. of a virus, a pure virus suspension must be available. Purified virus can be obtained from tissue culture medium, infected cells, etc.

High-speed equilibrium centrifugation [ 100,000 g] is used to obtain highly purified and concentrated preparations of viruses. The relative density of virus particles in a solution of sucrose or cesium chloride [CsCl], is constant for closely related viruses. The virus[es] is/are layered on top of the preformed gradient solution. As the tube is centrifuged, virus particles reach an equilibrium position at which their buoyant density [density at which a virus neither sinks nor floats when suspended in a density gradient] is equal to that of the surrounding sucrose or CsCl solution; the viral particles appear as a visible band. The virus band[s] is/are collected from the top or by piercing the bottom or side of the centrifuge tube.

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VIRUS QUANTITATION AND REPLICATION


Virus Quantitation and Titration A virus titration is a quantitative determination of viral activity, ie, the concentration of virus in the sample which can produce disease, lesions, or some recognizable effect in the host. A virus titer is the number of infectious units per ml of the sample.

A virus titer is determined by inoculating serial dilutions of virus into tissue cultures, embryonated eggs, or laboratory animals, and looking for evidence of virus multiplication. Titration procedures are widely used in viral studies to determine the following: infectious doses study virus multiplication vaccine doses virus concentration in various materials Viral Infectivity Assays

Suitable dilutions [usually 10-fold] of the stock virus are inoculated into the test system and the end point of the infectivity is determined using quantitative or quantal assays. Tenfold [10-fold; 1:10] Serial Dilutions

Quantitative Assays These assays measure the exact number of infectious virus particles in the sample. The most widely used is the monolayer plaque assay. Monolayer plaque assay. Viral plaques are colorless areas of necrotic cells surrounded by viable cells stained with a vital dye; hence a plaque assay can be used to determine titers only of viruses that cause visible cell damage. Since a single plaque can arise from a single infectious virus particle, ie, a plaque-form-

28 ing unit [PFU], the technique can be used both for accurate quantitative assay of virus infectivity and for purification of virus particles.

Monolayer cells are incubated with suitable dilutions of virus to allow virus adsorption to cells. The inoculum is removed and the cells overlaid with maintenance medium containing agar. The agar gel prevents progeny virions released from the original infected cells from establishing secondary sites of infection in uninfected cells. Following incubation [37oC; up to 2 weeks], the cells initially infected produce virus progeny that spread only to surrounding cells, producing a small area of infection, or plaque, which is visible to the naked eye. To enhance the contrast between the plaque and the surrounding monolayer, the cells may be stained with a vital dye, such as neutral red or crystal violet. Living cells absorb the stain, and plaques appear clear against a red [neutral red] or purple [crystal violet] background of healthy cells. To minimize errors in determining the virus titer, only those plates containing 20 to 100 plaques are usually counted. Since neutral red is nontoxic, staining can be done through the agar overlay, allowing the recovery of infectious virus from a plaque. Progeny virions are recovered by scraping the areas surrounding the degenerated cells under the agar and within the plaque area with the bent tip of a capillary pipette. The material is transferred from the pipette into the maintenance medium contained in an uninoculated tube of tissue culture. The tissue culture is incubated to obtain purified virus stock. Determination of Titer [Plaque-forming units/ml]

The number of plaques are counted, multiplied by the reciprocal of the dilution and then by a factor so that the titer can be expressed as PFU/ml. Plaque counts Average of 3 flasks 104 13

Dilution 10-4 10-5

Titer 1.04 x 106 1.3 x 106

Titer: 1.17 x 106 per 0.2 ml; to convert to1 ml, multiply by 5.0. Therefore, titer of virus suspension is 5.85 x 106 PFU/ml.

29 Pock Assay This involves the titration of certain viruses, eg, poxviruses and herpesviruses, on the chorioallantoic membrane [CAM] of the chick embryo.

The viruses are quantitated by relating the number of pocks [necrotic areas] counted to the viral dilution inoculated. The virus titer is reported as pockforming units/ml. Transformation Assay

Oncogenic viruses transform cells, so that they display reduced contact inhibition [a phenomenon in which cells stop dividing when their cell membranes make contact].

The transformed cells grow in an unrestrained fashion to produce a heapedup microtumor that stands out conspicuously against a background of normal cells in the monolayer cell culture. The virus titer is reported as focus-forming units [FFU]/ml.

Quantal Assay This method does not measure the exact number of infectious particles in the inoculum, rather, it determines only the presence or absence of infection. Being an all or none assay, it is not as precise as a quantitative assay.

Serial [eg, 10-fold] dilutions of virus are inoculated into several replicate cell cultures, embryonated eggs, or laboratory animals. Adequate time is allowed for virus to replicate and destroy the cultured cells or infect and kill the embryos or animals, as the case may be. The titer is expressed as the reciprocal of the highest dilution that infects or kills 50% of the subjects in the test system and is reported as follows: TCID50: Tissue culture infectivity dose50

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LD50: ELD50: PD50:

Lethal dose50 Embryo lethal dose50 Paralytic dose50

Statistical procedures are used to calculate the end point of quantal titrations, eg, Reed and Muench Method.
Tissue Culture Infectivity Data and Calculation of 50% End Point Using the Reed and Muench Method

Therefore, a virus suspension of 10-2.3 per 0.1 ml represents one TCID50; ie, at such dilution [concentration] 50% of the cultures inoculated will become infected. A dilution of 10-1.3 per 0.1 ml of virus suspension will contain 10 TCID50 in a volume of 0.1 ml. VIRUS REPLICATION

Viruses replicate only in living cells. The host cell provides the energy and synthetic machinery and low-molecular-weight precursors for the synthesis of viral proteins and nucleic acids.

The viral nucleic acid contains information necessary for programming the infected host cell to synthesize virus-specific macromolecules required for the production of viral progeny. An understanding of viral replication is essential to understanding pathogenesis, immunity, chemotherapy, the role of viruses in cancer, etc. The One-Step Growth Cycle

Permissive cell: A cell that contains the necessary intracellular components needed for virus replication [productive infection].

31 Nonpermissive cell: A cell type that does not allow a complete virus replication cycle [abortive or nonproductive infection]. Multiplicity of infection [MOI]: The number of infectious viruses inoculated per cell. To ensure that almost all cells are infected at least by 1 infectious unit, the study is usually conducted at a MOI of 5 to 10 PFU per cell.

Monolayer cell cultures are seeded with virus; time is allowed for cell infection. A high MOI is used so that all cells in the culture are infected simultaneously. Excess seed virus is washed off; maintenance medium is added and the cultures incubated. The increase in infectious virus over time is followed by sequential sampling and titration. Extracellular virions [viruses free in the maintenance medium] are titrated separately from intracellular virions. Intracellular virions are harvested by scraping the cells into fresh medium, and lysing the cells by three cycles of freeze-thawing.

The results obtained, allow determination of the following: Eclipse period. Refers to the time between the disappearance of infectious virions [uncoating] and the appearance, intracellularly, of the first progeny virions. The eclipse period generally ranges from 2 to 12 hours for viruses of different families. Enveloped viruses. Most enveloped viruses mature by budding from the plasma membrane of the host cell, hence the eclipse period ends when they are released extracellularly. Latent period. Time from uncoating to just prior to the first release of extracellular virions.

Eventually, as the cells become metabolically and structurally incapable of supporting more virus replication, virus production plateaus.

One-step growth cycle of a nonenveloped virus

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Viral Replication Cycle

General features of the viral replication cycle, using a nonenveloped DNA virus as a model. No topographical location for any step is implied. One step grades into the next such that, as the cycle progresses, several of these processes are proceeding simultaneously. Release occurs by cell lysis.

Although the details of virus replication vary from family to family, the general outline of the replication cycles is similar. For families of DNA viruses [except poxviruses, iridoviruses, and asfivirus] transcription and DNA replication take place in the cell nucleus, using the cellular RNA polymerase II and other cellular enzymes. Most RNA viruses, on the other hand, replicate in the cytoplasm. Attachment [Adsorption] For virus infection to occur, virus particles must be able to bind to host cells. Viruses have viral attachment proteins that bind to receptors on the plasma membrane of the cell [antibodies that combine with a VAP will neutralize infectivity of the virus]. The presence or absence of receptors plays an important determining role in cell tropism and viral pathogenesis. Each susceptible cell may contain up to 100,000 receptor sites for a given virus. However, in a number of cases, virus particles bound to antibodies can be taken up by Fc receptors on nonsusceptible cells.

Receptor sites are inherited characteristics of the host, thus, the receptor for a particular virus can vary from individual to individual. This may account for the individual differences in susceptibility to a particular virus.

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Cellular receptors recognized by viruses are usually proteins and glycoproteins but carbohydrates and occasionally glycoplipids are used as well. These receptors are molecules essential to the normal functioning of the cell, eg, hormone receptors, permeases, and members of the immunoglobulin superfamily, such as ICAM-1 [intercellular adhesion molecule-1]. Different viruses may use the same receptor, and related viruses may use different receptors. Some viruses use alternative receptors to enter different cells in different tissues and in different hosts [eg, vertebrate and arthropod]. Coreceptor. For a number of viruses, successful infection of a cell requires binding to both a cellular receptor and coreceptor, eg, HIV-1.
Examples of Animal Virus Receptors Virus Foot-and-mouth disease virus Rhinoviruses Rabies virus HIV-1; FIV Target Cell[s] Epithelial cells Epithelial cells Neuron
+ CD4 T cell

Receptor on Target Cell Integrins ICAM-1 Acetylcholine receptor CD4; chemokine coreceptor [CXCR4, CCR5] Sialic acid

Influenza A virus

Epithelial cells

Penetration [Engulfment or Viral Uptake] Receptor-mediated endocytosis [eg, viropexis]. The process involves the selective binding of ligands to specific cell membrane receptors. It is the predominant mechanism of viral entry for naked viruses and most enveloped viruses.

After binding of viruses to the cell surface, virions move down into clathrincoated pits [clathrin: a latticework of fibrillar protein that coats the cytoplasmic face of coated pits]. The pits fold inward to produce clathrin-coated vesicles

34 that enter the cytoplasm and, after removal of the clathrin coat, fuse with lysosomal vesicles to form endosomes. Enveloped viruses: Acidification within the endosome results in conformational change in envelope surface proteins, enabling the envelope to fuse with endosomal membrane and release of nucleocapsid into the cytoplasm. Naked virions: Release of nucleocapsid follows lysis of the endosome; eg, in adenoviruses, the low pH activates a virion surface protein(s) to lyse the endosomal membrane.

Surface fusion. Fusion of the viral envelope with the cell membrane is mediated in part by a viral-encoded protein, eg, fusion protein of Paramyxoviruses, and results in immediate entry of the viral nucleocapsid into the cytoplasm of the cell.

Ability of the viral envelope to fuse with host cell membrane can confer on the virion the ability to promote fusion between adjacent cells [syncytium]. Viral envelope proteins remain in the cell surface, thereby imparting to the cell a new antigenic specificity, and the infected cell can and does become a target for the immune mechanisms of the host [antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytolysis]. Uncoating

This is the physical separation of the viral nucleic acid from the envelope and/or capsid such that the viral genome can express its functions. Uncoating may begin simultaneously with penetration [fusion of envelope with cell membrane] or shortly after penetration. Uncoating results in the loss of virion infectivity.

Uncoating may occur in endosomal vesicles, cytoplasm, or the capsid may be transported along the cytoplasmic cytoskeleton from the site of entry to the nuclear pore to be uncoated. Strategies of Viral Replication

The replication cycles of viruses range from 6 to 8 hours [picornaviruses] to more than 40 hours [some herpesviruses]. Virus yield per cell ranges widely, but may be up to 100,000 particles or more. However, only 1% to 10% of the viral parti-

35 cles may be infectious. The rest are noninfectious [defective] particles. The key events in viral replication are: protein synthesis, replication of the viral genome, and assembly of the new components into progeny virions. Protein Synthesis Viral proteins (a) ensure the replication of viral genomes, (b) package the genome into virions, and (c) alter the structure and/or function of the infected cell. DNA Viruses For DNA viruses replicating in the nucleus, mRNA is transcribed by host cell DNA-dependent RNA polymerases. Cytoplasmic DNA viruses [Poxviruses, Iridoviruses, and Asfiviruses] carry their own DNA-dependent RNA polymerases.

Not all viral genes are expressed simultaneously or continuously throughout the replication cycle; early genes are transcribed first, followed by late genes. RNA Viruses

The RNA genome of a [+] strand virus is infectious and can function as mRNA. The RNA genome of a [-] strand virus cannot function as mRNA [complementary to mRNA], hence a virion-associated RNA-dependent RNA polymerase [carried in the capsid; host cell lacks enzyme] transcribes mRNA from the viral genome.

The [+] strand virion RNA of retroviruses is transcribed into DNA, which incorporates with host cell DNA [provirus]. The provirus DNA serves as a template for transcription of viral mRNAs by a cellular transcriptase.
Summary of Replication Strategies of RNA Viruses other than Retroviruses Genome RNA-dependent RNA Infectivity of RNA Initial event in cell polymerase [transcript- genome tase] in virion Positive sense RNA No Infectious Translation Negative sense RNA Yes Not infectious Transcription Double-stranded RNA Yes Not infectious Transcription

Transcription This is the process by which information contained in a nucleic acid molecule is transferred to messenger RNA [mRNA]. To synthesize viral proteins, the virus must present to the eukaryotic cell protein-synthesizing machinery, mRNA that the cell can recognize as such and translate. Primary viral RNA transcripts are processed into mRNA as follows:

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Capping: Addition of 7-methylguanosine to the 5 terminus by mRNA capping enzymes. Capping stabilizes the mRNA [decreases the degradation of mRNA by digestive enzymes] and also aids in aligning mRNA on the ribosomes during translation. Poly (A) tail: Sequence of 100 to 200 adenylate residues added to the 3 terminus of an RNA transcript by polyadenylate acid polymerase. The poly A tail acts as a signal allowing mRNA to be transported out of the nucleus and also aids in the binding of mRNA to host cell ribosomes. Splicing of viral pre-mRNA. Involves the deletion of the intervening noncoding sequences [introns] and the ligation of noncontiguous coding sequences [exons] by spliceosomes. A virus may synthesize a separate mRNA [monocistronic mRNA] corresponding to each gene in its genome or a polycistronic mRNA [representing several genes].
Polycistronic mRNA Translation Precursor polyprotein Proteases Functional proteins OR Endonucleases Monocistronic mRNAs Translation Functional proteins

Translation Processed viral mRNAs bind to ribosomes and are translated into proteins in the same fashion as cellular mRNAs.
Some Virus-Encoded Proteins

Enzymes required for transcription, replication of viral nucleic acid, and cleavage of proteins (proteases). Structural proteins of the virion including capsid and, for some viruses, core and/or envelope proteins. Regulatory proteins that control expression of the viral genome. Proteins down-regulating expression of cellular genes. Oncogene products (oncoproteins) and inactivators of cellular tumor suppressor proteins. Proteins that influence viral virulence, host range, tissue tropism, etc. Proteins that modulate cytokine responses. They enhance the spread

37
and titer of virus in the host by subverting the hosts innate and adaptive immune responses [see evasion of immune response].

Migration of Proteins. Newly synthesized viral proteins must migrate to the various sites in the cell where they are needed, eg, back into the nucleus in the case of viruses that replicate there. Replication of Viral Nucleic Acid DNA Viruses

Double-stranded DNA: Semiconservative replication, ie, both strands of a double-stranded DNA are used as templates such that progeny genome consists of one parental strand and one newly synthesized strand. Single-stranded DNA: Via a replicative form [replicative intermediate].
Some Enzymes required for Host and/or Viral DNA Replication

Helicase: Promotes unwinding of the DNA double helix. Single-stranded DNA-binding proteins. Helix-destabilizing proteins that keep the two separated DNA strands apart until each has been copied. A DNA polymerase to copy each strand from the origin of replication in a 5 to 3 direction. DNA ligase to join the Okazaki fragments together. Okazaki fragments are short fragments of DNA [1000-2000 nucleotide] synthesized as intermediates in the discontinuous replication of DNA and later joined together to form an intact strand.

RNA Viruses Single-stranded RNA. Replication of virion RNA requires first the synthesis of a complementary RNA copy [replicative form (RF)/replicative intermediate (RI)], which then serves as a template for the synthesis of progeny RNA.

One or several molecules of opposite sense RNA can be transcribed simultaneously from the RI by polymerase molecules at each growing point. Later, the RI is degraded. [-] strand RNA: The complementary RNA is of [+] sense. [+] strand RNA: The complementary RNA is of [-] sense.
(-) sense RNA genome (+) sense RNA genome

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Double-stranded RNA: Semiconservative replication. Retroviruses replicate via a DNA intermediate. Maturation and Release of Viruses Naked Icosahedral Virions

All nonenveloped [naked] animal viruses [RNA or DNA] have an icosahedral structure. The structural proteins of icosahedral viruses associate spontaneously to form a procapsid into which viral nucleic acid is packaged.

Proteolytic cleavage of one or more procapsid proteins may be required for the formation of infectious viral particles. The cleavage possibly causes rearrangement of the procapsid into a thermodynamically stable capsid in which the viral nucleic acid is shielded from host nucleases. Most naked virions accumulate within the cytoplasm or nucleus and are released only when the cell eventually lyses [burst process]. Evidence suggests that structural proteins of these viruses induce cell lysis via inhibition of host macromolecular metabolism. Enveloped Virions

All helical mammalian viruses are enveloped, as well as some icosahedral viruses. Enveloped viruses bud from the plasma membranes, from internal cytoplasmic membranes, such as the endoplasmic reticulum or Golgi apparatus, or from the nuclear membrane [herpesviruses].
An Illustration of Relationships between several lipid-containing Viruses and Host Cell Membranes

39 Budding from the Cytoplasmic Membrane Insertion of viral glycoproteins into the lipid bilayer occurs by lateral displacement of cellular proteins from a patch of cytoplasmic membrane. Viral nucleocapsid binds to virus-specified proteins [matrix protein] lining the cytoplasmic side of the patch. In the process, the virion is extruded or buds into the extracellular environment.

Release of each enveloped virion does not breach the integrity of the plasma membrane; hence thousands of viral particles can be shed over a period of several hours or days. Budding, therefore, is a more efficient mechanism of viral release in as much as it does not depend on disintegration of the infected cell. Viruses that mature and egress in this fashion vary considerably in their effects on host cell metabolism and integrity. They range from highly cytolytic viruses [eg, togaviruses, paramyxoviruses, etc.] to virtually noncytolytic viruses [eg, retroviruses]. Exocytosis

Flaviviruses, arteriviruses, coronaviruses, and bunyaviruses mature by budding through membranes of the Golgi complex or rough endoplasmic reticulum; vesicles containing the virus then migrate to the plasma membrane with which they fuse, thereby releasing the virions by exocytosis.

Herpesviruses. Maturation and envelopment of herpesviruses occur at the inner lamella of the nuclear membrane. The enveloped virions then pass directly from the space between the two lamellae of the nuclear membrane to the exterior of the cell via the cisternae of the endoplasmic reticulum.

40 Extracellular and Cell-to-Cell Spread of Viruses Extracellular Spread (Type I]) Virions are released from the cell to spread in the extracellular milieu. Many types of viruses spread by this route, and most spread by this mechanism, at least, some of the time.

Intercellular Spread (Type II) Virions spread from cell-to-cell through desmosomes of intercellular bridges [cell fusion] without contact with the extracellular milieu, eg, herpesviruses. Usually results in persistent infections.

Nuclear Spread (Type III) The viral genome is latent or integrated into the host genome and is passed from parent to progeny during cell division, eg, retro-viruses.

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EFFECTS OF VIRUSES ON HOST CELLS


When viruses replicate in cells, a complete spectrum of effects may be observed with different combination of interactions between virus and host cells in vitro and in vivo, due to biochemical effects of virus-specified products. These biochemical changes may lead to functional disturbances and, eventually, in many cases, to histopathological changes.

Viral effects on cells are classified as cytocidal [cytolytic, cytopathic], noncytocidal [nonlytic], or cell transformation. Not all viral infections, whether cytocidal or noncytocidal, necessarily lead to the production of new virions. Cell changes leading to cell death or transformation may occur in nonproductive infections.

Cytopathic Effect (CPE; Cell injury) CPE is defined as the visible or morphologic change(s) induced in a host cell by a virus that may result in host cell damage and/or death. Many of the CPEs are secondary effects of the virus metabolic needs and are not simply toxic effects of viral proteins on the host cell. However, there are some viral proteins that cause toxic effects to the host cell with no apparent purpose.

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CPEs are usually observed in monolayer cell cultures under low power of the light microscope. Viruses are detected, and often identified, by the particular CPE produced over time in a particular cell culture. CPEs observed in infected cells include cell lysis, cell rounding and detachment, inclusion bodies, syncytium formation, cytoplasmic vacuolation, cell transformation, and antigenic changes in the cell membranes of infected cells. Inclusion Bodies [Inclusions]

A morphologic change in cells infected by some viruses, is the formation of inclusion bodies [IBs; inclusions]. Inclusion bodies are intracellular structures which may be recognized by light microscopy following fixation and staining [hematoxylin and eosin] or by fluorescence microscopy. IBs are important because their presence or absence can help in the identification of the virus causing an infection.

IBs may be accumulations of viral components, eg, Negri bodies; the result of degenerative cellular changes, eg, the owls eye appearance of herpesvirus inclusions due to viral-induced chromatin condensation; or crystalline aggregates of virions, eg, adenovirus inclusions. IBs may be intranuclear or intracytoplasmic, single or multiple, large or small, round or irregular in shape, acidophilic [red, eosinophilic, Cowdry type A] or basophilic [blue]. Some viruses, eg, morbilliviruses [canine distemper, rinderpest, and measles] may produce both nuclear and cytoplasmic inclusion bodies in the same cell.

Inclusion bodies in virus-infected cells. (A) Vaccinia virusintracytoplasmic acidophilic inclusion. (B) Herpesvirus--intranuclear acidophilic inclusion; cell fusion produces syncytium. (C) Reovirus perinuclear intracytoplasmic acidophilic inclusion. (D) Adenovirus intranuclear basophilic inclusion. (E) Rabiesvirusintracytoplasmic acidophilic inclusion [Negri bodies]. (F) Morbillivirusintranuclear and intracytoplasmic acidophilic inclusions; cell fusion produces syncytium.

43 Mechanisms of Cell Damage in Cytocidal Viral Infections Viruses that induce cell death usually do so late in the replication cycle, when production of progeny virions is complete. Mechanisms of cell damage include the following:

Inhibition of host cell DNA, RNA, or protein synthesis. (1) Poxviruses produce a DNAse that degrades cellular DNA; (2) some viruses, eg, influenza viruses, encode proteins that inhibit both polyadenylation and splicing of host cell primary RNA transcripts needed to form mature mRNAs; and (3) viral proteins may sequester nuclear proteins needed for export of cellular mRNAs out of the nucleus, the mRNAs are subsequently degraded in the nucleus; however, export of viral mRNAs from the nucleus to the cytoplasm is not affected. Modification of the host cells translational apparatus such that viral mRNAs are selectively translated.

Some viral proteins cause the cells lysosomes to release their enzymes, resulting in destruction of intracellular contents and host cell death. Effects of viruses on the cell membrane Syncytia [multinucleated giant cells]. Result from the fusion of an infected cell with neighboring infected or uninfected cells. In general, giant cells are prone to premature cell death. Envelope proteins of some viruses facilitate fusion of the envelope with the cell membrane; this property can confer on the virus the ability to promote fusion between adjacent cells. In addition, viral glycoproteins synthesized within the infected cell can migrate to the cell surface and promote fusion with neighboring cells.

Altering membrane permeability. Viral proteins may disrupt membrane integrity, resulting in increased permeability to ions, eg, sodium, and normally excluded macromolecules or escape of intracellular molecules.

Changes in cell shape caused by damage to the cytoskeleton Cellular cytoskeleton consists of microfilaments, intermediate filaments, and microtubules that maintain the structural integrity of cells, and help in the transport and movement of organelles. Infection by many viruses leads to depolymerization of one or more cytoskeletal fiber systems.

Apoptosis [Programmed cell death]. Host defense against viral infection.

44 The biochemical alterations in the virus infected cell can cause the host cell to induce its own death by activating its apoptotic pathways. This usually happens early in the replication cycle, before progeny virus production is complete. Apoptotic cell death may arrest or at least slow the spread of virus throughout the body, allowing other host defenses to be marshaled. Apoptosis may also be triggered by some virus-encoded proteins, eg, VP2 protein of avibirnavirus. Conversely, some viruses have genes that encode anti-apoptotic proteins that promote cell survival until their replication cycle is complete and progeny virions have been released. Apoptosis may be classified as direct or indirect. Direct apoptosis: Apoptosis of infected cell; indirect apoptosis: apoptosis of uninfected cell.

Cytolysis by immunologic mechanisms, eg, ADCC Viral proteins [antigens] inserted into the host cell membrane during adsorption and penetration [surface fusion] or budding, may constitute targets for antibody-dependent cell-mediated cytotoxicity by NK cells. This may happen before significant progeny virus is produced, thus slowing the progress of infection and hastening recovery. Noncytocidal Changes in Virus-Infected Cells

Noncytocidal viruses infect cells and actively produce infectious viral particles without causing immediate host cell death. Though they often cause persistent infections, overall cellular metabolism may be little affected.

With few exceptions, eg, most retroviruses, slow, progressive changes occur that results eventually in cell death. In the host animal, cell replacement occurs so rapidly in most organs and tissues that the slow fallout of cells due to persistent infection may have little or no effect on overall function. Damage to the specialized functions of differentiated cells may affect regulatory and homeostatic functions of the host, eg, if a virus changes the production level of a hormone, or enzyme, the cells normally affected by the hormone or enzyme, will not function properly. CELL TRANSFORMATION

Cell transformation is the changing of a normal cell into a cancer cell. The development of cancer is a multistep process involving mutation and selection for cells with progressively increasing capacity for proliferation, survival, invasion, and metastasis.

45 Proto-Oncogenes Proto-oncogenes are genes whose protein products function in the signal transduction pathways that control normal cell growth, division, and differentiation. Proto-oncogenes were first discovered as passenger genes within the genome of acute transforming retroviruses and were thought to be part of the normal viral genome. However, it was later proven that they were not of viral origin and that retroviral oncogenes were actually derived from host cell proto-oncogenes via genetic recombination.

Proto-oncogene products include (1) secreted growth factor proteins, (2) transmembrane growth factor receptors, (3) intracellular signal transducers [eg, GTP-binding proteins, protein kinases, etc], (4) proteins that affect cell proliferation by activating nuclear transcription, etc. A proto-oncogene can be converted into a cellular oncogene by a point mutation, deletion mutation, through DNA rearrangement, or by insertion of a mobile genetic element such as retroviral DNA. Cellular Oncogenes

Cellular oncogenes are genes whose products can transform normal cells. They are abnormally expressed or mutated forms of the corresponding proto-oncogenes induced by carcinogens [chemical, physical, etc] or viruses.

Oncogenes frequently encode proteins [oncoproteins] that resemble protooncogene products that play various roles in the activation of cell proliferation. However, because oncoproteins function in an unregulated manner, they differ significantly from their normal counterparts. Some oncoproteins such as polypeptide growth factors, growth factor receptors, and nuclear transcription factors, drive the uncontrolled proliferation of cancer cells. Others contribute to other aspects of the behavior of cancer cells such as defective differentiation and failure to undergo apoptosis. Tumor Suppressor Genes

Tumor suppressor genes encode proteins that normally act to inhibit cell proliferation [by holding the cell cycle at the G1 phase] or survival, hence tumor development.

In many tumors, these genes are lost or inactivated, thereby removing negative regulators of cell proliferation and contributing to the abnormal proliferation of tumor cells. The best characterized of these proteins are retinoblastoma [Rb] protein and p53 protein [molecular mass of 53 kilodaltons].

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Rb protein. Rb protein was identified following genetic analysis of retinoblastoma, a rare ocular tumor of children [~1 child in 20,000]. Rb gene is normally expressed in almost all the cells of the body. Although retinoblastoma is rare, cancers involving the Rb gene are not. Rb protein inhibits the entry of cells into S phase by binding strongly to certain gene regulatory proteins, preventing them from acting in the nucleus to promote DNA replication. The state of phosphorylation determines the braking action of Rb protein. The Rb protein alternates between a phosphorylated state [promotes DNA replication] and an unphosphorylated state [inhibits DNA synthesis], in every cell cycle.

p53 protein. In addition to mediating cell cycle arrest [same as Rb protein], p53 is required for apoptosis induced by DNA damage [unrepaired DNA damage normally induces apoptosis of mammalian cells]. Cells lacking p53 fail to undergo apoptosis in response to agents that damage DNA, such as radiation. Tumor Viruses [Oncogenic Viruses]

All known oncogenic viruses either have a DNA genome or generate a DNA provirus after infection [retroviruses]. Transforming viruses usually have their genomes or truncated versions of them, integrated into the host cell genome. However, the genome of a few may remain episomal, replicating in step with host cell chromosome. DNA Tumor Viruses DNA tumor viruses are found among the families Polyoma-, Adeno-, Herpes-, Hepadna-, Papilloma-, and Poxviridae. DNA oncogenes are an essential part of the viral genome, encoding proteins required for viral replication. Unlike the oncogenes of retroviruses, DNA oncogenes have no counterpart in the normal host cell. DNA tumor viruses interact with cells in one of two ways:

Permissive cell. Productive infection, in which the virus completes its replication cycle, resulting in cell lysis. Nonpermissive cell. Nonproductive infection, in which the virus transforms the cell without completing its replication cycle.

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Schematic comparison of two types of interaction between a DNA tumor virus [polyomavirus] and a host cell

DNA oncogenic viruses activate the host cells replication machinery by blocking the action of key tumor suppressor genes. DNA oncoproteins bind to Rb and p53 proteins, inactivating them and so permitting the cell to replicate its DNA and divide (drives the cell from the resting [G1] phase into S phase). For example, transformation by the adenoviruses results from expression of two early-region genes, E1A and E1B, which are required for virus replication in permissive cells. In nonpermissive cells, these transforming proteins inactivate the Rb and p53 proteins, with E1A binding to Rb protein and E1B binding to p53 protein.

48 RNA Tumor Viruses All RNA tumor viruses belong to the family Retroviridae. They are the most important oncogenic viruses in animals; major causes of lymphomas and leukemias in many species of animals, including cats, cattle, primates, mice, and chickens. A characteristic property of RNA tumor viruses is that they are not lethal for the cells in which they replicate in, hence virus infection is both productive and oncogenic.

Acute transforming viruses [v-onc+]. They possess v-onc genes that are derived from host cell proto-oncogenes. Occasionally, a proto-oncogene becomes incorporated into a viral genome, yielding a new, highly oncogenic virus as the product of a virus-host recombination event. Such transforming retroviruses are usually defective, since a portion of their genome is deleted and replaced by cellular proto-oncogenes. Oncoproteins coded by v-onc genes have no role in virus replication. Chronic transforming retroviruses [v-onc-]. The viruses lack v-onc genes and are weakly oncogenic. Cell transformation may result from insertion of retroviral promoter and enhancer elements at sites close to, or even within, proto-oncogenes [insertional mutation], resulting in enhanced expression of the proto-oncogenes.

Some Characteristics of Transformed Cells

Altered cell morphology [less round] and chromosomal abnormalities [change in morphology and number]. Capacity to divide indefinitely in serial culture due to loss of contact inhibition. Most normal cells cease growing in vitro when they come close to another cell; transformation results in an abnormal, spindleshaped cell that does not recognize contact inhibition, resulting in unregulated cell growth. Loss of contact inhibition also results in piling up of cells to form a focus in monolayer cell culture. Induction of foci provides the basis for a quantitative assay for certain tumor viruses. Virus-specified tumor associated antigens Many tumor cells contain a virus-specific antigen on their cell surface, called tumor-specific transplantation antigen [TSTA], or an antigen in their nucleus, called the T antigen.

Capacity to produce malignant neoplasms when inoculated into isologous or severely immunocompromised animals.

49 Viral Genetics An understanding of viral genetics is crucial to identifying viral processes that may be appropriate targets for the development of antiviral therapy or vaccine development. For example, viruses that have stable antigens on their surfaces can be controlled by vaccination; other viruses that exist as many antigenic types or change constantly, are difficult to control by vaccination. Mutation. This is a heritable change in the nucleotide sequence of the genome of an organism. It is the most frequent cause of genetic change in viruses. Mutations may be lethal [the mutated virus is unable to replicate]; neutral; or afford the mutant virus some selective advantage.

The error rate in the replication of viral RNA is much higher than that of viral DNA because DNA replication is subject to proofreading exonuclease errorcorrection of DNA-dependent DNA polymerase. There is no cellular proofreading mechanism for RNA-dependent RNA polymerase. Classification of Mutants by Phenotypic Expression

Escape mutants. Mutations affecting antigenic determinants of virion surface proteins, affect sensitivity of progeny to neutralizing antibody. Such escape mutants are strongly favored when viruses replicate in the presence of antibody, resulting in persistent infections. Conditional-lethal mutants. Results from mutations that so affect a virus that it cannot grow under certain conditions [nonpermissive conditions], but can replicate under other, permissive, conditions.

Temperature-sensitive [Ts] mutants. Grow at low [permissive] temperature but not at high [nonpermissive] temperature. A point mutation in the genome, leading to an amino acid substitution in the translated polypeptide product, results in a structurally abnormal protein which, although functional at the permissive temperature, cannot maintain its structural integrity and functional conformation when the temperature is raised by a few degrees. Ts mutants have been used in attempts to produce attenuated live-virus vaccines, eg, feline infectious peritonitis.

Defective interfering [DI] mutants. A DI mutant is a virus that lacks one or more functional genes required for viral replication due to a deletion mutation. They require infectious homologous virus as helper for replication; in the process, they interfere with and usually decrease the yield of the helper virus. DI mutants have been demonstrated in most virus families.

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Another category of defective virus requires an unrelated replication-competent virus [heterologous virus] as helper, eg, hepatitis D virus replicates only in the presence of co-infecting hepatitis B virus. The interference with helper virus may result from successful competition by DI particles for factors involved in genome replication. Thus, DI particles may play a role in persistent infections by attenuating the lethality of the parental infectious virus. Interactions among Viruses

When two or more virus particles infect the same host cell, they may interact in a variety of ways. They must be sufficiently closely related, usually within the same genus or viral family, for most types of interactions to occur. Dual infection. If the viruses are dissimilar, they may replicate within the same cell as efficiently as in single infections. Genetic interactions. These are interactions involving the genomes of the parental virions and are important sources of genetic variability. The resulting progeny are genetically different from either parent.

Genetic recombination. This is the exchange of nucleotide sequences between different [but usually closely related] viruses. Recombination may occur during or following synthesis of the nucleic acid molecules. The recombinant virus is genetically stable, producing progeny like itself upon replication. Recombination can also occur between a virus and its host cell, eg, feline leukemia virus and host proto-oncogenes. Recombination occurs among DNA viruses and among RNA viruses.

Genetic Reassortment. This is the exchange of complete RNA molecules between genetically related viruses with segmented genomes. Both ssRNA and dsRNA segments can undergo reassortment. Since reassortment simply involves the exchange of RNA segments, rather than an actual crossover event, it can occur at high frequencies than classic recombination.

Nongenetic interactions. In nongenetic interactions, it is the products of the genes that interact rather than the genomes. Thus, progeny produced are similar to the parents.

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Phenotypic mixing or masking. When two closely related viruses infect the same cell, the two types of progeny genomes may become encapsidated not only by their own capsids but also by hybrid capsids, ie, capsids composed of proteins encoded by both genomes [phenotypic mixing] or by capsids specified entirely by the other genome [phenotypic masking; transcapsidation; or pseudotype]. The intermixed capsid proteins must be able to interact correctly to form a structurally intact capsid. Phenotypic mixing or its extreme form, transcapsidation, is most readily detected by antigenic analysis. Phenotypic mixing or masking also occurs among enveloped viruses, but here it involves not only viruses that are related but also viruses that are completely unrelated.

Schematic diagram of phenotypic mixing and neutralization of pseudotypes. (+, Viral lesions present: no neutralization. P, Partial neutralization: resistant fraction present or slower neutralization kinetics. -, No viral lesions: virus completely neutralized). A and B are the pure viral types; A(B) is A genome with B capsid pseudotype; B(A) is B genome with A capsid pseudotype.

Viral Interference Occurs when the multiplication of a superinfecting virus in cell culture or in the host is inhibited because of the presence of an initially infecting virus. Interference has been used as a basis for controlling outbreaks of infection with virulent strain of a virus by introducing into the population an attenuated strain that interferes with the spread of the virulent virus, eg, polio.

May result from the first virus causing the infected cell to produce an inhibitor, eg, interferon, that prevents replication of the second virus or one virus may compete with the second for components of the replication apparatus.

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PATHOGENESIS OF VIRAL INFECTIONS


Viral pathogenesis is concerned with the method by which viruses produce disease, ie, the mechanisms by which viruses injure discrete populations of cells in different organs and tissues to produce the signs and symptoms of disease in a particular host.

Majority of viral infections are subclinical and do not result in disease.


Only a very minor portion of viruses are mad dogs. Its usually of no advantage to a virus to cause disease, much less to kill the host. From the book, Deadly Feasts, by Richard Rhodes.

The same disease may be produced by different viruses; on the other hand, the same virus may produce a variety of diseases. The outcome of a virus infection in the host depends on the following: dose [number of infecting virus particles]. virulence of the virus; stability of virus in body; and speed of replication. speed of spread to target organs/tissues and extent of cellular damage. level of host defense [nonspecific and specific immunity]. secondary bacterial infections.

Pathogenicity. The ability of an organism to cause disease by overcoming the defenses of a host. Virulence. The relative capacity of a pathogen, compared to other pathogens, to produce disease in the infected host [eg, virus A is more virulent than virus B].

53 Virulence can be expressed as LD50 [lethal dose for 50% of the inoculated hosts] or ID50 [infectious dose for 50% of the inoculated host]. Susceptible cells. Virus infected cells whose infection may not be sufficient to cause clinically demonstrable disease. Target cells. Virus infected cells whose infection usually contributes to clinically demonstrable disease. Sometimes target cells are infected by virus produced in susceptible cells at the portal of entry. Clinical signs. Any objective evidence or manifestation of an illness. Clinical symptoms. Any subjective evidence of disease. Portals of Entry and Exit The portals of entry for microorganisms are skin and mucous membranes lining the respiratory, gastrointestinal, genitourinary tracts and the conjunctiva.

All pathogens have a preferred portal [or portals] of entry that is a prerequisite to their being able to cause disease. If they gain access to the body by another portal, disease might not occur. Pathogens have definite routes of exit which usually reflect the part of the body where infection takes place. Three common portals of exit are the respiratory tract via coughing or sneezing, the gastrointestinal tract via saliva, vomit or feces, and the urogenital tract. Skin

The skin is the largest organ in the body in terms of surface area. Unbroken skin is impenetrable by most microorganisms [some gain entry through hair follicles and sweat duct glands].

Breaches in the skin may be a result of minor abrasions, accidental inoculation [eg, contaminated needles, blood transfusion], arthropod bites, animal bites, surgical incisions, etc. Although skin lesions are produced in several generalized diseases, only a few viruses are shed from skin lesions in a way that leads to transmission. However, viruses are readily shed from vesicular or pustular lesions. Respiratory Tract

The respiratory tract is the most common portal of entry. Mucus produced by goblet cells trap particles, which are then carried to the throat by the upward

54 beating of cilia, to be swallowed. Additionally, the cooler temperature of the upper respiratory tract acts to inhibit the replication of many viruses.

Viruses entering the respiratory tract do so primarily in the form of either aerosolized droplets or saliva. Coughing and sneezing can generate large volumes of small aerosolized particles. The distribution of inhaled aerosol particles within the respiratory tract is largely a function of particle size. Large particles are filtered by the nasal turbinates, whereas small particles [< 5 m] are capable of reaching the alveolar spaces. Environmental factors such as ambient temperature, dessication and humidity also influence the stability of aerosolized viral particles. Gastrointestinal Tract

A number of viruses are acquired by ingestion. They may either be swallowed and reach the stomach and intestine directly or they may initially infect cells in the oropharynx, the resulting progeny being carried into the intestinal tract.

Protective mechanisms in the GIT include hydrochloric acid produced by gastric parietal cells, proteolytic enzymes, and bile salts [dissociates viral envelopes]. Thus, a virus infecting the GIT must be acid stable and resistant to inactivation by proteolytic enzymes and bile salts. The majority of viruses that enter via the GIT generally exhibit all of the properties mentioned. Enterotropic viruses such as coronaviruses and toroviruses are susceptible to inactivation by bile salts in vitro [enveloped viruses], however, the factors that enable them to survive in vivo and cause enteritis remain unknown. Other Routes

Other mucosal surfaces may provide sites for viral entry into the host. The genital tract [in coitus] is the route of entry of several important viruses, eg, equine arterivirus, bovine herpesvirus 1, and equine herpesvirus 3.

The conjunctiva may also provide a route for the entry of viruses that either produce local disease [conjunctivitis] or more rarely disseminate from this site to produce systemic disease. Mechanisms of Virus Spread in the Body

Following infection, viruses may remain localized to the body surface through which they entered, or they may cause generalized infections, which are usually associated with viremia and subsequent localization in target organs.

55 Local Spread on Epithelial Surfaces Many viruses replicate in epithelial cells at the site of entry and spread by sequential infection of neighboring cells, eg, papillomaviruses. Occasionally, there may be subepithelial and lymphatic spread, eg, some poxvirus infections.

Viruses that are ingested or inhaled can spread rapidly over the moist epithelial surfaces of the respiratory and alimentary tracts. Localization of infection to an epithelial surface cannot be equated with a lack of severity of disease, eg, large areas of intestinal epithelium may be damaged by rotaviruses, resulting in severe diarrhea. Subepithelial Invasion and Lymphatic Spread

A network of lymphatics lies beneath all cutaneous and mucosal epithelia. Viruses breaching the epithelium and its basement membrane to reach subepithelial tissues, are carried to local lymph nodes via the afferent lymphatic vessels.

Some viruses may be processed and their epitopes presented to helper T cells, initiating an immune response. Others may replicate in macrophages, dendritic cells, or lymphocytes; or may pass straight through lymph nodes to enter the bloodstream. There is often a local inflammatory response, the extent of which depends on the extent of tissue damage. Hematogenous Spread (Viremia)

Viruses that produce systemic disease must spread from their site of entry into the host to their ultimate target tissues. The blood is the most effective and rapid vehicle for the spread of virus through the body.

Viruses reach the bloodstream by direct inoculation [eg, arthropod bite], via the lymphatic system, by replicating in endothelial cells or by entering a subepithelial blood vessel. Once a virus reaches the bloodstream, it can localize in any part of the body within minutes.

56 Viremia. The presence of virus in the bloodstream.

Passive viremia. Direct inoculation of virus into the bloodstream via the bite of an arthropod vector, through iatrogenic inoculation with a contaminated needle, or by the transfusion of contaminated blood products. Primary viremia [active]. The release of progeny virions from the site of initial viral replication, eg, portal of entry, into the bloodstream. Secondary viremia [active]. The release of virus from a localized area of 2o viral multiplication into the bloodstream, which can, in turn, lead to the establishment of infection in yet other parts of the body.
Portal of entry [replication of virus] Regional lymph nodes [ replication] Primary viremia Incubation period Other organs [replication of virus] Secondary viremia vectors of arboviruses

Target organ(s); extensive replication of virus Tissue/organ damage: Clinical signs and symptoms

In the bloodstream, a virus can travel free in the plasma or present in, or adsorbed to, cellular elements [cell-associated viruses] or may both be free in the plasma and associated with cellular elements. Free in the plasma, eg, parvoviruses, flaviviruses, etc. This type of viremia is usually of short duration, with clearance of viruses coinciding with the appearance of neutralizing antibodies in the circulation. Cell-associated viruses. Viral particles may adsorb to cell or remain attached to or within the infected cell after replication. The viruses are often not cleared by antibodies and, therefore, tend to cause prolonged viremias, eg, lentiviruses multiply in macrophages and/or lymphocytes, producing in many instances lifelong viremias.

Tissue/organ invasion. If a circulating virus is to invade an organ or tissue, it must breach the blood-tissue junction. This usually occurs at the level of the capillaries, venules and sinusoids [lined with macrophages] where blood

57 flow is slowest and the basement membrane is thinnest. Viruses breach the blood-tissue junction by: [1] infecting endothelial cells and growing through the vessel wall, [2] passive transport across the vessel wall by leukocytes, and [3] transcytosis, ie, transport of virions in intracellular vesicles across the vessel wall and release of virions by exocytosis.

The magnitude and duration of viremia vary as a result of the dynamic interrelationship between the amount of virus entering the blood compartment and the efficiency with which the viruses are removed. Mononuclear phagocytes [esp. in the liver, bone marrow and spleen] and serum factors, including complement and antibody, act to aid in clearance of virus from the bloodstream.
Types of Interaction between Viruses and Macrophages, exemplified by Kupffer cells in the Liver Sinusoids

1. Macrophages may fail to phagocytose virions; [2] Virions may be phagocytosed and destroyed.

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3. Virions may be phagocytosed and then transferred passively to adjacent cells [hepatocytes]. 4A. Virions may be phagocytosed by macrophages and then may replicate in them. 4B. Phagocytosed virions may multiply in macrophages and in hepatocytes.

Neural Spread Viruses may reach the central nervous system [CNS] via the peripheral nerves. In general, viral invasion and replication in neurons is preceded by replication in nonneural cells, eg, epithelial cells. This is because nerve endings are rarely exposed to the infecting virus. The viruses may be transported within axons, in the endoneural space, in perineural lymphatics, or in infected Schwann cells. Some viruses use olfactory nerve endings in the nares as sites of entry. Following initial replication in olfactory neuroepithelial cells, progeny virions travel in axoplasm of olfactory nerves directly to the olfactory bulb of the brain. In addition to centripetal [afferent] movement of virus to the brain, viruses can move centrifugally [efferent] from the CNS, within peripheral nerves, to other locations in the body, such as the skin, salivary glands and mucous membranes. This is what happens in the reactivation of latent infections and in the production of recrudescent epithelial lesions. Localized and Systemic Acute Viral Infections
Important Features of Acute Viral Infections Localized Acute Infections Systemic Acute Infections Site of pathology Portal of entry Distant sites Incubation period Relatively short Relatively long Viremia No Yes Duration of immunity Variable; may be short Mostly lifelong Role of sIgA in host defense Very important Usually not important

59 Virus-Mediated Tissue and Organ Injury Infection of the Skin The outer layer of the epidermis is composed of the dead keratinized cells of the stratum corneum which do not support viral replication. Skin infection may be due to breaches in intact skin, allowing viral invasion from the outside or due to bloodborne spread of virions from an initial focus of infection, eg, sheeppox, or as a result of the inflammatory response initiated against viral antigens.

Since the epidermis is devoid of blood vessels, lymphatics, and nerve fibers, viruses that initiate epidermal infection are usually restricted to the site of entry and only rarely disseminate to produce systemic infections. Viruses infecting the dermis have access to blood vessels, lymphatics, macrophages, allowing for possible systemic spread. Different types of skin lesions are recognized: Rash. This is a temporary skin eruption produced when viruses spread extravascularly from dermal blood vessels. Skin rash has a characteristic distribution in many infectious diseases. Macule. This is a flat, reddened circumscribed lesion. It is caused by virus replication in the dermis and subsequent host inflammatory reaction to the infection. Noninfectious [devoid of virus]. Papule. A raised macule due to localized inflammatory reaction. Noninfectious. Vesicle. Virus replication spreads from the dermis to the epidermis, resulting in a small, circumscribed fluid-filled epidermal elevation [blister]. Contains infectious virions. Pustule. A vesicle with copious neutrophil infiltration. Localized erosion or sloughing of epithelium results in ulceration. Ulcer may or may not progress to scab formation.

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Virus-induced skin tumors. Sarcoids, myxomatosis, etc. Infection of the Respiratory Tract

In domestic animals, viruses may cause more than 90% of upper respiratory tract [nasal passages, pharynx, and trachea] infections, whilst triggering many mixed infections in the lower respiratory tract. Growth requirements for different viruses and other microorganisms may determine their site of infection in the respiratory tract, however, several overlapping infections can occur.

Virus infection may result in [1] local cessation of ciliary activity [ciliostasis], [2] loss of integrity of the overlying mucus layer, [3] destruction of epithelial cells, and [4] depressed phagocytic activity of alveolar macrophages. Infection of epithelial cells may not stop until virtually every columnar epithelial cell at that airway level is infected. The result is complete denuding of large areas of epithelial surface. Accumulation in the airways of transudates, exudates, inflammatory infiltrates, and necrotic epithelial cell debris leads to anoxia and respiratory distress. Total plugging of small airways can cause anoxic death, especially in animals with narrow airways and low lung capacity to dislodge debris. Viral-Bacterial Synergism in Respiratory Infections

Viruses alter the surface properties of respiratory epithelial cells so as to favor bacterial adherence and the growth of bacterial colonies. These microcolonies may resist phagocytosis and can more readily invade the lower respiratory tract.

Another effect of viral damage to epithelial cells is the release of iron, which enhances bacterial growth and colonization. Viruses can be directly immunosuppressive or can impair alveolar macrophage and neutrophil function in the lung and airways.

Interaction of viral and bacterial pathogens in respiratory disease. The percentage mortality is much higher in animals infected with both pathogenic virus and pathogenic bacteria than in those infected with virus or bacteria alone.

61 Infection of the Gastrointestinal Tract Most viruses that infect the GIT are ingested virions; however, some viremic infections reach the intestinal epithelium, resulting in enteritis and diarrhea, eg, parvovirus diarrhea. Severity of the enteritis caused by the various enteric viruses varies according to the immune status, age, and general condition of the animal at the time of infection.

Infection generally begins in the proximal part of the small intestine and spreads progressively to the jejunum and ileum and sometimes to the colon. Most intestinal viral infections involve the differentiated cells at the tips and upper parts of the villi. In parvovirus infections, it is crypt cells that are in S phase of the cell growth cycle that are destroyed. The pathophysiologic effects of viral diarrhea are a consequence of rapid destruction of mature enterocytes and their replacement by immature, cuboidal cells that cannot carry out normal absorptive and enzyme secretory functions. Because cuboidal cells are relatively resistant to viral infection, the disease is often self-limiting if dehydration is not so severe as to be fatal. Fluid loss is mainly a loss of extracellular fluid due to impaired absorption and osmotic loss due primarily to the presence of undigested lactose in the lumen [in newborn animals] rather than active secretion. However, occasionally, adenylate cyclase and cyclic AMP levels may increase, causing a hypersecretion of water and chlorides. Acidosis results from E. coli fermentation of lactose in undigested milk and loss of sodium, potassium, bicarbonate and chloride.

Structure of the Villus Epithelium of the Small Intestine with locations targeted by Enteroviruses

Left. Cross section through a villus and crypt of Lieberkhn showing the movement and maturation of dividing cells to form nondividing mature enterocytes. Right. Cells infected by various enterotropic viruses.

62 Infection of the Central Nervous System [CNS] Viral invasion of the CNS occurs via hematogenous spread [more common] or via peripheral nerves. Hematogenous invasion occurs across the blood-brain and blood-cerebrospinal fluid barriers. A consequence of these natural barriers is that most viruses fail to pass from the blood to the CNS.

Access from the blood may occur by: (1) growth of virus through the endothelium of small cerebral vessels, (2) passive transport across the vascular endothelium in intracellular vacuoles, (3) passage through the choroid plexus to the cerebrospinal fluid, or (4) being carried across by infected leukocytes.

Routes of viral invasion of the central nervous system.

There is a correlation between the level of viremia of a blood-borne neurotropic virus and its neuroinvasiveness; invasion occurs mostly during the high titer secondary viremic phase. Once the blood-brain barrier is breached, more extensive spread throughout the CNS is possible. Neurotropic viruses show marked preference for different neural cells, eg, rabies virus multiplies in neurons, whereas FIV invades astrocytes. Infection of neurons leads to the three histologic hallmarks of encephalitis: (1) neuronnal necrosis, (2) nonsuppurative encephalitis and (3) perivascular infiltration of mononuclear cells [perivascular cuffing]. Cerebrospinal fluid [CSF]. A marked increase in lymphocytes, mostly T cells, and monocytes; a slight increase in protein. The CSF usually remains clear.

63 Infection of the Vascular Endothelium When virus replicates in and damages vascular endothelium, its smoothness and its glycocalyx-thrombomodulin layer [prevents clot formation] are both lost, resulting in activation of both Factor XII [Hageman factor] and platelets and setting off the intrinsic pathway of clotting.

Platelet adhesion to subendothelial collagen is mediated by von Willebrand factor [vWF]. vWF is an adhesive glycoprotein synthesized in megakaryocytes and endothelial cells that circulates in blood complexed to factor VIII. vWF binds to receptors on subendothelial collagen and to vWF receptors on the platelet surface. Aggregation of platelets results in the release of procoagulants and clotting factors. If clotting occurs in the microvasculature, it may result in disseminated intravascular coagulation [DIC]. DIC is not a primary disease, rather, it is a complication of any condition associated with widespread activation of thrombin.

Severe endothelial wall damage can lead to edema and petechial and ecchymotic hemorrhage. Infection of the Embryo and Fetus

Majority of viral infections of the dam have no harmful effect on the fetus, however, some blood-borne viruses may cross the placenta to reach the fetal circulation, sometimes after 1o infection of the placenta.

Early death of fertilized ova, embryos, or fetuses, followed by resorption or unseen expulsion, may be classified as infertility. Abortion is usually indicative of severe damage to the fetus or its placental membranes or both. As a result of the intimate contact between the maternal and fetal placentae, disease of the latter is closely reflected in the former. The outcome of infections with teratogenic viruses [produce congenital anomalies] is influenced by the gestation period, being most damaging in the first and second trimesters. Persistently infected fetuses that remain infected into neonatal or adult life. Various viruses can induce immunologic tolerance or hyporesponsiveness,

64 resulting in little or no antibody production, eg, bovine viral diarrhea infection of cattle, etc.

When infections occur late in gestation, an effective immune response may eliminate the virus and there is no disease.

TYPES OF VIRAL INFECTIONS Inapparent (Subclinical; Asymptomatic) Infections An inapparent infection is one that does not cause a noticeable illness [too few cells are infected]. It occurs with most viral infections and can stimulate humoral and cellular immune responses. On the other hand, subclinical infections may also be an unrecognized source of virus spread. Factors leading to inapparent infections include:

Nature and dose of the virus. Attenuated and moderately virulent field strains can cause inapparent infections. Immune status of the host and failure of the virus to reach its target organ. Acute Viral Infection followed by Viral Clearance

Characterized by a short clinical course [days to weeks] followed by viral clearance by the host immune response. To maintain infection in a susceptible population, viruses that cause acute infections must be stable in the environment and/or be highly contagious.

65 Persistent Infections Following primary infection, if the host immune response does not effectively clear the virus, the virus persists in the host animal for long periods [months, years] with or without clinical disease.

Persistent infections can lead to continuing tissue injury, immunopathologic disease or to neoplasia. Persistent infections may lead to the maintenance [survival] of a virus in individual animals and herds, even following vaccination. Persistently infected animals are known as carriers; such carriers are very important in the long-distance spread of virus and in the reintroduction of virus into a given flock, herd, region, or country. Virus shedding may be continuous or intermittent. Acute infection followed by latent infection. Acute infection followed by latent infection in which viruses persist in a noninfectious form with intermittent periods of viral reactivation [switch from a latent to a productive infection] and shedding. During latency, the virus or its genome is maintained indefinitely in the cell, either by the integration of the viral nucleic acid into the host cell DNA or by carriage of the viral nucleic acid in an episomal form.

Acute infection followed by chronic infection. Acute infection followed by a chronic infection in which virus is continuously shed from or is present in infected tissues. Chronic infections can be established if the host immune response is unable to eliminate virus generated during an acute infection, eg, chronic foot-and-mouth disease. However, not all chronic infections start off as acute infections.

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Slow infections. Slow virus infections have a prolonged incubation period, lasting months or years, during which virus continues to multiply, leading to a slowly progressive lethal disease. Diseases caused by prions [infectious proteins] follow a similar pattern.

Mechanisms of Persistent Infections


Antigenic variation, eg, lentiviruses [eg, equine infectious anemia]. Retroviruses persist in the host as provirus and are transmitted vertically from generation to generation. Infection of cells of the immune system. Infected lymphocytes and monocytes transport virions to new sites where replication and viral shedding may occur. Infection of immunologically privileged sites. CNS. The blood-brain barrier limits contact between lymphocytes and CNS tissue; in addition, neurons do not express MHC class I proteins and hence cannot be directly recognized by cytolytic T lymphocytes.

Suppression of cell surface molecules required for T cell recognition, eg, down-regulation of MHC class I proteins. IMMUNOPATHOLOGY

In a number of viral infections, host immune responses result in substantial damage to host tissues, ie, immune-mediated diseases.

Chronic inflammation can result in extensive tissue damage, eg, fibrosis, and loss of organ or tissue function. Immune complexes formed in persistent viral infections such as equine infectious anemia and feline infectious peritonitis account for the vasculitis and glomerulonephritis observed in the two diseases. Autoimmune damage which is a result of molecular mimicry, eg, equine moon blindness.

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EPIDEMIOLOGY OF VIRAL DISEASES


Epidemiology is the branch of science that studies the frequency, distribution, and determinants of disease in populations. Purposes of Epidemiology

Acquisition of information on the characteristics of the causative agent, the host and host population, and behavioral, environmental, and ecological factors affecting disease transmission from one susceptible host to another. Planning and monitoring of disease control programs. Assessment of the economic effects of a disease and analysis of the costs and economic benefits of alternative control programs. Some Epidemiologic Terms

Arbovirus [Arthropod-borne virus]: Arboviruses multiply in the arthropod vector [eg, ticks, sandflies, and mosquitoes]; for each virus, there is a natural cycle involving vertebrates [various birds or mammals] and arthropods. Cases: Those animals suffering from a clinically diagnosable disease. Case-fatality rate: The percentage of deaths among the clinically ill animals. Carrier: Any animal that sheds an infectious agent without demonstrating clinical signs. Thus, a subclinically infected animal may be a carrier [healthy carrier], and may shed agent. Shedding by carriers may be continuous or intermittent. The periods for which animals are carriers vary, depending upon the pathogen.

Incubatory [acute] carriers: Animals that excrete agent during the diseases incubation period. Convalescent [chronic] carriers: Animals that shed agent when they are recovering from a disease.

Communicable period: The time during which the infectious agent may be transferred, directly or indirectly, from an infected animal to another animal. Contagious disease: An infectious disease in which the pathogen may be transmitted from one host to another by direct or indirect contact. Dead-end [Incidental, Accidental, or Terminal] host: One that is not involved in maintaining a virus; such a host does not usually transmit an infectious agent to other animals or vectors, eg, Eastern equine encephalitis.

68 Disease: Any disturbance of the normal structure or function of any part, organ, or system of the body that results in a characteristic set of symptoms and signs. Endemic: The constant or usually expected occurrence of a disease within a given geographic area or population. Epidemic: Major increase in disease incidence affecting either a large number of animals or spreading over a large area. Exotic: A disease not known to occur in a particular country or geographic area. Field isolate: Fresh virus isolate from the natural host. Fomites: Inanimate objects other than vehicles [air, food, milk, water, etc] involved in the transfer of infectious agents from one host to another.

Fomites include contaminated syringes or needles, bedding, water bowl, feed-bucket, restraint devices, etc.

Incidence: The number of new cases that occur in a population over a specified period of time. Incubation period: The period of time between infection and the development of clinical signs and symptoms. Infection: Invasion and multiplication of microorganisms in body tissues. Morbidity rate: Percentage of animals in a population that develop clinical disease attributable to a particular pathogen over a specified period of time. Mortality rate: Percentage of animals in a population that die from a particular disease over a specified period of time. Pandemic: A geographically widespread [sometimes global] epidemic. Pathogen: Any disease causing virus or microorganism Pathogenicity: Ability of an organism to cause disease. Population: Aggregation of animals. Prevalence: The number of occurrences of disease [old and new cases], infection, or related attributes [eg, presence of antibodies] in a population, at a particular point in time. Reservoir: The organism or environment that normally harbors a pathogen.

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A reservoir is essential for a microbes survival and reproduction. A host supplies an environment for maintenance of a microbe but is not necessary for the microbes survival. A reservoir may be animate or inanimate.

Screening: This is systematic diagnostic testing to detect asymptomatic or subclinical animals or individuals who appear healthy, for the purpose of control or prevention of the disease. Source: The place from which a pathogen passes directly to a susceptible host. Sporadic disease: A disease that occurs irregularly and hapharzadly. Sylvatic: Occurring in or affecting wild animals. Threshold level: The minimum concentration of a pathogen in the hosts circulation that allows successful transmission to an arthropod vector. Wild-type virus: Denotes the original virus circulating in nature, from which mutants arise. Zoonosis: A disease that primarily affects vertebrate animals but that can be transmitted to humans. Serologic Epidemiology Seroepidemiology is the use of serological data as the basis of epidemiological investigation. Antibodies provide evidence of current and previous exposure to infectious agents. Uses of seroepidemiology include:

Estimates of prevalence. Examination of appropriate numbers of sera can be used to determine the prevalence of a particular virus. Estimates of incidence (paired sera [acute/convalescent]). Four-fold or greater rise in antibody titer indicates a recent infection. Evaluation of eradication or immunization programs. Serological surveys can be used to detect and slaughter infected animals. Serological surveys are also valuable in determining the success of vaccination programs. Emergence of new diseases. Antibody data can be used to assess the impact and geographic distribution of new, emerging, and re-emerging viruses. Transmission of Viruses

Though the ability of a virus to multiply is of paramount importance, so is its ability to spread from host to host, otherwise it may die with the original host.

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A successful pathogen doesnt kill its host before it is transmitted. Disease transmission may be horizontal or vertical Horizontal [Lateral] Transmission

This is the transmission of infectious agents from any segment of a population to another. Horizontal transmission occurs by three principal routes:

Contact transmission. This is the spread of infectious agents by direct or indirect contact.
Infected host Direct Susceptible host

Indirect Intermediate object

Direct transmission. Involves close physical contact between the infected animal and a susceptible host, eg, licking, rubbing, biting, coitus, fecal-oral transmission, etc. Droplet transmission. Transmission in which the organisms are spread in droplet nuclei [saliva or mucus] that travel less than 1 meter from the source to the susceptible host. The droplets are discharged into the air by sneezing, coughing, etc. Due to the short distance traveled by the infectious agent, droplet transmission is not considered to be airborne transmission.

Indirect transmission. Involves an intermediate object, living or inanimate, that transmits infectious agent between infected and susceptible hosts. Airborne transmission. This is the spread of infectious agents by droplet nuclei in dust that travel more than 1 meter, sometimes for miles, from the reservoir to the host. The droplets are small enough [< 5m] to remain airborne for prolonged periods.

Vector transmission. An arthropod that carries pathogenic organisms from an infected host to another. Arthropod transmission is divided into: Mechanical transmission. This is the passive transport of an infectious agent on the feet or other body parts of the arthropod vector. If the vector makes contact with a hosts food, for example, the organisms can be

71 transferred to the food and later ingested by the host. A mechanical vector is nonspecific in its disease transmission, eg, housefly. Biological transmission. The infectious agent undergoes either a necessary part of its life-cycle, or multiplication, in the vector before transmission to the susceptible host. The time the pathogen spends in the arthropod vector is known as the extrinsic incubation period. If the pathogen multiplies in the gut of the arthropod, they can be passed with feces. If the arthropod defecates or vomits while biting a host, the parasite can enter the wound. Some pathogens migrate from the gut to the salivary gland; these are directly injected into the host.

Vehicle transmission. This is the transmission of infectious agents by a medium, such as water, food, air, blood, pus, intravenous fluids, drugs, etc. Iatrogenic Transmission

Infection that is transferred during surgical and medical practice. There are two main types: [1] introduction of pathogens by dirty instruments or by contaminated body surfaces and [2] introduction of pathogens contaminating prophylactic or therapeutic preparations, eg, Pseudomonas aeruginosa in intramammary drycow antibiotic preparations. Nosocomial Infections These are infections acquired within the veterinary hospital or clinic. These infections can only be prevented and controlled by careful attention to effective sanitation and management practices. Vertical Transmission These infections are transmitted from one generation to the next by infection of the embryo or fetus in utero, in ovo, during parturition, or postpartum [eg, milk, colostrum, and fecal contamination of teats].

Hereditarily transmitted diseases are carried within the genome of either parent. Congenitally transmitted diseases refer to diseases acquired either in utero or in ovo, rather than inherited. Transmission of several contagious diseases to newborn domestic animals can be prevented by feeding heat-treated colostrum [56oC for 1 hour] and pasteurized milk [72oC for 15 seconds].

72 Survival of Viruses in Nature Various strategies account for the maintenance of viruses in nature. These include:

Avoidance of a stage in the external environment. Vertical transmission; venereal transmission; or vector transmission.

Population size. A virus may disappear from a population if it exhausts its potential supply of susceptible hosts. Depending on the breeding characteristics [population turnover], duration of immunity, and the pattern of virus shedding, the critical population size varies with different viruses and with different hosts. Extension of host range. Many infectious agents can infect more than one host, eg, foot-and-mouth disease virus. Some infections of various hosts are inapparent, increasing the difficulty of control. Stability. When a virus is exposed to environmental influences such as temperature, humidity, dessication, etc, it must be able to survive and maintain its viability until it encounters a susceptible host. The length of time a virus can remain infective outside the host, is influenced by the nature of the envelope or capsid proteins. Persistent infections. Viruses may persist within the host, sometimes for life. Determinants of Disease

A determinant is any characteristic that affects the health of a population. Determinants are classified into those associated with the host, the agent, and the environment; the so-called epidemiologic triad.

Determinants associated with host, agent or environment, do not exert their effects in isolation, but interact to induce disease.

73 Host Factors A host describes a plant, animal, or arthropod that harbors or nourishes another organism. Replication or development of the agent usually occurs in the host.

Species, breed, sex, age, size and conformation, coat color, etc. Herd immunity. Refers to immunity conferred on a group or population as a result of the presence of immune individuals within that population. Herd immunity can function to prevent the successful entry of a pathogen into a group of animals, and/or it can minimize the extent and rapidity of the spread of that pathogen once it becomes established. The single most important component of herd immunity is the frequency of contact, ie, the likelihood in any time period that an infected individual will have contact with another individual sufficient to transmit the infection if the latter individual is susceptible. Another factor that also determines the level of herd immunity is the number of susceptible individuals in the group. Agent Factors

Infectivity. Describes the dose of an organism that is required to initiate infection. Varies between different strains of the same organism, and can depend upon the portal of entry, and the age and resistance of the host. Virulence. A less virulent virus is more efficient at infecting the population. Transmission efficiency. Affected by infectivity, virulence and stability. Exogenous pathogens. These are not usually present in the host. Endogenous pathogens. They are often found in healthy individuals, and usually do not cause disease unless the host is stressed or immunosuppressed. Environment Factors

The environment includes all the biotic [flora and fauna] and abiotic [air, soil, water, climate, etc] components of a place, be it a pen within a barn or a large geographic area.

Vegetation, extreme changes in the weather, eg, flooding, solar radiation, etc. Housing; overcrowding and poor ventilation. Stress factors, level of hygiene, and diet. Vectors and/or reservoir populations.

74 Course of a Typical Disease Once a pathogen overcomes the defenses of the susceptible host, the development of the disease follows a certain sequence that tends to be similar whether the disease is acute or chronic.

Incubation period. Influenced by a variety of factors including inoculum size, virulence of pathogen, resistance of host, and distance of site of inoculation from the target site. Prodromal period. A short period sometimes following IP in which first signs and symptoms of illness appear, eg, fever, general aches, malaise, etc. Acute period [period of illness]. When the disease is at its height. Decline period. During which the signs and symptoms subside. Convalescent period. The patient regains strength and the body returns to its prediseased stage.

Disease Control Strategies Disease prevention. Refers to those measures designed to exclude disease from an unaffected population; applied at the individual or population level. Disease control. Refers to the reduction of the morbidity/mortality from disease. Disease eradication. Describes efforts to eliminate selected organisms from a defined area. The following measures may be used either singly or in combination in preventing, controlling, or eradicating disease.

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Quarantine. This is the limitation on freedom of movement of animals, individuals, vehicles, etc, to prevent spread of a disease to other members of a population. The length of a quarantine period is usually based on the maximum incubation period of the disease in question. Test and Slaughter. This involves a method of case finding, usually by means of an immunologic screening test, and then the killing of test-positive animals. Employed most effectively in a disease with a low prevalence and for which a sensitive and specific diagnostic test exists.

Depopulation. Elimination of all susceptible hosts [infected, potentially infected, or contact animals] on a herd/flock or area basis. Vector and wildlife control. Physical and chemical disinfection. Chemotherapy and chemoprophylaxis. Vaccination. Restriction of movement of animals Import restrictions and strict quarantine for imported animals. Requiring diagnostic tests before animals are shipped. Sentinel Studies

Selected farms, abattoirs, veterinary practices or laboratories, termed sentinel units [they are designed to keep watch on a disease], may be used in the monitoring or surveillance of disease.

Seroconversion in sentinel animals; eg, chickens are used as sentinel flocks to monitor the activity of Eastern equine encephalitis virus. Sentinel animals are also used to monitor introduction of viruses to an area or elimination of viruses from an area. Notifiable Diseases

Usually for serious diseases that are of economic importance, there is State and Federal legislation making these diseases reportable to the appropriate authorities. Steps are rapidly taken to control the spread of the disease, eg, foot-andmouth disease.

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VIRAL CHEMOTHERAPY
Chemotherapy is defined as the treatment of infectious diseases by the administration of drugs that are inhibitory or lethal to the infectious agent[s].

The clinical use of antiviral drugs is limited due to the difficulty in achieving selective toxicity, ie, the drug must be harmful to the infectious agent without being harmful to the host. Viruses must utilize host cell biochemical machinery to synthesize new viral proteins and nucleic acids; thus, it is very difficult to inhibit viral functions without also inhibiting those of the host.

Clinical use of antibiotics and other antibacterial compounds during viral infections is mostly to prevent 2o bacterial infections. Acyclovir (Acycloguanosine; Zovirax; Valtrex)

Acyclovir is a synthetic nucleoside analog of deoxyguanosine; its antiviral activity is restricted to herpesviruses. It is administered as an inactive prodrug. Conversion of the prodrug to the active compound requires three kinases.

Acyclovir requires the herpesvirus-specified enzyme, thymidine kinase, to phosphorylate it to acyclovir monophosphate. Host cell kinases then form the di- and triphosphate forms. Acyclovir triphosphate is incorporated into the growing viral DNA, in place of deoxyguanosine, by the viral DNA polymerase. As a result, the viral DNA chain terminates and further DNA replication is blocked. Since herpesvirus thymidine kinase is not found in an uninfected cell, acyclovir cannot be phosphorylated and incorporated into host DNA; hence acyclovir is essentially nontoxic to normal cells. Mutants of herpesviruses that lack thymidine kinase fail to phosphorylate acyclovir and are resistant to it.

Acyclovir is available as a topical cream, oral, and IV preparation. Humans: Genital herpes: 200 mg capsules, PO. Mucocutaneous herpesvirus infections: 5% cutaneous ointment. Cats: Feline herpesvirus 1 corneal ulcers, ophthalmic ointment.

77 INFLUENZA CHEMOTHERAPY Amantadine (Symmetrel) This is a water-soluble cyclic amine which specifically inhibits most strains of influenza A viruses by blocking viral uncoating.

The M2 transmembrane channel is the target of amantadine, which acts by blocking the channel and abolishing its ion channel activity. As a result, uncoating of the virus is prevented. Amantadine is administered in the first 24 to 48 hours of infection for at least 10 days. It is most effective when given prophylactically to susceptible patients in anticipation of influenza A virus infection. Oseltamivir Phosphate (Tamiflu)

Tamiflu is an inhibitor of the neuraminidase enzyme synthesized by influenza A and B viruses. Neuraminidase is a glycoprotein on the surface of influenza viruses. It functions at the end of the viral life cycle by facilitating the release of progeny virions from infected cell surfaces during the budding process.

When taken within 30 h of onset of symptoms, the drug reduces the course of symptoms by about 3 days. AIDS CHEMOTHERAPY
Replicative cycle of HIV-1 indicating potential sites of action for HIV-1 inhibitors. RT, reverse transcriptase; cDNA, complementary DNA; mRNA, messenger RNA

Zidovudine (Azidothymidine, AZT, Retrovir) AZT was originally conceived as an anticancer drug in 1964 but found no use until 1985 when Mitsuya et al. discovered its activity against the HIV virus. It was the first anti-HIV drug approved by the FDA in 1987.

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AZT is phosphorylated by cellular enzymes to the triphosphate thymidine analog. This form inhibits viral reverse transcriptase as well as causing chain termination; when it is clipped on to a growing chain of DNA, the next link doesnt fit and DNA replication is foiled, hence blocking the synthesis of proviral DNA. Since reverse transcription of the viral genome takes place in the cytoplasm where the drug appears first and in the highest concentration, the RNAdependent DNA polymerase [reverse transcriptase] of the virus is 100 times more sensitive to inhibition by AZT than the cellular DNA polymerase, resulting in selective activity and low mammalian toxicity. AZT can be given orally or IV in humans. Major toxicities of AZT include anemia and granulocytopenia, which occur in up to 45% of treated patients. AZT is considered a maintenance drug, because, at best, it slows the progresssion of HIV infection but does not eradicate or eliminate the virus. Cats. AZT has been shown to reduce clinical signs when given to FIV-positive cats at a dose of 10 mg/kg/d twice a day, subcutaneously, for a period of 3 weeks. Other nucleoside analogs approved by the FDA for treatment of HIV include: Didanosine [ddI, 1991]; Zalcitabine [ddC, 1992]; Stavudine [d4T, 1994]; Lamivudine [3TC, 1995]; Tenofovir [TDF, 2001], etc. Protease Inhibitors

The drugs inhibit viral proteases responsible for the cleavage of viral polyproteins into functional proteins by binding to their active sites. The following protease inhibitors have received FDA approval:

Saquinavir [1995]; Indinavir [1996]; Ritonavir [1996]; Lopinavir [2000], etc.

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HOST RESPONSE TO VIRAL INFECTIONS


Subsequent to virus invasion of the body, host innate and adaptive immune responses to the virus are aimed at blocking infection and eliminating virus infected cells.
Virus Infection

Type I interferon MHC I

Intracellular growth

APCs

Envelope proteins in cell membrane ADCC

Progeny virions

Helper T cell

B cell

Antiviral state in uninfected cell

Tc cell

NK cell, Macrophage Antibodies

Target cell lysis

Extracellular virions [Neutralization]

APC, antigen-presenting cell; ADCC, antibody-dependent cell-mediated cytotoxicity; MHC, major histocompatibility complex protein; Tc, cytotoxic T cell.

Innate Immunity

Inhibition of virus infection by type I interferons. NK cell-mediated killing of infected cells.

Interferons and appear first, followed by a wave of NK cells, which together control virus replication but do not eliminate the virus. Virus elimination is accomplished when specific CD8+ T cells are produced.

80 Interferons [IFNs] Interferons are cytokines produced and secreted by somatic cells in response to a variety of stimuli, including virus infection. They possess potent antiviral, immunomodulating, and anticancer properties. Their antiviral activity was first reported by Isaacs and Lindenmann in 1957. There are 3 classes of IFN based on antigenic and chemical differences: IFN-, IFN-, and IFN-.

Being glycoproteins, interferons are orally inactive and must be given parenterally. Peak plasma concentrations occur 4-8 hours following SC or IM injection. Type I Interferons

They induce an antiviral state in uninfected cells via inhibition of viral protein synthesis; simultaneously, they induce apoptosis in the virus infected cell. They also stimulate production of MHC class I proteins and proteasome proteins.

IFN- [Leukocyte interferon]. Secreted by virus-infected macrophages and other leukocytes. Not host specific. IFN- [Fibroblast interferon]. Secreted by virus-infected fibroblasts and epithelial cells. Generally host species-specific. Interferons show no viral specificity, ie, IFN induced by a reovirus is effecttive against a variety of viruses. RNA viruses are stronger inducers of IFN than DNA viruses. Type I interferons are stable at pH 2.

Bifunctional role of type I IFNs in antiviral response; eliciting an antiviral state in uninfected cells and inducing apoptosis in infected cells.

IFN is released from cells within a few hours after viral invasion with high concentrations being achieved within a few days in vivo, at a time when the primary immune response is still relatively ineffectual.

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Virus-infected cell produces IFN which binds to a specific receptor on the cell membrane of uninfected cells, triggering intracellular synthesis of antiviral proteins: Protein kinase: Phosphorylates and inactivates a viral initiation protein, thereby preventing translation of viral mRNA. 2-5A synthetase: Activates a ribonuclease that degrades viral mRNA.

The antiviral state persists after IFN exposure and is lost slowly as a result of cell death or division. Type II Interferon [IFN-; Immune interferon]

IFN- is secreted by antigen, mitogen or cytokine stimulated T cells [Th and Tc] and NK cells. It is labile at pH 2 and demonstrates host specificity.

IFN- has no antiviral activity. It is mostly an immunoregulatory cytokine. Enhances expression of MHC class I and II proteins. NK cells

Lyses infected cells not expressing or expressing few MHC class I proteins. Lyses target cells via antibody dependent cell-mediated cytotoxicity [ADCC]. Adaptive Immunity

Protective immunity against viruses operates at two stages: (1) in the initial phase of infection, before a virus has invaded host cells and (2) after invasion into cells, when the virus is inaccessible to antibodies and phagocytes.

Adaptive immune responses are directed against viral capsid and envelope antigens. Internal viral antigens usually elicit protective CMI responses, whereas surface antigens elicit protective humoral and CMI responses. In contrast to the short-lived immune response against bacteria, antiviral immunity is, in many cases, very long lasting. This may be related to viral persistence within cells, perhaps in a slowly replicating or nonreplicating form. Viral infections usually do not provoke granulocytosis; leukocytosis observed is commonly due to lymphocytosis and other mononuclear cells. Antibody-Mediated [Humoral] Immunity

Antibodies are effective against viruses only during the extracellular stage of virus infection.

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Viruses may be extracellular early in the course of infection, before they enter host cells, or in the case of cytopathic viruses, when they are released from lysed cells. In generalized infections, the antibody response is of much greater intensity than that evoked by localized infections of superficial membranes. In such infections, a lot more viral antigen is produced that gains ready access to lymphoid tissues, evoking strong antibody-mediated immune responses. Superficial infections of the respiratory and GIT tracts may induce sIgA synthesis locally with little circulating IgG. Protective sIgA titers are maintained only for a few years. Antiviral Effects of Antibodies

Virus neutralization. Neutralizing antibodies inhibit virus replication by inhibiting viral attachment, penetration, or uncoating or all three processes. Opsonization [nonneutralizing antibodies]. Coating of virions by IgG may facilitate phagocytosis and intracellular killing by macrophages and, to a limited extent, by neutrophils. Clumping of viruses. Reduces the number of infectious units available for cell invasion. Complement activation. Opsonization (C3b, iC3b, C4b) and possible direct lysis of enveloped viruses. Lysis of virus-infected cells. Cells expressing viral antigens on their surfaces are susceptible to antibody-mediated destruction via ADCC [NK cells and macrophages] and complement-mediated cytolysis [C5b6789]. Cell-Mediated Immunity

The intracellular replicative steps of viruses and virus-infected cells are major targets for CMI. CMI responses are more important in recovery from infections with noncytolytic viruses.

In cytopathic infections, antibody seems to play a more important role in recovery. Cytotoxic T Cells (Cytolytic T Lymphocytes)

CTL-mediated cytotoxicity is the major mechanism through which cellular destructtion is achieved. CTLs recognize virus antigen expressed on cell membranes in association with MHC class I proteins.

83 Some Viral Strategies Used to Evade Host Immune Responses Antigenic plasticity or multiplicity

Plasticity. Rapidly changing surface antigenic structure by mutation, genetic reassortment or recombination, eg, lentiviruses and influenza A viruses. As a result, the virus becomes resistant to immunity generated by previous infection. Multiplicity. Antigenic variants with little or no cross-reactivity. There are so many existing serotypes of rhinoviruses [> 100 serotypes] that specific immunization against the common cold may not be a feasible preventive strategy.

Immunosppression. Immunocompetent cells are either lysed or inactivated, eg, HIV which survives by infecting and eliminating CD4+ T cells. Virokines. Epstein-Barr virus synthesizes a protein that is a homolog of IL-10 called vIL-10; like IL-10, it suppresses cytokine production by TH1 CD4+ cells. Viroceptors. Poxviruses encode proteins that are homologous to the receptors for several cytokines, including IFN-tumor necrosisfactor [TNF] and interleukin1. The secreted cytokine receptor homologues may bind cytokines and function as competitive antagonists of the cytokines. Down-regulation of class I MHC expression. Inhibition of class I MHC-associated presentation of endogenous protein antigens. As a result, cells infected by such viruses become insusceptible to killing by CD8+ T cells. Inhibition of complement activation. Vaccinia virus codes for a protein called vaccinia viral protein [VCP], which binds to C4b, inhibiting the classical complement pathway. A glycoprotein component of herpes simplex viruses bind to C3b, inhibiting both the classical and alternative pathways. Latency. Viruses may become latent [dormant] within infected cells. Viral antigens are scarce or absent on the cell surface. Evasion of neutralizing antibody. Large amounts of soluble viral proteins are produced that soak up antibody. Integration of the viral genome into the host cell genome, eg, retroviruses. Cell-to-cell spread [Type II spread]. Some viruses, eg, herpesviruses, lentiviruses, and morbilliviruses, cause adjacent cells to fuse together [membrane fusion], enabling the viral genome to spread from cell to cell without being exposed to the hosts immune mediators.

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LABORATORY DIAGNOSIS OF VIRAL DISEASES


Although many viral diseases can be diagnosed on the basis of clinical signs and symptoms, confirmation of the diagnoses requires the services of viral diagnostic laboratories. Viral diagnosis is based on:

Case history of the disease and clinical signs and symptoms. Gross pathology and histopathology. Demonstration, isolation, and identification of the causative virus. Selection, Collection, and Preservation of Specimens

Successful isolation of viruses from clinical specimens depends largely on the proper collection and handling of specimens. The best specimens are usually collected early in the illness when virus is being excreted at relatively high levels. Specimens collected during the first 72 hours of an illness are more likely to yield infectious virus than specimens collected later in the illness.

Specimens should be collected from apparently normal in-contact animals, sick, moribund, and dead animals, placed in sterile containers and shipped promptly to the diagnostic laboratory. The volume of the sample should be sufficient to permit direct testing, virus isolation, etc. The laboratory must be provided sufficient information to enable the microbiologist to select the most appropriate method[s] for a quick and accurate diagnosis. The information should include history, clinical findings, necropsy results if available, and a tentative clinical diagnosis.
Time Schedule for Collecting Specimens for Virus Studies

85 Selection of Specimens It is influenced by the clinical signs, together with a knowledge of the pathogenesis of the suspected disease.
Specimens Appropriate for Various Clinical Syndromes

Preservation of Specimens Because many viruses are labile, specimens for virus isolation should be collected into viral transport medium [VTM], refrigerated and transported to the laboratory without delay.

As a general rule, however, if a delay of more than 24 hours is anticipated, specimens should be frozen at 70oC for optimal preservation of infectious virions. Transport medium. Used to stabilize the infectivity of specimens before inoculation. Most consist of buffered isotonic saline at neutral pH, a high concentration of protein [albumin, gelatin, or fetal calf serum], and antibiotics and antifungal drugs to suppress the growth of bacteria and fungi. Specimen[s] for histopathologic examination should never be frozen, instead, they should be fixed in 10% buffered formalin or other fixatives. Methods of Viral Diagnosis

Detection of the virus itself or components of the virus; determines whether the patient is currently infected with a given virus.

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Detection and measurement of specific antibody. Indicates current infection or past infection with the virus in question. Preparation of Specimens for Inoculation

Tissues. Finely minced with scissors and homogenized in a glass or mechanical homogenizer. Tissue homogenates are diluted 1:10 and centrifuged at 1000 g for 15 minutes. Antibiotics are added to the supernatant to suppress bacterial and fungal contaminants. Feces. Dispersed on a vortex mixer, centrifuged and the supernatant incubated with antibiotics. Swabs. Processed by twirling them in the transport medium and expressing the fluid by pushing the swab firmly against the side of the container; or it may be vortexed. Specimen Inoculation Virus can be grown from the suitably prepared specimen by inoculation into: cell culturse; embryonated eggs; or laboratory animals. Detection of Viral Antigens in Clinical Specimens Direct Immunofluorescence. Viral antigens are detected by staining with a specific fluorescein-tagged antiviral antibody. Air-dried specimens for FA staining are usually fixed in either acetone or methanol for up to ten minutes in order to preserve viral antigens.

Touch preparations (made by grasping a small fragment of tissue with forceps and touching it gently [without rubbing or squashing] on several spots on a microscope slide) or frozen tissue sections are fixed in acetone and then stained. Indirect immunofluorescence can also be used.

Immunoperoxidase [IP] staining. Enzyme-labeled specific antibody is used to identify viral antigens. Formalin-fixed or fresh tissue specimens may be used.

Tissue sections are stained with horseradish peroxidase-labeled specific antibody. The enzyme reacts with an added substrate [hydrogen peroxide and a benzidine derivative]; the ensuing reaction results in the colorless soluble benzidine derivative being converted into a colored (brown) insoluble precipitate. The slides are examined using light microscope. Stained slides can be kept for extended periods of time for multiple examinations.

87 Latex agglutination. Antibody-coated latex particles are incubated with the clinical specimen in which antigen is sought and the particles agglutinate in a lattice formation if adequate antigen is present. Direct ELISA. Viruses and viral antigens in the specimen bind to the capture antibody bound to a solid phase. The bound viral antigen is then detected with a second antibody which is conjugated to horse radish peroxidase. The bound enzyme is then detected and can be quantitated by the development of color when hydrogen peroxide/chromogen solution is added. Agar gel immunodiffusion. Sample containing suspect virus and specific antibody are placed in opposite wells cut in an agarose gel. The reactants diffuse towards each other and form a visible line of precipitate if antigen is present. Nucleic Acid Tests [NATs] In general, specimens suitable for conventional virologic techniques, can also be used for NATs. However, specimens collected in heparin-containing tubes, eg, whole blood or collected using alginate swabs, cannot be used for PCR. Both heparin and alginate inhibit Taq [Thermus aquaticus] polymerase activity.

Nucleic acid hybridization. Viral RNA or DNA in specimens can be detected by hybridization using labeled viral DNA or RNA probes. Polymerase chain reaction [PCR]. The technique allows the in vitro amplification of viral DNA or RNA. The amplified nucleic acid can then be detected by standard hybridization procedures. RT-PCR [Reverse transcriptase-PCR]. Detects either mRNA or ribosomal RNA [rRNA], after RT makes a copy of DNA [cDNA] complementary to the RNA sequence. RT-PCR is used to detect RNA viruses, organism-specific RNA of pathogens, including bacteria and DNA viruses, etc. Electron Microscopy

Electron microscopy can be used to demonstrate virus[es] in diagnostic specimens and to detect viruses that cannot be grown in vitro. Negative staining: This is a simple and rapid method for the detection and recognition of virus particles. The technique was introduced in 1959 by Horne and Wildy, who described the use of heavy metal salts for enhancing the contrasts in virus-particle images.

The technique is very useful for identifying enteric viruses, however, for it to work, large numbers of virus particles [>107particles/ml] must be present in the clinical specimen.

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The viral particles are mixed with a solution of a heavy metal salt highly opaque to electrons, eg, sodium phosphotungstate, osmium tetroxide or uranyl acetate. The mixture is then spread in a thin layer on a carboncoated copper grid and dried. The parts of the particles that are not penetrated by the salt stand out as electronluscent areas on an opaque background.

Immunoelectron microscopy [IEM]. The use of specific antibody results in clumping of virus particles, facilitating their recovery by centrifugation and direct identification of the virus in question.

Identification of Viral Isolates Hemadsorption/Hemadsorption-inhibition assay. Monolayer cell cultures infected with some enveloped viruses, eg, paramyxoviruses, togaviruses, etc, adsorb erythrocytes of certain species to their plasma membranes [hemadsorption]. The phenomenon is due to insertion of viral glycoprotein peplomeres [see virus replication] into the cytoplasmic membrane at sites of virus budding.

Virus identification is carried out by treating the infected cell culture with specific antibodies prior to adding the erythrocytes [hemadsorption-inihibition].

Virus neutralization. The assay is based on the reaction of specific antibody with virus to render it noninfectious for the indicator system, eg, cell culture, embryonated eggs or laboratory animal.

89 Complement fixation test. Suspect virus-specific antibody complex binds guinea pig complement, which is thereafter unavailable for the lysis of hemolysinsensitized sheep red blood cells. Immunofluorescence staining of infected monolayer cells. Direct ELISA is also employed in the identification of virus isolates. Hemagglutination/hemagglutination inhibition test. Many viruses [naked and enveloped] can agglutinate RBCs of various species of mammals and birds.

When specific antibody and virus are mixed prior to the addition of erythrocytes, hemagglutination is inhibited; thus, the specificity of the antibody used serves to identify the virus isolate.

Detection and Quantitation of Antiviral Antibodies Serologic Assays In the assays, two-fold serial dilution of suspect serum is used; a constant amount of virus is added to each of the dilutions. The titer is the highest dilution giving the desired result. Antibodies can be detected and/or quantitated using any of the assays below:

Hemagglutination-inhibition test Complement fixation test Immunodiffusion test Indirect ELISA test Indirect immunofluorescence test [IFA] Virus neutralization test IgM Class-Specific Antibody Assay

Demonstration of specific IgM antibodies in a single acute-phase serum can be used for rapid diagnosis of viral diseases. In viral infections, IgM antibodies usually appear within 7 to 10 days after infection but drop to low levels within 1 to 2 months and usually disappear within 3 months.

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Thus, the presence of specific viral IgM antibody implies a current or recent infection with the virus. Since maternal IgM cannot cross the placenta in domestic animals, if found in the serum of a newborn animal prior to intake of colostrum, they are diagnostic of intrauterine infection. A common method is the IgM antibody capture assay, in which the viral antigen is bound on a solid phase substrate; the test serum is allowed to react with the substrate and then specific IgM antibodies captured by the antigen are detected with labeled anti-IgM antibody matched to the species from which the specimen was obtained.

Viral Genotyping Viral genotyping involves the partial or complete sequencing of the nucleotides that make up the viral genome. Knowledge of viral genotypes has facilitated the development of antiviral drugs and in the design of vaccines.

Disease diagnosis. The nucleotide sequence of the virus of interest is compared to a reference wild-type viral genotype. It is the most accurate way to type and subtype viruses. Molecular epidemiology. The genotyping of a virus isolate provides information on the source and epidemiology of the viral disease. Drug resistance. DNA sequence analysis is the gold standard for identifying critical changes in the nucleotide sequence that may confer decreased susceptibility to a drug. A change in a single base in HIV protease gene can lead to amino acid substitutions that can disrupt the function of the resulting protease, preventing binding to a protease inhibitor and leading to drug resistance in the patient.

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POXVIRIDAE
Properties of Poxviruses

The largest and most complex of all viruses. Most poxviruses are brick shaped; 250 x 200 x 200 nm in size. Parapoxviruses are ovoid shaped; 260 x160 nm in size. Virions have a complex structure with a core, lateral bodies, outer membrane, and sometimes an envelope. Core: Dumbbell-shaped; contains viral DNA together with several viral proteins. Lateral bodies: Unknown nature. Envelope: May or may not be present.

Genome consists of a linear dsDNA [largest of any animal virus]. Encode all of the enzymes required for transcription and replication of the viral genome. Several poxvirus genes encode viroceptors that are homologous to some cytokine receptors, eg, IFN-,IL-1 receptors, etc; they modulate host response to infection. Cytoplasmic replication; a cell membrane-derived envelope encloses some of the mature virions. Both enveloped and nonenveloped virions are infectious. Virions produce eosinophilic intracytoplasmic inclusion bodies.

Antigenic Characteristics All poxviruses share group-specific nucleoprotein [NP] antigen which is exposed following alkaline digestion of the virion.

Genetic recombination among the viruses within a genus, results in extensive serological cross-reactions and cross-protection; none between different genera.

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A hemagglutinin is produced only by the Orthopoxviruses. Subfamilies and Genera

Subfamily Chordopoxvirinae. Contains poxviruses of vertebrates. Genus Orthopoxvirus Genus Parapoxvirus Genus Avipoxvirus Genus Capripoxvirus Genus Leporipoxvirus Genus Suipoxvirus Genus Molluscipoxvirus Genus Yabapoxvirus Vaccinia virus Pseudocowpox virus Fowlpox virus Sheeppox virus Leporipoxvirus Swinepox virus Myxoma virus Yaba monkey tumor virus

Subfamily Entomopoxvirinae. Contains viruses of insects. Epidemiology

Poxviruses are resistant to ambient temperatures and can survive for many months or years in dried scabs.

Poxviruses are transmitted between animals by several routes: Introduction of virus into small skin abrasions. Aerosol infection of the respiratory tract. Mechanical transmission by biting arthropods. Pathogenesis and Immunity

Poxviruses are highly epitheliotropic, causing cutaneous [all poxviruses infect the skin] and, occasionally, systemic diseases in birds and wild and domestic mammals. Many poxviruses are host specific, but the orthopoxviruses affect a wide range of species.

Following cutaneous introduction or inhalation, the virus usually gains access to the systemic circulation via the lymphatic system, although multiplication at the site of injection in the skin may lead to direct entry into the blood and a primary viremia. A secondary viremia disseminates the virus back to the skin and to other target organs.

Poxviruses induce lesions by a variety of mechanisms:

93 Degenerative changes in the epithelium. The lesions start of as erythematous macules, become papular, and then vesicular. Vesicles develop into umbilicated pustules [so-called pock lesion]. The pustules rupture and form a crust [scab]. Lesions heal and often leave a residual scar. Rupture of a pustule predisposes to 2o bacterial infection. Proliferative lesions. Poxviruses that encode epidermal growth factor may induce cellular hyperplasia.

Immunity. Poxviruses encode proteins which may counteract host defenses. immunity varies from short-lived in some poxvirus infections, eg, parapoxvirus infections, to prolonged in others. Diagnosis

Virus isolation. Scrapings from skin lesions; vesicular fluids and crusts. Chorioallantoic membrane [CAM]. Pock lesions; except parapoxviruses [do not replicate in embryonated eggs]. Cell culture. Poxviruses grow in a variety of cell cultures.

Virus identification. Negative stain electron microscopy; FAT, etc. Histopathology. ORTHOPOXVIRUS DISEASES Cowpox

Distribution. The disease is endemic only in Europe and east Asia. Hosts. Cattle, wild and domestic cats, humans, zoo animals, including elephants, rhinoceros, etc.

Rodents serve as reservoir hosts.

Etiologic agent. Orthopoxvirus. One serotype. Transmission. Infection of cattle and domestic cats occurs through contact with rodents. In dairy herds the virus spreads by the process of milking. Clinical features

Disease in cows. IP: 3-7 days. Lesions are observed on the teats and udder.

94 Ulcerated pustules eventually give rise to thick red scabs. Secondary bacterial infections of teat lesions are very common. Uncomplicated teat lesions usually heal in 3 to 4 weeks.

Disease in cats. More severe than in cattle or humans. Scabs are more widespread and 2o bacterial infection, which may result in pneumonia, is very common. Cats usually recover in 6 to 8 weeks.

Disease in humans. Maculopapular lesions are first observed on the hands or the face, accompanied by nausea, fever, and lymphadenopathy.

Immunity. Recovering animals have long lasting immunity.

Other Orthopoxviruses Variola virus [smallpox in humans]; Camelpox virus [camels]; Monkeypox virus [squirrels, monkeys, rabbits, great apes, humans]; Ectromelia virus [Mice, voles]; Horsepox [horses, cattle, humans]; Rabbitpox [rabbits]; Vaccinia virus [humans, cattle, buffalo, swine, rabbits]; Uasin Gishu disease virus [horses].

PARAPOXVIRUS DISEASES Parapoxviruses infect a range of species, but are most important in cattle, sheep, goats, and camels. Parapoxviruses of domestic animals are zoonotic. Pseudocowpox The disease occurs as a common endemic infection in cattle worldwide. It is seen as a chronic infection [low, but progressive morbidity] in many dairy herds and occasionally in beef herds. Etiologic agent. Bovine parapoxvirus 2. One serotype. Transmission. Infected cattle are the sources of infection. Cross-suckling of calves and improperly disinfected teat clusters of milking machines. Possible mechanical transfer of virus by biting flies. Clinical features. Infections are generally mild. Morbidity in a susceptible herd approaches 100%; however, virus spread is slow such that at any point in time, only about 10-15% of cows may have lesions.

The lesions are characterized by hyperplasia of squamous epithelium. Thick scabs [0.5-25 cm] develop from ruptured pustules. The scabs become elevated as a result of developing granulation tissue. Within 7 to 10 days, the

95 scabs drop off, leaving a dark red horseshoe-shaped ring of small scabs surrounding small, wart-like granulomas.

Lesions affect the teats, udder, and perineum. Similar lesions are observed on the muzzle and in the mouths of nursing calves.

Immunity. Short-lived immunity of 4 to 6 months duration. Humans. A mild skin lesion commonly referred to as milkers nodule. Contagious Ecthyma Contagious ecthyma is an acute infection of sheep and goats of all breeds, however, it is predominantly a disease of lambs and kids. The disease is worldwide. Synonyms. Scabby mouth, contagious pustular dermatitis, sore mouth, and orf [human disease]. Etiologic agent. Ovine parapoxvirus. One serotype. Transmission. The virus can survive in the environment indefinitely in scabs. Infection is established through cutaneous abrasions.

Oral lesions in lambs or kids result from nursing dams with teat lesions.

Pathogenesis. The lesions are characterized by vesiculopapular eruptions followed by development of pustules and yellow-brown scabs.

Dermal tissue may proliferate resulting in a verrucose mass under the scabs.

Clinical features. IP: 4-7 days. High morbidity [90%] but low mortality.

The primary lesion develops on the skin of the lips and frequently extends to the mucosa of the mouth. In severe cases, lesions may be seen on the genitals, perineum, and feet [if severe, can lead to lameness]. Extensive lesions on the face and oral cavity are painful to the animal and may lead to anorexia and weight loss. Within 1-4 weeks, the scabs drop off and the underlying tissues heal without scarring.

Mortality often results from the invasion of 1o lesions by the larvae of the screwworm fly or by bacteria such as Fusobacterium necrophorum.

Immunity. Sheep are susceptible to reinfection; chronic infections can occur.

96 Vaccination. Ewes are vaccinated several weeks before lambing, using nonattenuated virus vaccines from infected scabs or from virus grown in cell culture.

Vaccine is brushed over lightly scarified area of the skin, usually the inside of the thigh, where a localized lesion develops. Vaccine confers short-lived immunity. Lambs and kids. Vaccinated at 1 month of age and revaccinated 2-3 months later, followed by annual revaccination.

Humans. Maculopapular lesions and nodular lesions are observed 2 to 4 days following infection. The lesions last for 4 to 9 weeks and heal without scarring.
Other Parapoxviruses Bovine parapoxvirus 1 [Bovine papular stomatitis in calves; humans].

CAPRIPOXVIRUS DISEASES Diseases caused by capripoxviruses include sheeppox, goatpox, and lumpy skin disease. Sheeppox is the most important poxvirus disease of domestic animals. Sheeppox Distribution. Endemic in Africa, the Middle East, Asia, and parts of Europe. Etiologic agent. Ovine capripoxvirus. One serotype. Transmission. Aerosolization and by direct contact with saliva, nasal secretions, or scabs shed by sick animals. Scabs remain infective for up to 6 months.

Infection can also occur through skin abrasions; as well as mechanical transmission by biting arthropods, eg, Stomoxys calcitrans.

Pathogenesis. Sheeppox is a systemic disease.

Inhalation is followed by a leukocyte-associated viremia, which leads to virus localization in the skin and, to a lesser extent, internal organs. Severe necrotizing vasculitis developing in arterioles and postcapillary venules in the skin, results in ischemic necrosis of the dermis and overlying epidermis and intense neutrophil infiltration in and around the affected blood vessels. The lesions may be due to immune complex deposition [type III hypersensitivity], since virus does not multiply in endothelial cells.

97 Clinical features. IP: 12-14 days. Morbidity: Up to 80%. Malignant form. Seen in lambs and susceptible nonnative breeds, eg, merino.

Initial signs are fever, salivation, lacrimation, hyperpnea, edema of the eyelids, and serous nasal discharge later becoming mucopurulent. A day or two later, cutaneous nodules develop, which may be widely distributed over the body. The nodules usually scab and persist for 3 to 4 weeks, healing to leave a permanent depressed scar. Mortality rate: Up to 50%; case fatality rate in lambs: up to 100%.

Benign form [Mild]. More common in adult sheep or resistant breeds.

Mostly skin lesions and a comparatively no to mild systemic reaction.

Immunity. Recovered sheep have a solid immunity, lasting probably for life. Prevention and control. Notifiable disease in most countries of the world.

Importation of sheep from infected areas to disease-free countries is prohibited. Quarantine and slaughter should an outbreak occur. Attenuated and inactivated vaccines are employed in endemic areas to control the disease. Goatpox

Goatpox occurs in Africa, Asia, and parts of Europe. The disease is similar to sheeppox, but is generally milder with a low mortality rate [5%]. Goatpox is a reportable disease. A strain of goatpox virus causes a more severe disease in sheep.
Other Capripoxviruses Bovine capripoxvirus [Lumpy skin disease in cattle and buffalo]

SUIPOXVIRUS DISEASES Swinepox Suipoxvirus is the principal cause of pox lesions in swine. Similar disease is caused by Vaccinia virus. All age groups are susceptible to swinepox, however, most outbreaks occur in young growing pigs. The disease occurs worldwide.

98 Transmission. Direct contact associated with skin injury. The virus can persist in shed scabs for up to a year.

Mechanical transmission by the pig louse, Haematopinus suis. It can carry the virus for weeks or months. Mosquitoes and biting flies have also been implicated in the transmission of the disease.

Clinical features. IP: 3-7 days. Course: 3 to 4 weeks.

Lesions are observed primarily on the abdomen and inner aspects of the thighs but may occur on other areas. Udder and teat lesions occur in some sows that nurse infected piglets.

Congenital infection has been reported in naive pregnant gilts and sows that develop viremia with infection of the fetal membranes. Affected piglets have lesions all over the body, tongue and hard palate. They may be stillborn or die within a few days after birth.

Control. Eradication of lice from the piggery. There is no commercially available vaccine. Recovered pigs are solidly immune. AVIPOXVIRUS DISEASES Avipoxviruses cause diseases in chickens, turkeys, guinea fowl, peacocks, pheasants, and other avian species. Fowlpox Distribution. Worldwide. Hosts. Chickens, turkeys, pigeons, pheasants, etc. Etiologic agent. Avipoxvirus. Several types. First virus to be grown in embryonated eggs.

Avipoxvirus is extremely resistant to dessication and can survive in exfoliated scabs for prolonged periods. Inclusion bodies. Bollinger bodies: Large intracytoplasmic inclusions. Borrel bodies [elementary bodies] occur inside the Bollinger bodies. Borrel bodies are minute spherical bodies obtained by tryptic digestion of Bollinger bodies.

99 Transmission

Infection occurs through small abrasions in the mouth or through injuries to the comb, wattles, or skin as a result of fighting, pecking or other injuries. Mechanical transmission by mosquitoes, ticks, biting flies, and lice.

Clinical features. IP: 4-14 days. There are 2 forms of the disease.

Dry form [cutaneous form]. Small papules on the comb, wattles, and around the beak; lesions may develop on the legs and feet and around the cloaca. The nodules become yellowish and progress to a thick scab. Egg production drops markedly. Affected birds recover in about 4 weeks.

Wet form [diphtheritic form]. Involves infection of the mucous membranes of the mouth, pharynx, larynx, and sometimes the trachea. The lesions coalesce resulting in a necrotic pseudomembrane which may prevent feeding. Death results from suffocation by occlusion of the larynx. Mortality can reach 50%.

Immunity. Recovered birds are solidly immune. Prevention and control. Control of mosquitoes and other biting insects.

Attenuated fowlpox virus, turkeypox virus or pigeonpox virus vaccines are applied by scarification of the skin of the thigh or by the wing web stab method [an applicator with two slotted needles is dipped in vaccine and thrust through the wing web]. Vaccinated birds are examined 7-10 days later for swelling and scab formation [take] at the site of vaccination. ULCERATIVE DERMATOSIS of SHEEP (UNCLASSIFIED POXVIRUS)

Ulcerative dermatosis is a contagious, poxvirus disease of sheep manifested in two distinct forms: One characterized by formation of crusted ulcers around the mouth and nose or on the legs, and the other as venereally transmitted ulceration of the prepuce and penis or vulva. The disease occurs worldwide. Synonyms. Lip-and-leg ulceration, ovine venereal disease, and venereal balanoposthitis and vulvitis. Etiologic agent. Ovine poxvirus [unclassified].

100 Transmission. Infection results from viral contact with damaged skin or by coitus. Morbidity rate is usually 15-20% but up to 60% of a flock may be affected. Clinical features. The lesion is a raw, granulating ulcer.

Face lesions occur on the upper lip, on the chin, and on the nose. Foot lesions occur in the interdigital space and above the coronet. Recovery takes 2 to 8 weeks.

Ewes. Lesions occur as edema, ulceration, and scabbing of the lips of the vulva. Rams. Lesions partially or completely surround the preputial orifice and may become so severe as to produce phimosis [constriction of the preputial orifice; difficulty in urination]. Rarely, the ulcerative process may extend to the glans penis.

Reproductive loss results from incapacitation of rams and interference with feeding and breeding.

Diagnosis. Recognition of the characteristic ulcerative lesions on examination of the penis and prepuce of rams intended for breeding purposes.

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CIRCOVIRIDAE
Properties of Circoviruses

Nonenveloped icosahedral virions, 17 to 22 nm in diameter. Genome is a circular, single-stranded DNA. Replicate in the nucleus of dividing cells [presence of cellular enzymes expressed during the S phase of cell growth]. Large, grape-like basophilic intracytoplasmic or intranuclear IBs. Virions are highly resistant to inactivation in the environment..

Genus Circovirus

Porcine circovirus type 1 [Nonpathogenic] Porcine circovirus type 2 Chicken infectious anemia virus

Genus Gyrovirus

Post-Weaning Multisystemic Wasting Syndrome (PMWS) The disease was first described in Western Canada in 1991. It is mostly a disease of growing pigs 4 to 14 weeks of age, characterized by progressive weight loss, respiratory distress, diarrhea, skin discoloration, etc. Distribution. The disease has been reported in North America, Europe, Asia, and other parts of the world. Hosts. The disease affects only swine. Etiologic agent. Porcine circovirus type 2 [PCV2]. Strains vary in virulence. Transmission. Fecal-oral transmission. PCV2 is found in blood, saliva, feces, urine, and semen. Subclinically infected and convalescent swine may carry the virus for several months, hence serving as sources of infection.

Venereal [AI; coitus] and transplacental transmission.

Pathogenesis. Initial virus replication occurs in tonsillar macrophages, followed by invasion of regional lymph nodes and viremia.

It appears the main target cells are monocytes/macrophages, hepatocytes, dendritic cells, Kupffer cells, and cardiac myofibers. Organs and tissues affected include the lung, liver, spleen, Peyers patches, etc. Depletion of lymphocytes [T- and B cells]; however, PCV2 is not found in lymphocytes, hence the cause of the lymphopenia is not known.

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In utero infection. Infection during the first and second trimesters may result in fetal death and resorption, aborted fetuses with severe cardiac congestion or mummified fetuses [SMEDI; stillbirth, mummification, embryonic death, and infertility]. Infection during the last trimester has minimal effects on the fetuses.

Clinical findings. Subclinical infection is most common. Morbidity rate varies from 10 to 30%; case-fatality rate may be up to 80 to 90%.

Co-infection. Pigs co-infected with other pathogens such as porcine parvovirus, porcine arterivirus [PRRS], Mycoplasma hyopneumoniae, etc, present with severe disease and have more pronounced microscopic lesions. The most common clinical signs are wasting [progressive weight loss], and dyspnea. Other findings include diarrhea, skin discoloration, and congenital tremors [chronic contractions of the skeletal muscles].

Diagnosis. Samples: Blood, tonsils, lymph nodes, spleen, ileum.


Clinical signs suggestive of PMWS. Characteristic microscopic lesions: Lymph node depletion; basophilic intracytoplasmic or intranuclear IBs [aggregations of ingested virions]; lymphohistio cytic to granulomatous inflammation observed in the tonsils, lungs, liver, Peyers patches, lymph nodes, etc. Antigen detection. Detection of PCV2 in the tissues using ELISA, PCR, immunoperoxidase and immunofluorescence staining. Most pigs are usually seropositive, thus antibody detection is not of much value.

Control. Vaccination. Inactivated PCV2 vaccine [Circovac]. Sows and gilts are vaccinated before farrowing, ensuring specific passive protection of neonatal piglets through colostrum uptake. Porcine Dermatitis and Nephropathy Syndrome (PDNS) PDNS, first described in 1993, is associated with PCV2. The disease occurs sporadically, with morbidity rates of 1% or 2%. Affected pigs are usually older than PMWS pigs. The two main features of the disease are:

Necrotizing skin lesions. Occur mostly in the perineal area and on the hind limbs. May extend forward along the abdomen. Systemic necrotizing vasculitis and fibrinous glomerulonephritis.

103 Chicken Infectious Anemia (CIA) CIA was first identified in young chicks in 1979 in Japan. Since then it has been reported in all commercial chicken flocks worldwide. The disease is characterized by growth retardation, aplastic anemia, and generalized lymphoid depletion. Hosts. Disease occurs in chickens less than 3 weeks of age. Infection, but not disease, occurs in other age groups. Etiologic agent. Avian circovirus. One serotype. Strains vary in virulence. Transmission. Fecal-oral transmission.

Vertical transmission of virus through the hatching egg occurs during the 1 to 3 week viremic period following infection of antibody-negative breeder hens.

Pathogenesis. Infection is followed by viremia. The virus can be detected in most tissues and feces for about 3 or 4 weeks.

The principal target cells are precursor T cells in the thymus and hemocytoblasts in the bone marrow. Replication of the virus results in cell death by apoptosis [caused by a virus encoded protein called apoptin]. Immunosuppression and anemia. Pathology. Atrophy of the thymus and less commonly the bursa of Fabricius, pale bone marrow, and subcutaneous and skeletal hemorrhages.

Clinical signs. IP: 10 to 14 days.

Chicks are anorexic, pale, and show depressed weight gain. Mortality ranges between 10% to 50%. Surviving chicks recover slowly from the disease.

Diagnosis. Clinical signs and lesions.

Detection of viral antigen. PCR; in situ hybridization using DNA probes; immunoperoxidase and immunofluorescence staining. Antibody detection. ELISA, IFA, and virus neutralization test.

Control. MLV vaccines are available for vaccination of antibody-negative breeder hens prior to the start of egg production [prevents transovarian transmission]. The vaccine may be injected or added to the drinking water.

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HERPESVIRIDAE
Properties of Herpesviruses

Enveloped virion, 150 nm in diameter, containing an icosahedral capsid. Linear ds DNA genome. Replication occurs in the nucleus. The envelope is acquired by budding through the inner layer of the nuclear membrane. Most herpesviruses produce eosinophilic intranuclear inclusion bodies. All species persist indefinitely in infected hosts, with reactivation and intermittent or continuous viral shedding. Virus-specific proteins present in host cell membranes result in cell fusion [syncytium formation]. The viruses are fragile and do not survive well outside the body.

Pathogenesis and Immunity Other than sheep, at least one major disease of each domestic animal is caused by a herpesvirus. Many herpesviruses are host specific but some may affect a range of species.

Herpesviruses are fragile and do not survive well outside the host, thus transmission requires close contact, especially mucosal contact [eg, coitus, licking, etc.]. Short-distance droplet spread is a major means of transmission. The diseases vary from acute inflammatory conditions to persistent infection with periodic or continuous shedding. Some herpesviruses are oncogenic. Shedding of virus in oral, nasal, or genital secretions serves as the source of infection for other animals. Reactivation of latent herpesvirus infections is frequently observed in immunosuppressed hosts. Both humoral and cell-mediated immune responses are generated during herpesvirus infections. Neutralizing antibody is primarily directed at envelope glycoprotein peplomers, and reaches a maximum titer about 14 days after infection. Viral antigens on the surface of infected cells are targets for cell-mediated immune lysis.

105 Diagnosis

Demonstration of viral antigens. Immunofluorescence staining [FAT] of tissues or smears; electron microscopic examination of specimens. Detection of viral nucleic acid using PCR. Detection of viral antibodies using ELISA, virus neutralization, etc.; four-fold increase in antibody titer [seroconversion]. Virus isolation. Herpesviruses can be grown in embryonated hens egg or in cell cultures derived from their natural hosts. Gross and histopathology [intranuclear inclusions, syncytia, etc]. SUBFAMILY ALPHAHERPESVIRINAE

Most -herpesviruses grow rapidly, lyse infected cells, and establish latent infections primarily in sensory ganglia. Latent -herpesvirus genomes are maintained in host cells in episomal [extrachromosomal] form; rarely, they may be integrated into host DNA.

Optimum replication occurs at temperatures lower than the normal adult body temperature. Hence most -herpesviruses produce localized lesions, especially in the skin, central nervous system, or on the mucosae of the respiratory and genital tracts. Alphaherpesvirus infections in neonates less than 3 months of age are characterized by generalized infections, with foci of necrosis in multiple organs and tissues [low level maternal antibodies]. In pregnant animals, a mononuclear cell-associated viremia may result in the transfer of virus across the placenta, leading to abortion. Some -herpesviruses have moderately wide host range.
Alphaherpesvirus Diseases in Domestic Animals Virus Human herpesvirus 1 Human herpesvirus 2 Varicella:Zostervirus (Human herpesvirus 3) Bovine herpesvirus 1 Disease Cold sores [fever blisters] Genital infections Chickenpox and shingles Infectious bovine rhinotracheitis, infectious pustular vulvovaginitis, infectious balanoposthitis, abortion

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Bovine herpesvirus 2 Bovine herpesvirus 5 Equine herpesvirus 1 Equine herpesvirus 3 Equine herpesvirus 4 Porcine herpesvirus 1 Feline herpesvirus 1 Canine herpesvirus 1 Caprine herpesvirus 1 Gallid herpesvirus 1 Gallid herpesvirus 2 Gallid herpesvirus 3 Meleagrid herpesvirus 1

Bovine mammilitis, pseudo-lumpy skin disease Encephalitis Abortion, respiratory disease, encephalitis, perinatal foal mortality Coital exanthema Rhinopneumonitis Pseudorabies Feline viral rhinotracheitis Hemorrhagic disease in puppies Conjunctivitis, respiratory and enteric disease Infectious laryngotracheitis of chickens Mareks disease of chickens (serotype 1) Nonpathogenic Mareks disease virus [serotype 2] Nonpathogenic turkey herpesvirus

Infectious Bovine Rhinotracheitis Bovine herpesvirus 1 [BHV-1] is associated with several diseases in cattle, including rhinotracheitis, genital infections, conjunctivitis, abortion, generalized disease of neonatal calves, and possibly encephalomyelitis. The virus occurs worldwide. Etiologic agent. Bovine herpesvirus 1. One serotype. Two subtypes of BHV-1 have been described:

BHV-1.1 [respiratory subtype], BHV-1.2 [genital subtype] and BHV-1.3 [encephalitic subtype; renamed bovine herpesvirus 5].

Transmission

Rhinotracheitis, conjunctivitis. Aerosol derived from ocular and nasal secretions. Genital infections. Primarily by coitus; artificial insemination [contaminated semen].

Pathogenesis. Viral spread from the initial focus of infection may occur via a mononuclear cell-associated viremia.

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The virus can persist in the animal for years after an infection. These latent infections are potential sources for new outbreaks. Thus, all seropositive animals are considered potential carriers. Latent virus is reactivated with injections of corticosteroids or by various stresses that result in active shedding of virus. Sites of latency: Trigeminal nerve [respiratory form]; sciatic nerve [genital form]. Lesions. In both the respiratory and genital forms of the disease, the primary lesions are focal areas of epithelial cell necrosis.

Clinical Features Respiratory disease [necrotic rhinitis, red nose, dust pneumonia]. Most common in feedlot cattle, seen about 7-14 days after cattle are introduced to a feedlot. Epidemic outbreaks have also occurred in dairy cattle.

High morbidity [100%] but low mortality [ 10%]. Fever, depression, anorexia, salivation, and a profuse, mucopurulent, nasal discharge. Conjunctivitis may or may not be present. Diffuse hemorrhages and erosions in muzzle [Red nose], turbinates, nasal mucosa and gums, and necrosis of larynx. Acute, uncomplicated cases may last for 5-10 days. Death is usually the result of a 2o bacterial bronchopneumonia.

Conjunctivitis. May occur as the 1o clinical feature in some infections or may accompany rhinotracheitis. The conjunctiva is inflamed, edematous and accompanied by profuse mucopurulent discharges. Maybe unilateral or bilateral. Abortion. May occur concurrently with respiratory disease but can occur up to 100 days after infection. Abortion can occur regardless of the severity of the disease in the dam. Generalized disease of neonatal calves [10 days of age; infected in utero or early after birth]. Fever, sudden anorexia, excessive salivation, depression, dyspnea [bronchopneumonia], diarrhea [gastroenteritis], etc. It is often fatal. Genital disease. No abortions are seen.

Cows [Infectious pustular vulvovaginitis, IPV]. IP: 1-3 days after coitus. Pain, dysuria, frequent urination and tail swishing, edematous swelling of the vulva, and slight vulval discharge.

108 Vaginal mucosa is bright red with many small pustules that may become confluent to form a fibrinous pseudomembrane. Uncomplicated lesions heal by 10-14 days.

Bulls [Infectious balanoposthitis]. Inflammation of the glans penis and preputial lining with pustule formation.

Control. Modified live vaccines, inactivated virus vaccines and subunit vaccines containing the major surface glycoproteins [gB, gC, and gD] of BHV-1, are currently available.

IM and intranasal modified live vaccines are available, but the IM types may cause abortion in pregnant cows. The intranasal vaccines are more highly attenuated and can be used in breeding herds. Bovine Mammillitis and Pseudo-Lumpy Skin Disease

Etiologic agent. Both diseases are caused by Bovine herpesvirus 2 [BHV-2]. Distribution. Mammillitis: worldwide; Pseudo-lumpy skin disease: endemic in southern Africa. In most countries, BHV-2 causes only mammillitis. Transmission. Mechanically by arthropods and trauma to the skin. Carrier status in recovered animals. Clinical features Mammillitis

Ulcerative lesions are observed on the teats, but in severe cases, most of the skin of the udder may be affected. Normally, healing occurs in 3-4 weeks. Reduction in milk yield [10%] due to difficulty in milking the affected cows and intercurrent mastitis.

Pseudo-lumpy skin disease. IP: 5 to 9 days.

Mild fever followed by sudden appearance of skin nodules over entire body. Healing, without scar formation, is complete within a few weeks.

Control. Isolation of affected animals; control of arthropod vectors. Pseudorabies (Aujeszkys disease, Mad itch) Pseudorabies is primarily a disease of swine and, secondarily, a variety of domestic animals. The disease occurs worldwide.

109 Etiologic agent. Porcine herpesvirus 1. One serotype, however, variations in virulence has been reported among various strains.

The virus may survive in the environment for a few hours to 2-3 days.

Hosts. Recovered adult pigs are the primary hosts and reservoirs of pseudorabies virus and serve as the principal source of virus for a variety of secondary hosts. Rodents [eg, brown rats] may possibly serve as minor reservoirs.

Secondary hosts. Ruminants, dogs, cats, horses, feral animals, etc.

Transmission. Spread of the virus from swine to other species, and to other swine, is the most important single factor in the transmission of pseudorabies. Swine. Virus is shed in nasal secretions, saliva, and milk. Some swine that have recovered may shed virus continuously in their nasal secretions.

Licking [abraded skin], biting, aerosols, and ingestion of infected carcasses and contaminated water and feed, serve as the means of transmission.

Dogs and cats. Ingestion of infected pig carcasses and rodents. Ruminants. Direct contact with pigs; oral and nasal routes. Pathogenesis. Following oral or intranasal infection, virus replicates in the epithelium of the nasopharynx and tonsils. Virus spread to regional lymph nodes and the CNS occurs along axons of the various cranial nerves, eg, olfactory, trigeminal, etc.

A brief viremia is associated with virulent strains of the virus, with localization of virus in many organs. Characterized by inflammation and focal areas of necrosis in multiple organs, eg, spleen, liver, etc.

CNS. There is ganglioneuritis at many sites, diffuse nonsuppurative meningoencephalitis, marked perivascular cuffing and extensive necrosis of neuronal and glial cells. Pseudorabies in Swine

Clinical features Weaned, Growing, and Adult swine. IP: About 30 hours.

Sneezing, coughing, fever, profuse salivation, constipation, and vomiting.

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Pigs are listless, depressed; there is incoordination, circling, and convulsions, followed by mortality [< 2%]. Pruritus, a dominant feature of the disease in 2o hosts, is rare in pigs.

Piglets born to nonimmune sows

Sneezing, coughing, encephalitis, prostration and death. Death may occur within 24 hours following parturition. Mortality rate approaches 100%. Maternal antibody is protective, hence disease in piglets born to recovered or vaccinated sows is less severe with recovery as the outcome.

Nonimmune pregnant sows [SMEDI]. Virus may cross the placenta to infect some or all of the fetuses. Up to 50% may abort.

Infection day 30 of gestation results in death and resorption of embryos. Infection in late pregnancy. Delivery of mummified, macerated, stillborn, weak, and normal swine. Less than 20% of sows aborting are infertile on the next breeding but do eventually conceive. Pseudorabies in Secondary Hosts

In secondary hosts the disease is characterized by a severe local pruritus with a high mortality. Cattle [Mad itch]

Intense pruritus. Cattle may become frenzied. Pruritus of the head and neck is usually due to respiratory infection. Progressive involvement of the CNS: paralysis of pharynx, salivation, and forced respiration. Death, from respiratory failure, occurs within a few hours or may take as long as 6 days.

Dogs and cats

Dogs. Frenzy associated with pruritus, paralysis of the jaws and pharynx with drooling of saliva. Unlike rabies, there is no aggression. Cats. The disease is so rapid that pruritus may not be observed.

Diagnosis

History [contact with pigs] and clinical signs [death following intense pruritus].

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Virus isolation Pigs: brain, spinal cord, tonsils, lungs, liver. Secondary hosts: brain, spinal cord, lungs [catle].

Fluorescent antibody staining of frozen tissue sections [brain, tonsils]. Serology. Detection of antibodies using serum neutralization, ELISA, and latex agglutination tests.

Control. Pseudorabies is a reportable disease.

Vaccination of swine in endemic areas. Vaccines do not prevent infection or the establishment of latent infection by the wild-type virus. Both modified live virus and inactivated vaccines are used. A pseudorabies vaccine in which both the thymidine kinase gene and a glycoprotein gene have been removed, is currently available. Equine Herpesvirus 1 (Equine abortion virus; EHV-1)

EHV-1 is the most virulent equine herpesvirus and is associated with abortion storm, respiratory disease, perinatal foal disease, and encephalitis in horses. It is the most important cause of viral abortion in horses. It is antigenically related to equine herpesvirus 4. EHV-1 is endemic in equine populations worldwide. Transmission. Inhalation of infectious aerosols; direct or indirect contact with nasal discharges, aborted fetuses, placenta or placental fluids. Pathogenesis. Following inhalation, the virus replicates in the epithelial cells of the respiratory tract and rapidly localizes in the regional lymph nodes within 2 to 3 days of infection.

EHV-1 has a predilection for circulating leukocytes and endothelial cells. Immunosuppression. Attributed to inhibition of antigen presentation by infected host cells. EHV-1 codes for a protein that inhibits TAP protein, thereby blocking antigen delivery to class I MHC molecules in the ER.

Pregnant mare

Macrophage-associated viremia results in infection of the endothelial cells of blood vessels within the uterine wall, other organs, and the developing fetus, resulting in abortion.

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Most abortions occur during the last trimester [esp. between the 8th and 10th months], but can occur as early as the fourth month of gestation. Prior to abortion, the mare may show signs of respiratory tract infection. Reproductive efficiency is not compromised.

Encephalomyelitis. Caused by some strains of EHV-1.

Neurologic signs may occur as the primary disease or in association with respiratory disease and/or abortion. EHV-1 does not infect neurons or glial cells; instead, CNS lesions are due to viral infection and multiplication in the endothelial cells lining arterioles of the brain and spinal cord. Lesions are characterized by vasculitis of the small blood vessels in the CNS, followed by hypoxic degeneration [infarction] and hemorrhage throughout the brain and spinal cord. The severity of the disease can vary from slight hind limb incoordination of a transient nature to quadriplegia and recumbency resulting in death. Some animals recover completely, others may suffer permanent impairment of locomotor function. Case fatality is 2-3%.

Perinatal foal mortality. Fatal generalized disease characterized by septicemia and respiratory distress due to interstitial pneumonia. Control. EHV-1 abortion and respiratory disease are controlled using modified live and inactivated EHV-1 vaccines. The inactivated vaccine may be given during the 5th, 7th, and 9th months of pregnancy. Immunity is short-lived. Equine Herpesvirus 4 (EHV-4) EHV-4 is usually associated with respiratory disease [rhinopneumonitis] but occasionally has been recovered from individual cases of abortion. Distribution. Worldwide. Transmission. Inhalation of droplets from infected horses and older horses in which inapparent virus shedding occurs, following reactivation of the latent virus. Clinical features. Disease is observed mainly in foals over 2 months old, weanlings, and yearlings.

Fever, anorexia, and a profuse serous nasal discharge later becoming mucopurulent. Most affected foals recover completely. Occasionally, young foals may suffer a fatal bronchopneumonia if there is intercurrent infection, stress, overcrowding, etc.

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In partially immune, older animals, infections are usually subclinical.

Control. Combined inactivated EHV-1 and EHV-4 vaccines are used to control respiratory disease and abortion. Immunity is short-lived [2-4 months]. Hemorrhagic Disease of Puppies (Fading puppy syndrome) First recognized in the U.S.A. in 1965. It is a highly fatal, generalized hemorrhagic disease of puppies under 4 weeks of age. Disease has also been reported in some wild Canidae [wolves, coyotes]. Disease occurs worldwide. Etiologic agent. Canine herpesvirus 1. One serotype. Transmission. Newborn puppies can acquire the virus in utero; from contact with infected oral, nasal, or vaginal secretions of the dam; or from contact with secretions of infected littermates.

Older dogs. Contact with saliva, nasal secretions and urine of infected dogs or puppies. Venereal transmission.

Pathogenesis. Puppies.

Initial replication occurs in the nasal epithelium, pharynx and tonsils, followed by macrophage-associated viremia and virus replication in endothelial cells. Large ecchymotic hemorrhages and necrosis are observed in multiple organ systems. Neonatal puppies infected < 1 week of age are most susceptible to fatal generalized infections; puppies > 3 weeks of age at the time of infection are relatively resistant and generally develop mild or inapparent infection. CHV-1 multiples optimally at 33oC. The hypothalamic thermoregulatory centers of puppies are not fully developed until about 2-3 weeks of age. The neonatal pup is dependent on ambient temperature and maternal contact for the maintenance of its normal body temperature. Therefore, the more severe the hypothermia, the more severe and rapid is the course of the disease.

Clinical features Puppies. IP: 3-8 days. Course: 1-2 days.

Painful crying, abdominal pain, anorexia, dyspnea, and soft, odorless yellowish green stool. No elevation in body temperature.

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Meningoencephalitis occurs in oronasally infected puppies. Virus may travel up the nerve axons to the CNS [under normal circumstances, puppies generally die from systemic illness before neurologic signs are manifest].

Older dogs. Mild respiratory infection [rhinitis and pharyngitis].

Bitch. Vesicular vaginitis characterized by discharges. Primary in utero infection may result in abortion, mummification, stillbirths and infertility [bitch produces healthy litters in later pregnancies]. Male dogs. Balanoposthitis.

Histopathology. Focal areas of perivascular necrosis and hemorrhage involving multiple organs.

Acidophilic or basophilic intranuclear inclusions, depending on the stage of cellular infection and method of fixation.

Control. The low incidence of severe disease in pups [prevalence of the virus, based on antibody surveys, is < 20%] and the mild nature of the disease in older dogs, has resulted in lack of availability of vaccines.

Reduce hypothermia by providing heated whelping box or by putting puppies under an infrared lamp. Isolation of affected bitch and her litter. Puppies are infective for 2-3 days. Feline Rhinotracheitis

Feline herpesvirus 1 [feline rhinotracheitis virus] was first isolated in 1957. It is one of the two common causes of acute respiratory disease of kittens. Distribution. Worldwide. Prevalence of antibody in colony cats is over 70%; household cats: less than 50%. Etiologic agent. Feline herpesvirus 1. One serotype. Hosts. All members of the family Felidae appear to be susceptible. Transmission. FRV is shed primarily in ocular, nasal, and oral secretions.

The natural routes of infection for FRV are nasal, oral, and conjunctival. Recovered cats carry the virus for some time, with intermittent shedding, and can transmit the disease to susceptible cats.

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Epidemiology of the Carrier State in Feline Rhinotracheitis Virus Infection

Pathogenesis. Virus replication occurs primarily in the mucosae of the nasal septum, turbinates, nasopharynx, and tonsils. Clinical features. IP: 2-6 days but may be longer if exposure rate is low. Neonates

Fever, sneezing, coughing, profuse serous nasal and ocular discharge [may become mucopurulent], dyspnea, anorexia, weight loss, and profuse frothy salivation. Keratitis with punctate corneal ulcers. Oral ulcers are rare. Kittens [ 4 weeks]: extensive rhinotracheitis and associated bronchopneumonia from 2o bacterial infection may be fatal.

Older Kittens [ 6 months]: mild or subclinical disease. Pregnant queen. Abortion about the 6th week of pregnancy may occur.

Virus has not been isolated from aborted fetuses. It is thought to be due to the debilitating effects of the respiratory disease rather than a direct effect of the virus on the fetus.

116 Pathology. Necrosis of epithelia of the nasal cavity, pharynx, epiglottis, tonsils, larynx, and trachea. Osteolytic changes in the turbinate bones may be observed. Diagnosis. History and clinical signs.

Gross and histopathology [inclusion bodies; syncytia, etc.] Virus isolation. Ocular or pharyngeal swab. Serology. Four-fold increase in virus neutralizing antibody titer.

Differential diagnosis

Feline rhinotracheitis: Hypersalivation, sneezing, keratitis. Feline calicivirus: Ulcers of the tongue, palate, and pharynx; lameness Feline pneumonitis [Chlamydophila felis]: Conjunctivitis, ocular discharges.

Control

Inactivated and attenuated live-virus vaccines, alone or in combination with other antigens, are available. A genetically engineered vaccine comprised of a gene deletion mutant of feline herpesvirus 1 into which the gene encoding the capsid protein of feline calicivirus has been inserted, is being developed. Infectious Laryngotracheitis (ILT)

ILT is an acute, highly contagious disease of chickens characterized by severe dyspnea, coughing, and rales. The disease was first identified in chickens in the U.S.A. in 1926. The disease has been reported worldwide. Synonyms. Infectious tracheitis; fowl diphtheria. Hosts. Chickens and pheasants. Etiologic agent. Gallid herpesvirus 1. One serotype, however, strains of the virus differ significantly in virulence. Transmission. Introduced into a flock by carrier birds. Mostly via inhalation; occasionally via ingestion. Pathogenesis. Severe laryngotracheitis characterized by necrosis, ulceration, hemorrhage, and the formation of diphtheritic membranes.

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The diphtheritic membrane may form a second tube for the length of the trachea, restricting and occasionally totally occluding air flow. Death results from asphyxiation.

Clinical features. IP: 2 to 8 days.


Most common in chickens 4 to 18 months of age. Marked respiratory distress; extension and slinging of neck during inspiration. Head depressed, resting on breast during exhalation; coughing, rattling. Birds may cough up bloody mucus which stains walls and posts. Morbidity: 100%; Mortality: 20-70%, depending on virulence of the strain.

Diagnosis

Fluorescent antibody staining of smears and tissues. Virus isolation. Nasal mucosa. Chorioallantoic membrane of embryonated eggs [stunted embryos which die 2-12 days postinoculation]. GHV-1 grows well in various cell cultures.

Detection of neutralizing antibody using pock or plaque reduction assays.

Control. Complete depopulation and disinfection of premises have been used to eradicate the disease.

Vaccination of replacement pullets, 6 weeks of age or older, in endemic areas using modified live-virus vaccine. Mareks Disease (MD)

Mareks disease is one of the most important diseases of chickens. The disease occurs worldwide. Synonyms. Fowl paralysis; range paralysis; polyneuritis; neurolymphomatosis. Hosts. Primarily chickens; turkeys, pheasants, quail, and game fowl are susceptible. MD in turkeys is not a major problem. However, outbreaks of MD in commercial turkey flocks, with mortality ranging from 40 to 80%, have been reported in Israel, Germany, France, and the United Kingdom. Etiologic agent. Gallid herpesvirus 2 [serotype 1]. Four pathotypes of serotype 1 are currently recognized:

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Mild [mMDV]. Mostly associated with neural MD. Disease is preventable with HVT [turkey herpesvirus] vaccine. Virulent [vMDV]. Associated with a high incidence of neural and visceral lymphomas. Disease is preventable with HVT vaccine. Very virulent [vvMDV]. Associated with high incidence of neural and visceral lymphomas. The viruses are oncogenic in HVT vaccinated chickens. Disease is preventable with bivalent vaccines. Very virulent plus [vv+MDV]. Associated with a high incidence of lymphomas. They are oncogenic in chickens vaccinated with bivalent vaccines.
MDV Lesions induced in Lymphatic Organs (Spleen, BF, Thymus) 8 Days Post-Infection of 24-day-old Chickens with different Pathotypes of MDV

The virus is slowly cytopathic and remains highly cell associated [macrophage-associated] so that cell-free infectious virus is almost impossible to obtain, except in dander from feather follicles.

Transmission. This is a highly contagious disease. Infection follows inhalation of infectious feather debris, dust or chicken dander.

Cell-free virus released from the feather follicles are highly infectious but labile. Viruses in desquamated cells [dander] are less infectious but can survive in poultry house dust and litter for several months.

119 Pathogenesis. Subclinical infection with virus shedding is more common.

Genetic susceptibility. Susceptibility among chickens can be traced to different MHC class II haplotypes. B19 haplotype chickens are very susceptible to MD, whereas, B21 haplotype chickens are genetically resistant to MD. Virus replication in the respiratory tract is followed by macrophage-associated viremia. Various overlapping virus-cell interactions have been identified: Fully productive infection. Characterized by production of fully infectious virions [enveloped virions] and cell death [cytolysis]. Occurs only in feather follicle epithelium. Productive-restrictive infection. Characterized by production of naked virions [not infectious] and viral antigens and cell death. Occurs in B cells and activated T cells [profound immunosuppression]. Non-productive latent infection. Viral genome persists in lymphoid cells [T cells] but no antigens are expressed. Non-productive neoplastic transformation. Some latently infected T cells undergo neoplastic transformation.

Lesions in Mareks disease result from the infiltration and in situ proliferation of the transformed T lymphocytes; also, cell lysis in productive infections results in a marked inflammatory response.

120 Clinical features. Mareks disease is a progressive disease with overlapping signs; outbreaks are usually observed in chickens 2 to 5 months old. Mortality may reach 80%. Neurolymphomatosis [classical Mareks disease]

Enlargement of one or more peripheral nerve trunks. The nerves may be 3x their normal diameter, show loss of striations, and are edematous, gray, or yellowish in appearance. Enlargement of nerves is usually unilateral. Lameness, droopy wings, splayed legs [one leg is held forward and the other backwards], incoordination, and limberneck are commonly observed.

Visceral lymphomatosis

Lymphocytic tumors are present in the heart, liver, kidney, spleen, etc.

Ocular lymphomatosis

Graying of the iris [gray eye, cats eye, pearl eye] of one or both eyes resulting from T cell infiltration; there is partial or total blindness.

Cutaneous lymphomatosis

Plucking of the feathers reveals round, nodular lesions in the skin. Productive infection of the epithelial cells at the base of feather follicles is associated with the release of cell-free virus.

Diagnosis. History, age, clinical signs, and gross postmortem findings are adequate for diagnosis.

Detection of viral antigen using, PCR, fluorescent antibody test, etc. Detection of antibodies using AGID, IFA, and virus neutralization tests. Virus isolation. Buffy coat or spleen cells. Cell cultures, CAM or yolk sac routes of embryonated hens egg.

Control. Reportable disease. Vaccination is the main method of control.

Avirulent turkey herpesvirus [HVT; heterologous] vaccine is available. Bivalent vaccines consisting of [1] HVT and CV1988/Rispens strain of serotype 1 or [2] HVT and either the SB-1 or 301B/1 strains of serotype 2 Mareks disease virus [Gallid herpesvirus 3; avirulent strain] are also available.

121 SUBFAMILY BETAHERPESVIRINAE Betaherpesviruses are slow-replicating viruses and cell lysis does not occur until several days after infection; hence they are associated with chronic infections that may last several months before clinical recovery.

Infected cells often are enlarged [cytomegaly], hence cytomegaloviruses. Virus is maintained in latent form in secretory glands [eg, salivary glands], and lymphoreticular cells [macrophages, lymphocytes, etc]. Betaherpesviruses are often associated with continuous viral excretion. Inclusion Body Rhinitis

Inclusion body rhinitis is endemic in many swine herds worldwide. In a swine herd up to 90% of swine may carry the virus. Etiologic agent. Porcine herpesvirus 2 [Porcine cytomegalovirus]. One serotype. Highly cell-associated. Transmission. Mainly through inhalation.

Transplacental transmission in susceptible [naive] sows.

Pathogenesis. The primary site of virus replication is in the nasal mucous glands and other epithelial cells of the upper respiratory tract.

Viremia may follow virus replication, with localization of the virus in macrophages, epithelial-type cells, and in endothelial cells. Endothelial cell damage and necrosis account for the petechial hemorrhages and edema observed in the disease. Anemia observed in some neonates has been attributed to bone marrow damage. If severe, can increase mortality up to 50%. In general, mortality is usually due to widespread damage caused by the virus or secondary infections.

Clinical features. Disease is seen in pigs 2 to 10 weeks old. However, it is most severe in piglets less than 3 weeks of age.

Sneezing, coughing, serous nasal and ocular discharges, and depression. Discharge becomes mucopurulent and may block the nasal passages. Interferes with suckling, resulting in rapid weight loss and possibly death.

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Transplacental infection is associated with mummification, stillbirths, neonatal death, and runt pigs [SMEDI]. Older pigs [> 4 months old]: Subclinical infection.

Pathology. Widespread petechiae and edema seen most commonly in the thoracic cavity and subcutaneous tissues.

Basophilic intranuclear inclusion bodies and cytomegaly.

Diagnosis. Clinical signs; gross and histopathology.


Virus isolation from nasal mucosa, lung, and kidney. Detection of antibodies using ELISA or IFA tests.

Control. Good management and abstention from introducing new stock during the mating period and the first month of pregnancy should be practiced. SUBFAMILY GAMMAHERPESVIRINAE Member viruses are lymphotropic [replicate in T or B lymphocytes]; some cause lymphoid tumors [eg, Burkitts lymphoma and nasopharyngeal carcinoma in humans].

Some gammaherpesviruses are slowly cytopathic for epithelial and fibroblastic cells, causing cell death without production of virions. Some gammaherpesviruses are shed continuously from epithelial surfaces. Latency occurs in lymphoid tissue. Malignant Catarrhal Fever [MCF]

MCF is a sporadic but highly fatal disease of domesticated and exotic ruminant species. The virus infects primarily lymphoid tissues and epithelial cells of the respiratory and gastrointestinal tracts.

The disease has been characterized as a sheep-associated form [Europe and North America] and wildebeest- and hartebeest-derived forms [Africa].

Etiologic agents

African form. Alcelaphine herpesvirus 1 [formerly bovine herpesvirus 3]. First isolated from wildebeest. Alcelaphine herpesvirus 2 [hartebeest-associated malignant catarrhal fever]. Highly cell-associated viruses in cattle.

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American and European form. Ovine herpesvirus 2. There is close antigenic association between the American strain and the African strains. MCF has been reported in feedlot cattle in North America, in the absence of contact with sheep. The identity and source of virus in this third form is unknown. However, recrudescence of latent infections is common and may account for cases with no known contact with reservoirs.

Hosts. Cattle, deer, buffalo, rabbits, and occasionally pigs. Reservoirs. Wildebeest, hartebeest [Africa]; sheep [U.S.A.]. Inapparent infections occur in the reservoirs. Transmission

African form. Close contact between cattle and wildebeest, especially during calving. AHV-1 occurs in nasal and ocular secretions of young wildebeest in a cell-free state. American form. Close contact between cattle and sheep during lambing. Cattle have cell-associated virus, but not cell-free virus, in secretions, and this may explain the noncontagious nature of MCF when contact occurs with MCF-affected cattle [dead-end hosts].

Pathogenesis. Virus infection is followed by cell-associated viremia. Damage to organs and tissues appear to be immune-mediated since virus is usually absent from the sites of lesions.

CD8+ T cells are the predominant cells associated with the widespread necrotizing vasculitis. The vascular lesions account for the gross lesions observed, eg, epithelial erosions, keratoconjunctivitis, and encephalitis.

Clinical features. IP: 3 to 8 weeks.

Morbidity: With a few exceptions, morbidity is usually low; only a few cows in the herd show clinical signs. The disease is invariably fatal [~ 100%].

Peracute form. Characteristic signs and lesions may not develop, since the disease lasts only for 1 to 3 days.

Severe inflammation of the oral and nasal mucosa and hemorrhagic gastroenteritis.

124 Acute form

Fever, depression, profuse nasal and ocular discharges, generalized lymphadenopathy, and extensive mucosal erosions of GIT [bloody diarrhea] and respiratory tract. Bilateral ophthalmia. Photophobia, corneal opacity which begins peripherally and progresses centripetally, often leading to blindness. CNS [edema of the meninges]. Incoordination, muscle tremors, head pressing, stupor, convulsions, etc.

Diagnosis. History, clinical signs [esp. bilateral ophthalmia], gross and histopathology, etc.

Antigen detection. Detection of viral DNA by PCR. Antibody detection. Competitive-inhibition ELISA, virus neutralization test, etc.

Immunity. The rare bovine that recovers from MCF might be immune for life. Control. Separation of cattle from sheep, hartebeest, and/or wildebeest. Incidence is usually too low to justify the development of a vaccine.

Other Gammaherpesviruses Equine herpesvirus 2 [Respiratory disease; conjunctivitis and corneal edema in foals] Equine herpesvirus 5 [Pathogenicity unknown] Bovine herpesvirus 4 [Pathogenicity unknown]

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ASFARVIRIDAE [ASFAR: African swine fever and related viruses]


Properties of Asfarviruses

Large, spherical enveloped virion, 175-215 nm in diameter; icosahedral capsid. Genome is a linear, ds DNA [codes for up to 200 proteins]. Replicates in cytoplasm; virions mature by budding from plasma membrane. Forms acidophilic, intracytoplasmic inclusion bodies. Virions are thermolabile and sensitive to lipid solvents.

African Swine Fever (ASF) (African Pig Disease; Warthog Disease) The disease was first reported in 1921 in Kenya. It is an acute-to-chronic disease, characterized by high fever, cutaneous hyperemia, edema, hemorrhage in internal organs [esp. lymph nodes], and abortions. Distribution. Sub-Saharan Africa, several European countries, the Dominican Republic [1978], Haiti [1979], Cuba [1971, 1980], and Brazil. It remains endemic only in Africa and parts of southern Europe. Hosts. All breeds and types of domestic pigs and European wild boar. Inapparent infections occur in warthogs, bush pigs, and giant forest hogs, which act as reservoir hosts. Etiologic agent. Asfivirus. Five distinct genotypes have been identified by restriction endonuclease studies of viral DNA. All European and the Americas isolates fall within one group, whereas African isolates show greater variation.

Variations in virulence. Some strains cause severe disease with near 100% mortality; others cause transient disease or inapparent infections. Very stable virus. May remain viable in soil, blood, bone marrow, or pork at room temperature for several months. Survives for 15 weeks or longer in chilled carcasses and up to 5 to 6 months in processed meat products. Epidemiology

Sylvatic cycle. Asfivirus is maintained in a sylvatic cycle involving asymptomatic infection in wild pigs and argasid ticks [soft ticks, Ornithodoros sp.].

After primary infection, young warthogs develop a viremia with high enough virus titers to infect some of the ticks feeding on them.

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Ornithodoros sp., are both biologic vectors and reservoirs of the virus. The virus replicates in the tick, resulting in transstadial, transovarial, and sexual transmission [male to female]. Infected ticks may live for several years and are capable of transmitting disease to swine at each blood meal. Asfivirus is the only arbovirus among DNA viruses.

Domestic cycle. Outbreaks result from tick bites, ingestion of infected uncooked garbage or tissues of acutely infected pigs and warthogs, or through aerosols.

Pigs that have recovered from clinical disease may remain infected for long periods of time. Such carrier pigs are a major source of virus dissemination [virus is shed in all body secretions and excretions, eg, urine, feces, etc.].

Pathogenesis The primary effects of ASF virus infection are hemorrhages and apoptosis of host cells. Following ingestion or inhalation, initial virus replication occurs in the pharyngeal mucosa, tonsils and the regional lymph nodes, followed by viremia.

During the viremic phase, the virions multiply in endothelial cells, megakaryocytes and leukocytes [esp. macrophages]. Destruction of lymphoid tissue is due to apoptosis of infected mononuclear phagocytes and uninfected T and B lymphocytes (indirect apoptosis; due to cytokines [eg, TNF- and apoptotic mediators [eg, virus-encoded p54 protein], released by infected macrophages). There is marked leukopenia, lymphopenia and immunosuppression. Gross lesions are most prominent in the lymphatic and vascular systems. Vascular damage, eg, degeneration of vascular endothelium, disseminated intravascular coagulation [DIC], thrombocytopenia [destruction of megakaryo-

127 cytes], and impaired blood coagulation, result in edema and hemorrhage in many organs and tissues. Clinical Features. Peracute. Pigs may die suddenly or there may be a 1-3 day course of high fever, hyperpnea, and cutaneous hyperemia before death. Acute. IP: 4-10 days. Mortality: 95-100% in primary exposure; then reduces to 20-40%.

Fever, inappetance, dyspnea, coughing, and severe leukopenia. Diarrhea is seldom seen in uncomplicated ASF [virus does not multiply in intestinal epithelial cells]. Pregnant sows often abort regardless of the stage of pregnancy. Maybe due to causes other than transplacental transmission because many aborted fetuses are free of virus. Sows that abort often die in 3 to 6 days.

Subcute/chronic infections [endemic areas]. Necrotic skin lesions, arthritis, pneumonia [2o bacterial infection], and growth retardation. Diagnosis. Virus isolation: Blood, spleen, visceral lymph nodes, tonsils.

Hemadsorption can be demonstrated within a few days following inoculation of cell culture. Antigen detection in tissues. FAT; immunodiffusion using tissue suspensions as the source of antigen; PCR to detect viral DNA; or direct ELISA.

Immunity. Although infected pigs produce virus-specific antibodies, sera from infected swine do not neutralize the virus, hence the humoral immune response does not seem to have any substantial protective value.

Resistance to infection may be related to the level of virus-specific CD8+ T cell cytotoxicity and antibody-dependent cell-mediated cytotoxicity [ADCC]. Attempts to develop a vaccine have been unsuccessful.

Prevention and Control. In endemic countries, management system avoids feeding uncooked waste food scraps. Double fencing to prevent access of ticks and warthogs to domestic swine. ASF-free countries. Notifiable disease. Prohibition of importation of live swine and swine products from infected countries. Monitoring the efficient destruction of all waste food scraps from ships and aircraft involved in international commerce. Eradication in case of ASF outbreak.

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PARVOVIRIDAE [Parvus, small]


Properties of Parvoviruses

Nonenveloped icosahedral virion; 25 nm in diameter. [-] strand single stranded DNA genome. Replicate in nucleus of dividing cells. Produce large intranuclear inclusion bodies. Most member viruses hemagglutinate red blood cells. Viruses are very stable.

Subfamilies and Genera

Subfamily Parvovirinae. Contains parvoviruses of vertebrates. Genus Parvovirus. Replicate autonomously. Cannot grow in stationary cells. Replicate in cells undergoing mitosis [do not code for enzymes required for replication, but rely on the enzymes in the replicating cell to perform this function]. Genus Dependovirus. Consists of defective viruses which are unable to replicate except in the presence of a helper virus, usually an adenovirus [adeno-associated viruses]. Genus Erythrovirus. Replicate autonomously. Includes human parvovirus B19 [erythema infectiosum in children and aplastic anemia].

Subfamily Densovirinae. Contains parvoviruses of insects.

Resistance. Parvoviruses are remarkably stable to environmental conditions [extremes of pH and heat]; hence, disinfection of contaminated premises is difficult.
Diseases caused by Parvoviruses Genus Parvovirus Virus Feline parvovirus Canine parvovirus 1 Canine parvovirus 2a, 2b and 2c Disease Feline panleukopenia Mild diarrhea Enteritis, leukopenia, myocarditis, etc Reproductive disorders [SMEDI] Panleukopenia, enteritis

Porcine parvovirus

Mink parvovirus

129 Pathogenesis The pathogenesis of parvoviruses is determined by the requirement for mitotic cells for viral replication.

Fetus or newborn. Pantropic. Virus may infect a wide range of cells in various tissues and organs. Older neonates. Narrower range of cells affected. All age groups. The continuous division of cells of the lymphoid tissue, bone marrow and intestinal epithelium renders them highly susceptible to parvoviruses, with resultant leukopenia and enteritis. Feline Panleukopenia

Feline panleukopenia is a highly contagious, often fatal, disease of cats. It is most severe in kittens. Synonyms. Feline distemper; feline infectious enteritis. Etiologic agent. Feline parvovirus. One serotype. The virus is antigenically related to canine parvovirus 2 and mink parvovirus. Distribution. Worldwide. Hosts. All members of the cat family, both domestic and wild, are susceptible as well as members of related families such as raccoon, mink, etc. Epidemiology. The virus is ubiquitous because of its contagious nature and capacity for persistence in the environment.

Most infections are subclinical because 75-85% of unvaccinated, clinically healthy cats have demonstrable antibody titers by one year of age. Unvaccinated kittens with maternal antibody are protected for up to 3 months. Recovered cats are solidly immune, however, they may excrete virus in their feces and urine for up to 6 weeks or longer. The extreme resistance of the virus to inactivation and the very high rates of viral excretion [up to 109ID50/g of feces] result in high levels of environmental contamination; virus may survive 1 yr in a suitable environment and can be transported via fomites [shoes, food dishes, bedding, infected cages, etc]. Owners losing a kitten to feline panleukopenia should not introduce a new kitten into the household without having it vaccinated.

130 Transmission. Cats are infected via the oronasal route by exposure to infected animals or their secretions [urine, saliva, feces, and vomitus] or to fomites.
Pathogenesis of Feline Panleukopenia

Panleukopenia. Panleukopenia results from destruction of various white blood cell elements [lymphocytes, neutrophils, monocytes], including both those present in the circulation [a consequence of virus adsorption and immune cytolysis] and those in the lymphoid organs, eg, thymus, bone marrow, spleen, Peyers patches, etc.

Thrombocytopenia [due to bone marrow injury] may accompany leukopenia.

Enteritis. Feline parvovirus multiplies in the rapidly dividing intestinal epithelial cells in the crypts of Lieberkhn. Normally the epithelial cells at the tips of the intestinal villi are continuously lost into the lumen of the gut and are replaced by dividing cells from the crypts.

The normal loss of cells from villus tips continues, however, failure in replacing them with cells from the crypts leads to greatly shortened, nonabsorptive villi and hence to diarrhea.

131 In utero Infection

Early in utero infection may produce a range of reproductive disorders in the pregnant queen, including early fetal death and resorption with infertility, abortions, or the birth of mummified fetuses. Towards the end of gestation, infections may result in birth of live kittens with various degrees of damage to the late developing neural tissues. Feline parvovirus produces variable effects on kittens from the same litter.

Central Nervous System Infection The CNS, optic nerve, and retina are susceptible to damage by FPV during prenatal or early neonatal development. Of these, cerebellar hypoplasia is most common.

Cerebellar hypoplasia. Observed in fetuses infected during the last 2 weeks of pregnancy and the first 2 weeks of life.

A. Fetal cerebellum consists of an external germinal [granular] layer, the cells of which undergo rapid cell division and migrate to form internal germinal [granular] and Purkinjes cell layers. B. Normal postnatal cerebellar cortex consisting of an outer germinal layer with stellate cells, a middle Purkinjes cell layer, and a deeper granular layer. C. Neonatal cerebellum damaged by FPV is reduced in size and has disordered cell layers.

Disseminated intravascular coagulation [DIC] Kittens with feline panleukopenia are susceptible to 2o bacterial infections with enteric microflora. Gram-negative endotoxemia, with or without bacteremia, is a common sequela of systemic FPV infection. Endotoxin [LPS] induces tissue factor [Factor III] expression on endothelial cells. Tissue factor is a potent activator of coagulation.

132 Clinical signs. Most common in kittens 3 to 5 months of age. Incubation period [IP]: 5 days [ranges from 2-10 days].

Fever, depression, anorexia, a rough coat, repeated vomiting, profuse persistent, frequently bloody diarrhea. Dehydration, 2o bacterial infections, and DIC are the major causes of death. Mortality rate: 25-90%. Cerebellar hypoplasia. Kitten is permanently ataxic. Usually it is not apparent until the kitten begins to walk at 3-4 weeks of age.

Diagnosis. Clinical signs, hematological examination, and postmortem findings.

Virus isolation in cell culture. Swabs from pharynx or rectum; spleen, ileum or mesenteric lymph node. Direct ELISA or immunofluorescence for the detection of antigen in tissues. PCR assay for the detection of viral DNA in tissues. Direct hemagglutination of pig or rhesus RBCs by virus present in feces. Paired serum samples. Virus neutralization test, ELISA, or hemagglutination inhibition test to check for 4-fold increase in antibody titer.

Treatment. Supportive measures.

Good nursing care, fluid therapy and withholding food in early stages of the disease to lessen vomiting and slow intestinal mitotic activity. Administration of broad-spectrum antibiotics.

Control

Large catteries. Strict hygiene and quarantine of incoming animals. Disinfection. The virus survives disinfection with 70% alcohol, various dilutions of organic iodines, phenolics, and quaternary ammonium compounds [QUATS]. FPV is inactivated by bleach [6% sodium hypochlorite], 4% formaldehyde, and 1% glutaraldehyde in 10 minutes at room temperature.

Immunity. Neutralizing antibodies can be detected within 3 to 5 days of infection. Immunity after natural infection appears to be lifelong.

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Attenuated [modified] live-virus vaccine and inactivated vaccine are used. MLV should not be administered to cats that are pregnant, immunosuppressed, or sick, or to kittens < 4 weeks old. Canine Parvovirus

Canine parvovirus disease was first recognized in 1978, when it caused a worldwide pandemic [severe gastrointestinal illness]. Etiologic agent. Canine parvovirus 2 [subtypes 2a, 2b and 2c]. In North America, disease is caused mostly by CPV-2b. CPV-2c was first identified in Italy in 2000. Since 2006, it has been found in Arizona, California, Georgia, Oklahoma, and Texas. All 3 subtypes are circulating in Europe and other parts of the world.

Canine parvovirus 1 was isolated from feces of dogs in 1967 but it is not a major cause of disease.

Hosts. Domestic and wild Canidae. Self-limiting infection in cats with no clinical signs. Epidemiology. Similar to that of FPV. CPV-2 can persist on fomites for 5 months or longer. Disease now has lower morbidity and mortality rates as immunity has built-up in the dog population. Transmission. Similar to feline panleukopenia. Pathogenesis. Similar to feline panleukopenia. Panleukopenia; enteritis; DIC; etc. CPV DNA was detected in the brain tissue of two puppies with cerebellar hypoplasia; however, supporting evidence implicating CPV as the cause was underwhelming. Clinical features. IP: 3-8 days. Three age-related syndromes have been recognized:

2-12 days. Generalized neonatal disease. Uncommon. 3-8 weeks. Myocarditis. However, myocarditis can develop from infection in utero. Usually all pups in a litter are affected. Myocardial necrosis with acute cardiopulmonary failure; characterized by sudden death or a pup may succumb after a short episode of illness. It is less common now. 2-4 months. Panleukopenia/enteritis. Most common.

Diagnosis. Same as feline panleukopenia.

Direct ELISA kit for virus detection in feces.

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IgM-capture ELISA test is used to determine recent infection.

Control. Same as feline panleukopenia. Immunity. Same as feline panleukopenia. Immunity after natural infection appears to be lifelong. Threshold antibody titer is 1:80.

Attenuated live-virus and inactivated vaccines are available. Porcine Parvovirus [PPV]

Porcine parvovirus occurs worldwide and is endemic in many swine herds. Virtually all sows in a herd are naturally infected before their second pregnancy, hence reproductive problems are observed mostly in primiparous gilts.

The virus has been isolated in association with reproductive failure in swine: stillbirth, mummification, embryonic death, and infertility [SMEDI]. There is only one serotype.

Transmission. Oronasal in the dam followed by transplacental transmission.

Venereal transmission is also possible because infected boars shed virus in the semen for a couple of weeks.

Pathogenesis. Oronasal infection of the nonimmune pregnant dam is followed by local replication and viremia.

Transplacental infection. Each embryo or fetus has a separate placenta, therefore, not all embryos or fetuses are infected at the same time. However, infection of a fetus or embryo is followed by virus spread through the uterus and infection of some or all of the remaining embryos or fetuses occurs. As a result, death at different stages of pregnancy is typical of PPV infection. PPV has a predilection for the mitotically active cells of fetal tissues, including capillary endothelial cells and leukocytes. Widespread endothelial cell damage is reflected in damage to many fetal organs and tissues.

Clinical features. The hallmark of PPV infection is small litters with dead fetuses [mostly mummified; some are stillborn] and healthy piglets. Abortions are uncommon.

Embryo/fetus [< 30 days]. Embryo or fetus dies and is resorbed; return to estrus of the dam.

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Early fetus [30-70 days]. [mummification].

Fetuses die and become relatively dehydrated

Late fetus [> 70 days to term]. Most infected fetuses frequently develop lesions but may survive in utero and produce antibody to PPV [immunocompetence of pig fetuses starts at 55-70 days]. Immunotolerant piglets. Some infected fetuses can be born as infected, immunotolerant piglets that can pass the virus continuously or intermittently. Boars, sows, and gilts. Mostly inapparent or subclinical infections.

Diagnosis. FA staining of frozen sections of fetal tissues; PCR for antigen detection in the fetus; ELISA and HI for antibody detection. Immunity. Immunity is lifelong. Maternally-derived immunity may persist for about 4 months; however, in some pigs it can persist for 6 to 9 months, interfering with active immunity. Consequently some gilts can be seronegative at mating time and are susceptible to infection. Control. Inactivated and attenuated vaccines are in wide use.

Nonimmune swine may be exposed to virulent virus several weeks before breeding to establish immunity, eg, commingling gilts and boars with other swine that may be shedding PPV [eg, older breeding stock]. Unlike most parvoviruses, swine parvovirus causes persistent infection with chronic shedding.

Fetal Mummification Fetal death in domestic animals occurring in midgestation or the last trimester of the gestation period, may be followed by abortion or fetal maceration. If not, autolytic changes in the fetus, absorption of placental and fetal fluids, involution of the maternal placenta, and fetal mummification occur. Fetal mummification is uncommon during the first trimester because fetal death, prior to development of the fetal bones, is usually followed by resorption of the fetal and placental tissues.

Conditions that favor fetal mummification include: [1] persistence of the corpus luteum of pregnancy, and [2] the maintenance of the dead fetus within the uterus by the presence of a normal viable fetus or fetuses in multiparous animals.

Other viral causes of SMEDI include porcine enterovirus A and B, porcine arterivirus [porcine reproductive and respiratory syndrome], porcine cytomegalovirus [IBR], porcine herpesvirus 1 [pseudorabies], etc.

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PAPILLOMAVIRIDAE
Properties of Papillomaviruses

Nonenveloped icosahedral virions, 55 nm in diameter. Genome consists of circular dsDNA. Virus replication depends on the differentiated state of host cells. Episomal DNA of papillomavirus may be oncogenic. Papillomaviruses have not been grown in culture, but will transform cultured cells.

Genus

Papillomavirus [Papilli = pustule; oma = tumorous] Cause papillomas [warts] in their natural hosts. Papillomaviruses are resistant to ether, high temperatures, and low pH.
Diseases Caused by Papillomaviruses Virus Bovine papillomaviruses 1-10 Host Cattle Disease Cutaneous fibropapilloma and papilloma, teat papilloma, and intestinal papilloma [type 4] Sarcoid Cutaneous fibropapilloma Cutaneous papilloma Oral papilloma [type 1], endophytic papilloma [type 2], pigmented cutaneous plaques [types 3 and 4] Cutaneous papilloma Cutaneous fibropapilloma Cutaneous papilloma

Bovine papillomavirus 1 and 2 Feline papillomavirus Equine papillomaviruses 1 and 2 Canine papillomavirus 1-4

Horses Cat Horses Dogs

Porcine genital papillomavirus Ovine papillomavirus Cottontail rabbit papillomavirus [Shope papillomavirus] and rabbit papillomavirus Human papillomavirus [>100 types]

Swine Sheep Rabbits

Humans

Cutaneous and mucosal papillomas Papilloma

Avian papillomavirus

Parrots

137 Papillomatosis Papillomaviruses produce warts on the skin and mucous membranes in many different species; however, only those affecting cattle, horses, and dogs are of veterinary significance.

Papillomaviruses are usually host specific and produce proliferative lesions in specific anatomical sites. Papillomas develop after the introduction of virus through abrasions of the skin and mucous membranes. Pathogenesis
Diagram of the Stages of Infection of the Host by Papillomavirus

Virus infection initially occurs in the actively dividing basal keratinocytes in the stratum germinativum. Virus-induced hyperplasia [due to a virus encoded protein] results in increased basal cell division. As the cells move to the outer layers of the epidermis and differentiate, synthesis of capsid proteins and productive replication of the viral genome to thousands of copies per cell take place. Virus accumulation and associated cytopathology [degeneration and hyperkeratinization] are most noticeable in the stratum granulosum. Virions are shed with desquamated cells of the stratum corneum of the skin or nonkeratinized cells of mucosal surfaces. Most warts usually regress spontaneously after several weeks or months.

138 Bovine Papillomatosis Warts are more commonly seen in cattle than in any other domestic animal. All ages are susceptible, but the incidence is highest in calves and yearlings. Etiologic agents. Bovine papillomaviruses 1-10. Do not cross-protect.

Types 1, 2 and 5. Infect keratinocytes and dermal fibroblasts to produce fibropapillomas. Types 3, 4, 6, 9, and 10. Infect keratinocytes to produce cutaneous papillomas.

Transmission. The virus is transmitted between animals by contaminated halters, nose leads, grooming and earmarking equipment, rubbing posts, and other articles contaminated by contact with diseased cattle.

Venereal. Papilloma seen on the end of the penis of bulls and in the vagina of cows.

Clinical features. IP: 4-8 weeks. Fibropapilloma. Consists of a fibrous core covered to a variable depth with stratified squamous epithelium, the outer layers of which are hyperkeratinized.

The lesions vary from small firm nodules to large cauliflower-like [verruca vulgaris] growths. They usually regress within 6 to 12 months. Fibropapillomas can occur on the udder and teats and around the genitalia [penis, vulva, and vagina], but are most common on the head, neck, and shoulders. Young bulls. Fibropapillomas are found frequently on the penis but not on the prepuce. The wart grows rapidly and may surround the tip of the penis.

Cutaneous papilloma. Lack a fibrous core. It is usually flat with a broad base; they tend to persist. Malignancy. BPV-2 and BPV-4 infections may lead to bladder cancer and squamous cell carcinoma, respectively, in cattle ingesting bracken fern [Pteridium aquilinum]. The chemical, quercetin, contained in bracken fern appears to be a major contributing factor [both co-carcinogen and immunosuppressive agent] in the transition from benign fibropapilloma [BPV-2] or papilloma [BPV-4] to invasive carcinoma of the alimentary tract or bladder [chronic endemic hematuria]. Diagnosis. Lesions are characteristic.

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Virus particles can be demonstrated in lesion biopsies by TEM. Detection of papillomavirus DNA using DNA hybridization assays and PCR.

Treatment. For lesions that must be removed for esthetic or health reasons, surgical excision or cryosurgery with liquid nitrogen is very effective. Dry ice [CO2, -80oC] may also be used.

Topical agents may be tried when surgery is impractical. Podophyllin [50% podophyllin; 2% podophyllin in 25% salicylic acid] and undiluted medical grade DMSO. They are usually applied once daily until remission occurs. Bovine interferon- has been used to treat cattle.

Control. Commercial and autogenous vaccines may be used when warts are a problem in a herd, however, results are variable. Canine Oral Papillomatosis (Canine Papillomavirus 1) Oral papillomatosis is a contagious, self-limiting disease affecting the oral cavity of young dogs. The condition can involve all young dogs in a kennel. Clinical features. IP: 4-8 weeks. The warts begin as smooth white elevations, often around the lips, but later become roughened and cauliflower-like. They occur anywhere on the oral mucosa, in the cheeks, tongue, palate or pharynx, but do not extend below the epiglottis or into the esophagus.

Halitosis, hemorrhage, ptyalism, and discomfort may be observed in affected dogs. In some dogs the warts are so numerous [50-100] as to interfere with mastication and deglutition. Spontaneous regression is the norm, however, some warts may persist indefinitely [2o bacterial infection of such ulcerated warts may occur]. Dogs that have recovered from a crop of warts are refractory to reinfection.

Treatment. Surgical excision, cryosurgery, and electrosurgery may be used to remove the warts. Autogenous vaccines may be tried, however, their efficacy is questionable. Equine Papillomatosis Warts appear as small, elevated, keratinized papillomas around the lips, muzzle, external nares, and legs of 1-3-year-old horses.

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There may be a few or as many as 100 or more. Growth occurs for 4-8 weeks, and then in most horses, warts spontaneously regress within 1-9 months. Treatment and control are the same as for cattle. Equine Sarcoid

Sarcoids commonly occur in horses, donkeys and mules between 1 to 6 years of age. They are locally invasive benign fibroblastic skin tumors. Sarcoids are associated with bovine papillomavirus 1 or 2.

Equine sarcoid is the most common neoplasm in horses, representing about 20% of all equine tumors diagnosed at necropsy. Breed susceptibility, associated with the type of major histocompatibility complex, has been defined. Arabians, Appaloosa and Quarter horses are at more risk than are Standardbreds and Thoroughbreds. Sarcoids commonly occur on traumatized areas. The lesion is characterized by an irregular mass of proliferating dermal fibroblasts, accompanied by characteristic proliferation of the overlying epidermis. They may be single or multiple, occurring most frequently on the lower legs, head, and ventral abdomen. The growths may reach the size of a mans fist, variable in shape and bulging under the skin. The skin is thickened and roughened, becoming ulcerated.

Sarcoids do not metastasize; however, they persist for life [spontaneous regression is rare] and they are locally invasive, often recurring within 6 months after surgical removal [difficulty in identifying the junction of the sarcoid and the surrounding tissues].

Diagnosis. Clinical features and histologic examination of biopsy material. Treatment. Surgical excision, hyperthermia, radiation therapy, laser surgery, immunotherapy, and cryosurgery have all been tried.

Preparation of Autogenous Bovine and Equine Wart Vaccines Tissue homogenate [grind a crop of warts] is diluted 1:10 in saline and is filtered through gauze. The final solution is centrifuged at 300 rpm for 5 minutes. Formaldehyde is added to the supernatant fluid to a final concentration of 0.5% formaldehyde. The vaccine can be stored for up to two to four weeks at 4oC until used. Cattle: Inject 5-25 ml [depending on size] SC, and repeat 10-14 days later. Horses: Inject 2 ml ID at 14-day intervals for three to four injections.

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ADENOVIRIDAE
Properties of Adenoviruses

Nonenveloped hexagonal virions with icosahedral capsid; 80-100 nm in diameter. Genome consists of a double-stranded, linear DNA. Virions replicate in the nucleus; release of progeny virions occurs via cell lysis. Virions produce basophilic intranuclear inclusion bodies. The replication and assembly process are inefficient, with the result that only about 10% of nucleic acid produced in infected cells are encapsidated. DNA, protein and numerous defective particles accumulate to form nuclear inclusion bodies. Virions agglutinate red blood cells. Virions are stable in the environment but are inactivated by common disinfectants.

Virion showing Penton Fibers projecting from the Vertices; Crystalline array of Virions in Host Cell Nucleus

Penton Base and Fiber The fibers mediate the attachment of adenoviruses to cell surface receptors; induce neutralizing antibodies.

Hemagglutination. Occurs when the tips of penton fibers bind to an appropriate receptor on the surface of the RBC. The pentons and fibers carry serotype-specific antigens.

Genera Mastadenovirus [Mammalian adenoviruses]. A single penton fiber projects from each vertex.

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Aviadenovirus [Avian adenoviruses]. Each penton fiber is bifurcated, giving the appearance of two fibers extending from each penton base.
Diseases Associated with Mastadenoviruses Host Species Dogs Serotypes 2 Diseases Infectious canine hepatitis [canine adenovirus 1] Infectious canine tracheobronchitis [CAV-2] Cattle Sheep Goats Swine Horses Rabbits 10 7 2 4 2 1 Asymptomatic or mild upper respiratory disease Asymptomatic or mild upper respiratory disease Asymptomatic or mild upper respiratory disease Asymptomatic or mild upper respiratory disease Asymptomatic or mild upper respiratory disease Diarrhea

Pathogenesis All adenoviruses have a narrow host range, ie, are highly host specific. Adenoviruses produce mostly subclinical infections, with occasional upper respiratory tract and gastrointestinal tract diseases.

The penton and fiber proteins of the capsid are toxic to the cell. The toxic activity can result in: [1] inhibition of cellular macromolecular synthesis [eg, inhibition of cellular mRNA export from the nucleus and protein synthesis], [2] cell rounding and [3] tissue damage. Immunosuppression. Adenoviruses encode proteins [eg, E1A, E1B, E3, etc.] that suppress host immune and inflammatory responses: down-regulation of class I MHC expression; blocking of cellular apoptotic pathway; blocking of interferon-induced protein kinase R-mediated inhibition of viral protein synthesis, etc.

All adenovirus infections are associated with long periods of latency. Virus persists in lymphoid and other tissue, such as tonsils, adenoids, and Peyers patches, and can be reactivated in immunocompromised patients. Several adenoviruses are pathogenic in immunodeficient animals such as certain Arabian foals with severe combined immunodeficiency [SCID]. Immunity. Antibody is critical for recovery from infection.

143 Infectious Canine Hepatitis (ICH; Rubarths disease) Rubarths disease. ICH was first distinguished from canine distemper by Rubarth in 1947 using ferrets; ferrets are resistant to ICH but susceptible to canine distemper. ICH occurs worldwide. Hosts. Family Canidae [domestic and wild] and Ursidae [bears]. Etiologic agent. Canine adenovirus 1 [One serotype]. CAV-1 is antigenically related to, but distinct, from CAV-2.

Stable virus [survives for days at room temperature and months at < 4oC]. CAV-1 is susceptible to 1-3% of sodium hypochlorite [household bleach].

Transmission. Virus is present in respiratory secretions, urine, feces, and saliva. Recovered dogs shed virus in their urine for 6 months.

Ingestion of urine, feces, or saliva of infected dogs is the main route of infection. However, virus may be acquired via conjunctival or aerosol routes. Pathogenesis

144 Initial infection occurs in the tonsillar crypts and Peyers patches. There is viremia of 4 to 8 days duration and infection of macrophages, endothelial and parenchymal cells in many tissues, leading to hemorrhages and necrosis. Liver, kidneys, spleen, and lungs are the main target organs.

Initial cellular injury of the liver, kidney, and eye is associated with cytotoxic effects of virus. A sufficient neutralizing antibody response by day 7 PI [> 1:500] clears the virus from the blood and liver and restricts the extent of hepatic damage. Antibody titer < 1:4: Widespread hepatic necrosis. Partial immunity [> 1:16, <1:500]. Dogs may develop chronic active hepatitis and hepatic fibrosis.

Corneal edema [Blue eye]. Occurs in about 20% of natural infections and less than 1% of dogs after SC-MLV CAV-1 vaccination.

On or about day 4, virus enters the aqueous humor from the blood and replicates in corneal endothelial cells. By day 7. Severe anterior uveitis and corneal edema develop. Circulating immune complexes deposited on corneal endothelium [immune complex hypersensitivity] results in complement activation, neutrophil chemotaxis, frustrated phagocytosis, and corneal endothelial damage. Disruption of the corneal endothelium results in accumulation of edematous fluid in the corneal stroma.

From days 8-21, macrophages remove immune complexes and corneal endothelium regenerates, re-establishing the hydrostatic gradient.

Kidney [glomerulonephritis]. Chronic kidney lesions result from immune complex reactions after recovery from acute or subclinical disease. Disseminated intravascular coagulation [DIC]. A frequent complication of ICH, it begins in the early viremic phase of the disease.

DIC may be triggered by exposure of subendothelial collagen and subsequent platelet aggregation and/or inability of the damaged liver to remove activated clotting factors.

Clinical features. IP: 4-9 days. ICH is most frequently seen in dogs less than one year of age, although unvaccinated dogs of all ages can be affected.

Most infections are asymptomatic. In systemic infections, there are 3 overlapping syndromes which are usually seen in puppies less than 6 months old.

145 Peracute disease. Dog is found dead either without apparent preceding illness or after an illness lasting only 3 or 4 hours. Due to massive destruction of hepatocytes. Acute disease. Dogs that survive the viremic phase. Fever, depression, vomiting, bloody diarrhea, petechial and ecchymotic hemorrhages of the gums, pale mucous membranes, and jaundice. Mild cases.

Clinical signs in uncomplicated ICH last 5-7 days prior to improvement. Encephalitis. Common in foxes [fox encephalitis] but less common in dogs.

Diagnosis. Based on clinical signs and laboratory findings.


Virus isolation in cell culture: Urine, blood, tissue homogenates, etc. Antigen detection in tissues: Fluorescent antibody test; PCR. Paired sera: Employs virus neutralization, ELISA, or hemagglutination-inhibition tests. Gross and histopathology; hematology [leukopenia].

Immunity. Recovered animals are immune to the systemic form of the disease but may not resist an aerosol challenge and may develop respiratory disease.

Maternal antibody interferes with active immunization until puppies are 9 to 12 weeks of age. Both inactivated virus and attenuated [modified] virus CAV-1 vaccines are used. Annual revaccination is recommended. MLV: Vaccine virus localizes in the kidney and causes mild subclinical interstitial nephritis and persistent shedding of virus. Less than 1% of puppies may develop corneal edema.

Attenuated CAV-2 vaccines provide cross-protection against CAV-1. Canine Infectious Tracheobronchitis [ITB, Kennel Cough]

Kennel cough is mostly a self-limiting upper respiratory disease of dogs. Etiologic agents. Kennel cough has multiple etiologies: CAV-2, canine parainfluenza virus 5, Bordetella bronchiseptica, and Mycoplasma cynos.

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Other contributing agents include CAV-1 and canine morbillivirus [canine distemper].

Transmission. This is a highly contagious disease. Transmission is mostly via aerosolized microdroplets. Clinical features. IP: 3-10 days. Uncomplicated ITB. Mild respiratory disease. Laryngitis, tonsillitis, pharyngitis, tracheitis, and bronchitis.

Paroxysms of dry, hacking, mostly nonproductive cough, followed by retching. Rhinitis with serous to mucopurulent nasal discharges. Usually resolves spontaneously within 2 weeks or less.

Diagnosis. Similar to CAV-1. Palpation of trachea stimulates coughing spell. Immunity. CAV-2 vaccination [MLV] is routinely incorporated into vaccine protocols recommended for all dogs. Equine Adenovirus Infections Occurs worldwide and has been recognized in Arabian foals in the U.S.A. since 1970. Arabian foals are known to be very susceptible due to their combined T and B cell immunodeficiency.

As maternal antibody wanes, the foals become increasingly susceptible to a wide range of pathogens before they die, usually within 3 months. EAV-1 causes bronchiolitis and pneumonia. Diseases Associated with Aviadenoviruses [12 Serotypes] Species Chickens Ducks Quail Turkeys Disease[s] Inclusion body hepatitis; egg drop syndrome76 Hepatitis [rarely] Bronchitis Bronchitis; hemorrhagic enteritis; marble spleen disease

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RETROVIRIDAE
Properties of Retroviruses

Spherical enveloped virions with an icosahedral capsid; 80-100 nm in diameter. The genome is diploid, consisting of 2 identical haploid molecules, noncovalently linked at their 5 ends. Each haploid segment is a linear [+] sense ssRNA [does not serve as mRNA immediately after infection]. Virions contain reverse transcriptase [RNA-dependent DNA polymerase] which transcribes DNA from virion RNA. Virion DNA integrates into cellular chromosome as a provirus. Replication takes place in both cytoplasm and nucleus; virions assemble at, and bud from, cytoplasmic membrane. No inclusion bodies. Some retroviruses are oncogenic; many produce latent infections. Retroviruses are noncytopathic. Retroviruses are sensitive to heat and detergents; because of their diploid genomes, they are more resistant than other viruses to UV light.

Arrangement of Retroviral Genomes All retroviruses have gag, pol, and env genes. Some acquire an oncogene, and as a consequence, are usually defective in their replication.

gag gene [group-specific antigen]: encodes the virion core [capsid] proteins.

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pol gene [polymerase]: encodes reverse transcriptase and integrase. env gene [envelope]: encodes the envelope glycoprotein peplomers. Virus Replication

Adsorption, penetration by fusion or receptor-mediated endocytosis, and uncoating. Viral RNA is released into the cytoplasm.

Parental RNA is copied to ssDNA [RNA:DNA hybrid] by reverse transcriptase. ssDNA is made double-stranded, again by reverse transcriptase. dsDNA moves to the nucleus where it is integrated as a provirus at different sites in the cellular DNA. Provirus is replicated with host genome and is passed to daughter cells [type III spread]. Using host cell polymerases, provirus serves as a template for the synthesis of mRNA [for protein synthesis] and [+] sense RNA molecules which are encapsidated into progeny virions. Maturation of virions occurs by budding through plasma membrane. Endogenous Retroviruses

These nonpathogenic retroviruses occur widely in the genome of vertebrates. They are transmitted only as provirus in the germ line DNA [ova or sperm] from parent to offspring. Endogenous retroviruses are regulated by host cellular regulatory genes and are usually completely silent.

In cats, endogenous retroviral DNA [eg, RD 114] sometimes recombines with feline leukemia subtype-A virus [FeLV-A] proviral DNA to produce recombinant FeLK-B virus. Morphologic Distinction of Retrovirus Particles

Based on electron microscopic observations, retroviruses were classified on the basis of the morphology and position of the nucleocapsid core, as follows: immature intracytoplasmic A-type particles bud through the plasma membrane into mature B-type [contains an eccentric core]; C-type [centrally located electron-dense core]; and D-type [contains a tubular core] particles.

149 Oncogenic Retroviruses Oncogenic retroviruses are the major cause of leukemias, lymphomas, and sarcomas in many animal species.

Malignant tumors arising from cells of mesenchymal origin are known as sarcomas, and those from leukocytes as lymphomas [if solid tumors] or leukemia [if circulating cells are involved]. Antibodies directed against antigens expressed on the surface of cells during maturation are cytotoxic [complement-mediated and ADCC], and may prevent tumor formation. Acute-transforming viruses [see Effects of Viruses on Host Cells]. Viral genome contains viral oncogene [v-onc+]. Acquisition of the v-onc gene is associated with deletions elsewhere in the genome, so that most v-onc+ viruses are replication defective, except Rous sarcoma virus. The viruses produce tumors within a very short time after infection in a high percentage of infected hosts, eg, feline sarcoma virus.

Chronic-transforming viruses [see Effects of Viruses on Host Cells]. The viruses are v-onc- and cause cancers late after infection and in only a relatively low percentage of infected hosts.
Diseases Caused by Retroviruses

Genus Alpharetrovirus

Virus Avian leukosis viruses Avian sarcoma virus Rous sarcoma virus

Hosts Chicken Chicken Chicken

Disease[s] Leukemia, lymphomas Sarcoma Sarcoma

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Betaretrovirus Pulmonary adenomatosis virus Ovine Pulmonary adenomatosis [Jaagsiekte] Feline leukemia and feline sarcoma Sarcoma Bovine leukemia Maedi/Visna Arthritis, encephalitis, etc. Equine infectious anemia AIDS

Gammaretrovirus

Feline leukemia and sarcoma viruses Porcine type C virus

Cats

Swine Bovine Sheep/goats Goats

Deltaretrovirus Lentivirus

Bovine leukemia virus Maedi/Visna virus Caprine arthritis-encephalomyelitis virus Equine infectious anemia virus

Horses

Human immunodeficiency viruses 1 and 2 Feline immunodeficiency virus

Humans

Cats

Feline immunodeficiency Immunodeficiency Immunodeficiency

Simian immunodeficiency virus Bovine Immunodeficiency virus

Monkeys Bovine

Bovine Leukemia (Enzootic Bovine Leukosis) Bovine leukemia is a highly fatal, systemic, malignant neoplasia of the reticuloendothelial system of cattle, characterized by the development of aggregations of neoplastic lymphocytes in various organs. Distribution. Worldwide. In the U.S.A., EBL prevalence in dairy cattle ranges from 10-50%; in beef cattle it ranges from 1 to 20%. Etiologic agent. Deltaretrovirus. The virus is found associated with B lymphocytes [free virus is rarely or never found in the blood]. It is v-onc-.

The transactivating gene [tax] encodes p34 transactivator protein that up-regulates proto-oncogene promoter sequence.

Transmission. Primarily by transfer of infected lymphocytes between animals. Infected lymphocytes are found in the blood, milk, and in tumor masses. The virus is labile outside the host, therefore, environmental contamination is not a source of infection.

Blood transfer from infected animal to a susceptible animal. Amounts of blood as small as 0.1 ml are capable of transmitting the infection.

151 Blood transfusions, trauma, hypodermic needles, mechanically by bloodsucking insects, etc.

Congenital infection, either in utero or through colostrum and milk containing infected lymphocytes, occurs in 4-8% of calves born to infected cows.

Pathogenesis. The major target cells are B lymphocytes. There are 4 possible outcomes following virus infection:

Failure of the animal to become infected due to genetic resistance. Susceptibility or resistance to persistent lymphocytosis, is related to the class II genes of the bovine major histocompatibility complex, BoLA. Cattle with BoLA-Aw 7 alleles are resistant; cattle with BoLA-Aw 12 alleles are susceptible.

Asymptomatic infection and the development of only an antibody response. The animal becomes seropositive 4 to 12 weeks after exposure. Seropositive animals with persistent lymphocytosis characterized by a benign proliferation of B lymphocytes [~ 33% of infected animals]. Seropositive animals that develop lymphosarcoma [malignant lymphoma]. Account for < 1-2% annually of infected cattle. Observed in cattle between 4 and 8 years of age.

Clinical signs. Varies, depending on the organ or tissues affected, but weight loss, anorexia, and decreased milk production are frequently observed. Diagnosis. BLV infection must be distinguished from clinical disease.

Clinical history and histopathologic examination of affected tissues. Persistent virus infection leads to a marked antibody response to the envelope glycoprotein gp51 and the major core protein p24, which can be detected by a number of serologic tests: Agar gel immunodiffusion test [AGID], ELISA, Western blot [WB], and IFA.

Prevention and control. EBL can be eradicated from a herd by repeated serologic testing of animals over 6 months of age, at 2- to 3-month intervals. Sporadic Bovine Leukosis (SBL) SBL is a noninfectious lymphosarcoma observed in cattle under 3 years of age. Etiologic agent is unknown. It occurs in 3 forms:

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Juvenile multicentric form. Observed in cattle < two years of age. It is characterized by progressive weight loss and multiple lymph node enlargement. Thymic form. In yearlings between 6 months to 2 years of age. It is characterized by a swelling in the neck, causing bloat and edema. Cutaneous form. Observed in cattle 1-4 years of age and is characterized by multiple skin nodules and internal metastatic tumors. Feline Leukemia and Sarcoma

Feline leukemia virus is a naturally occurring, highly contagious virus associated with neoplastic and nonneoplastic diseases, such as profound anemia, immunosuppression, enteritis, and reproductive failure. Disease occurs worldwide. Hosts. Domestic cats and some wild cats. Etiologic agent. Gammaretrovirus. The virus is noncytopathic and v-onc-.

p27 protein. Structural component of the inner viral core and is the major feline leukemia virus [FeLV] group-specific antigen. It is produced within virus infected leukocytes and platelets, excreted in saliva and tears, and found free in the plasma. Detected by IFA and ELISA tests. gp70 protein. An envelope protein responsible for viral attachment. Based on the differences in the gp70 protein, there are 3 main subgroups of FeLV [FeLV-A, FeLV-B, FeLV-C]. Neutralizing antibodies against gp70 proteins protect against viremia; the antibo-dies are subgroup specific. FeLV-A. Found in all naturally infected viremic cats. Subgroup A viruses are highly contagious and are the only subgroup viruses that are transmitted horizontally from cat to cat. FeLV-B. Found in ~ 50% of naturally infected cats along with FeLV-A. It arises through recombination between the env genes of FeLV-A and endogenous FeLV-related proviral DNA. Cats infected with both FeLV-A and FeLV-B have a higher risk of developing tumors than those infected with FeLV-A alone.

FeLV-C. Subgroup C viruses are rare. They arise de novo in a FeLVA infected cat as a result of mutations in the receptor-binding region of

153 the FeLV-A env gene. They cause a rapidly fatal nonregenerative anemia, hence they are not transmitted to other cats.

p15E. Envelope protein associated with FeLV-induced immunosuppression. It suppresses lymphocyte blastogenesis; blocks the response of T cells to interleukin 1 and 2, and suppresses the response of cats to FOCMA. FeLV is extremely labile once outside the cat and is rapidly inactivated by most household disinfectants and detergents. In a dry environment, the virus is inactivated in 3 to 5 minutes. In a moist environment, the virus may survive for 24 to 48 hours at room temperature. The virus is maintained in nature because viremic cats may live for several years and because shedding of virus appears to be constant. If all FeLVpositive cats are removed from a household, a new cat may safely be obtained after a waiting period of 3 to 4 weeks.

Transmission. Prolonged, direct exposure is usually required for transmission. Viremic cats shed virus continuously.

Occurs primarily via the saliva, where the virus concentration is very high. Even though virus multiplication occurs in several organs and tissues, it is less likely to spread via feces, urine, and fleas. Iatrogenic transmission could occur via contaminated needles, instruments, fomites, or blood transfusion. In utero infections occur but more kittens are infected when the viremic queen licks them and nurses them.

Pathogenesis. The virus multiplies in T and B lymphocytes and myeloid cells.

Following oronasal exposure, the virus replicates in localized lymphoid tissues. A low grade [transient] viremia occurs, spreading the virus to the spleen, lymph nodes, intestines, and bone marrow. ELISA test is . Infection of leukocyte and platelet precursors in the bone marrow and the subsequent release of infected cells into the circulation, results in a persistent 2o viremia. The IFA test is positive at this point. In persistently viremic cats, infection proceeds to extensive involvement of the bone marrow, pharynx, esophagus, stomach, bladder, respiratory tract, etc. FOCMA antigen [feline oncornavirus membrane-associated antigen]. This is a tumor-specific antigen present only on the membrane of cells transformed by either FeLV or FeSV.

154 FOCMA antibody lyses tumor cells via ADCC and complement activation. Thus, cats with high FOCMA antibodies are resistant to the development of leukemia and lymphoma, regardless of whether they are positive or negative for FeLV. FOCMA antibody does not neutralize virus, hence cats with increased FOCMA Ab titers may still be viremic and die of nonmalignant disease.

Clinical and Pathological features. IP: 3 months to 3 years. Within 6 weeks postinfection [PI], one of three host-virus relationships develops: Self-limiting infection. Majority of cats [92-96%].

Cats develop neutralizing and FOCMA antibodies; there is no viremia and cat does not shed virus. Cat does not develop FeLV-related disease, however, because a DNA copy of the FeLV genome stably integrates into the host cell chromosomal DNA, malignant transformation some time in the future cannot be excluded.

Persistent active infection

Persistent viremia; serum lacks neutralizing and FOCMA antibodies; terminates in FeLV-related disease. Immunosuppression is the most common sequel to persistent FeLV viremia and accounts for most FeLV-related deaths.

Latent infections

The cats immune system may hold the virus in check [not absolutely defeating it, but not been overwhelmed by it either]. While the virus is latent, the cat does not have FeLV signs and does not shed the virus, so there is no risk of infecting other cats. Although most latently infected cats remain asymptomatic, occasionally [because of other stress factors such as disease, pregnancy, or a new household cat], they convert to a state of persistent viremia. Latent infections are detected following bone marrow culture and reactivation. Neoplastic Diseases

All hematopoietic cell lines are susceptible to transformation by the virus. Thus, lymphoid and myeloid [including granulocytic, erythroid, and megakaryocytic] types occur.

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Lymphosarcoma [malignant lymphoma]. Consists primarily of solid masses of proliferating lymphocytes. Accounts for 30% of all feline tumors. Three major forms are described based on the anatomic location of the tumors. Multicentric. Tumors are observed in multiple lymphoid and nonlymphoid organs and tissues. Predominantly, T cell tumor. Thymic [mediastenal] lymphosarcoma. Observed primarily in kittens. A T cell tumor. Alimentary lymphosarcoma. Usually observed in older cats; it involves the lymphoid tissues of the GIT [GALT] and/or mesenteric lymph nodes. B cell tumor. Cats are usually feline leukemia negative.

Myeloproliferative disease. These are a group of primary bone marrow disorders characterized by abnormal proliferation of one or more hematopoietic cell lines. The diseases are characterized by the presence of large numbers of neoplastic cells in the bone marrow, a nonregenerative anemia, and immunosuppression. Erythromyelosis. Erythroid cell lines. Granulocytic leukemia. Granulocytic myeloid cell, usually a neutrophil. Erythroleukemia. Both erythroid and granulocytic myeloid precursors become neoplastic. Myelofibrosis. Proliferation of fibroblasts and cancellous bone resulting in medullary osteosclerosis and myelofibrosis. Nonneoplastic Diseases

Immunopathological diseases Immune complex disease. Immune complexes formed in the presence of moderate antigen excess [abundant FeLV antigens relative to anti-FeLV IgG antibodies], predispose cats to systemic vasculitis, glomerulonephritis, polyarthritis, and other immune disorders. Immunosuppression. Some persistently viremic cats suffer from a severe lymphopenia [primarily due to a loss of circulating T cells], neutropenia or both. It is characterized by: Hypocomplementemia due to immune complex hypersensitivity; reactivation of latent infections; and increased susceptibility to 2o infections.

156 Some commonly observed conditions include chronic gingivitis and stomatitis, feline infectious peritonitis, hemobartenollosis, etc.

Reproductive disorders Infertility, resorption or abortion of fetuses and endometritis have been observed in viremic queens. Most kittens born to viremic queens become viremic and die at an early age of fading kitten syndrome, characterized by failure to nurse, hypothermia, and thymic atrophy within the first 2 weeks of life.

Diagnosis. Virus isolation is rarely attempted.

FeLeuk test. Detects the group-specific antigen, p27, within the viral core. ELISA. Tests for presence of circulating p27 antigen in plasma, whole blood, or serum. Saliva and tears may also be used. ELISA-positive cats should be retested within 2-3 months to determine if viremia is transient or persistent. IFA test. Tests for p27 antigen associated with leukocytes and platelets in fixed blood smears. 97% of IFA-positive cats remain positive for life. Both ELISA and IFA detect viremia and a positive test only indicates increased risk of FeLV-related disease[s].

Control. Cats positive for FeLV are potential spreaders, therefore, IFA-positive cats should be isolated from other cats.

Vaccination. Vaccination reduces the incidence of disease by about 80%. Available vaccines include inactivated whole virus vaccines and genetically engineered vaccines such as gp70 subunit vaccine and live canarypox vector vaccine [PUREVAX Recombinant Leukemia vaccine]. Feline Sarcoma Virus [FeSV]

FeSV is associated with multiple fibrosarcomas in young cats. The virus arises de novo as a result of a recombinational event between FeLV-A and host cell proto-oncogenes [see acute-transforming viruses].

FeSV, which is v-onc+, is a defective virus. Replication requires helper FeLV because a portion of its genetic information, eg, env gene, is lost during recombination. Thus, all strains causing fibrosarcomas are pseudotypes, with envelope provided by FeLV.

157 Clinical features. Fibrosarcomas express FOCMA antigens. They are locally invasive and metastasize to the lungs and other sites [solitary fibrosarcomas in old cats > 5 years old, are not caused by FeSV]. Avian Leukosis The viruses causing avian leukosis are endemic in all chicken populations worldwide. Incidence is 3-20% in most chicken populations. Etiologic agent. Alpharetrovirus. The virus is v-onc-. The viruses are classified into 10 subgroups on the basis of differences in viral envelope antigens.

Subgroups A and B. Associated with most field outbreaks of leukosis. Subgroups C and D. Recovered infrequently. Subgroup E. Genetically inherited [endogenous], nononcogenic virus. Subgroup J: Associated with myeloid leukosis.

Transmission

If virus is transmitted congenitally via the egg [vertical], or horizontally within the first few days of life [< 5 days], the chicken becomes viremic for life because of the development of immunological tolerance. The birds excrete virus in the saliva and feces. If horizontal transmission occurs beyond 5 or 6 days after hatching, the chicken develops neutralizing antibodies, the viremia is transient, and the chicken is unlikely to develop leukemia.

Pathogenesis

The primary target cells are lymphoblasts with B lymphocyte markers in the bursa of Fabricius. Bursectomy prevents the development of lymphoid leukosis.

Clinical features. Disease occurs sporadically in birds over 14 weeks of age.

Lymphoid leukosis [syn. visceral lymphomatosis; big liver disease]. Observed in chickens 14 to 30 weeks of age. Lymphoid cell infiltrations of various organs, eg, bursa of Fabricius, liver, spleen, kidney, etc.

Osteopetrosis [Thick leg]. Characterized by a proliferation of periosteal osteoblasts of the long bones of the limbs. The enlargement is usually bilateral [lesions are not neoplastic].

158 Diagnosis. Based on flock history and tumors in multiple organs without nerve or ocular involvement [Mareks disease]. Control. Terminate breeder flocks that break out with lymphoid leukosis. Genus Lentivirus (Latin, lentus slow) Lentiviruses are associated with chronic infections characterized by persistent, cell-associated viremia. Antigenic variation, a result of frequent random mutations, is commonly observed in lentivirus infections. Feline Immunodeficiency Disease The virus causing feline immunodeficiency disease was first isolated in 1987 from a large, multiple-cat household located in Petaluma, California. The discovery was precipitated by the cattery owners recognition of an immunodeficiencylike syndrome among a group of FeLV-negative cats. Etiologic agent. Feline lentivirus. Five subtypes, based on envelope proteins, have been identified: Subtypes A, B, C, D, and E. Subtypes A and B predominate in North America. There is no cross-protection among subtypes. Host range. Domestic cats and some wild felidae, eg, cheetahs. Transmission. Virus is shed mainly in the saliva. Infection is lifelong.

The principal mode of transmission is via cat bites, hence disease is most common in free-roaming cats and uncommon in closed purebred catteries. One bite is sufficient to transmit the virus. The virus is transmitted both in utero and postpartum via colostrum and milk.

Pathogenesis. Viremia following initial bite wound. The hallmark of feline immunodeficiency is the progressive disruption of normal immune function.

The virus has tropism for helper T cells, brain macrophages, peritoneal macrophages, and astrocytes. The resulting immunosuppression subjects the cat to opportunistic infections, such as toxoplasmosis, fungal diseases, etc.

Clinical features. The disease is more common in cats 3 to 5 years of age.

Acute phase. Cats are either asymptomatic or may show, transiently, clinical signs of fever, malaise, lymphadenopathy, and diarrhea. Antibody to the virus can be detected, however, it is ineffective in eliminating the virus.

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Latent phase. A latent [asymptomatic] phase follows the acute phase, during which no signs of illness are observed; may range from months to years Terminal phase. Chronic diseases characterized by a variety of clinical findings that usually are a result of opportunistic bacterial and fungal infections, eg, chronic stomatitis and gingivitis, chronic respiratory disease, chronic diarrhea and wasting, dermatitis, neurologic signs, etc.

Diagnosis. Detection of FIV-specific antibodies in the blood using IFA, ELISA or Western blot. Antibodies appear within 2-4 weeks of infection and persist for life.

If positive results are obtained in kittens <12 weeks old, kittens must be retested after the age of 12 weeks to assure that antibodies detected were not maternally derived. Detection of FIV core antigen, p24, by ELISA or proviral DNA by the PCR in peripheral blood leukocytes of infected cats.

Control. Preventing contact between FIV-negative and FIV-positive cats.

A number of vaccines are available, but their efficacy are in doubt.

Cat-to-human transmission. The virus appears to be specie-specific. FIV antibodies have not been detected in people having had close contact with FIVinfected cats, or in people having inadvertently injected themselves with viruscontaining materials. Equine Infectious Anemia [EIA, Swamp fever] EIA in Equidae is characterized by a long relapsing illness after an initial acute attack. Disease occurs worldwide. Etiologic agent. Equine lentivirus. One serotype. Transmission. Occurs via transfer of blood cells from an infected horse.

Mechanical transmission by tabanids [horseflies, breeze flies], stable flies [Stomoxys spp.], mosquitoes, and probably Culicoides spp. The virus is infective for up to 30 minutes on a flys mouth parts. Vertical transmission via placenta and through colostrum and milk.

Pathogenesis

The virus replicates initially in macrophages and then in lymphocytes. Lifelong, cell associated viremia develops in all infected horses.

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Persistent Ag-Ab complexes [immune complex hypersensitivity] result in damage to vascular endothelium [vasculitis], followed by inflammatory changes in parenchymatous organs, especially, the liver. Vasculitis in the CNS results in ataxia, spinal leptomeningitis, and encephalomyelitis. Anemia. Viral antigens adsorb to RBC, bind with EIA antibody, and trigger erythrophagocytosis by mononuclear phagocytes and complement-mediated hemolysis [type II hypersensitivity]. Glomerulonephritis. Immune complex-mediated disease.

Clinical features. IP: 2-4 weeks but can be up to 3 months.

Acute. Fever, severe anemia, jaundice, blood-stained feces, tachypnea, and petechial hemorrhages of the mucosae. Mortality rate is ~ 80%. Subacute. Moderate fever followed by recovery. Recovered viremic horses may experience recurrent episodes of disease. Chronic. Varies from mild signs of illness and failure to thrive to episodic or persistent fever, cachexia, and ventral edema.

Diagnosis. Agar gel immunodiffusion test [AGID, Coggins test].

Detects antibodies to the major group-specific antigen [p26] of EIA virus. Results are valid for 6 months or 1 year from the date the blood is collected. It detects all infected animals except those in the early incubation period, the first 2 to 3 weeks after infection. A horse suspected of being exposed should be retested after 4 to 6 weeks. Foals that have been nursed by infected dams may be temporarily positive, however, they should be negative by 6 months of age if not infected.

ELISA tests. ELISAs detect antibodies sooner and at lower concentrations than the AGID test. However, false-positive results have been noted with the ELISA tests, therefore, positive ELISAs are confirmed using the AGID test.

Control. Identifying positive horses by the Coggins test. Destruction [not mandatory] or isolation of positive reactors.

Horses imported to the U.S.A. and some other countries are required to have a negative test certificate.

161 Caprine Arthritis-Encephalomyelitis (CAE) CAE is characterized by polyarthritis [most common form], mastitis, encephalomyelitis, and interstitial pneumonia. A 1981 serologic survey of 1160 goats from 24 states within the USA, revealed that 81% of the goats tested were seropositive. The disease occurs in goats worldwide. Etiologic agent. Caprine lentivirus. Transmission. The main means of transmission is via colostrum and milk from the doe to the newborn.

Kids infected at birth remain persistently infected for life.

Pathogenesis. Persistent virus infection of monocytes and macrophages due to ineffective neutralizing antibody response [continual emerging of new antigenic variants]. Chronic inflammatory changes observed in tissues have been attributed to immune complex hypersensitivity reactions.

Arthritis. The form seen most often clinically. Seen in goats 12 months or older. It is characterized by unilateral or bilateral swelling of mostly carpal joints [big knee]. Other joints, such as the tarsal joint, may also be affected. Pain and thickening of the joint capsules [hyperplastic synovitis] restrict the movement of the goat. Advanced cases are accompanied by emaciation, rough hair coat, and carpal hygroma.

Encephalomyelitis. Occurs most often in kids 1 to 5 months of age. It is a progressive leukoencephalomyelitis associated with ascending paresis and paralysis. The goats are afebrile, alert, and maintain a good appetite. Other signs include upward deviation of the head, twisting of the neck, and paddling movements of the feet. Signs are irreversible; most kids are usually euthanized.

Interstitial pneumonia. Mostly in adults. It is insidious in onset and is progressive. There is gradual weight loss and respiratory distress. Indurative mastitis. Presents as a diffuse swelling of the mammary gland with associated firmness [fibrosis; hard bag, hard udder]. There is no systemic illness.

162 Diagnosis. Based on clinical and serologic evaluation.

Detection of CAEV antibodies using agar gel immunodiffusion test [AGID], ELISA, or IFA test.

Control. All kids must be immediately removed from the dam at birth and provided colostrum from known virus-free does.

Colostrum from CAEV-positive does can be used if it is treated at 56oC for 1 hour. Alternatively, kids can be reared on pasteurized milk. Test and removal. Goats are tested every 6 months, and those testing positive are removed. A positive serologic result indicates a goat is infected for life and a potential continual source for transmission of the virus. Maedi/Visna Disease

Maedi/Visna is a disease of adult sheep and, to some extent, goats. Distribution. Worldwide. In the U.S.A., it is prevalent in many of the major sheep-producing areas, especially in the Midwestern and northwestern states. Etiologic agent. Ovine lentivirus. The maedi and visna viruses are regarded as variants of a single agent; visna is considered a 2o encephalitic form of maedi. Transmission. Aerosolization; ingestion of feces or urine contaminated water.

Ewe to lamb via colostrum and milk. Intrauterine infection may occur infrequently. Biting arthropods and surgical equipment readily transmit the virus mechanically from viremic sheep [lifelong viremia].

Pathogenesis. A lifelong mononuclear cell-associated viremia is observed.

Certain breeds of sheep are more susceptible: Icelandic, Finnish, Border Leicester and Texel breeds. Goats are susceptible to infection by the virus. A lymphocyte-associated viremia occurs. Infection and seroconversion [most sheep seroconvert 2 to 3 weeks after infection] are common, but clinical disease occurs infrequently [5%].

Clinical features. Onset of disease is insidious, with IP varying from months to years [9 years], hence disease is seen in sheep 2 years and older.

163 Maedi [shortness of breath]; Ovine progressive pneumonia, OPP]

Coughing, progressive weight loss and emaciation; dyspnea; pregnant ewes may abort or deliver weak lambs. Disease is fatal within 3 to 8 months, especially with 2o bacterial infection.

Visna [wasting]

Slowly progressive ataxia, trembling, paresis or total paralysis. Appetite is maintained and sheep remain alert, however, unattended sheep may die of starvation. Sheep assisted with eating and drinking may survive for 1-2 years. Lesions. Diffuse, demyelinating encephalomyelitis.

Arthritis. A polyarthritis resulting in severe lameness, especially of the carpal and tarsal joints, may occur. Mastitis. A noninflammatory, indurative mastitis with reduction in milk production, which results in poor growth of lambs, is commonly observed. Diagnosis. Clinical history, virus isolation, and histopathological lesions.

Detection of antibodies using ELISA, AGID, and Western blot.

Immunity. Viral persistence occurs in the presence of antibodies and cellmediated immune responses.

In infected sheep, antigenic variation in the envelope antigen may be an important mechanism for circumventing viral elimination.

Control

Noninfected flock. The virus can be prevented from entering a flock by pretesting and quarantine of incoming animals. Retesting of flock every 6 months is essential to maintaining a virus-free status. Infected flock. birth. Removal of seropositive animals and/or isolating lambs at

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RHABDOVIRIDAE [Rhabdos, rod]


Properties of Rhabdoviruses

Bullet-shaped, enveloped virions with helical nucleocapsid; 70 nm in diameter and 170 nm long. Genome is a [-] sense ssRNA. Envelope contains large glycoprotein peplomers [G protein]. Neutralizing antibodies are directed against G protein epitopes. Cytoplasmic replication; virions bud from the cytoplasmic membrane. Some rhabdoviruses cause rapid cytopathology, eg, vesicular stomatitis virus, others are noncytopathic, eg, rabies virus. During replication, defective interfering particles [DI] are commonly formed. DI particles are deletion mutants with greatly truncated genome [T particles] that interfere with replication of normal infectious virions. Rhabdoviruses are thermolabile, sensitive to UV light, and are readily inactivated by detergent-based disinfectants, eg, QUATS [Zephiran], Cidex, and Nolvasan.

Genus Lyssavirus Genus Vesiculovirus. Genus Ephemerovirus. Genus Novirhabdovirus Genus Cytorhabdovirus Genus Nucleorhabdovirus

Rabies virus Vesicular stomatitis virus Bovine ephemeral fever virus Infectious hematopoietic necrosis virus of fish

GENUS LYSSAVIRUS [Gr. frenzy] Lyssaviruses are relatively noncytopathogenic, thus encephalitis and death occur in many cases of infection with little or no cell destruction.
Genotype 1 2 3 4 5 and 6 7 Classification of the Genus Lyssavirus Serotype Description of Strains 1 Classical rabies virus 2 Lagos bat viruses 1, 2, and 3 3 Mokola 1, 2, 3, and 5 4 Duvenhage 1, 2, and 3 5 European bat Lyssavirus types 1 and 2 6 Australian bat Lyssavirus

Rabies [Latin rage] Rabies is one of the oldest and most feared diseases of humans and animals. It is the most lethal of all infectious diseases.

165 Distribution. Worldwide. Japan, New Zealand, Hawaii, Antarctica, certain parts of Europe, Great Britain, and most of the Caribbean are rabies-free.

Australia was rabies-free until the isolation of the Australian bat lyssavirus. In North America and Europe, wildlife rabies is increasing in importance. Cattle rabies is important in Central and South America. Worldwide, dog rabies is the cause of most of the estimated 40-50,000 cases of human rabies each year.

Hosts. All warm-blooded animals have variable degrees of susceptibility.

Foxes, coyotes, jackals, wolves, and certain rodents are among the most susceptible animal groups. Skunks, bats, raccoons, rabbits, cattle, some members of the families Felidae and Viverridae [civet, mongoose, etc] have a high susceptibility. Domestic dogs, sheep, goats, horses, and nonhuman primates are moderately susceptible.

Etiologic agent. Worldwide seven genotypes of lyssavirus have been identified. Classical rabies is caused by genotype 1 lyssavirus [one serotype]. In North America, six strains of genotype 1 have been identified.

Two skunk strains; an arctic fox and red fox strain; gray fox strain; dog/coyote strain; and a raccoon strain. The virus is extremely labile when exposed to UV light and heat. At 20oC, the virus cannot survive for more than 24 hours in carcasses.

Transmission. Mostly by bite or scratch of a rabid animal with the virus in its saliva. Aerosol transmission in cave dwelling bats; human rabies from corneal transplantation has been reported.

Skunks have been most important in the perpetuation of wildlife rabies in the United States. They account for most cases of cattle rabies. High prevalence of rabies among skunks. Excretion of large quantities of virus in their saliva during the prolonged period [4-18 days] of clinical disease.

Bats. Many species of bats harbor different variants of rabies virus; many do so asymptomatically. Subclinical infection can progress to more advanced

166 clinical disease during times of stress, etc. Rabid bats may develop paresis or paralysis. In some cases, the bats may become aggressive. For decades, cases of rabies in insectivorous bats have been reported in North America. Vampire bats play a major role in human and animal rabies in Mexico, Central America, and parts of South America. In Latin America, about 250-500,000 cases of cattle rabies annually are attributed to vampire bats. In 1996, the Australian bat lyssavirus was isolated from a fruit eating bat in Queensland. Two fatal human cases have been reported.

Pathogenesis. Infection may start in striated muscles or cells of the subepithelial tissues near the site of inoculation until virions reach a sufficient concentration to reach motor or sensory nerve endings in the muscle or skin.

The virus is shed from myocytes into extracellular spaces, bind to acetylcholine receptors [other receptors may be used, eg, ganglioside and phospholipid receptors] facilitating its entry into nerve endings. The second phase of infection begins when the virus progresses centripetally to the CNS via the axoplasm of the peripheral and central nerves. The virus reaches the limbic system where it replicates extensively, leading to the furious form of rabies. Spread within the CNS continues, with replication in the neocortex, resulting in the dumb or paralytic form of rabies. Late in infection, the virus moves centrifugally from the CNS down peripheral nerves to a variety of organs including the adrenal cortex, pancreas, and the salivary glands [via cranial nerves]. Extensive replication in the salivary glands results in high concentrations of virus in the saliva. Pathology. Histopathologic examination of the brain reveals that many neurons are infected, but there is no frank cytopathology and little inflammatory cell infiltration [minimal target damage but lethal neurologic dysfunction]. Immunology. Humoral and cell-mediated immune responses cannot be detected during the time the virus moves from the site of the bite to the CNS. Infection is noncytopathic in muscle and nerve cells, hence very little viral antigen is released to stimulate host defense mechanisms. Neurons do not express MHC class I proteins. Antibodies appear in serum and later in the cerebrospinal fluid after neurologic signs appear.

167 Clinical features. IP: 14 to 90 days, but may be longer. The IP is influenced by dose, strain of virus, degree of innervation and site of inoculation [intraaxonal movement is about 10-100 mm per day].

Prodromal period. Period of virus shedding and change in temperament before obvious clinical disease is observed. Two clinical forms are observed: an initial furious form and a terminal dumb form. Death results from respiratory failure. A higher proportion of dogs, cats, and horses exhibit fury than is the case for cattle or other ruminants or laboratory animals. In dogs and cats, rare individuals survive more than 10 days after virus is first shed into the environment.

Diagnosis

Direct FAT to demonstrate rabies antigen in touch impressions of brain tissue [medulla, cerebellum, and hippocampus]. Reverse transcription-polymerase chain reaction [RT-PCR] to test for the presence of viral RNA in the brain of the suspect animal. Antemortem diagnosis [only in human cases]. FAT or RT-PCR is performed using skin biopsy, corneal impression, or saliva. Demonstration of Negri bodies in the thalamus, hypothalamus, pons, cerebral cortex, and dorsal horns of the spinal cord. Not all virus-positive brains show Negri bodies [75% in humans; lower in some wildlife species like bats]. Virus isolation by intracerebral inoculation of weanling mice with fresh homogenized tissue. Control mice are inoculated with extracted tissue incubated with specific neutralizing antibody. Mice develop encephalitis within 14 days.

Control of rabies

Rabies-free countries. Quarantine involving segregation of dogs and cats in licensed premises for 6 months. Endemic countries. Vaccination of dogs and cats; wildlife vaccination. Post-exposure treatment in humans Wash wound thoroughly with aqueous soap solution or QUAT.

168 Human rabies immune globulin [HRIG] is used at 20 IU/kg of body weight, half infiltrated around the wound, half injected intramuscularly. In the U.S., two human rabies vaccines are licensed: human diploid cellculture vaccine [HDCV] and rhesus kidney cell-culture adjuvanted vaccine [RVA]. The protocol consists of five 1.0 ml doses given IM in the deltoid area, one each on days 0, 3, 7, 14, and 28. If a person was vaccinated previously, no HRIG is given, instead, two doses of vaccine are given, one each on days 0 and 3. GENUS VESICULOVIRUS Vesicular Stomatitis Vesicular stomatitis [VS] is a sporadic disease of cattle and other ruminants, horses, swine, and humans characterized by fever and vesicle formation. Distribution. Occurs primarily in the Americas and certain areas of the Caribbean. Etiologic agent. Vesiculovirus. Two serotypes and several subtypes exist. There is no cross-protection among the serotypes.

Indiana serotype (VS-IN; 3 subtypes: Fort Lupton, Alagaos [Brazil], and Coccal [Trinidad]) and New Jersey serotype [VS-NJ]. The New Jersey serotype is the more virulent and has the widest distribution. The virus can remain stable for days or weeks in cool water, soil, and on vegetation. It is inactivated in 10 mins by 1% formalin, roccal [QUAT], etc.

Transmission. Contact with contaminated milking machines [teat and udder lesions]. Ingestion of contaminated fomites [mouth lesions].

Arthropods. Though the virus replicates in black flies, sandflies, leaf hoppers, culicoides and mosquitoes, mechanical transmission is believed to be more significant than biological transmission. Houseflies are mechanical vectors.

Pathogenesis. Virus enters the body through breaks in the mucosa and skin or by the bites of flying arthropods.

Vesicle formation and epithelial loss follow epithelial cell destruction and interstitial edema, which separates the epithelium from underlying tissues. Spread of lesions occurs by local extension. No viremia or carrier state in cattle, horses, and swine.

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Immunity following recovery is short-lived [ 6 months], however, antibody titers can persist for 8 to 10 years. Reinfection has been reported in the presence of a high antibody titer.

Clinical features. IP: 1 to 5 days. Morbidity: 5-10%; occasionally, up to 80%.

Vesicular lesions on the tongue, lips, gums, teats, and coronary bands. Oral lesions are accompanied by profuse salivation and anorexia. Uncomplicated cases fully recover in 2 weeks.

Diagnosis. Virus isolation from vesicular fluids and tissue scrapings. Virus grows well in cell cultures, embryonated eggs and suckling mice [intracerebral inoculation].

Paired acute and convalescent serum samples. Antibodies are quantitated using CFT, ELISA or VN test.

Prevention and control. Vesicular stomatitis is a reportable disease.

Quarantine of affected premises. Movement of cattle, swine, and horses from affected premises is prohibited until 30 days after the last clinical signs or the disease are evident, with the exception of animals going directly to slaughter. USDA approved autogenous killed vaccines are available only during an outbreak, however, their efficacy is in doubt. Horses. There are no laboratory tests to differentiate between a positive serological reaction due to vaccination or natural exposure. The American Horse Council and AAEP recommend that horse owners and veterinarians carefully consider the potential repercussions from use of VS vaccines [some states and countries require negative blood tests for horses entering their borders].

Humans. The virus causes a flulike disease of one weeks duration. GENUS EPHEMEROVIRUS Bovine Ephemeral Fever (3-day sickness/3-day stiff-sickness) BEF is a benign, noncontagious, arthropod-transmitted acute febrile disease of cattle and water buffalo. Distribution. Africa, Middle East, Asia, and Australia. BEF has never been reported in North America.

170 Etiologic agent. Bovine ephemerovirus. One serotype. Strains vary in virulence. Transmission. Biologic vectors include mosquitoes [Aedes spp., Anopheles spp., and Culicine spp.] and the biting midge Culicoides brevitarsis. Pathogenesis. Following inoculation, virus replication occurs mostly in monocytes and macrophages in the lungs, spleen and lymph nodes.

There is generalized inflammation and immune complex-mediated vasculitis and thrombosis of the small blood vessels. There is also increase in plasma fibrinogen leading to serofibrinous polyserositis involving the pleural, pericardial, and peritoneal surfaces and the joints. Pulmonary edema, emphysema and bronchiolitis. Marked neutrophilia with a left shift [abnormal level of band neutrophils in the circulation] and a significant drop in plasma calcium [hypocalcemia].

Clinical findings. IP: 2-4 days. Morbidity: 25% to 100%. Mortality: 1-2%.

Sudden onset, inappetance, fever, and sudden and severe drop in milk production [up to 80%] in lactating cows. Excessive salivation, lacrimation, serous nasal discharge, drooling and dyspnea. Abortion [probably fever-induced] is observed in 5% of pregnant cows. Muscle stiffness with shifting lameness and reluctance to move. Recovery is dramatic and complete in 3 days [range, 2-5 days]; however, some animals remain recumbent for long periods after the temperature returns to normal.

Diagnosis. Specimens: Buffy coat cells, spleen, lung, pericardium, etc.

Virus isolation. Cell cultures derived from Aedes albopictus or IC inoculation of suckling mice. Antigen detection. FA staining of blood or tissue smears. Paired serum samples: ELISA, complement fixation or VN tests.

Control. Vector control.

Attenuated and inactivated vaccines are used in endemic areas. Recovering animals have long-lasting immunity.

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PICORNAVIRIDAE
Properties of Picornaviruses

Virions are nonenveloped with an icosahedral symmetry; 27 nm in diameter. Genome is linear, [+] sense ssRNA. Cytoplasmic replication. Virion RNA acts as mRNA and is translated into a polyprotein, which is then cleaved to yield individual proteins. Virions do not produce inclusion bodies. Most picornaviruses cause rapid cell death with characteristic CPE. There are multiple serotypes that all produce the identical disease with very little cross-protection between types, eg, foot-and-mouth disease..

Genera. A major difference among the various genera is their stability at low pH.

Genus Apthovirus. Member viruses are unstable below pH 7. Genus Enterovirus. Most member viruses are stable at pH 3. Genus Cardiovirus: Member viruses are stable at pH 3. Genus Hepatovirus. Member viruses are stable at pH 3. Simian hepatitis A virus and human hepatitis A virus.

Genus Teschovirus. Member viruses are stable at pH 3. Genus Tremovirus. Most member viruses are stable at pH 3. Genus Erbovirus. Member viruses are unstable below pH 7.

Stability. The stability of picornaviruses to environmental conditions is important in the epidemiology of the diseases they cause and in the choice of methods of disinfection.

If protected by mucus or feces and shielded from strong sunlight, they are relatively heat stable at normal ambient temperatures. Aerosols of aphthoviruses and rhinoviruses are usually less stable, but under conditions of high humidity, they may remain viable for several hours. Enteroviruses survive for several days and weeks in feces.

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Diseases Caused by Picornaviruses Genus Aphthovirus Virus Foot-and-mouth disease viruses Equine rhinitis A virus Bovine rhinitis B virus Teschovirus Porcine teschovirus 1 Porcine teschovirus 2-11 Enterovirus Porcine enterovirus B [PEV-9 and PEV-10] Bovine enteroviruses 1-2 Host[s] Ruminants and swine Disease Foot-and-mouth disease

Horses, camelids Cattle Swine Swine Swine

Mild respiratory disease Mild respiratory infection Polioencephalomyelitis Subclinical infections Subclinical infections

Bovine

Mild enteric and respiretory disease Vesicular disease

Swine vesicular disease virus Human rhinoviruses A [ 74 serotypes]; B [ 25 serotypes]; and C Erbovirus Equine rhinitis B virus

Swine

Humans

Common cold

Horses

Mild rhinitis

Cardiovirus

Encephalomyocarditis virus

Rodents, swine, elephants and mammals in contact with rodents Chickens

Encephalomyocarditis in swine and elephants; rarely in other species

Tremovirus

Avian encephalomyelitis virus Duck hepatitis A virus Bovine kobuvirus

Encephalomyelitis

Avihepatovirus
Kobuvirus

Ducks Cattle

Hepatitis Enteritis

Foot-and-Mouth Disease (FMD) Synonyms. Aphthous fever; epidemic aphthae [aphtha [Gr]: small ulcer] Distribution. Currently, North and Central America, Oceania, and the Caribbean are disease-free.

FMD is endemic in Asia, Africa, the Middle East, and most of South America. An outbreak of FMD in Britain in February 2001 resulted in the spread of the disease to other members of the European Community.

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FMD was last reported in 1929 in the U.S.A, 1952 in Canada, and 1954 in Mexico.

Hosts. All cloven-hoofed domestic and wild animals, including cattle, sheep, goats, swine, buffalo, etc. Horses are genetically resistant to FMD. Etiologic agent. Aphthovirus. Seven serotypes [A, O, C, SAT-1, SAT-2, and SAT-3 [Southern African territories] and ASIA-1 and 80 subtypes [many subtypes within each serotype due to antigenic drift, ie, point mutations] have been identified. There is no cross-protection among serotypes.
Asia: O, A, C, Asia-1 Europe: O, A, C Africa: O, A, C, SAT-1-3 South America: O, A, C

Under natural conditions, the virus has been shown to survive for 14 days in a stall and for as long as 20 weeks on sacks and hay. The virus in muscle is inactivated within 48 hours of slaughter but survives for longer periods in bone marrow, viscera, and blood clots. Virus infectivity is destroyed by high temperature [>56oC] and UV light. Because the virus is sensitive to acid and alkaline pH [stable at pH 6-9] sodium hydroxide, sodium carbonate [soda ash], and citric or acetic acid are effective disinfectants.

Transmission. Mostly through inhalation. Infected animals generate large volumes of infectious aerosols, especially aerosols produced by swine.

Direct contact between infected and susceptible animals, by contaminated animal products [meat, milk, semen, etc] and mechanically by viral contamination of vehicles, equipment, farm products, etc; also mechanical spread of the virus by birds, rodents and arthropods has been reported. Garbage containing uncooked meat scraps and bones from infected animals has been a source of infection in pigs. The virus can be spread over long distances, eg, country to country, by asymptomatic carriers or animals incubating the disease [virus shedding begins 24 hours prior to observance of clinical signs].

Pathogenesis. Following inhalation, initial virus replication occurs in mucosal epithelial cells of the pharynx, followed by invasion of the regional lymph nodes and viremic spread to multiple organs and tissues.

174 Virus replication and cell lysis is most prominent in the epithelium of the mouth and feet, dorsum of the snout of pigs and the teats.

Initial hyperemic areas develop into vesicles, some of which may coalesce to form large blisters. The vesicles are filled with clear yellow fluid. Necrotizing myocardial lesions, characterized by small gray foci and streaks of irregular size and shape in the myocardium, giving the myocardium a striped appearance, the so-called tiger heart, are the most common cause of fatal FMD in young calves, lambs, goats, pigs, and buffaloes. Myocardial lesions may also be seen in adult animals infected with serotype O virus.

Carrier state. Virus can persist in the nasopharynx for up to 2 years in cattle and up to 6 months in sheep. There is no carrier state in swine.

Clinical features. Clinical signs are most severe in cattle and swine. Sheep and goats develop subclinical infections. Cattle. IP: 2 to 8 days; Course: 2-3 weeks. Morbidity: high; Mortality: < 5%.

Fever, anorexia, and depression. Within 24 hours, infected animals salivate profusely, smack their lips, and are lame. Vesicles are present on the tongue, lips, gums and palate, teats, rumen pillars, coronary band, and interdigital areas. Ulceration of vesicles lead to 2o bacterial infection. Abortion in pregnant cow [probably fever induced, etc; virus does not cross the placenta] and mastitis with about 25% drop in milk production.

Swine. Aerosols contain extremely high levels of virus.


Fever and lameness because of pain caused by blisters on their feet. Vesicles or erosions on the snout, lips, and tongue, on the coronary band, and between the claws on the feet.

Sheep, goats, and other wild ruminants. Mostly asymptomatic. The disease is usually mild and is characterized by foot lesions and lameness. Diagnosis. FMD must be distinguished from other vesicular diseases [vesicular stomatitis, swine vesicular disease, vesicular exanthema, bluetongue, bovine viral diarrhea-mucosal disease, etc].

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Virus isolation: Vesicular fluid, blood, esophageal and pharyngeal fluids, etc. Cell cultures of bovine origin.

An ELISA test is available for rapid identification of virus in vesicular fluid or tissues. An ELISA test is also available for antibody detection. Virus identification. Virus neutralization, RT-PCR, AGID, and complement fixation tests can be used.

Immunity. Cattle recovering from FMD are usually immune to reinfection with the same virus type for a year or longer, but immunity is not lifelong [possibly due to antigenic variation].

Immunity is type-specific, thus recovered animals can be infected immediately with any of the other serotypes and develop disease.

Control. FMD is a notifiable disease. Endemic countries. Vaccination using inactivated and modified live-virus vaccines. Imposition of quarantine in an outbreak. Disease-free countries

Quarantine and slaughter of all susceptible animals and herds in contact with, or within a certain radius of, the infected herd. Disposal of carcasses by burning or deep burial and decontamination of premises. Federal law prohibits the importation of animals or animal products from infected countries. Products that have been processed so that they are free of the virus, such as by cooking or drying, may be permitted entry.

Zoonosis. Most infections in humans are subclinical. Fever, anorexia, and vesicular lesions on the skin and/or mucous membranes, followed by recovery. Avian Encephalomyelitis (Epidemic Tremor) Epidemic tremor was first described in the New England states in 1932 but is now worldwide. Disease is observed mostly in chickens 1 to 3 weeks of age; mild encephalomyelitis is observed in turkey poults, quail, and pheasants. Etiologic agent. Avian tremovirus. One serotype. Strains vary in virulence. Transmission. Mainly by fecal-oral route. Transmission through the eggs may occur during a short viremic phase [approximately 1 week] in infected breeder hens [no disease in adult hens except for a transient drop in egg production].

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Lateral spread from infected chicks to hatch mates in the hatcher or during brooding.

Clinical features. IP: 1-7 days.

Ingestion of virus is followed by replication in the epithelial cells of the alimentary tract and viremic spread to other organs and the CNS. Reduction in hatchability of fertile eggs [death of a proportion of infected embryos during incubation]. The main signs in young chicks are unsteadiness, sitting on hocks, tremors especially of the head and neck, prostration, blindness, paralysis, coma, and death. Mortality rate may exceed 50% in an initial outbreak. Recovered birds may have CNS deficiencies and may have to be destroyed.

Diagnosis

Demonstration of viral antigen in brain using immunofluorescent stain. Virus isolation. Brain. Yolk sac of embryonated eggs or cell culture. Pathology. No gross lesions are seen at necropsy.

Control. Either depopulation or vaccination.

An attenuated live-virus vaccine administered in the drinking water to breeding hens prevents vertical transmission and ensures passively immune progeny. Maternal antibodies protect chicks for the first few weeks of life. Encephalomyocarditis

Rodents are the natural hosts of the virus. The virus is transmitted from rodents to domestic animals, wildlife species and humans; however, it is a significant pathogen only in pigs [severe disease in young pigs] and wildlife, eg, elephants. The virus is found worldwide. Etiologic agent. Cardiovirus. One serotype. Transmission. Swine are infected by eating feed or drinking water contaminated with rodent urine and feces. Pathogenesis. Following oral ingestion, virus replication occurs in epithelial cells of the alimentary tract, followed by invasion of the regional lymph nodes and viremia.

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High virus titers can be demonstrated in the myocardium, spleen, and mesenteric lymph nodes. Transplacental infection may occur in fetuses near full term with many of them developing myocardial lesions.

Clinical features. Subclinical in weaned pigs and adults.


Mid-to-near term abortions, stillbirths, and mummified fetuses [SMEDI]. Young and growing pigs. Sudden death, suggesting myocardial failure and encephalomyelitis. There may be anorexia, depression, and difficulty in breathing. Mortality approaches 100%.

Diagnosis. Pathognomonic cardiac lesions [myocardial infarcts, esp. in the right ventricle].

Fluorescent antibody or immunohistochemical staining of affected tissues. Virus isolation. Heart and brain. Detection of antibodies. Serum neutralization test, ELISA, AGID, latex agglutination, and hemagglutination inhibition test.

Prevention and control. Rodent control.

Outbreaks usually run short courses with immunity occurring in surviving animals. An inactivated vaccine aids in the prevention of reproductive failures in swine.

178

PARAMYXOVIRIDAE
Properties of Paramyxoviruses

Virions are enveloped, pleomorphic [spherical or filamentous]; and 150-300 nm in diameter. Genome consists of a linear [-] sense ssRNA. Cytoplasmic replication; budding from the cytoplasmic membrane. Syncytium formation; intracytoplasmic and intranuclear inclusion bodies [genus Morbillivirus]; others, intracytoplasmic inclusion bodies. All eosinophilic. Sensitive to heat [56oC] or dessication and are readily destroyed by lipid solvents and common disinfectants.

Functions and Terminology of Virion Proteins

Hemagglutinin, H. Attachment to host cells. Elicits neutralizing antibodies in the host that inhibit attachment of virus to cellular receptors. Neuraminidase, N. Virion release from cells; destruction of mucin inhibitors. Fusion protein, F. Cell penetration [fusion of envelope with cell membrane]; cell fusion [syncytium formation], and direct cell-to-cell spread via fusion [plays a key role in persistent infections]. Elicits neutralizing antibodies that prevent fusion of envelope with host cell membrane.

Subfamilies and Genera

Subfamily Paramyxovirinae

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Genus Respirovirus Genus Morbillivirus Genus Rubulavirus Genus Avulavirus Genus Henipavirus

Human parainfluenza virus 1 Measles virus Mumps virus Newcastle disease virus Hendra virus

Subfamily Pneumovirinae Genus Pneumovirus Genus Metapneumovirus Human respiratory syncytial virus Turkey rhinotracheitis virus

Diseases Caused by Paramyxoviruses Genus Respirovirus Avulavirus Virus Bovine parainfluenza virus 3 Avian paramyxovirus 1 Avian paramyxovirus 2-9 Rubulavirus Canine parainfluenza virus 5 Porcine rubulavirus Morbillivirus Bovine morbillivirus Canine morbillivirus Peste des petits ruminants virus Human morbillivirus Henipavirus Hendra virus Nipah virus Pneumovirus Metapneumovirus Bovine pneumovirus Turkey metapneumovirus [turkey rhinotracheitis virus] Host[s] Cattle, sheep Avian Avian Dogs Swine Cattle Dogs, etc Sheep, goats Humans Horses, humans Swine, humans Cattle, sheep Turkeys Chickens
Disease[s]

Respiratory disease Newcastle disease Respiratory disease Respiratory disease Encephalitis, reproductive problems, etc Rinderpest Canine distemper Rinderpest-like disease Measles Respiratory disease Encephalitis Respiratory syncytial disease Respiratory disease Swollen head syndrome

Genus Respirovirus

Virions possess hemaglutinin-neuraminidase protein; form intracytoplasmic inclusion bodies. Virions hemagglutinate and hemadsorb.

180 Bovine Parainfluenza Virus 3 Disease Etiologic agent. Bovine parainfluenza virus 3 (PI-3). One serotype. Hosts. Cattle and sheep. Transmission. Mainly through aerosols and ingestion of fomites contaminated with nasal discharges. Clinical features. Uncomplicated PI-3 virus infection is a mild respiratory disease followed by recovery. High morbidity but low or no mortality.

Calves and lambs. Fever, lacrimation, serous nasal discharge, dyspnea and coughing. Usually, complete recovery occurs in 3 to 4 days. Bovine pneumonia [Shipping fever] complex. PI-3 virus is a major contributor to shipping fever complex [Mannheimia haemolytica, etc]

Diagnosis. Virus isolation from nasal discharges.


Virus identification in nasal discharges, etc, by FAT and ELISA. Paired sera [acute/convalescent]. Antibodies can be quantitated using VN test, Hemagglutination inhibition test or ELISA test.

Control. A number of attenuated [modified] virus vaccines are available commercially. Vaccines may be given intranasally or parenterally. They induce protective mucosal IgA antibodies. Genus Avulavirus

Virions possess hemaglutinin-neuraminidase protein; form intracytoplasmic inclusion bodies. Virions hemagglutinate and hemadsorb. Newcastle Disease

Newcastle disease is a highly contagious disease of domestic poultry and several avian species, including several pet birds. The disease was first observed in Java [Indonesia], in 1926 and in the same year it spread to England. Currently, the disease occurs worldwide. Etiologic agent. Avian paramyxovirus1. There is only one serotype. Strains of the virus vary in virulence depending on the cleavability and activation of their precursor fusion glycoproteins by host enzymes.

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Avirulent or low-virulent strains. The trypsin-like protease that cleaves their precursor F glycoproteins has limited tissue distribution. The enzyme is usually present extracellularly or in epithelial cells of only the respiratory and alimentary tracts. This leads to decreased production of infectious progeny virions. Lentogenic strains. Less virulent strains.

Virulent strains. The endoproteases that cleave their precursor F glycoproteins have widespread tissue distribution. The enzymes are found intracellularly in cells lining mucous membranes. Thus, virus replication produces high levels of virulent progeny virions that are able to infect more cell types and cause widespread tissue damage, viremia, and systemic disease. Mesogenic strains. Moderately virulent strains. Velogenic strains. Highly virulent strains. They are subdivided into viscerotropic and neurotropic strains. Velogenic strains are not present in the USA but are frequently brought in via illegally imported game chickens and exotic birds [eg, psittacines].

Hosts. Gallinaceous birds [domestic chickens, turkeys, guinea fowl, peacocks] and pheasants, quail, pigeons, parrots, cockatoos, cockatiels, parakeets, etc. Transmission. Virus is shed during the incubation period, during the clinical stage, and for limited periods during convalescence. The virus is present in respiratory discharges, feces, eggs, and all parts of the carcass.

Chickens are readily infected via aerosols and ingestion of contaminated food and water. Birds may shed virus in all secretions and excretions for at least 4 weeks following recovery. A persistent carrier state has been demonstrated in psittacine and in certain other wild birds.

Pathogenesis and Immunity. Initial virus replication occurs in the mucosal epithelium of the upper respiratory and intestinal tracts, followed by viremia.

1o viremia disseminates the virus to the spleen and bone marrow, eventually resulting in a 2o viremia, with infection of other target organs, eg, lungs, intestine, and CNS. Neutralizing antibody [hemagglutination inhibition antibodies] can be detected within 4-6 days of infection and persists for at least 2 years.

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Mucosal immunity is provided by sIgA whereas IgG in the circulatory system blocks viremia.

Clinical forms or pathotypes Viscerotropic velogenic ND [Exotic ND; VVND].

A peracute, lethal disease of chickens of all ages characterized by hemorrhagic and ulcer-like lesions in the digestive tract. Respiratory distress and CNS lesions are not as marked. Mortality approaches 100% in all age groups.

Neurotropic velogenic ND [Exotic ND; Pneumoencephalitis].

This is an acute and frequently lethal infection of chickens of all ages. It is characterized by lesions in the respiratory tract and CNS. Hemorrhages are conspicuously absent from the digestive tract. Mortality approaches 100%.

Mesogenic pathotypes

An acute respiratory and occasionally lethal nervous infection of young chickens. Mortality approaches 25% in young chickens, rare in adults.

Lentogenic pathotypes

A mild to inapparent respiratory infection of chickens.

Diagnosis

FAT staining of tracheal sections or smears. Virus isolation from lungs, spleen, and brain, using the allantoic route, in embryonated eggs. Virus identification: HI tests; hemadorption inhibition test; or VN test.

Control. It is a notifiable disease. Quarantine and depopulation of infected farms. Vaccination. Naturally occurring lentogenic strains [B1 or La Sota] are used. They are administered via aerosols, drinking water, eye or nostril droplets, or beak dipping. An inactivated oil emulsion is also available. Maternal antibodies protect baby chicks for 3 to 4 weeks after hatching. Humans. Individuals may develop conjunctivitis, which is usually mild and persists for 1 to 2 days but on occasion is quite severe and may lead to lasting impairment of vision.

183 Genus Rubulavirus


Virions possess hemaglutinin-neuraminidase protein. Virions produce eosinophilic intracytoplasmic inclusion bodies. Virions hemagglutinate and hemadsorb. Canine Parainfluenza Virus 5 (CPiV-5)

CPiV-5 is part of the kennel cough syndrome. The virus is present in dog populations worldwide.

CPiV-5 is antigenically related to simian virus 5 [SV-5].

Transmission. Occurs by aerosolized microdroplets. Clinical features. IP: 3 to 10 days.

The virus does not multiply in macrophages, hence infections are restricted to the URT. Damage to the tracheal epithelium results in 2o bacterial infections. In uncomplicated cases, there is a sudden onset of dry, hacking cough, fever, and serous nasal discharge that may last for 3 to 14 days.

Diagnosis. Virus isolation from nasal or throat swabs. Control. Intranasal and parenteral vaccines, in combination with other antigens, are available. Genus Morbillivirus

Hemagglutinin protein but not neuraminidase is found in measles virus. Animal morbillivirus glycoprotein peplomers do not possess hemagglutinin-neuraminidase activity. Virions produce intranuclear and intracytoplasmic inclusion bodies. Canine Distemper (Hardpad disease)

Canine distemper is a highly contagious, systemic, febrile disease of unvaccinated dogs and other carnivores worldwide. Hosts. Domestic and wild dogs; family Procyonidae [raccoon, panda]; family Ursidae [bears] and family Mustelidae [ferrets, etc]. CNS infections in exotic Felidae [eg, lions, cheetahs, jaguars, ocelots, etc] have been reported.

184 Etiologic agent. Canine morbillivirus. Only one antigenic type. The virus is related to measles and rinderpest viruses.

The severity and type of disease is influenced by the strain of virus. Snyder Hill, R252, and A75/17 strains, are highly virulent and neurotropic. Other strains vary in their ability to cause CNS lesions. The virus is extremely fragile and is readily inactivated within a few hours at room temperature. In warm climates, the virus does not persist in kennels after infected dogs have been removed. Between 0oC and 4oC, the virus survives in the environment for weeks. Ordinary disinfectants readily inactivate the virus.

Transmission. Inhalation of infected droplets. The virus is found in all secretions and excretions from the 5th day after infection, sometimes for weeks [up to 60 to 90 days, although shorter periods of shedding are more common]. Pathogenesis. The virus multiplies in macrophages, epithelial cells, T and B lymphocytes [immunosuppression], and nervous tissue.

185

Initial replication of virus occurs in the cells of the URT or in conjunctival epithelium, with resultant spread to, and multiplication in, regional lymph nodes. A lymphocyte-associated 1o viremia spreads the virus to the reticuloendothelial system. Further replication at these sites results in a lymphocyte- and monocyte-associated 2o viremia. Humoral and cell-mediated immune responses influence the outcome of infection. In dogs with neutralizing antibody titer of > 1:100 on the 8th or 9th day, there is no further spread of virus, and virus disappears rapidly from the lymphatic tissues; the infection remains subclinical. If a titer of > 1:100 has not been attained by day 14, virus spreads throughout the body.

The brain is sometimes infected, usually when infection in visceral organs is subsiding. Some dogs develop polioencephalomyelitis [Snyder Hill, A75/17] or demyelinating encephalomyelitis [R252]. Late complications. Observed in old dogs. They may also be seen in dogs with no history of earlier acute or subacute disease. Hardpad disease. Hyperkeratosis of foot pads and the nose due to virus multiplication in the skin. Old dog encephalitis. In a minority of dogs, the virus persists in the brain and cause neurological damage more than 24 months after apparent recovery. This may be due to the very slow replication and spread of the virus in the brain.

Clincal features. IP: 3 to 7 days. Most infections are subclinical [50 to 70%]. The prevalence rate is highest in dogs between 3 to 6 months of age, coinciding with the loss of maternal antibodies.

Peracute cases with sudden death are rare. Acute cases. Some dogs show primarily respiratory or intestinal signs. CNS signs depend on infection with neurotropic strains. Mortality rate: 30 to 80%. Surviving dogs often have permanent CNS sequelae.

Diagnosis. Virus isolation is very difficult but can be done.

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Demonstration of viral antigen by immunoperoxidase or FA staining of impresssion smears of conjunctiva or peripheral blood lymphocytes [antemortem] or lung, stomach, intestinal, and bladder tissue [postmortem]. Rising antibody titer in paired serum samples using VN, CFT, or ELISA tests.

Immunity. Dogs surviving distemper have prolonged [almost lifelong] immunity to reinfection. Upon exposure, dogs can be treated with hyperimmune serum or immune globulin.

Puppies can be vaccinated with modified live-virus vaccine at 6 weeks of age and then at 2- to 4-week intervals until 16 weeks of age. Modified live measles vaccine. Canine morbillivirus and human measles virus share a common fusion [F] protein antigen. The vaccine offers protection in young puppies with high levels of maternal antibodies. It is used only as a replacement for the first vaccination in 6- to 12-week-old puppies. Puppies younger than 6 weeks of age with very high maternal antibody concentrations do not respond well to either distemper or measles vaccination. The vaccine should be administered IM for optimum response. Peste des Petits Ruminants (PPR, Goat Plague or Kata [for Catarrh])

PPR is an acute to subacute contagious disease of domestic and wild small ruminants characterized by erosive stomatitis, diarrhea, pneumonia, etc. Distribution. PPR is endemic in Africa, Asia, and the Middle East. Hosts. Sheep and goats; however, PPR is more severe in goats than in sheep. Severe disease can occur in wild small ungulates, eg, gazelles, white-tailed deer, etc.

Subclinical infection without virus shedding occurs in swine and cattle.

Etiologic agent. Caprine morbillivirus. Only one serotype. The virus is rapidly inactivated in the environment. Transmission. Close contact between infected and susceptible animal is necessary for virus transmission.

Mainly by inhalation of respiratory tract excretions; ingestion of contaminated feed and water, or through the conjunctiva. PPR virus is present in saliva, feces, urine and in nasal, pharyngeal, and conjunctival secretions.

187

Animals incubating the disease shed virus 1 to 2 days before clinical signs occur. There is no carrier state in sheep or goats.

Pathogenesis. Following inhalation, virus replication occurs in the epithelium of the URT, tonsils, and in lymphocytes in the regional lymph nodes.

Viremia disseminates the virus to other lymphoid tissues and to the mucosae of the respiratory and alimentary tracts. Infected cells undergo necrosis, resulting in mucosal erosions and lymphopenia.

Clinical findings. IP: 3 to 6 days. Morbidity: Approaches 100%. Disease may be acute [mainly in goats] or subacute [sheep].

High fever, anorexia, depression, and serous nasal and ocular discharges that later become mucopurulent; profuse salivation and diarrhea [sometimes hemorrhagic]; and pneumonia. Pregnant animals may abort. Erosions: Lips, gum, tongue, soft and hard palates, intestinal mucosa, etc. Case-fatality rates: Goats [55-85%] and sheep [10%]. PPR is rapidly fatal in young animals under 6 months of age.

Diagnosis. Virus can be isolated from lymphoid tissues, buffy coat cells, and all body secretions and excretions.

Gross and histopathology. Antigen detection. RT-PCR, Immunohistochemical staining, ELISA, etc. Virus can be identified using VN and ELISA tests; paired serum samples using VN and ELISA tests.

Control. PPR is a reportable disease. Kids and lambs are vaccinated at 3-4 months of age with a modified live virus vaccine. Genus Pneumovirus

No hemagglutin-neuraminidase protein. Virions possess glycoprotein [G protein] peplomers. Virions produce intracytoplasmic inclusion bodies. Bovine Respiratory Syncytial Disease

This is a mild to severe respiratory tract disease in cattle and sheep. The disease occurs worldwide.

188 Etiologic agent. Bovine pneumovirus. Subtype A and B based on antigenic differences of the G protein. Cross-protection occurs between the subtypes. Strains vary in virulence.

The virus is extremely fragile. It is thermolabile and sensitive to freeze-thaw cycles, hence specimens should not be frozen [can be refrigerated].

Transmission. Aerosolization of respiratory tract excretions.

The virus persists in herds, possibly in a few carrier animals or through continuous subclinical infections.

Pathogenesis. The virus causes complete loss of the ciliated epithelium, so that pulmonary clearance is compromised, with subsequent 2o bacterial infections [Mannheimia haemolytica, Histophilus somni, etc].

Immunosuppression due to leukopenia and neutropenia.

Clinical features. IP: 5 to 7 days. Disease may be acute, mild, or inapparent.

Cattle of all ages, types, and breeds are susceptible, however, incidence is highest in weaned calves and young cattle. Fever, rhinitis, nasal and lacrimal discharges, tracheitis, bronchitis, bronchiolitis, and nonproductive cough; mild interstitial pneumonia, interstitial emphysema and interstitial edema [extensive pulmonary mast cell degranulation leading to increase in vascular permeability]. Morbidity is very high [30-50% or higher]; case fatality rate is usually low [35%]. A high case fatality rate in cases progressing to severe bronchopneumonia due to 2o infections.

Immunity. Immunity following recovery from disease is short-lived and multiple reinfections are common; however, clinical disease is generally mild. Diagnosis. Virus isolation is very difficult because of the viruss fragility.

Demonstration of viral antigen. ELISA; FA staining of lung lavage. Gross and histopathology [syncytia, inclusion bodies]. Paired serum samples: VN and ELISA tests.

Control. Inactivated and modified live virus vaccines are available.

189

ORTHOMYXOVIRIDAE
Properties of Influenza Virus

Pleomorphic, spherical or filamentous, enveloped virions; 80-120 nm in diameter. Genome consists of linear [-] sense ssRNA, divided into 6-8 segments. The envelope contains two peplomers: the rod-shaped hemagglutinin glycoprotein peplomer and the mushroom shaped neuraminidase protein. Virions require host DNA transcription in order to replicate. Transcription and RNA replication occur in the nucleus; capped 5 termini of cellular mRNAs are cannibalized as primers for viral mRNA transcription. Virions mature by budding from plasma membrane; no inclusion bodies. Genetic reassortment and defective interfering particles frequently occur. Influenza viruses are very labile under ordinary environmental conditions.

Genera

Genus Influenzavirus A. Contains pathogens that infect humans, horses, swine, dogs, fowl, mink, seals, and whales. Genus Influenzavirus B. Contains only human pathogens. Genus Influenzavirus C. Contains pathogens of humans and swine but the viruses rarely cause disease. Genus Thogotovirus. Tick-borne viruses causing diseases in humans and livestock in Europe, Africa, and Asia.
Schematic Representation of an Influenza A virion

HA: Hemagglutinin NA: Neuraminidase M1: Matrix protein RNP: Ribonucleoprotein M2: Transmembrane ion channel

190 Classification

Type or species. Based on antigenic characteristics of ribonucleoprotein [RNP] and matrix protein [M1]. Subtypes. Based on the two envelope proteins, hemagglutinin and neuraminidase. Hemagglutinin [HA, H]. Bind to sialic acid moieties of glycoprotein or glycolipid receptors on cell surface. Antibodies to HA proteins neutralize infectivity of the virions. Neuraminidase [NA]. Enhances the release of virions by cleaving sialic acid receptors [receptor destroying enzyme,] on the cell membrane which would otherwise recapture the progeny virions and hold them at the cell surface. Neuraminidase also lowers the viscosity of mucus, thus facilitating viral transport within the respiratory tract. Antibodies to NA decrease the amount of virions released from infected cells but do not neutralize free virions.

Replication

Adsorption and penetration by endocytosis. Uncoating. The low pH within endosomes triggers a conformational change in the HA, resulting in fusion of the viral envelope with endosomal membrane. Simultaneously, protons pass into the virion through the M2 ion channel, dissociating M1 from the RNP and releasing the RNP into the cytoplasm. The RNPs are transported into the nucleus where transcription and replication of viral RNA occur. Cap snatching. A viral endonuclease cleaves the 5methylguanosine cap plus about 10 to 15 nucleotides from host cell mRNA, and this is used as a primer for transcription by the viral RNA polymerase. Each gene is transcribed separately. Following RNA replication, M1 protein enters the nucleus and binds to the nucleoprotein-transcriptase-negative sense RNA complex, down regulating transcription and permitting its export from the nucleus into the cytoplasm, prior to assembly into virions. HA, NA, and M2 migrate preferentially to plasma membrane on the apical surface of the cell. M1 recognizes and binds to the cytoplasmic portion of the HA and precipitates budding.

191 Influenza A Viruses Phylogenetic analysis indicates that all of the influenza viruses of mammals, including humans, originated from the avian influenza gene pool.

HA/NA subtypes. In avian species, 16 HA [HA1-HA16] and 9 NA [NA1-NA9] have been identified. The following subtypes have been associated with disease in humans and domestic animals: Horses: H7N7 and H3N8. Swine: H1N1, H1N2 and H3N2. Humans: H1N1, H2N2, H3N2, and H5N1. Poultry: H5N2, H7N1, H5N1, H5N9, H7N3, etc.

Nomenclature. Influenza viruses are codified as to type, host species [omitted if human], geographic origin, strain number, year of isolation, followed in parenthesis by the subtype. For example: A/equine/Prague/1/56 (H7N7) and A/equine/Miami/2/63 (H3N8) Genetic Changes in Influenza A Viruses

Antigenic shift [major antigenic change]. This involves the acquisition of a gene for a completely new HA or NA, as a result of genetic reassortment.

Antigenic shift in nature occurs when the prevailing human strain of influenza A virus concurrently infects a pig infected with an avian influenza A virus [influenza A viruses from birds grow poorly in humans and vice versa; however, both avian and human influenza viruses can replicate in pigs]. Only a reassortant containing mainly genes derived from the human virus but the HA gene derived from the avian virus is able to infect humans and possibly initiate a pandemic.

Antigenic drift [minor change]. Results from point mutations within a HA or NA subtype.

Mutations affecting neutralizing epitopes produce strains each antigenically slightly different from its predecessor. Equine Influenza

Equine influenza is an acute, extremely contagious, febrile respiratory disease of horses, donkeys and mules. Etiologic agent. Two antigenically distinct influenza virus A have been identified in horse populations worldwide.

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Equine influenza A/H7N7; [previously equine influenza A1]. The last major disease outbreak associated with H7N7 occurred in 1979. Equine influenza A/H3N8; [previously equine influenza A2]. Has been associated with all recent outbreaks of equine influenza. The virus has undergone a minor antigenic drift since its isolation in 1963. The virus is very unstable under ordinary environmental conditions.

Distribution. Worldwide except New Zealand, Iceland, etc. Transmission. Aerosolized infectious exudates. No carrier state [horses may shed virus for up to a week following recovery]. Pathogenesis. The virus replicates in the epithelial cells of the respiratory tract. Systemic findings have been attributed to cytokine response to infection.

Lesions observed may include laryngitis, tracheitis, bronchitis, interstitial pneumonia, pleuropneumonia, and interstitial myocarditis [rare].

Clinical features. IP: 1-3 days.

Sudden onset, rapid spread, high fever, conjunctivitis, reddening of the nasal mucosa, and increased nasal and conjunctival exudates. Dry, hacking, paroxysmal cough; abortion in mares with prolonged fever. Recovery occurs in about 7 to 10 days. Mortality is rare; it is more frequent if there is intercurrent bacterial infection.

Diagnosis

Virus isolation and identification: Nasal mucus and lungs. The virus grows well in embryonated eggs [allantoic or amniotic routes] and cell culture. Virus is identified using hemagglutination inhibition test. Paired serum samples: HI test.

Control. Horses are vaccinated with a bivalent inactivated A/equine (H7N7) and A/equine (H3N8) antigens or with modified live virus vaccine [Flu Avert I.N.]. Canine Influenza Canine influenza is a highly contagious respiratory disease that present with clinical signs that mimic kennel cough [eg, fever, coughing, nasal discharges, etc].

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In January 2004, an outbreak of respiratory disease was reported in 22 of 80 greyhounds from a Florida racing kennel. The dogs presented with signs of kennel cough. Eight of the dogs died from hemorrhagic pneumonia. All 80 of the greyhounds were diagnosed either with equine influenza virus A [H3N8] or had antibodies to the virus. From June to August of 2004, outbreaks of similar respiratory disease were reported at 14 tracks in 6 states [Alabama, Arkansas, Florida, Kansas, Texas, and West Virginia]. Serologic evidence indicates that the influenza virus is circulating in other dog breeds.

Etiologic agent. Canine influenza A virus [H3N8]. The virus is a mutated strain of equine influenza virus A [H3N8].

The CIV has been confirmed in 30 states and Washington, DC.

Transmission. CIV is transmitted mainly through aerosolized respiratory discharges. Virus is shed during the incubation period and for 7 to 10 days from the onset of clinical signs.

Direct dog-to-dog contact [infected dog coughing and/or sneezing on another dog]. Asymptomatic dogs can shed and spread the virus.

Pathogenesis. Virus replication occurs in the epithelial cells of the nasal, tracheal, and bronchial airways.

Host inflammatory response results in rhinitis, tracheitis, bronchitis, and bronchiolitis. Damage to the respiratory mucosa predisposes the respiratory tract to 2o bacterial infections, leading to mucopurulent nasal discharges and coughing.

Clinical signs. Morbidity: ~100% infection with up to 80% morbidity. All dogs, regardless of age or breed, are susceptible to CIV. Two forms of the disease have been reported:

Mild form. Majority of infected dogs show this form. Low-grade fever, nasal discharge [may be purulent], and protracted soft, moist cough [may last up to 21 days; some dogs may have a dry cough].

Severe form. Mortality: up to 8%. High fever [104oF to 106oF], increased respiratory rate with dyspnea, consolidation of lung lobes and other indications of pneumonia.

194 Diagnosis. Clinical diagnosis is difficult.


Virus isolation: nasal and pharyngeal swabs. Serology. Paired acute and convalescent serum samples. HI test.

Control. On May 27, 2009, the USDA licensed the first CIV vaccine for dogs [Intervet/Schering-Plough Animal Health].

The vaccine does not prevent infection; however, it significantly reduces the severity and duration of clinical illness. The vaccine also reduces the amount of virus shed and the period of shedding. Swine Influenza (Swine flu, Hog flu)

Swine influenza is an acute, highly contagious respiratory disease that occurs in all swine producing areas in the world. Etiologic agent. Influenza A virus subtypes H1N1, H1N2, and H3N2.

Two distinct antigenic variants of H1N1 are currently co-circulating in swine in different parts of the world. Influenza B and C viruses have been isolated from swine, however, they are yet to be associated with disease in swine.

Hosts. Swine, humans, and turkeys. Transmission. Outbreaks were once most common in late fall and winter but now occur throughout the year.

Aerosolization and pig-to-pig contact. Carrier pigs [may harbor virus for up to 3 months] are usually responsible for introducing the virus into previously uninfected herds.

Pathogenesis. Virus replication occurs in the epithelial cells of the nasal, tracheal, and bronchial airways. Infection progresses and affects the entire airway within a few hours.

Factors affecting the severity of the disease include 2o bacterial infections and migration of Ascaris suum larvae through the lungs.

Clinical features. IP: 1-3 days. It is a herd disease.

Fever, rhinitis, nasal discharge, sneezing, conjunctivitis, and weight loss; paroxysmal cough, rapid and labored breathing, anorexia, and prostration.

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In uncomplicated cases, swine recover in 3 to 6 days; mortality is < 1%. Intercurrent bacterial infections: mortality rate is higher.

Diagnosis. Same as equine influenza virus.

Virus may be identified using FA, hemadsorption inhibition test, HI, or ELISA tests. PCR can be used to detect virus in nasal swabs or lungs.

Control. Maintaining a closed herd to avoid introducing carrier pigs.

Attenuated and inactivated swine influenza vaccines are commercially available.


H1N1 Swine Flu Confirmed in a Cat November 5, 2009, Ames, Iowa. Two members of an Iowa family came down with swine flu. A couple of days later, their 13-year-old shorthair cat fell sick. It was later confirmed that the cat had swine flu. The cat recovered after being treated at Iowa State University College of Veterinary Medicine.

Ferrets. H1N1 has also been confirmed in two ferrets: one in Oregon [the ferret recovered] and the other in Nebraska [the ferret died].

Avian Influenza (Fowl plague) Avian influenza is characterized by respiratory, enteric and/or nervous system lesions in many kinds of poultry and birds. The most severe form, fowl plague, is characterized by a short course and extremely high mortality. Etiologic agent. Influenza A virus. The virus is unstable and the genome prone to change. A large number of different strains of influenza A virus have been isolated from birds, ranging from relative avirulence to highly virulent. Some avian influenza viruses are zoonotic.

A/chicken/Scotland/59 (H5N1): More virulent for chickens than turkeys. A/turkey/Ontario/7732/66 (H5N9): More virulent for turkeys than chickens.

Hosts. Many avian species can be infected with influenza viruses that may or may not cause disease. Turkeys are more commonly affected than chickens. Reservoir hosts. Migratory water fowl, especially ducks [refractory to the most virulent viruses] are the natural reservoirs. Transmission. Aerosolization; ingestion of feces contaminated water or feed.

196 Pathogenesis. tracts.

Virus replication occurs in both the respiratory and intestinal

In infections caused by highly virulent strains (highly pathogenic avian influenza A virus [HPAI]), there is viremia and multifocal lymphoid and visceral necrosis, resulting in myocarditis, encephalitis, myositis, etc. Neutralizing antibodies [hemagglutination inhibition Abs] are detectable within a couple of days after onset of disease and may persist for up to 18 months.

Clinical features. IP: Variable, from a few hours to a few days.


Coughing, gasping, blood-stained oral and nasal discharges, and diarrhea. Combs and wattles are cyanotic and there may be edema of the head; varying degrees of airsacculitis. Layers: drop in egg production and quality of the egg. Morbidity and mortality rates are variable, depending on virulence of the virus.

Diagnosis. Virus isolation: cloacal swab, using embryonated hens egg. Virus is identified using ELISA, HI, and single radial diffusion tests. Control. Quarantine and eradication. An interval must pass between slaughter and repopulation [determined by using sentinel birds].

Vaccination. There are currently no vaccines approved for use in birds in the United States. Eradication of HPAI is the goal.

197

REOVIRIDAE (Respiratory Enteric Orphan)


Properties of Reoviruses

Nonenveloped, spherical virions; 80 nm in diameter. Virions possess 3 concentric capsid layers, all icosahedral: outer, middle, and inner capsids. Genome consists of dsRNA, divided into 10-12 segments. Cytoplasmic replication, with formation of large intracytoplasmic perinuclear inclusion bodies. Genetic reassortment occurs between viruses within each genus or serogroup.

Genera

Genus Orthoreovirus. Nearly smooth outer capsid. Virions are stable over a wide pH range. Genus Rotavirus. Wheel-shaped outer capsid. The outer and inner capsids are readily dissociated from the core. This is necessary for infectivity. Rotaviruses are resistant over a wide pH range.

Genus Orbivirus. Outer capsid is shaped like a ring. Orbiviruses are stable over a narrow pH range [pH 6-8]. Genus Coltivirus: Colorado tick fever virus in humans. Genus Aquareovirus: Reoviruses of fish and shellfish.
Diseases Associated with Members of the Family Reoviridae Virus Host[s] Diseases Mild or inapparent upSeveral mammalian Mammalian reoviruses per respiratory infecspecies 1-3 tions Avian reoviruses 1-11 Chickens, turkeys and geese Sheep, deer and cattle Cattle Horses, mules, etc Deer Arthritis, enteritis, nephrosis, etc Bluetongue Similar to bluetongue African horse sickness Epizootic hemorrhagic disease

Genus Orthoreovirus

Orbivirus

Ovine orbivirus Ibaraki virus Equine orbivirus Epizootic hemorrhagic disease virus

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Equine encephalosis viruses 1-5 Rotavirus Rotaviruses Horses Abortion and encephalitis Enteritis

Almost all species

Bluetongue (Soremuzzle) Bluetongue is an arthropod-borne disease of sheep, wild ruminants, and rarely cattle and goats, characterized by congestion, edema, and hemorrhage. Disease occurs worldwide. Etiologic agent. Ovine orbivirus. More than 24 serotypes have been identified worldwide. Immunity is serotype specific. Serotypes 2, 10, 11, 13, and 17 are found in the United States. Hosts. Mostly sheep and the white-tailed deer. Varies from subclinical to severe depending on strain of virus, breed of sheep, age, and other stressors. Transmission. Bluetongue is a noncontagious disease.

Culicoides spp. [biting midges, no-see-ums], especially Culicoides variipennis var sonorensis, are the biological vectors. Culicoides spp. usually feed at dawn and dusk and they require standing water in which to breed. Transplacental transmission occurs in cattle and sheep. Semen that contains RBC or WBC infected with the virus may infect susceptible cattle.

Pathogenesis. The virus replicates in WBCs and endothelial cells of the blood vessels. Infection, but not replication, occurs in RBCs. Damage to endothelial cells is followed by DIC and degenerative and necrotic changes in various organs and tissues.

Viremia may last for 14-28 days in sheep and 70-90 days in cattle.

Clinical features. Similar in sheep and white-tailed deer. IP: 6-10 days. Morbidity: Up to 80%; mortality: up to 50%.

Fever, ulcerated mouth, cyanosis of tongue [bluetongue], profuse salivation and frothing, and nasal discharge [serous, later becoming mucopurulent]. Coronitis and laminitis resulting in lameness and recumbency. Fetal infection. Fetal death and/or abortion; CNS malformations such as hydranencephaly; porencephaly, etc. Cattle. Cattle are the reservoir and amplification host of the virus [minimal infection of endothelial cells]. Oral lesions may resemble FMD, etc.

199 Diagnosis. Season of the year.

Virus isolation. Heart blood [washed RBCs or buffy coat], spleen, liver, etc. IV inoculation into 10- or 11-day-old chicken embryos or cell culture.

Antigen and nucleic acid detection. ELISA, FAT, immunoperoxidase, immune electron microscopy, PCR, etc. Seroconversion [acute and convalescent sera].

Prevention and control. Bluetongue is a notifiable disease. Sheep should be put in barns at dusk [prime culicoides feeding period].

Vaccination. Attenuated monovalent virus vaccines to serotypes 10, 11, and 17 are used in the United States. Attenuated vaccines should not be used in pregnant animals; or during the vector season because the vectors may pick up the vaccine virus and transmit it to other animals. African Horse Sickness (AHS)

AHS is an acute or subacute, arthropod-borne disease of Equidae characterized by clinical signs and lesions associated with respiratory and cardiac impairment. Distribution. Endemic only in sub-Saharan Africa; outbreaks have occurred in the Middle East, North Africa, the Indian subcontinent, Spain, and Portugal. The disease has never been reported in the Western Hemisphere. Etiologic agent. Equine orbivirus. Nine serotypes are recognized. Hosts. Horses, donkeys, and mules. Dogs become infected after eating infected horse meat. Transmission. The principal vectors are Culicoides spp. Pathogenesis. Following the bite of an infective arthropod, the virus replicates in the local lymph nodes, resulting in a transient 1o viremia and infection of various organs and tissues in the reticuloendothelial system.

A 2o viremia results in vasculitis of small and medium-sized blood vessels.

Clinical findings Pulmonary form [Acute form]. IP: 3 to 5 days. Morbidity: high; Mortality: 95%.

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Fever, hyperpnea, spasmodic coughing; profuse sweating and frothy discharge from the nostrils. Conjunctiva is congested and the supraorbital fossa may be edematous.

Cardiac form. IP: 7 to 14 days. Morbidity: high; Mortality: 50-70%.

Fever, edema of the supraorbital fossa, eyelids, lips, tongue, intermandibular space, and subcutaneously towards the chest.

Mild form. IP: 4 to 14 days.

Fever which may last for a few days, congested mucous membranes, anorexia, and depression followed by recovery.

Diagnosis. AHS is a notifiable disease.

Virus isolation. Blood, spleen suspension, lungs, nasal swab. Intracerebral inoculation of 2 to 6-day-old mice. Identification of virus is done using serum neutralization assay.

Prevention and control. In endemic areas horses are vaccinated using attenuated live polyvalent vaccines. Immunity is serotype specific.

Horses should be protected from flies during disease outbreaks. U.S.A: A sixty-day quarantine on all horses imported from Africa, Asia, and the Mediterranean. Genus Rotavirus

Rotaviruses are a major cause of enteritis involving the small intestine in neonates of all domestic animals, including humans. Rotaviruses are host-specific; however, occasionally, cross-species infection can occur among certain species.

Rotavirus enteritis is a disease of neonates, 1 to 8 weeks of age. Severe disease occurs in neonates less than 3 weeks of age, however, only rarely is the disease seen during the first week of life. The epidemiology, clinical signs, and diagnosis are similar in all species. Rotaviruses are divided into 7 antigenically-distinct serogroups [A through G] based on differences in the group-specific inner capsid antigen VP6. Most rotaviruses affecting domestic animals belong to serogroup A. Within a serogroup, a number of serotypes are recognized based on the antigenicity of the outer capsid glycoprotein VP7 and spike protein VP4.

201 VP4 [protease-sensitive protein; P serotype]. 12 P [P1-P12] serotypes are recognized within group A rotavirus. Type-specific neutralizing antibodies inhibit viral attachment to host cell. VP7 [glycoprotein; G serotype]. Within Group A rotavirus, 14 G [G1-G14] serotypes are recognized. Serotype specific neutralizing antibodies inhibit virus uncoating.

Epidemiology Ingestion of contaminated food, water, or fomites. Fecal excretion of rotaviruses is extremely high [1 x 1011 viral particles/gram of feces]. Maximum shedding occurs on the 3rd and 4th days following infection.

Rotaviruses can survive in feces for several months leading to gross contamination of the rearing pens. A carrier state has been reported in some recovered animals. Pathogenesis

Cell attachment and penetration are the functions of the spike protein VP4 and its cleavage products VP5 and VP8.

Cleavage of VP4 is necessary for infectivity and is carried out by the pancreatic enzymes trypsin and chymotrypsin. VP4 neutralizing antibodies can bind to and neutralize VP5 and VP8 proteins. Rotaviruses infect the mature enterocytes at the apices of the villi of the small intestine, causing villus atrophy. Villi are shortened and covered with immature cuboidal cells. Cuboidal cells. Reduced absorptive surface; reduced enzymatic [disaccharidase] activity. However, they are relatively more resistant to viral infection, resulting in a self-limiting infection. In suckling animals, undigested lactose in milk promotes bacterial growth and exerts an osmotic effect. Bacterial fermentation of lactose contributes to acidosis.

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Clinical features. White scours or milk scours in calves, piglets, foals, and lambs. IP: 16-24 hours. Morbidity: 100%; Mortality: 0-50%.

Reduced colostral intake, pathogenic E. coli, poor hygiene, etc, contribute to the severity of the disease. Dehydration and/or 2o bacterial infection may result in death but most recover within 3 to 4 days.

Diagnosis. Fresh feces and segments of intestine.

Electron microscopy [Negative stain]. 1 x 105 viral particles/gm of feces; can be enhanced by using immunoelectron microscopy.

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Antigen detection. FA staining of frozen sections of intestine; ELISA, or latex agglutination tests can also be used.

Prevention and control. To prevent infection of newborn animals, antibody must be present continuously in the lumen of the gut.

This does not occur for more than about 7 days unless the dam is hyperimmunized against the virus, hence dams are vaccinated with inactivated vaccine to promote higher levels of antibody in the colostrum and milk. Avian Reoviruses (11 Serotypes)

Avian reoviruses cause arthritis/tenosynovitis in chickens. They have also been isolated from several other diseases in chickens and turkeys; however, their etiologic significance in the diseases is yet to be defined. Viral Arthritis and Tenosynovitis Occurs primarily in meat-type chickens [broilers] between 4 and 8 weeks of age; however, disease has been diagnosed in layers and turkeys. Etiologic agent. Avian reovirus. Transmission. The virus is acquired via the fecal-oral route; transovarial transmission and aerosol transmission may also occur. Pathogenesis. Following ingestion, the initial site of virus replication is the epithelium of the intestinal tract and the bursa of Fabricius.

Viremia, which occurs within 24 to 48 hours postinfection, disseminates the virus to multiple organs and tissues, including the hock joint. The virus can persist in the tendons for up to 22 weeks.

Clinical features. Inapparent infection occurs in many birds. Morbidity: ~ 100%; Mortality: <6%.

Swelling of one or both of the hock joints and of the associated tendons. Birds become lame and are reluctant to move. Chronic tendonitis may result, leading to rupture of the gastrocnemius tendon.

Diagnosis. Virus isolation from feces, cloaca, synovial fluid, and trachea. Control. Vaccination of breeder flocks to ensure that progeny chicks possess an adequate degree of passive immunity.

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CORONAVIRIDAE
Properties of Coronaviruses and Toroviruses

Pleomorphic or spherical virions [Genus Coronavirus] 80-220 nm in diameter; or disc, kidney, or rod-shaped [Genus Torovirus], 120-140 nm in diameter. Virions possess envelope with large, widely spaced, club-shaped peplomeres. Viral genome is a linear [+] sense ssRNA. Virions replicate in the cytoplasm and acquire their envelope from the endoplasmic reticulum; release occurs via exocytosis. No inclusion bodies. Coronaviruses cause persistent infections. Group II coronaviruses and toroviruses hemagglutinate and hemadsorb.

S protein [glycoprotein peplomer]. Mediates viral attachment and membrane fusion; induces neutrallizing antibodies. M glycoprotein. Transmembrane matrix protein. N nucleoprotein. Core nucleoprotein. HE [hemagglutinin-esterase] protein. Found in group II coronaviruses and toroviruses. Binds to neuraminic acid on cell membranes. Neutralizing antibodies are produced against HE protein.

Genera

Genus Coronavirus: Contains several pathogens of mammals and birds. The genus is subdivided into 3 groups based on genetic and serologic properties. Genus Torovirus: Contains two animal viruses.
Antigenic Groups and Diseases Caused by Coronaviruses Antigenic Group Group I Coronaviruses [Mammalian] Virus Porcine coronavirus Disease[s] Transmissible gastroenteritis [TGE] Asymptomatic infections Enteritis

Feline coronavirus

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Feline infectious peritonitis Canine coronavirus Human coronavirus 229-E Group II Coronaviruses [Mammalian and avian] Bovine coronavirus Porcine hemagglutinating encephalomyelitis virus Bluecomb virus of turkeys Group III [Avian] Infectious bronchitis virus Enteritis Common cold Gastroenteritis Vomiting, wasting, and encephalomyelitis Enteritis Tracheobronchitis, nephritis

SARS [Severe acute respiratory syndrome]: SARS-CoV

Transmissible Gastroenteritis (TGE) of Swine TGE is a contagious enteric disease affecting swine of all ages, with diarrhea and vomiting the prominent clinical signs. It is one of the major causes of death in young piglets. The disease occurs worldwide. Host. Swine. Dogs and cats can become infected and shed virus in their stools for up to 2 weeks. Etiologic agent. Porcine coronavirus. One serotype. The virus is antigenically related to canine coronavirus and feline coronavirus.

The virus is inactivated by high temperatures [> 32oC], sunlight, and a variety of chemical disinfectants. Porcine respiratory coronavirus. PRC is a deletion mutant of the virus causing TGE. The mutant has lost its enterotropism and has acquired an affinity for the respiratory tract. Mild respiratory disease without diarrhea has been reported in swine in Europe and the United States.

Transmission

Highly contagious disease with morbidity approaching 100%. Within a herd. Fecal-oral cycle. Infected pigs shed virus for 2-3 weeks. Herd-to-herd. Newly introduced infected animals, especially feeder pigs [chronic carrier pigs shed virus for up to 10 weeks]; mechanical spread of virus via fecal contamination of footwear, clothes, etc. Dogs, cats, foxes, and starlings, may be involved in the herd-to-herd spread of the virus during epidemics.

206 Pathogenesis. The virus is acid labile; however, in young piglets their gastric secretions are not yet as acidic as those of older animals and their diet of milk buffers the gastric acid, thus protecting the virus.

The virus infects and destroys the villous enterocytes of the small intestine, which results in villous atrophy. Villous atrophy results in impaired digestion, accumulation of fluids in the intestine and diarrhea.

Clinical features. IP: 18 to 72 hours. The clinical signs, duration of the disease, and mortality are inversely related to age. Epidemic TGE: Occurs after introduction of the virus into a susceptible [seronegative] herd. Course: 2 to 4 weeks.

Inappetance, diarrhea, and vomiting. Mortality in piglets under 2 weeks of age may approach 100%. Lactating sows often develop anorexia and agalactia.

Endemic TGE. Persists in partially immune herds with frequent or continuous farrowings or frequent addition of susceptible swine, eg, feeder pig operations.

Pigs from 1 to 3 weeks of age: Diarrhea. Mortality: 10-20%.

Diagnosis. Clinical signs and herd history.

Detection of virus in stool: ELISA or immune electron microscopy. Virus isolation in cell culture. Detection of viral antigen in mucosal smears or sections of small intestine using fluorescent antibodies or immunoperoxidase stain. Detection of rising antibody titer in paired sera [four-fold increase].

Prevention and control. Good sanitation and management practices.

Sows are vaccinated with attenuated vaccines 3 weeks before farrowing to ensure high levels of colostral antibodies. Alternatively, virulent virus may be administered to pregnant sows [there is no viremia], to boost antibody levels in colostrum and milk. Feline Infectious Peritonitis (FIP)

FIP is a contagious, progressive, debilitating, fatal disease of domestic and wild cats worldwide. All age groups are susceptible, however, most cases of FIP are seen in cats under 2 years of age.

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Catteries. More than 90% of cats are seropositive. Clinical disease is much lower [<10%].

Etiologic agent. Feline coronavirus (FCoV). Two serotypes of FCoV have been identified, serotypes I and II, based on cytopathogenicity and degree of neutralization by antisera to canine coronavirus [CCV].

Serotype II is a recombinant of FCoV and CCV. Both serotypes I and II can mutate and cause feline infectious peritonitis, however, serotype I appears to be more prevalent in field infections. FIPV is an escape mutant of FCoV [avirulent or causes a mild enteritis]. Feline coronaviruses cross-react with porcine coronavirus [TGE] and canine coronavirus. FCoVs are fairly stable in the environment and can survive for several weeks. The viruses are destroyed by household bleach [Clorox] at 1:32 dilution.

Transmission. FCoV is shed mainly in the feces, however, in early infection it may be found in the urine, saliva, and possibly respiratory secretions.

Primarily fecal-oral transmission; aerosol transmission is also possible. In utero infection is suggested by the observance of FIP in some stillborn kittens.

Pathogenesis. In vivo mutations of avirulent FCoV results in the highly pathogenic FIPV. Mutations in the gene encoding the virion S glycoprotein results in the ability of the mutant[s] to infect and multiply in monocytes and macrophages.

Multiplication in monocytes/macrophages results in dissemination of virus to multiple organs and tissues and production of high virus titers. In vivo mutations can occur in multiple organs and tissues, including the intestinal tract and regional lymph nodes. The course of FIPV infection is determined by the nature of the immune response. Cats that produce humoral but fail to mount a cell-mediated immune response develop effusive disease. FIP antibodies are not protective; additionally, they enhance virus uptake by macrophages and monocytes, enhancing replication in these cells. A soluble protein coded by some mutant strains induce T cell apoptosis, thus impairing cell-mediated immune responses

208 Cats that mount a rapid cell-mediated response usually do not develop disease, however, they harbor the virus latently and persistently. Cats producing a partially protective cell-mediated response develop the noneffusive form.

Intercurrent diseases, especially feline leukemia and feline immunodeficiency, stress, parasites, etc., may exacerbate the impact of FIPV infection.

Gross lesions observed in FIP are multiple grayish-white nodules, which, as the disease progresses, may become chronic pyogranulomatous lesions [foci of lymphocytes, macrophages, neutrophils, and plasma cells]. The lesions are observed in various organs and tissues, including liver, lungs, kidney, pleura, pericardium, meninges, brain, uvea, etc.

Clinical features. Disease is sporadic, insidious and characterized by anorexia, chronic fever, depression, and progressive debilitation. Classic [effusive, wet, diphtheritic] form. 75% of FIP cases. IP: 1-2 months.

Deposition of immune complexes (virus-antibody) [type III hypersensitivity] in small blood vessels results in complement activation and C5a recruitment of neutrophils.

209

Frustrated phagocytosis results in vascular and perivascular lesions, with leakage of plasma proteins and fluids into body cavities. Most prominent is the peritoneal effusion; there may also be pleural and pericardial effusions. The abdomen is distended with a viscous yellow ascitic fluid; may result in adhesion of intestinal tract and visceral organs. Variable course, from 5 to 7 weeks. Mortality rate: 100%

Noneffusive [dry, parenchymatous] form. 25% of FIP cases. IP: Several weeks to months. Fluid accumulation is minimal.

Characterized by multiple pyogranulomatous lesions. The CMI response of the cat produces localized perivascular infiltrations of inflammatory cells in the parenchyma of multiple organs. The cellular infiltrates cause local tissue necrosis and functional disruptions. Meningoencephalitis [ataxia, tremors, convulsions, and paralysis]. Bilateral ocular involvement [granulomatous uveitis] Pyogranulomatous pneumonia Hepatomegaly, renal insufficiency, etc.

Protracted course; some cats may survive for as long as a year or more.

Diagnosis. History and characteristic clinical signs.


Biopsy of affected organs to show pyogranulomatous lesions [more reliable]. Hypergammaglobulinemia. Serology. Detection of coronavirus Ab titer using IFA and ELISA tests. Antibody titer in cats with FIP usually increases from 1:100 to 1:3200 or greater. Some cats with FIP remain seronegative or have very low Ab titer and some healthy cats with no clinical signs of FIP have high titers.

Control. Segregation of infected cats from susceptible cats. Detection of carrier cats requires positive fecal RT-PCR results for 9 months.

Vaccination. Primucell FIP vaccine [Ts, 33oC] modified live virus vaccine. It is instilled intranasally where conditions favor a low level of viral replication [CMI response] without antibody or low antibody formation. Thus, local immunity is produced at the site where FCoV first enters the body, the oropharynx. However, field reports are variable concerning the efficacy of the vaccine.

210 Bovine Coronavirus Enteritis Etiologic agent. Bovine coronavirus . One serotype. Does not react with any other coronaviruses. May be associated with winter dysentery in adult cattle.

The virus is moderately labile and may survive for 1 to 2 days at room temperature.

Transmission. Fecal-oral. Long-term persistent infections do occur, producing inapparent persistent carriers. Pathogenesis. Similar to rotavirus diarrhea but more severe.

The virus multiplies in the absorptive epithelial cells of the villi of the small intestine and in the superficial and crypt epithelial cells of the colon. Diarrhea is due to the destruction of the absorptive epithelium of the small intestine and, to a lesser extent, the large intestine.

Clinical features. Commonly seen in calves about 1 to 4 weeks of age.


Diarrhea may last for 4 or 5 days. Colibacillosis often a 2o complication. Morbidity: up to 100%; mortality is between 0 and 50%.

Diagnosis. Similar to rotavirus diarrhea. Control. Management practices to reduce exposure are important.

Dam is immunized to promote elevated antibody levels in the colostrum. Encephalomyelitis-Vomiting and Wasting Disease Complex of Swine

The disease in suckling piglets is caused by a neurotropic porcine coronavirus [hemagglutinating encephalomyelitis virus, HEV]. The disease has been reported in North America, Europe, and Australia. Hosts. The disease is observed in pigs less than 4 weeks of age. Transmission. Spread occurs mainly via aerosols; virus may also be ingested. Pathogenesis. Virus multiplication occurs mostly in the nasal mucosa, tonsils, and lungs, and to a limited extent, the small intestines.

Virus spread to the CNS occurs via peripheral nerves. The site and extent of CNS damage apparently determines the type and severity of clinical signs.

211 Vomiting is due to virus replication in the vagal sensory ganglion [ganglion distale vagi] and disturbance of the vomiting center. Infection of cerebral and cerebellar neurons produces motor disturbances, etc.

Infection of other organs does not contribute significantly to the pathogenesis of the disease.

Clinical features. Most infections are subclinical. Differences in pathogenicity of virus strains, age, and susceptibility of the litter influence which form the disease takes. However, the two forms of the disease can occur in the same outbreak.

Acute form [encephalitic form]. IP: 4-8 days. Course: 2-3 days. Occurs in piglets under two weeks of age. Anorexia, hyperesthesia, muscle tremors, incoordination, collapse, and paddling movements. Vomiting may occur for 1-2 days, but is rarely severe. Morbidity and mortality may approach 100%. Death may occur within 24 hours after the initial appearance of clinical signs.

Vomiting and wasting disease [VWD]. IP: 4-7 days. Reported in piglets under 4 weeks of age. Anorexia; repeated retching and vomiting leads to emaciation. Disease may last for several days to weeks; morbidity and mortality approaches 100%.

Diagnosis. Specimen: Brain stem.


Demonstration of HEV-infected neurons by FAT. Virus isolation. Cell cultures of porcine origin. Virus may be isolated from the brain of piglets with acute encephalomyelitis. VWD. Due to the chronic course of the disease, virus isolation is usually not possible.

Virus identification. Virus is identified using hemagglutination inhibition test, FAT, neutralization test, RT-PCR, electron microscopy, etc. Serology. Demonstration of four-fold increase in antibody titers in surviving piglets.

212 Pathology. Gross lesions are not observed at necropsy.

Histopathology. Nonsuppurative encephalomyelitis, perivascular cuffing, neuronal necrosis, etc.

Control. Virus infection is widespread in swine breeding herds; maternal antibodies protect piglets for 4 to 15 weeks. There is no vaccine available. Avian Infectious Bronchitis (Gasping disease) Avian infectious bronchitis is a highly contagious disease of chickens, usually manifested as a respiratory condition, especially in young chickens. The disease occurs worldwide. Hosts. Chickens of all ages but severe disease occurs in young chickens. Etiologic agent. Avian coronavirus. Eight serotypes have been identified. Some may cross-protect. Some strains are nephropathogenic.

The virus can survive on fomites for several days and possibly weeks.

Transmission. Mainly via aerosols of contaminated droplets; ingestion of fecescontaminated feed and water.

A short-term carrier state [up to 50 days] may be observed in some birds.

Pathogenesis.

Initial replication occurs in the respiratory tract, followed by viremia, which distributes the virus to multiple organs and tissues. Some strains cause permanent damage to the oviduct if infection occurs in chickens less than 2 weeks of age. Nephropathogenic strains [Gray, Holte, and Australian T]. Enlarged kidneys with tubules filled with urates [uric acid].

Clinical features. IP: 18-36 hours. Young chicks. Depression, sneezing, coughing, and tracheal rales. Growth may be stunted.

Course: 7-21 days, depending on severity of disease. Mortality is usually 2530%, but can reach 75% with infection by highly virulent strains. Intercurrent infection with E. coli and/or Mycoplasma spp., increases the mortality rate.

213 Layers. Less severe respiratory signs; marked drop in egg production [20-50%].

Eggs: Soft-shelled, misshaped and rough-shelled. [watery albumen].

Internal quality is poor

Diagnosis. Clinical signs.


FAT staining of tracheal tissue smears. Virus isolation. Specimens: Tracheal swab, lung, bronchi, kidneys, etc. Embryonated eggs: Allantoic sac route. Dwarfing or stunting of some embryos; urates in the mesonephros.

Virus identification using AGID, electron microscopy, DNA probes, or PCR.

Immunity. Chickens that recover from natural infection are solidly immune to reinfection with the homologous virus strain for at least 6 to 8 months. Control. Many vaccines are available commercially for use in chickens. Genus Torovirus Contains elongated [rod shape] or kidney shaped viruses. peplomers are responsible for hemagglutinating activity. The glycoprotein

Negatively stained toroviruses

Bovine Torovirus (Breda virus) The virus is associated with enteritis in neonatal calves. From serologic evidence, Breda virus infections are common in the United States, Canada, and in several European countries. Approximately 90-95% of cattle sera are positive for Breda virus antibody. Serotypes. Two serotypes based on hemagglutination inhibition reactions.

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Serotype 1. Represented by Iowa isolate 1 Serotype 2. Represented by Iowa isolate 2 and the Ohio isolate.

Transmission. Fecal-oral route. Pathogenesis. Following ingestion, the virus replicates in the epithelial cells of both the small and large intestines.

Both crypt and villous epithelial cells are infected, including the dome epithelial cells covering Peyers patches. Infected cells slough off, the villi become stunted and fused and crypts dilated.

Clinical features. IP: 48 to 72 hours.

Calves may show mild to severe diarrhea of 4 to 5 days duration, followed by rapid recovery or death after severe loss of weight and dehydration. Both the small and large intestines are infected; thus the diarrhea is usually more watery than that seen in uncomplicated rotavirus diarrhea.

Diagnosis. Fecal samples are taken during the first 3 days of diarrhea.

Immunoelectron microscopy, ELISA, HI tests, and FAT are used for antigen identification in feces.

Control. Good management practices. More than 90% of neonatal calves have high maternal antibodies to the virus.

Maternal immunity wanes after a few months, followed by active seroconversion between 7 and 24 months of age. There is no vaccine for Breda virus.

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TOGAVIRIDAE
Properties of Togaviruses

Spherical, enveloped virions with icosahedral capsid, 70 nm in diameter. Genome is a linear [+] sense ssRNA. Cytoplasmic replication; followed by budding from cytoplasmic membrane. Virions do not form inclusion bodies. The envelope glycoprotein peplomer is a heterodimer consisting of: E1 protein [binds to host cell receptor] and E2 protein [facilitates virus entry into host cell by binding to cellular proteins, followed by receptor-mediated endocytosis]. Neutralizing antibodies are produced against E1 and E2 proteins. The virions are not very stable in the environment and are readily inactivated by common disinfectants. Togaviruses hemagglutinate RBCs of various species.

Genus Alphavirus. Arthropod-borne viruses. In vertebrates, alphaviruses cause cytolytic infections. Infection in invertebrates is noncytolytic and is persistent.
Diseases in Domestic Animals Genus Alphavirus Virus WEE, EEE, VEE viruses Highlands J virus Semliki Forest virus Getah virus Rubivirus Rubella virus Host[s] Horses, humans Horses Horses Horses Humans Disease Encephalitis Encephalitis Febrile disease Febrile disease German measles

Western, Eastern, and Venezuelan Equine Encephalitis (Equine Sleeping Sickness) WEE, EEE, and VEE are infectious diseases affecting horses characterized by signs of deranged consciousness, motor irritation and paralysis. The diseases are limited to the Americas. Etiologic agents. Western, Eastern, Venezuelan strains of equine alphavirus.

WEE virus. Several antigenic variants of WEE virus have been identified. Least virulent of the alphaviruses. Case fatality rate in horses is 20-40%.

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EEE virus. One EEE virus strain; however, two antigenic variants have been identified: North American and South American variants. Most virulent for all species; case fatality rate is about 90-98%.

VEE virus. VEE viruses are classified into six subtypes [I-VI]. There are six serotypes of subtype I [IAB-IF]. Endemic subtypes [II-VI; and ID-IF] are avirulent or of low pathogenicity for equines and are maintained in rodent-mosquito-rodent sylvatic cycles. Epidemic serotypes IAB, IC and IE [isolated only during epidemic outbreaks] cause diseases in horses and humans. Case fatality rate is 5080%. Infections occur in dogs, but clinical disease is rare. Phylogenetic evidence suggests that epidemic serotypes IAB and IC are mutants of less virulent endemic ID strains.
Subtypes IIX Subtype I

III

IV

VI

IAB

IC

IE

ID

IF

Endemic subtypes X Everglades virus

Epidemic serotypes

Endemic serotypes

Epidemiology. Under natural conditions, these are diseases of various species of birds and rodents and only accidental infections occur in humans, horses, mules, and donkeys. All three diseases occur only in the Americas. WEE. The virus was first isolated from the brain of an encephalitic horse in Merced County, CA in 1930. Since then it has been isolated from virtually all countries in the western Hemisphere.

In the USA, it is predominantly seen west of the Mississippi river as a disease in humans and horses but has also been isolated in the Eastern USA. The virus is maintained in an endemic cycle involving domestic and passerine birds and Culex tarsalis.

217 Occasionally, small mammals become amplifying hosts.

Because WEE virus can replicate in mosquitoes at cooler temperatures than other arthropodborne viruses, epidemic disease in horses and humans may occur early in the summer.

EEE. The virus was first isolated from horses in New Jersey in 1933. EEE is found in the eastern USA, Central and South America, and the Caribbean.

The virus is maintained in a sylvatic cycle involving wild birds [asymptomatic infection] and Culiseta melanura mosquitoes [the mosquitoes feed almost exclusively on birds]. Other species of mosquitoes [Aedes spp., etc] which feed on wild birds transmit the virus to humans, horses, and pheasants. EEE virus causes a devastating disease with high mortality in pheasants [5070%], chickens [80%], humans, and horses.

VEE. The virus was isolated in 1938 in Venezuela. The virus occurs in Venezuela, South and Central America. Epidemic types of VEE virus infection of horses have not been reported in the USA since the last major outbreak in 1971 in Texas. In 1995, a severe outbreak occurred in Mexico.

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An endemic type of VEE virus [Subtype II, Everglades virus] is present only in South Florida. The endemic variants are maintained in a sylvatic cycle involving rodents and other small mammals and Culex spp. of mosquitoes.

Transmission. The diseases are seasonal and occur from June to October, paralleling the mosquito season.

Mosquitoes become infective 7-20 days after feeding on a reservoir host or horse experiencing a high-titer viremia. WEE and EEE. Humans and horses are considered dead-end hosts because the transitory viremia rarely exceeds threshold dose for mosquitoes. Some reports indicate that a few horses develop high-titer viremia in EEE infections and thus can transmit the virus to mosquitoes that feed on them.

VEE-induced viremia in horses [1 x 108-10 particles per ml of blood] persists throughout the course of the disease and exceeds the threshold dose for mosquito vectors, therefore, humans and horses are not dead-end hosts for VEE.

Pathogenesis. Alphavirus infections are characterized by biphasic illness: an early self-limiting viral replication phase in extraneural peripheral tissues followed by an often fatal central nervous system [CNS] phase.

Following the bite of a mosquito, viral replication occurs in cells near the entry site, including fibroblasts, fibrocytes and Langerhans cells. Infected Langerhans cells may then transport the virus to the regional lymph nodes. The ensuing 1o viremia leads to viral invasion of extraneural tissues and organs, eg, myeloid and lymphoid tissues, skeletal muscle myocytes, etc. Osteoblasts and dendritic cells are major cellular targets of alphaviruses and are the cells that mostly account for the high-titer secondary viremias.

Innate responses, principally type 1 interferon production, account for the selflimiting viral replication in peripheral tissues. Hematogenous spread of the virus to the CNS results in viral replication in neurons and neuronal necrosis. Antibodies are principally responsible for viral clearance from the CNS.

Lesions [nonsuppurative encephalomyelitis] in the CNS are localized mainly in the gray matter of the cerebral cortex, thalamus and hypothalamus, with minor involvement of the medulla and spinal cord.

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Congenital [transplacental] transmission of the VEE virus has been reported in pregnant mares infected near term.

Clinical features: IP: 1-2 weeks. Course: about 7 days.

Infection may be subclinical, characterized by only a transient fever and anorexia if invasion of the CNS does not occur. Severe disease: depression, anorexia, tachycardia, occasional diarrhea, and fever. Animal walks aimlessly into objects [impaired vision], adopts a sleepy attitude, and terminally, there is paralysis of portions of the bodylower lip becomes pendulous, tongue protrudes, or difficulty in walking.

Diagnosis. Specimens: Blood, brain.

Serology. IgM capture ELISA to detect IgM in a single serum sample [IgM antibodies are detectable prior to the 2nd week of illness]. Paired serum samples. Acute and convalescent sera taken 10-14 days apart.

Virus isolation. The viruses are extremely labile and disappear from infected tissues within a few hours of death. Intracerebral inoculation of suckling [2-4-day-old] mice; Aedes albopictus cell culture or vero cells can be used. Virus isolates can be identified using hemagglutination-inhibition test, ELISA, virus neutralization test, PCR, IFA, etc.

Control. Recovery confers immunity for 2 years or longer.

EEE, WEE, and VEE are controlled with the use of bivalent or trivalent inactivated vaccines. The vaccines are given annually in the spring in 2 doses 7 to 10 days apart. An attenuated virus vaccine for VEE is also available. In areas where mosquitoes are active year round, foals are vaccinated when 3, 4, and 6 months of age and annually thereafter.

Humans. Clinical signs are similar to that observed in horses. Case fatality rate in EEE is about 50-75%; WEE is about 3-10%; and VEE is about 20-30%.

Abortions and fetal deaths have been reported in pregnant women.

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FLAVIVIRIDAE
Properties of Flaviviruses

Spherical, enveloped virions, with icosahedral capsid; 50 nm in diameter. Genome is a linear [+] sense ssRNA. Cytoplasmic replication. Virions do not form inclusion bodies. The virions are not very stable in the environment and are readily inactivated by heat and ordinary disinfectants.

Genus Flavivirus: Most members of the genus are maintained in nature in arthropod-vertebrate-arthropod cycles; mostly ticks and mosquitoes. Genus Pestivirus: Pestiviruses are closely related antigenically. Unlike flaviviruses, they do not multiply in invertebrates. Genus hepacivirus. Contains human hepatitis C and G viruses.
Diseases Caused by Flaviviruses and Pestiviruses Genus Flavivirus Host[s] Sheep Sheep Swine Avian; mammals Pestivirus Bovine Swine Sheep and lambs Disease[s] Louping ill Wesselsbron disease Japanese encephalitis; abortion West Nile meningoencephalitis Bovine viral diarrhea-Mucosal disease Hog cholera Border disease

Other flaviviruses cause important human diseases: Yellow fever virus, St. Louis encephalitis virus, Dengue fever virus, etc.

DISEASES CAUSED BY PESTIVIRUSES Bovine Viral Diarrhea-Mucosal Disease [BVD-MD] BVD and MD are related but clinically different diseases caused by the same virus. The diseases in cattle and other ruminants are characterized by gastroenteritis with severe diarrhea and ulcerations of the muzzle, nasal, and oral cavities. The diseases occur worldwide in both beef and dairy cattle.

221 Etiologic agent. Bovine pestivirus. Based on genotypic differences [in the 5 untranslated region of the viral genome], BVDV isolates are divided into genotypes I and II. Considerable antigenic diversity and antigenic cross-reactivity exist among Type I isolates. Type II isolates are antigenically distinct.

Genotype I and II isolates are divided into two biotypes: noncytopathic [NCP] and cytopathic [CP] strains, based on cytopathic effects when grown in tissue culture and is not related to virulence in vivo. Noncytopathic strains. The majority of field isolates are NCP strains [approximately 95%]. Genotype I and II NCP strains are associated with enteric, congenital, reproductive and other diseases. Only the NCP biotype can cross the placenta and establish persistent infection in the fetus. Cytopathic strains. It is believed that all CP strains arise from mutation or genomic recombination of NCP strains. Genotype I and II CP strains are associated only with mucosal disease in calves already persistently infected with the NCP biotype. There is antigenic similarity between bovine pestivirus, porcine pestivirus, and ovine pestivirus.

Hosts. Natural infections occur mostly in cattle. Sheep, goats, deer, bison, and other wild ruminants and swine can be infected with the virus. Transmission. This is a highly contagious disease. The natural reservoir for the virus is the young cattle persistently infected [lifelong] with the NCP strain that constantly sheds the virus. Epidemiologic data in the US indicate that between 0.4 to 1.7% of cattle are persistently infected.

Ingestion of feed, water, and fomites contaminated with urine, oral and nasal secretions, or feces from infected cattle. Viruses can also be inhaled. Transplacental transmission occurs regularly.

Pathogenesis. Initial virus replication occurs in the oronasal mucosa followed by viremia and dissemination of virus throughout the body. Virus replication occurs in lymphoid tissues, especially Peyers patches, resulting in necrosis of leukocytes [leukopenia] and accompanying immunosuppression.

Depending on the virulence of the virus, erosive or ulcerative lesions extending from the mouth through the esophagus, forestomachs, abomasum, and intestine may be observed. In respiratory infections, the trachea is the primary site of lesions and tracheal ulcers may be seen.

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Transplacental infection. Nonimmune cow infected with NCP strain. Conception up to 30 days of gestation. Embryonic death with return of dam to estrus. 45 to 125 days of gestation. Fetal death and abortion, mummification, however, as high as 70% of fetuses can survive the infection; or immunologically tolerant persistently infected [PI] fetus. 125 to 175 days of gestation. Congenital defects including cerebellar hypoplasia, hydranencephaly, microencephalopathy, ocular abnormalities such as retinal atrophy, optic nerve neuritis and microphthalmia with retinal dysplasia. 180 days of gestation to term. The immune system in the fetus is fully developed [by about 125 days], hence an immune response is mounted by the fetus with elimination of the virus. At birth, the calf is seropositive but is virus free.

Clinical features. The outcome of infection is dictated by the age, pregnancy status, immunological status and virulence of infecting virus.

Inapparent or subclinical BVD. This is the most common form of BVD frequently observed in immunocompetent seronegative cattle 8-24 months of age. It is mostly inapparent or may present as one of high morbidity and low case-fatality rate. IP: 5-7 days. Mild fever and leukopenia followed by recovery. Some animals may have diarrhea, nasal and ocular discharges, and an erosive stomatitis.

Peracute BVD. Caused by highly virulent strains of NCP Type II isolates. The disease occurs in immunocompetent seronegative cattle. It is a severe form of the enteric disease with a high case-fatality among clinically ill animals. Thrombocytopenia and hemorrhagic syndrome. Observed in adult cattle and veal calves presenting with peracute BVD. It is characterized by fever, bloody diarrhea, hyphema, petechial and ecchymotic hemorrhages, epistaxis, etc. Case-fatality rate is about 25%. Pathogenesis of Mucosal Disease (MD)

MD usually occurs in cattle between 6 to 24 months of age. It is the result of PI immunotolerant calf becoming co-infected with a homologous CP strain [genetically and antigenically similar to the persistent NCP strain] or mutation, in vivo, of the persistent NCP strain to produce a homologous CP strain.

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Both NCP and CP viruses are isolated from cattle with mucosal disease. Infection with a heterologous CP strain results in antibody production and no mucosal disease.

Persistently Infected Calf and Mucosal Disease

Acute mucosal disease. It is a disease of sudden onset, with morbidity rate of 5 to 44% and case-fatality rate of 100%.

Fever, anorexia, profuse watery diarrhea [sometimes hemorrhagic], nasal discharge, erosive or ulcerative stomatitis, dehydration, and emaciation. Death occurs in 5-7 days after onset of clinical signs.

Chronic mucosal disease. Animals that survive acute mucosal disease may develop chronic mucosal disease.

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The animals are usually unthrifty and eventually die from chronic inanition [nutritional deprivation; wasting] within 18 months.

Diagnosis. Virus isolation. Buffy coat cells, blood, feces, nasal exudates, lymphoid tissues, and smears from ulcerative lesions.

Antigen detection. FA stain; PCR; nucleic acid hybridization probes, and immunoperoxidase staining. Paired acute and convalescent sera are tested using virus neutralization test.

Control. Recovery confers lifelong immunity. Colostral antibodies decline by 3 to 8 months in calves.

Both type I and type II inactivated and modified live virus vaccines are available. Because the virus is both fetotropic and immunosuppressive, MLV vaccines should be used only in nonpregnant cattle. Monoclonal antigen-capture ELISA test. Rapid and accurate detection of pestivirus-specific antigens in tissue samples, peripheral blood leukocytes, and blood clots of PI cattle. Herd Chek BVDV Antigen Serum Test Kit [IDEXX]. An enzyme immunoassay used to detect BVDV antigen in PI animals using plasma, serum, or whole blood. Detects Type I and Type II isolates; rapid testing methodresults are available in 2 hours. Hog Cholera

Hog cholera is a highly contagious febrile disease of pigs and is economically the most important disease of swine worldwide. Synonyms. Classical swine fever, swine fever, and European swine fever. Distribution. The disease is endemic in Asia, Africa, and South and Central America. It occurs intermittently in several European countries.

Hog cholera has been eradicated in the United States [1978], Canada, Australia, Great Britain, New Zealand, and some European countries.

Etiologic agent. Porcine pestivirus. There is only one antigenic type.

The virus produces little or no CPE in cell culture. Virus presence is detected using FA stain. The Virus does not hemagglutinate or hemadsorb.

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The virus is moderately fragile and does not persist in the environment or spread long distances by the airborne route.

Transmission. Virus may be ingested or inhaled. Carriage of virus between herds by inapparently infected pigs is significant in farm to farm spread.

Virus is found in lacrimal and nasal discharges, saliva, urine, feces, and tissues of infected pigs. Uncooked garbage and kitchen scrap feeding [FDA regulations require that garbage be cooked for at least 30 min before being fed to pigs]. Transplacental infection in pregnant sows.

Pathogenesis. Initial virus replication occurs in the tonsils with invasion of the regional lymph nodes.

1o viremia results in 2o replication in endothelial cells, bone marrow, and lymphoid organshemorrhages, edema, leukopenia, and thrombocytopenia.

Clinical features. Disease occurs in domestic and wild pigs. Peracute. Young swinedie without clinical signs. Acute. IP: 2 to 4 days. Course: 7-10 days. Mortality rate: 100%.

High fever, depression, anorexia, vomiting, diarrhea and/or constipation. CNS [hemorrhage/encephalomyelitis]: paresis, paralysis, circling, tremors, and occasionally, convulsions.

Subacute/chronic infections. Virus strains of low to moderate virulence.

Abortion, fetal death and mummification, and congenital anomalies [cerebellar hypoplasia]. Surviving piglets may be persistently infected [lifelong]. Immune complex-mediated glomerulonephritis [type III hypersensitivity]; secondary bacterial pneumonia and enteritis.

Pathology. Peracute. No gross lesions.

Acute. Petechial and ecchymotic hemorrhages, turkey egg kidneys, congestion and infarction of the spleen, etc. Subacute/chronic. Necrotic ulceration of the mucosa of the large intestine [so-called button ulcers in the colon].

226 Diagnosis. Virus isolation: tonsil, blood, spleen, lymph nodes, etc.

Antigen detection: FA or immunoperoxidase staining; ELISA. Serology: Virus neutralization assay, ELISA, etc.

Control. Hog cholera is a notifiable disease.

Endemic areas: Routine vaccination using an attenuated live virus vaccine. Quarantine in an outbreak. Hog cholera-free countries. Eradication. Detection and quarantine of infected herds, followed by slaughter and disposal of infected herds. DISEASES CAUSED BY FLAVIVIRUSES West Nile Meningoencephalitis

WNV was originally isolated in 1937 from a febrile woman in the West Nile area of Uganda, East Africa. Distribution. Africa, Middle East, Southern Europe, Parts of Asia, and Australia. The first outbreak of disease in the Americas occurred in New York City in 1999. West Nile virus infection has been reported in the Caribbean.

In August 1999, six individuals were admitted to Flushing Hospital in Queens, NY, with encephalitis. It was later confirmed that they were infected with West Nile virus [WNV]. During that same August, an animal pathologist at the Bronx Zoo observed an extensive epornitic of unknown etiology among migratory wild birds and the zoos avian collection. The epornitic turned out to be West Nile infection.

Hosts. Birds [including geese, raptors, and corvids], humans, horses, sheep, etc, and canidae. Cats can seroconvert without clinical disease. Etiologic agent. Flavivirus. Based on genetic analysis, WNV isolates are divided into two lineages.

Lineage type I WNV. Endemic in Europe, Israel, North America, and central and North Africa. Disease in horses is more severe. Lineage type II WNV. Endemic in central and southern Africa. Disease in horses is generally mild.

Epidemiology. Wild birds are the natural reservoirs of the virus.

227 The virus is maintained in a mosquito-bird-mosquito sylvatic cycle. WNV has been isolated from at least 37 mosquito species.

Viremia in amplifying hosts may last up to 7 to 14 days. During this time, mosquitoes feeding on the avian host become infected by the virus. Mammals are considered incidental or dead-end hosts, since virus titer during the viremic phase of the disease is too low and of short duration [ 2 days] for transmission to mosquitoes. In North America, the principal amplifying host is the house sparrow [Passer domesticus] and the principal vector is Culex pipiens. It has been determined that as few as 7 infected mosquitoes are sufficient to infect a seronegative horse. Other means of transmission include blood transfusion, transplacental transfer, breast milk and organ transplantation.

Pathogenesis. The degree of virulence of WNV among vertebrates is variable, ranging from subclinical to lethal infections.

Following virus inoculation, initial WNV replication occurs in epidermal Langerhans cells. The cells migrate to and seed draining lymph nodes, resulting in a primary viremia [2-5 days post-inoculation] and subsequent infection of peripheral organs and tissues.

WNV infects parenchymal cells of multiple organs, including the spleen, kidney, liver, and central nervous system, causing cell necrosis. Neuronal damage is most prevalent in the brain stem and anterior-horn neurons of the spinal cord. Meningitis, encephalitis, myelitis, etc.

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WNV is cleared from all organs and tissues within 2 to 3 weeks after infection. However, persistent viral infection in the brains of immunocompromised animals and humans has been reported.

Clinical signs. IP: 8-15 days; course: 2-3 weeks.

Horses of all ages and breeds are susceptible to infection. Biphasic fever, depression, weakness, muscle fasciculation, incoordination, etc. Severely affected horses are recumbent with flaccid paralysis. Mortality in horses with neurologic signs is 22 to 40%.

Humans. Inapparent infection is more common, however, it can result in fatal encephalitis [mortality: 10%].

Diagnosis. Virus isolation using Vero cells.

Antigen detection in tissues. Immunoperoxidase staining, FA stain; viral genome by reverse transcriptase-polymerase chain reaction [RT-PCR]. Antibody detection. ELISA, serum neutralization [plaque reduction neutralization], IgM capture assay, and paired acute and convalescent sera.

Control. Mosquito control.

A modified live, canarypox vectored vaccine [Recombitek, Merial], an inactivated vaccine [West Nile InnovatorTM, Fort Dodge Animal Health] and West Nile Innovator DNA vaccine are available for use in horses. Louping Ill [Ovine encephalomyelitis]

Louping ill is an infectious encephalomyelitis of sheep that has so far been reported only in Europe. The disease gets its name from the peculiar leaping gait of ataxic sheep. Hosts. Mostly sheep; occasionally seen in goats, horses, cattle, dogs and pigs. Transmission. The virus is transmitted between sheep by the bite of the tick Ixodes ricinus [reservoir and biological vector]. Transtadial transmission only. Pathogenesis. Following the tick bite, virus replication occurs in the regional lymph node to produce a viremia which peaks at 2 to 4 days, declining with the development of neutralizing antibody.

In most animals, CNS invasion occurs during the early viremic phase.

229

CNS. Virus replication results in severe inflammation throughout the CNS and necrosis of brainstem and ventral horn neurons. Lesions. Non-suppurative encephalitis.

Clinical findings. IP: 2 to 4 days.

In most sheep, infection is subclinical. Disease is seen most frequently in sheep less than 2 years of age. Biphasic febrile response; second peak coincides with the development of CNS signs. Cerebellar ataxia, tremors, hyperexcitability, and paralysis. Mortality rate in newly infected sheep is 60%; 5-10% in subsequent outbreaks.

Diagnosis. Clinical history.


Virus isolation from brain and spinal cord. Antigen detection; and detection of antibody using virus neutralization [VN] or hemagglutination inhibition assays. Necropsy findings. Gross changes are not observed in the brain. Perivascular cuffing, neuronal necrosis, etc.

Control. Tick control and immunization of lambs using an inactivated vaccine. Humans. The virus is transmitted to humans by ticks; during postmortem examination; handling of infected tissue or sheep; and inhalation of aerosol droplets.

Drinking of raw milk. Infected sheep and dairy goats excrete virus in their milk. The disease is biphasic; the first phase is influenzalike and the second phase is a meningoencephalitic syndrome that usually resolves without sequelae in 4 to 10 days.

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ARTERIVIRIDAE
Properties of Arteriviruses

Spherical, enveloped virions with icosahedral capsid; 50-70 nm in diameter. Linear [+] sense ssRNA genome. Cytoplasmic replication; envelope is acquired when virions bud into the endoplasmic reticulum, from where they are released via exocytosis. Do not form inclusion bodies. Arteriviruses frequently establish persistent infections. The viruses are moderately resistant in the environment. They are sensitive to drying, heat, and common disinfectants.

Diseases Associated with Arteriviruses Virus Equine arterivirus Porcine arterivirus Host Horse Swine Disease[s] Equine viral arteritis Porcine reproductive and respiratory syndrome [PRRS] Simian hemorrhagic fever None, but elevates lactic dehydrogenase levels in mice

Simian arterivirus Mice arterivirus

Monkeys Mice

Equine Viral Arteritis (EVA) EVA is an acute, systemic to subclinical respiratory disease of horses characterized by generalized vascular necrosis and by abortion storm.

EVA was first recognized in 1953 following an outbreak of severe respiratory disease and abortion on a breeding farm near Bucyrus, Ohio. The disease occurs worldwide.

Etiologic agent. Equine arterivirus. One serotype, however, several strains with variable degrees of virulence have been identified. The Bucyrus strain is the prototype strain. Hosts. Equine [reported mainly in standardbred and thoroughbred horses]. Transmission. Occurs through respiratory, sexual, and transplacental routes. Sources for aerosol transmission are aborted fetuses, fetal membranes and amniotic fluids of mares, and genital fluids of infected stallions [droplets remain infectious for up to 8 to 10 days in the environment].

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Venereal transmission from infected stallions to mares. Carrier stallions. On breeding farms the stallion plays an important role in the dissemination and perpetuation of EVA from year to year. A high proportion [30-60%] of stallions infected with EVA become long-term carriers Infection is confined to the reproductive tract, with the highest concentration of virus in the accessory sex glands and in the vas deferens. The carrier state is testosterone dependent, hence persistent virus shedding does not occur in geldings or mares. If persistently infected stallions are castrated, virus shedding in semen ceases; on the other hand, castrated horses given testosterone continue to shed virus.

Mares. Although the virus can be readily isolated from vaginal swabs obtained from mares during the early stages of infection, venereal transmission from acutely infected mares to stallions has not been documented. No carrier state in mares.

Transplacental transmission of equine arterivirus to a fetus late in gestation can occur and may result in a foal with congenitally acquired EAV infection.

Pathogenesis. Following inhalation, initial virus replication occurs in alveolar macrophages followed by invasion of the draining bronchial lymph nodes.

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Primary viremia results in virus replication in selected epithelia, endothelial cells, mesothelium, and smooth muscle of the media of small arteries. Damage to the blood vessels leads to generalized edema and hemorrhage. Secondary sites of replication include the kidneys, liver, and seminiferous tubules [temporary infertility in stallions]. Viruses can be found in almost all body fluids and tissues, except the brain. Virus can cross the placenta and infect the fetus, resulting in death. Abortion results from severe necrotizing myometritis and placental detachment. Abortion usually occurs within 7 to 10 days after the onset of maternal illness.

Clinical features. IP: 3 to 14 days. Most infections are asymptomatic.

Fever, depression, anorexia, lacrimation and conjunctivitis, diarrhea, petechiae of nasal mucosa and conjunctiva, and skin rash. Edema of the limbs, head, trunk and external genitalia in the stallion. 40-80% of mares may abort [abortion storm]. Abortion may occur anytime from 3 months to over 10 months of gestation. Disease is rarely fatal. Neonatal foals. Disease is characterized by fever, limb and facial edema, a progressive bronchointerstitial pneumonia and high mortality.

Diagnosis

Virus isolation: Various body fluids and tissues. Cell cultures: Rabbit kidney or equine kidney cell cultures.

Serology. FA staining of infected tissue culture; rising antibody titer using virus neutralization tests.

Control. Vaccination of horses with an attenuated live-virus vaccine or inactivated vaccine produces long lasting immunity.

Colts to be used for breeding are vaccinated at 6 to 8 months of age to prevent the establishment of persistent infections. Noncarrier stallions should be vaccinated annually at least 4 weeks before the breeding season. Carrier stallions can be mated only to mares that were vaccinated more than 3 weeks previously or are EVA seropositive; the bred mares are isolated from unvaccinated or seronegative horses for 3 weeks.

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Porcine Reproductive and Respiratory Syndrome (PRRS) PRRS is characterized by abortion or premature farrowing in sows or gilts, pneumonia in young swine, and preweaning mortality. Synonyms. Swine mystery disease [SMD]; swine infertility and respiratory syndrome [SIRS]. Distribution. The disease was first recognized in 1987-88 in swine herds in North Carolina, Iowa, and Minnesota and in 1991 in the Netherlands. However, it is now recognized as a major disease in swine populations worldwide. Etiologic agent. Porcine arterivirus. There are 2 genotypes: Lelystad virus [Netherlands, Europe] and VR2332 virus [North America]. Both genotypes are now endemic in the United States. Viral strains vary in virulence. Transmission. Virus is maintained in persistently infected swine for several months. Transmission occurs via aerosols, by contact and infected semen.

Virus is shed in nasopharyngeal secretions, urine, feces, semen [up to ~ 43 days], and mammary secretions.

234 Pathogenesis. Initial replication of virus occurs in pulmonary intravascular macrophages [PIM] and in alveolar macrophages, followed by a persistent viremia [up to 210 days], which disseminates the virus to various organs and tissues. The virus has a predilection for lymphoid tissues, eg, tonsils, spleen, etc. Clinical features. IP: 2 to 7 days. Adult swine. Mainly subclinical to a slight febrile response and anorexia of short duration. Mortality rates are low. Pregnant sows. Reproductive problems [SMEDI] include late-term abortions [virus is known to cross the placenta after the 72nd day of gestation], stillbirths and mummified fetuses, premature farrowings, and the birth of weak piglets.

Sows are resistant to infection with homologous PRRS but may abort again if infected with a heterologous strain [lack of cross protection]. Gross and histopathologic lesions are observed in the umbilical cord [arteritis leading to hypoxia] but not in stillborn or aborted fetuses.

Piglets. Young piglets exhibit depression, severe respiratory distress [interstitial pneumonia] with coughing and sneezing, conjunctivitis, and a poor growth rate.

Mortality rates may approach 100%. Secondary bacterial infections are common and may contribute to mortality.

Diagnosis. Virus is rapidly inactivated in aborted fetuses, hence specimens for virus isolation are collected from moribund live piglets.

IFA examination of infected cell cultures or frozen sections of lung and fetal tissues. Detection of IgG antibodies using ELISA or IFA in affected pigs or sows that have aborted; detection of virus in serum using PCR.

Control. Killed and attenuated vaccines for control of reproductive and/or respiratory forms of PRRS are available. The vaccines have been effective in controlling outbreaks and preventing economic losses.

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BUNYAVIRIDAE
Properties of Bunyaviruses

Spherical enveloped virions, 80-100 nm in diameter. Virions possess a segmented, [-] sense ssRNA genome. Cytoplasmic replication; budding into Golgi vesicles. Capped 5 termini of cellular mRNAs cannibalized [cap snatching] as primers for mRNA transcription. Genetic reassortment occurs between closely related viruses. Cytopathic virions. Several species hemagglutinate. Bunyaviruses are readily inactivated by detergents and common disinfectants.

Genus Orthobunyavirus; type species: Bunyamwera virus Genus Hantavirus; type species: Hantaan virus Genus Nairovirus; type species: Dugbe virus Genus Phlebovirus; type species: Rift Valley fever virus Genus Uukuvirus; type species: Uukuniemi virus
Diseases Associated with Bunyaviruses Genus Phlebovirus Orthobunyavirus Virus Rift Valley fever virus Akabane virus Host[s] Ruminants, humans Cattle, sheep, goats Disease[s] Hepatitis, abortion Arthrogryposis, hydraencephaly Arthrogryposis, Hemorrhagic enteritis

Cache Valley virus Nairovirus Nairobi sheep disease virus Sin Nombre, etc

Cattle, sheep Sheep, goats, humans

Hantavirus

Rodents, humans

Hemorrhagic disease, pulmonary syndrome Encephalitis, etc

Uukuvirus

Uukuniemi virus, etc

Several vertebrate hosts

Rift Valley Fever (RVF) RVF is an acute, arthropod-borne infection primarily infecting sheep, goats, cattle, and humans. It is characterized by hepatitis, abortion, and encephalitis. Distribution. Endemic in certain parts of Africa and the Arabian peninsular.

236 Etiologic agent. Phlebovirus. One serotype.

In target tissues, the virus replicates very quickly and to very high titer. It is rapidly cytopathic.

Transmission. The virus is transmitted by many species of mosquitoes, including Culex spp. and Aedes spp.

Interepidemic maintenance is via transovarial transmission in Aedes mosquitoes. During the rainy season, infected mosquitoes develop and transmit the virus to ruminants, which amplify the virus. Mechanical transmission occurs via Culicoides spp. and sandflies. Aerosol transmission occurs in humans.

Pathogenesis

Following arthropod inoculation or via the oropharynx, local replication is followed by a primary viremia that may last for 2 to 5 days. Virus replication in the liver and other major organs, leads to widespread cell necrosis. Extensive necrosis of hepatocytes occurs throughout the liver. There may be acidophilic intranuclear inclusion bodies in hepatocytes.

Clinical features. IP: Lambs/calves: 12 hours; sheep/cattle: 30 to 72 hours.

Sheep and goats. Fever, anorexia, bloody diarrhea, and mucopurulent nasal discharge. 90-100% of pregnant females abort [abortion storm]. Young animals surviving the hepatic infection may present with encephalomyelitis characterized by neuronal necrosis and perivascular cuffing. Mortality in kids and lambs occurs in 36 hours and is about 95 to 100%; it is 20-30% in adult sheep and goats.

Cattle. Disease is less severe, however, 90-100% of pregnant cows abort. Mortality rate in calves is about 70% and about 10% in cows.

Diagnosis. Based on clinical signs, virus isolation, and seroconversion.


Virus can be isolated in mice or cell culture. Serology. IgM capture enzyme immunoassay on single acute sera; ELISA, virus neutralization, or hemagglutination-inhibition assays on paired sera.

237 Prevention and control. Sheep and cattle are vaccinated using attenuated and inactivated vaccines. Humans. IP: 2-6 days. Humans may be infected by aerosol in the laboratory and during slaughter of viremic animals.

Clinical signs resemble influenza or dengue fever: sudden onset, fever, chills, malaise, headache, vomiting, etc. The disease may last for 4 to 6 days. Mortality rate may approach 10%. An inactivated vaccine is used in individuals at risk.

Akabane Disease Akabane disease is a congenital disease of cattle, sheep, and goats characterized by abortions, stillbirths, and birth of calves, etc. with skeletal abnormalities [eg, arthrogryposis] and neurological disorders [eg, hydranencephaly].

238 Distribution. The disease has been reported in Australia, Japan, Israel, Taiwan, and Turkey; the virus has been isolated in Kenya and South Africa. Etiologic agent. Orthobunyavirus. Transmission. The virus is transmitted by mosquitoes, Aedes spp. and Culex spp., and Culicoides spp. Pathogenesis. Following inoculation by the mosquito, the virus is carried to the placenta by maternal circulation, where it infects the developing fetus [there is no disease in the dam].

The main fetal infection is a necrotizing nonsuppurative encephalomyelitis and polymyositis. Severely affected fetuses die and are aborted. Surviving fetuses may develop hydranencephaly, arthrogryposis, cavitations of the cerebrum, torticollis, scoliosis, and kyphosis.

Diagnosis. Based on pathologic and serologic evidence.

Pathology. Samples of brain and cervical, thoracic, and lumbar regions of the spinal cord are submitted in 10% neutral formalin for histologic examination. Serology. Presence of virus-neutralizing antibodies in aborted fetuses or in precolostrum serum samples from the affected offspring.

Prevention. An inactivated vaccine is available in Japan and Australia. Cache Valley Disease Cache Valley disease is caused by ovine orthobunyavirus. The disease is characterized by abortions, stillbirths and congenital defects in sheep.

Cache Valley disease was first reported in Cache Valley, Utah. The virus has been isolated from mosquito pools in several states in the USA and in several provinces in Canada and Mexico. Cache Valley disease is similar to Akabane disease.

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CALICIVIRIDAE
Properties of Caliciviruses

Nonenveloped spherical virions; 40 nm in diameter. Icosahedral capsid with cup-shaped depressions. Genome is a linear [+] sense ssRNA. Replication occurs in cytoplasm; virions do not produce inclusion bodies. Caliciviruses are rapidly cytopathic. The virions are relatively resistant to heat and detergent-based disinfectants, but are intermediate in their pH stability [>99% are inactivated at pH 3].

Diseases Associated with Caliciviruses Genus Calicivirus Virus Feline calicivirus Porcine calicivirus Norwalk virus [humans] Hepatitis E virus [humans] Chicken calicivirus Bovine enteric calicivirus Porcine enteric calicivirus Rabbit calicivirus Disease[s] Respiratory disease Vesicular exanthema Diarrhea, vomiting, fever, etc. Hepatitis E Stunting, mortality in chicks Diarrhea, anorexia Diarrhea, anorexia Rabbit hemorrhagic disease

Feline Calicivirus [FCV] Infection The virus is one of the two major causes of respiratory disease in kittens; the other is feline herpesvirus 1. Disease occurs worldwide. Etiologic agent. Feline calicivirus.

Multiple strains of FCV exist which vary slightly in antigenicity, however, extensive cross-protection occurs, allowing for the use of monotypic vaccines. The vaccine, however, may not fully protect against all strains of FCV.

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Different strains of FCV vary in virulence. Some strains are associated mainly with subclinical infection or upper respiratory disease. Highly virulent strains regularly produce pneumonia, especially in young kittens. Two strains cause transient lameness [limping syndrome] with or without oral or respiratory disease in 8- to 12-wk-old kittens. There is transient fever, alternating leg lameness, and pain on palpation of affected joints.

FCV can persist for up to a week in a damp environment.

Transmission. The natural routes of infection are nasal, oral, and conjunctival.

The virus persists in tonsillar and other oropharyngeal tissues, hence a high percentage of recovered cats remain persistently infected and shed virus more or less continuously in the saliva for many months, possibly for life.
Epidemiology of the Carrier State in FCV Infection

241 Pathogenesis. Virus replication occurs in the oral and respiratory tissues.

Lesions are mostly confined to the oral cavity, respiratory tract, and eyes. Oral ulcers are the most prominent feature of FCV infection. In infections with highly virulent strains, there may be pulmonary edema and interstitial pneumonia.

Clinical features. Disease is rare in cats > 1 year of age.

Acute infection. IP: 3 to 6 days. Course: 1-3 weeks or longer. Fever, oral ulcers [hypersalivation], mild conjunctivitis and ocular and nasal discharges.

Coinfection with an immunosuppressive virus such as FeLV or FIV may further increase the susceptibility to disease of individual cats.

Diagnosis. Isolation of virus, followed by identification using ELISA or FAT. Control. Modified live-virus and inactivated FCV vaccines are widely used, usually in combination with feline herpesvirus 1. Both vaccines protect against acute disease caused by many, but not all, strains of FCV.

Vaccination reduces the severity of disease, but will not protect against viral infection or eliminate or prevent the development of carrier state. Virulent Systemic Feline Calicivirus (VS-FCV) (Hemorrhagic Fever Syndrome)

In 1998, 4 shelter cats introduced a highly virulent, vaccine-resistant, strain of calicivirus into a veterinary clinic in Los Angeles. The virus spread to two other clinics and a rescue organization. Of 54 suspected cases, 59% of adult cats and 14% of kittens under one year of age, died.

Other outbreaks of VS-FCV infection have been reported in Massachusetts, Pennsylvania, Tennessee, and Nevada.

Pathogenesis. Virus replication in epithelial cells results in epithelial cell necrosis.

Systemic vascular damage leading to severe edema, hemorrhage, cutaneous ulceration, etc, stem from virus replication in endothelial cells.

Clinical features. Sudden death, fever, ocular and nasal discharge and ulceration of the oral cavity.

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Hemorrhages from the nose, bloody feces, facial and limb edema, hair loss and ulcerative dermatitis of the face and feet. Mortality ranges from 33% to 50%.

Pathology. Necropsy findings include hepatomegaly, pneumonia, pericarditis, and pancreatitis. Control. Vaccination. CaliciVaxTM, from Fort Dodge Animal Health. The vaccine provides protection against VS-FCV and the traditional calicivirus. Vesicular Exanthema of Swine (Porcine Calicivirus) The disease was first recognized in swine in southern California in 1932. The disease was eradicated in 1956, however, the virus is still endemic in marine mammals, eg, sea lions.

VE is clinically indistinguishable from foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis. San Miguel Sea Lion Virus

This calicivirus was first isolated in 1972 from California sea lions inhabiting San Miguel Island. The animals showed several signs of disease including abortion and vesicular lesions of the flippers.

The virus produced vesicular lesions similar to VE lesions in experimentally infected swine. It appears sea lions and other marine mammals, are the reservoir population of the virus that causes vesicular exanthema in swine.

243

BIRNAVIRIDAE
Properties of Birnaviruses

Nonenveloped icosahedral virions, 60 nm in diameter. Genome consists of two molecules of linear dsRNA. Cytoplasmic replication; no inclusion bodies. Neutralizing antibodies are produced to the major capsid protein VP2. Virions are relatively heat stable; stable at pH 3 to pH 9.

Genus Avibirnavirus Genus Aquabirnavirus Genus Entomobirnavirus

Infectious bursal disease virus Infectious pancreatic necrosis virus Drosophila X virus

Infectious Bursal Disease (IBD, Gumboro disease) IBD was first identified in chickens in 1962 in an outbreak in Gumboro, Delaware. The disease in chickens is characterized by necrosis and depletion of lymphoid elements of bursa of Fabricius. The disease occurs worldwide. Etiologic agent. Avibirnavirus. There are two serotypes which show minimal cross-protection.

Serotype 1. Causes IBD in chickens. Strains vary in virulence. Serotype 2. Causes inapparent infections in chicks and turkey poults. The virus is extremely stable and persists for over 4 months in pens.

Transmission. This is a highly contagious disease. The virus is excreted in the feces for up to 14 days. Infection occurs when maternally-derived antibody levels begin to wane at two to three weeks of age. Pathogenesis. Following oral uptake, the virus replicates in gut-associated macrophages and lymphoid cells in the cecal tonsils, duodenum and jejunum. From here, progeny virions enter the circulatory system, leading to 1o viremia.

Within a few hours, viral antigen can be detected in the bursal lymphoid cells. Virions released from the bursa produce a pronounced 2o viremia, resulting in localization in various organs and tissues.

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Maximum virus replication occurs in pre-B cells and IgM+ B cells [immature B cells] in the BF. Lymphoid follicles of the bursa become totally necrotic as a result of both necrosis and apoptosis [the major antigenic protein VP2 induces apoptosis in infected cells]. Very virulent strains also produce depletion of cells in the spleen, thymus, and bone marrow.

The predilection of the virus for bursal cells leads to a severe long-lasting immunosuppression in subclinically infected or recovering chickens. The humoral immune response is most severely affected; the cell-mediated immune response is affected to a lesser extent [NO produced by activated macrophages can promote destruction of both virus-infected and virus-free cells]. Chickens immunosuppressed by early IBDV infection do not respond well to vaccination and are predisposed to infections with opportunistic viruses and bacteria.

Clinical features. IP: 2 to 3 days. Disease is most severe in chickens 3 to 6 weeks old [bursa reaches maximum size]; chicks 1 to 3 weeks old are less susceptible, whereas birds older than 6 weeks, rarely develop signs of disease.

Sudden onset, depression, anorexia, incoordination, whitish, watery diarrhea, soiled vent feathers, dehydration, and vent picking. Clinical disease lasts 3 or 4 days; surviving birds recover completely. Morbidity: up to 100%; mortality: 30-90%, decreasing as birds get older. Lesions. BF enlarges to 5x its normal size, becomes edematous, reddened, and hemorrhagic. During the terminal phase of the disease, it may become atrophic. The kidneys are usually enlarged, with accumulation of urates due to the dehydration.

Diagnosis. Characteristic lesions in the BF.


Virus isolation using CAM of embryonated eggs or cell culture. Detection of viral antigen in BF using AGID test or immunofluorescent stain. Detection of antibodies using ELISA and VN tests.

Control. Depopulation and careful disinfection of premises.

Breeder hens are vaccinated to ensure high levels of IBDV antibodies in the eggs. Maternal antibodies provide protection for 4-7 weeks after hatching.

245 BORNAVIRIDAE
Properties of Bornaviruses

Spherical enveloped virions, 90 nm in diameter. Genome is a linear [-] sense ssRNA. Replication takes place in the nucleus; virions produce intranuclear inclusions. In cell culture, virus spread occurs via cell-to-cell contact, resulting in persistent infections. Virions are susceptible to heat, acid, lipid solvents, and common disinfectants.

Borna Disease Borna disease is a neurologic, usually fatal, disease of horses first recognized in 1895 in Borna, Germany. Borna disease has been reported in several countries in Europe and in Japan. Antibodies to BDV in cerebrospinal fluid or serum have been detected in horses in the United States and other countries. Etiologic agent. Bornavirus. Hosts. The disease occurs naturally in horses, sheep, cattle, cats, rabbits, ostriches, and humans. Transmission. Via inhalation or ingestion. Virus is excreted in saliva, nasal secretions and conjunctival fluid. Pathogenesis. Virus spread to the brain occurs via the olfactory and trigeminal nerves.

In the brain, the virus multiplies in neurons, astrocytes, and oligodendrocytes. There is severe meningoencephalitis and to a lesser extent myelitis. Intranuclear eosinophilic inclusion bodies [Joest-Degen bodies] can be demonstrated in infected neurons. Clinically ill animals may have undetectable to very low antibody titers.

Clinical features. IP: Average 4 weeks [may be a few days to six months].

Horses show mild fever, fatigue, anorexia, coughing, attacks of colic, icterus; followed by somnolence, hyperesthesia, ataxia, paresis and paralysis. Terminally, there may be nystagmus and blindness. Course of disease is 3 to 20 days and is usually fatal; surviving horses may have permanent sensory and/or motor deficits.

246 Diagnosis. Demonstration of antibodies in the serum or CSF using IFA.


Virus can be isolated in cell culture. Detection of viral RNA in conjunctival or nasal secretions and saliva using reverse transcriptase-PCR [RT-PCR]. Pathology. No gross findings.

Control. Identification and quarantine of carrier animals. Humans. The virus has been associated with behavioral and neuropsychiatric disorders in humans.

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ASTROVIRIDAE (astron, star)


Properties of Astroviruses

Nonenveloped icosahedral virions, 28 to 30 nm in diameter. Linear [+] sense ssRNA genome. Some virions have smooth surfaces; others have surfaces with five- or sixpointed star. Replication occurs in the cytoplasm; virions are released by cell lysis. Virions are host specific. They are moderately stable in the environment.

Astrovirus Gastroenteritis Astroviruses are associated with mild, self-limiting enteritis in calves, lambs, piglets, puppies, kittens, and humans. Disease occurs worldwide. Transmission. Fecal-oral transmission. Clinical features. IP: 1 to 4 days. Many infections are subclinical. Course of the disease may be I to 4 days or longer.

Calves. The virus destroys dome M cells [overlies Peyers patches] and absorptive enterocytes, resulting in watery diarrhea. Germinal centers of underlying lymphoid tissue become depleted.

Lambs. Mature enterocytes of villi are affected, causing stunting and fusion.

Diagnosis. Same as other enteric conditions.

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PRION DISEASES
A prion [proteinaceous infectious particle] is a small [30 nm], infectious, abnormal isoform of a normal cellular protein, with no nucleic acid.

Cellular prion protein [PrPc; also called PrPsen for protease-sensitive form] is a glycoprotein encoded by a cellular PrP gene located on chromosome 20 in humans and chromosome 13 in sheep and cattle. It has been suggested that because of their location on the cell surface, cellular prion proteins may be associated with cell adhesion and recognition or the uptake of various molecules. Prions are found in many tissues, especially in neurons and lymphocytes.

The amino acid sequence of PrPc [208-254 AAs, depending on the specie] and the abnormal isoform of the protein, called PrPsc [the term is derived from the scrapie isoform of the prion protein, but is in general use for all prion diseases; also called PrPres for protease-resistant form], in a particular host are identical, only the conformation of PrPsc is different. Characteristics of PrPsc

Prions are very resistant to ultraviolet [UV] and ionizing irradiation, proteases, boiling, 3.7% formaldehyde, etc.

Infectivity of prions may be reduced by 90 to 95% after several hours of treatment with phenol, household bleach [0.5% sodium hypochlorite], autoclaving [132oC for 90 minutes], and iodine disinfectants. Prions do not induce inflammatory or immune responses in their hosts.

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A. Susceptibility of PrPc and PrPsc to protease digestion. B. Structure of normal prion protein [PrPc] with predominantly helices and PrPsc [the abnormal isoform] with predominantly helices.

The Conformational Conversion of PrPc to PrPsc

Sporadic spongiform encephalopathy. The great majority of all spongiform encephalopathies are due to spontaneous misfolding of cellular prion proteins. This form is exemplified by sporadic Creutzfeldt-Jakob disease [CJD], which occurs in individuals between 50 to 70 years of age. Genetic or familial spongiform encephalopathy. This form is associated with specific mutations within the gene encoding PrPc. It is exemplified by familial CJD, which is an inherited disease. Infectious [or transmissible/acquired] spongiform encephalopathy. Infection may be acquired as a result of feeding meat and bone meal to animals, iatrogenically via the transplantation of infected corneas, the use of purified hormones, etc. The exogenous PrPsc binds to PrPc and induces a conformational change that converts PrPc to PrPsc. This form is exemplified by Kuru and vCJD.

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Transmissible Spongiform Encephalopathies Prions are responsible for several neurodegenerative diseases in humans and animals, termed transmissible spongiform encephalopathies. The characteristic pathology includes vacuolization [a spongiform degeneration in the gray matter of the brain], severe astrocytosis [astroglial hypertrophy and proliferation], and loss of neurons. The diseases are always fatal.

The spectrum of prion diseases has been attributed to variations in prion proteins. Each strain represents a unique conformation of PrPsc [differences in amino acid substitutions]. Prions from one species are more effective in transmitting disease to the same species, so-called species barrier. However, rarely, cross-species infection can occur, eg, new variant CJD.
Prion Diseases of Animals and Humans Host Humans Disease[s] Kuru; Creutzfeld-Jakob disease; new variant CJD, familial CJD, fatal familial insomnia; etc Scrapie Bovine spongiform encephalopathy Feline spongiform encephalopathy Transmissible mink encephalopathy Chronic wasting disease

Sheep and goats Cattle Cats Mink Mule deer, elk

251 Scrapie Scrapie is a chronic, fatal, neurodegenerative disease of adult sheep and occasionally goats. The name scrapie comes from the fact that affected sheep scratch on posts, etc., to relieve the intense pruritus associated with the disease.
Part of a report written in 1772 by the Reverend Thomas Comber, on the subject of an ovine disease that he referred to as rickets: The principal Symptom of the first Stage of this Distemper, is a kind of lightheadedness, which makes the affected Sheep appear much wilder than usual, when his Master or Shepherd, as well as a Stranger, approaches him. He bounces up suddenly from his Laire, and runs to a Distance, as though he were pursued by Dogs, &c. In the second Stage of the Distemper, the principal Symptom of the Sheep is his rubbing himself against Trees, Post, &c. with such Fury as to pull off his wool and tear away his Flesh. The distressed Animal has now a violent Itching in his Skin but it does not appear that there is ever any cutaneous Eruption. The third and last Stage of this dreadful Malady seems to be only the Progress of Dissolution, after an unfavourable Crisis. The poor Animal, as condemned by Nature, appears stupid, separates from the Flock, walks irregularly, (whence probably the Name of this Disease, Rickets) generally lies, and eats little. These Symptoms increase in Degree till Death, which follows a general Consumption. I do not find, Sir, that this Distemper is infectious: but alas! It is hereditary, and equally from Sire and Dam; and, like other hereditary Distempers, may lie latent one Generation.and then revives with all its former Fury It is an incontrovertible Point, that whatever Sheep is once seized by this Distemper, never recovers; and it seems almost as incontrovertible, that whatever Sheep escapes it in his first Years, never takes it This Distemper is generally said to be of about forty Years standing in England; and the Shepherds of this County pretend to trace it from the neighbouring County of Lincoln hither. From the book HOW THE COWS TURNED MAD by Maxime Schwartz

Distribution. Worldwide, except Australia and New Zealand. Transmission. Contaminated pastures remain infectious for years.

Ingestion of PrPsc contained in feed, infected placenta, and fetal tissues. Possible entry of scrapie prion protein through skin abrasions. Transmission from ewe to lamb at birth from infected placenta. Vertical transmission has not been demonstrated in goats.

Genetic susceptibility or resistance in sheep. Scrapie has been reported in most breeds of sheep, however, there are breed and individual differences in susceptibility.

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Susceptibility has been traced to certain polymorphisms in the sheep prion protein [PrP] genes that code for cellular prion protein [PrPc]. Genes occur in pairs [diploid], hence a sheep has two PrPc genes; if the genes are the same [homozygous], the sheep produces only one type of PrPc; if they are different [heterozygous], two types of PrPc are produced.

PrPc has 254 amino acids. The conversion to scrapie prion protein [PrPsc] is associated with amino acid changes in three locations [amino acid number 136, 154, and 171]. Since an amino acid is determined by a group of three nucleotides called a codon, the three important locations on the PrPc genes are referred to as codon 136, codon 154, and codon 171. Codon 136. Valine [V] is linked to susceptibility; Alanine [A] is linked to resistance. Genotypes AA, VV or AV. Codon 154. Histidine [H] is linked to susceptibility; Arginine [R] is linked to resistance. Least significant of the three codons. Codon 171. Has the most significant effect on scrapie susceptibility or resistance in sheep in the USA. Glutamine [Q] is linked to susceptibility; Arginine [R] is linked to resistance. Genotypes QQ, RR, or QR. QQ sheep are highly susceptible to infection if exposed to scrapie agent; QR sheep are rarely susceptible if exposed; and RR sheep are highly resistant.

Clinical features. IP: 2 to 4 years. Seen in animals between 2-5 years of age.

Suffolk, Cheviot, Hampshire, and Swaledale breeds have a higher incidence of natural scrapie. Earliest clinical sign seen is pruritus: sheep rub the affected parts against objects and bite the flanks [patchy wool loss]. Muscle tremors, weaving gait, staring eyes, and eventual hindquarter paralysis. Death may occur within 6 weeks to 5 months of developing signs.

Diagnosis. History, age of the animal and clinical signs.

Antemortem diagnosis. Demonstration of PrPsc in tonsillar biopsy or biopsy of lymphoid follicles in the third eyelid, using immunohistochemical methods. Presence of PrPSC proteins in the CSF is considered diagnostic. Brain material can be inoculated into mice.

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Histopathologic examination of the brain. Neuronal vacuolation and degenerative changes in the brain. EM demonstration of scrapie-associated fibrils [plaques].
Brain of sheep showing extensive spongiform changes and SAF [plaques]

Immunity. In natural infections, there is complete absence of inflammatory, humoral or cellular immune responses. Prevention and control. Scrapie is a reportable disease.

USDA Genetics Based Flock Clean-Up and Monitoring Plan. The program targets scrapie infected and source flocks. Sheep in the flock are genotyped and sheep with susceptible genotypes are removed. The flock is then placed under surveillance for five years. Flocks exposed to scrapie are monitored using the eyelid biopsy test [PrPSC can be detected in scrapie positive sheep by 14 months] and if scrapie is detected, another round of genetic typing is carried out. Bovine Spongiform Encephalopathy (BSE)

BSE is a sporadic, slowly progressive, neurologic disorder of adult cattle first recognized in the United Kingdom in 1986. Susceptibility of cattle to BSE is not related to sex, breed, or genotype.

BSE prions have an unusually broad host range, infecting a number of animals, including domestic and wild cats and humans.

Synonyms. Mad cow disease and Raging cow disease. Distribution. Great Britain, Belgium, France, Ireland, Portugal, and other European countries; Canada, USA, and Japan.

254 Transmission. Traced to contamination of meat-and-bone meal produced from meat and offal from slaughtered and dead sheep.

Possible vertical transmission from cow to calf.

Clinical features. IP: 2 to 8 years. Course: 2 to 3 weeks to over a year.

Most cattle affected have been 3 to 5 years of age. There is apprehension, hyperesthesia, and mild incoordination. Later, there is kicking, aggressive behavior, hypermetria [goose-stepping], and increasing incoordination with falling and difficulty in rising. Terminally there is frenzied behavior, head tremors, and animals become recumbent. There is no pruritus.

Diagnosis. Histologic examination of the brain: lesions in the gray matter include neuronal vacuolation, neuronal degeneration and loss, and astrocytic hypertrophy and hyperplasia.

EM demonstration of SAF in the brain.

Control. In 1988 Great Britain banned the inclusion of all ruminant-derived protein, including meat-and-bone meal, in ruminant rations.

In the U.S.A., brains of cattle dying from undiagnosed neurologic disease are submitted to the National Veterinary Services Laboratory for BSE screening.

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INTRODUCTION TO SEROLOGY
Serology is the study of antigen-antibody [Ag-Ab] interactions in vitro. The principle of all antigen-antibody reactions lies in the specific combination of epitopes on the antigen with the hypervariable regions or complementarity-determining regions of the antibody molecule.

Serologic assays differ in their sensitivity, specificity, and speed; some are strictly qualitative; others may be qualitative and/or quantitative. Serologic Applications

Serologic assays can be used for the detection and measurement of antibodies or antigens. Disease diagnosis Detection of antibody titer rise: 4-fold increase between acute and convalescent serum samples collected 2-4 weeks apart against an etiologic agent. Acute samples should be frozen and submitted at the same time as the convalescent sample to avoid differences due to laboratory techniques. The detection of circulating antigen in certain infections, eg, hepatitis B surface antigen by serum of known antibody; the matching of red blood cells against serum antibodies of a recipient.

Monitoring the level of the humoral [antibody] immune response. Seroepidemiology. A population survey using a serologic test to screen for exposure and immunity to an infectious disease. Collection and Handling of Serologic Samples

Virtually all serologic tests require serum or plasma as the sample. The most practical method of collection is the Vacutainer System [Becton-Dickinson, Rutherford, NJ].

A red-top vacuum tube is used when serum is required and a lavender-top tube is used when plasma is required.

Serum. This is the fluid portion of blood remaining after the blood cells and materials responsible for clotting are removed. When collecting serum, allow the blood sample to clot for 20 to 30 minutes at room temperature and then centrifuge for 10 minutes at a speed not to exceed 1500 rpm.

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After centrifugation, a small pipette is used to aspirate the serum off the packed erythrocytes. Place the aspirate into a transfer tube or other sealable tube and label clearly. The serum may be submitted immediately or frozen or refrigerated for later use.

Normal serum. Serum devoid of antibodies. Antiserum. Serum containing antibodies. Hyperimmune serum. Serum containing high concentrations of antibodies.

Plasma. This is the noncellular portion of blood. It is obtained by collecting blood in an anticoagulant, followed by centrifugation.

Terminology Agglutinin. Antibody that aggregates or clumps particulate antigen. Antiglobulin (antispecie antibody). Antibody made against an immunoglobulin, usually by injecting immunoglobulin into an animal of another species. Lysin. Antibody that causes lysis of cell membrane. Neutralizing antibody. Antibody that renders the microbe [commonly viruses] noninfectious. Precipitin. Antibody that forms precipitates with soluble antigen. Seroconversion. The appearance of antibody in the blood in response to infection, disease, or immunization, ie, seronegative to seropositive.

Seronegative. Animal with no detectable antigen-specific antibodies.

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Seropositive. Animal with detectable antigen-specific antibodies. However, classification of an animal as seropositive is usually based on the titer being above a certain threshold level [cut-off point]. For example, an animal with titer >1:4 may be classified as positive, whereas an animal with titer 1:4 may be classified as negative.

Serotype. The type of a microorganism as determined by its distinct antigenic properties, eg, E. coli O157:H7.

Surface antigens of a typical bacterial cell

Subtype. Variation within a serotype, eg, Canine parvovirus 2a and 2b. Serogroup. A group of bacteria containing a common antigen or a group of viral species that are closely related antigenically. Sensitivity. The minimum concentration of a substance that can be reliably measured by a serologic test. Measurement of false-negative.
Relative Sensitivity of Tests Measuring Antibody Test ~ Detectable Amount (g/ml) ELISA 0.0005 Radioimmunoassay (RIA) 0.00005 Precipitation reaction in fluids 20.0 Double diffusion in agar gel 3-20 Complement fixation 0.05 Bacterial agglutination 0.05 Passive agglutination 0.001-0.01 Hemagglutination inhibition 0.005 Virus neutralization 0.00005 Antitoxin neutralization 0.06

Specificity. The specificity of an antibody refers to its capacity to discriminate between ligands of similar structure by combining with them at different extents. The greater the differences in affinity for two closely related structures, the more specific the antibody.

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Although Ag-Ab reactions are highly specific, in some cases antibody elicited by one antigen can cross-react with an unrelated antigen. Such cross-reactivity occurs if two different antigens share an identical epitope or if antibodies specific for one epitope also binds to an unrelated epitope possessing similar chemical properties. Measurement of false-positive.

Heterophile antigens

Titer. The reciprocal of the highest dilution [lowest concentration] of serum [antibody] that produces a test reaction.

Therefore, if the highest dilution that produces a test reaction is 1 in16, then the titer is 1/16. This means that the undiluted serum contains 16 times the antibody for the reaction. Two-Fold Serial Dilution of Serum (Antibodies)

In certain situations it is desirable to determine the concentration [titer] of antibody present in a serum sample, eg, following vaccination.

Titration is accomplished by serially diluting the serum in a series of decreasing concentrations in a test tube. Antibody is detected using a suitable fixed amount of antigen.

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ANTIGEN AND ANTIBODY IMMUNOASSAYS


TESTS INVOLVING ENZYME LABELING In these tests, an enzyme covalently conjugated to an antibody reacts with a colorless substrate [chromogen] to generate a colored reaction product. ELISA assays possess both high specificity and high sensitivity.

Enzymes employed in ELISA tests include horseradish peroxidase and alkaline phosphatase. Direct ELISA (Sandwich ELISA)

The sandwich ELISA can be used to detect or quantitate an antigen. Monoclonal antibody is bound to the walls of wells in a microtiter plate, to a membrane, or to a plastic wand.

Add sample [that may contain antigen] to the antibody-coated wells; incubate for 1 hour at room temperature. If antigen is present in the sample it will be trapped by the antigen-binding sites on the fixed antibody. After washing unbound materials away, a secondary [labeled] antibody containing a conjugated enzyme [horseradish peroxidase] is added; incubate for 1 hour at room temperature. The 2o antibody is also specific for the antigen so it will bind to any remaining exposed antigenic determinants. Following a wash the enzyme activity of the bound material in each microtiter well is determined by adding the substrate of the enzyme [hydrogen peroxide] and a chromogen [in the presence of oxygen it changes from colorless to a colored compound]. The color formed can be read in a spectrophotometer and is directly proportional to the amount of antigen originally present.

260 Canine Parvovirus Antigen Test Kit (Snap Test)

Indirect ELISA An indirect ELISA is used to detect or quantitate antibody. Serum or some other sample containing 1o antibody is added to an antigen-coated microtiter well and allowed to react with the bound anti-gen.

After any free primary antibody is washed away, the presence of antibody bound to the antigen is detected by adding an enzyme-conjugated 2o antiisotype antibody [anti-species antibody], which binds to the 1o antibody. Any free 2o antibody is then washed away, and a substrate and chromogen mixture is added. The intensity of the color is proportional to the amount of enzymelinked 2o antibody that is bound, which in turn is proportional to the amount of 1o antibody present in the sample under test.

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IMMUNOFLUORESCENCE The technique of immunofluorescence was introduced in 1941 by Coons, who employed -anthracine, a blue fluorescing compound, coupled to pneumococcus antiserum to detect bacterial antigens in tissue sections.

Fluorochromes are chemical substances that are capable of absorbing a short wavelength of light and instantaneously emitting a longer wavelength light. The emitted light is generally viewed with a fluorescence microscope, which is equipped with a UV light source and excitation filters. The most commonly used fluorescent dyes are fluorescein isothiocyanate [FITC] and rhodamine. Both dyes can be conjugated to the Fc region of an antibody molecule without affecting the specificity of the antibody. FITC absorbs blue light [490 nm] and emits an intense yellow-green fluorescence [517 nm]. Rhodamine absorbs in the yellow-green range [515 nm] and emits a deep red fluorescence [546 nm].

Fluorochrome-labeled antibodies may be employed in various techniques, the most important of which are the direct and indirect fluorescent antibody tests. Direct Fluorescent Antibody Test (FAT)

Direct FAT is used to detect the presence of antigen, eg, bacterial cultures and viral or other antigens, in a smear or fresh or fixed tissue.

The antibody directed against the antigen is labeled with FITC. A tissue section or smear containing the organism is fixed with acetone and incubated with labeled antibody, and then washed to remove unbound antibody. When examined with a fluorescence microscope, the antigenic particles bound to the labeled antibodies are seen to fluoresce.

262 Indirect Fluorescent Antibody Test (IFAT) The IFA test may be used for the detection and quantitation of antibodies in serum or for the demonstration and identification of antigens in tissues or cell cultures. Three reactants are needed:

Antigen A; unlabeled1o antibody A; and a fluorochrome-labeled anti-isotype 2o antibody. The IFA test has two advantages over direct staining: The 1o antibody does not need to be conjugated with label; this eliminates the need to purify and individually conjugate each serum sample. Indirect methods increase the sensitivity of staining because multiple fluorochrome anti-isotype antibodies can bind to each 1o antibody molecule.
Detection of Bacterial Surface Antigens Using IFA Test

Testing for Antibody Known antigen is employed as a tissue smear, section, or cell culture on a slide or coverslip. This is incubated in a serum suspected of containing antibodies to that antigen, and the serum is then washed off, leaving only specific antibodies bound to the antigen.

The bound antibodies may be visualized by incubating the smear in FITClabeled antiglobulin serum. When the slide is washed [to remove unbound antiglobulin] and examined, fluorescence indicates that antibody was present in the test serum. PRECIPITATION REACTIONS

The interaction between an antibody and a soluble antigen in aqueous solution forms a lattice that eventually develops into a visible precipitate. Formation of an Ag-Ab lattice depends on the valency of both the antibody and antigen:

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The antibody must be bivalent; a precipitate will not form with monovalent Fab fragments. The antigen must either be multivalent, ie, it must have multiple copies of the same epitope, or polyvalent, ie, have different epitopes that react with different antibodies present in polyclonal antisera. Precipitation is a consequence of the growth of Ag-Ab aggregates in such a way that each one Ag molecule is linked to more than one Ab molecule and each Ab molecule is linked to more than one Ag molecule. The precipitate develops as neighboring antibody molecules within the lattice form ionic bonds with each other, causing the lattice to lose its charge and thus become insoluble. Precipitation Reactions in Solution

A simple way to demonstrate the presence of antibody against an antigen in solution is to layer a small volume of one over the other in a test tube. At the interface, precipitation will occur, forming a ring. This gives a qualitative evidence of an Ag-Ab reaction.

A quantitative precipitation reaction can be performed by placing a constant amount of antibody in a series of tubes and adding increasing amounts of antigen to the tubes. After the precipitate forms, each tube is centrifuged to pellet the precipitate, the supernatant is poured off, and the amount of precipitate is measured. Plotting the amount of precipitate against increasing antigen concentrations yields a precipitin curve. When the supernatants are examined (each supernatant is divided into aliquots, to one is added a small amount of fresh antigen [to detect excess antibody], and to the other a small amount of fresh antiserum [to detect excess antigen]), it is observed that excess of either antibody or antigen interferes with maximal precipitation. Prozone. Uncombined antibody is present in the supernatant. Due to the presence of excess antibody, each antigen molecule is covered with antibody, preventing cross-linkage and thus precipitation. Equivalence zone. Both antigen and antibody are completely precipitated and no uncombined Ag or Ab is present in the supernatant. The ratio of Ab to Ag is optimal such that extensive cross-linking and lattice

264 formation occurs. As this lattice grows in size it becomes insoluble and eventually precipitates.

Zone of antigen excess (postzone). Uncombined Ag is present in the supernatant. In this zone, precipitation is partly or completely inhibited because each Ab molecule is bound to a pair of Ag molecules; further cross-linkage is impossible since these complexes are small and soluble, hence no precipitation occurs.

Agar Gel Immunodiffusion Reactions When Ag and Ab diffuse toward one another in an agar matrix or when antibody is incorporated into the agar and antigen diffuses into the antibody-containing matrix, a visible line of precipitation will form.

As in a precipitation reaction in solution, visible precipitation occurs in the region of equivalence, whereas no visible precipitate forms in regions of Ab or Ag excess. Ouchterlony Test (Double Diffusion Test)

First described by Elek and Ouchterlony in 1948, the test is based on the principle that Ag and Ab diffuse through a semisolid medium [eg, agar] and form stable immune complexes which then can be analyzed visually.

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Molten agar is poured on a clean glass slide or into Petri dish and allowed to harden. Small wells, about 5 mm in diameter and 1 cm apart, are punched out of the agar. Samples containing Ag and Ab are placed in opposing wells and allowed to diffuse toward one another in a moist chamber for 18-24 hours. The resultant precipitation lines that represent Ag-Ab complexes are analyzed visually in indirect light with the aid of a magnifying lens.

Applications Identifying the presence of either soluble Ag or Ab in body fluids, eg, Coggins test [detects presence of Abs against equine infectious anemia virus in horses]. Comparing antigens. The pattern of the precipitin lines that form when two different antigen preparations are placed in adjacent wells, indicates whether or not they share epitopes.

Identity occurs when two Ags share identical epitopes. The antiserum forms a single precipitin line with each Ag that grow toward each other and fuse to give a single curved line of identity. Nonidentity occurs when two Ags are unrelated [ie, share no common epitopes]. The antiserum forms independent precipitin lines that cross.

266 Partial identity occurs when two Ags share common epitope[s] but one or the other also has unique epitopes. The antiserum forms a line of identity with the common epitope[s] and a curved spur with the unique epitope[s]. AGGLUTINATION REACTIONS Latex Agglutination In latex agglutination, antigen or antibody is adsorbed to the surface of latex polystyrene beads and suspended in water. The addition of specific Ab or Ag results in the formation of Ag-Ab complexes, causing agglutination [clumping].

The reaction results in changes in the appearance of the latex suspension from smooth and milky to clumpy [latex particles have clustered together]. If no reaction occurs, the mixture remains evenly dispersed.
Detection of Bacteria in Cerebrospinal Fluid Using Latex Agglutination Test

Latex agglutination test kits are available for the detection of rheumatoid arthritis factor in canine serum, K99+ [F5] E. coli in stools of diarrhetic calves, S. aureus and Streptococcus agalactiae in bovine mastitis, etc.

267 Hemagglutination (HA)/Hemagglutination Inhibition (HI) Tests A number of viruses are capable of binding and agglutinating mammalian and avian erythrocytes. Antibodies directed against these viruses may inhibit this hemagglutination by blocking their viral attachment proteins.

Virus-induced hemagglutination may be used as a preliminary test when attempting to identify a virus, eg, canine parvovirus. Inhibition of hemagglutination by specific antibody may be used to identify a specific virus or to measure antibody levels in serum. Preparation of 10% RBC Suspension

Collect blood using sodium citrate as anticoagulant: I part of 5% sodium citrate to 5 parts of blood. Mix the blood-citrate solution by repeated inversions of the stoppered tube used to collect the blood.

Wash the cells 3 times in 0.85% saline; gently mix the citrate-blood suspension with ~ 5 volumes of the saline, sediment the suspension at 1500 rpm for 10 min, pour off the supernatant, add fresh saline, and repeat twice more. Drain off the last wash; resuspend the washed cells in saline to give a 10% stock solution. This may be kept at 4oC for up to 5 or 6 days. For the HA/HI tests, 0.5% [1:200] RBC suspension is used. Schematic Representation of Viral Hemagglutination Test

Schematic Representation of Viral Hemagglutination Inhibition Test

268 Hemagglutination Inhibition Antibody Titer Determination

Neutralization Tests Neutralization tests are used to estimate the ability of antibody to neutralize the biological activity of viable viruses in vitro. Virus Neutralization Test These are widely employed in the quantitation of antiviral antibodies, or virus identification. They utilize the ability of antibody-containing sera to block the infectivity of viral agent upon inoculation of the mixture into susceptible hosts or cell cultures. Virus identification. Incubate virus with antiserum at room temperature for 1 hour. Control virus is incubated with normal serum.

Inoculate mixtures into indicator system: embryonated eggs, monolayer cell culture, or laboratory animals. Observe for lack of mortality or cytopathic effect [CPE] in test sample.

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Complete inhibition of CPE at a challenge dose of 10 or 100 TCID50 by a known antiserum type is considered a positive serum neutralization test and indicates the identity of the virus.

Neutralizing antibody titer

Serially dilute serum [2-fold] and add constant virus dilution [10 TCID50] to each serial dilution. Incubate mixture at room temperature for 1 hour. Inoculate serum-virus mixture into indicator system and determine Ab titer. Complement-Fixation Test (CFT)

The CFT is one of the most widely applicable of all serologic tests. Once the required reagents (antigen, complement, sheep RBCs, and antibody against the erythrocytes [hemolysin]) are prepared and standardized, the CFT may be used to measure serum antibody levels and/or diagnosis of viral, bacterial, and fungal diseases. Titration of Complement Sensitized sheep RBCs [antibody-coated sheep RBCs] are added to varying amounts of the complement source in microtiter wells.

After a suitable incubation period at 37oC, the wells containing the various complement dilutions are examined for hemolysis after centrifuging the microtiter plate. The amount of complement needed for the complement-fixation test is the dilution of complement necessary to achieve lysis of half the RBCs added, ie, the CH50 dilution.

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First, antigen and the serum under test [deprived of its complement by heating at 56oC] are incubated in the presence of normal guinea pig serum, which provides a source of complement. After allowing the Ag-Ab-complement mixture to react for a short period, the amount of free complement remaining in the mixture is measured by adding an indicator system (antibody-coated sheep RBCs [sensitized RBCs]). Negative result. Lysis of sensitized RBCs [seen as the development of a transparent red solution]. Positive result. Absence of lysis [seen as a cloudy red cell suspension].

IDENTIFICATION OF PROTEINS
A number of techniques are available that can be used to identify and characterize specific proteins in a complex mixture of proteins. One such technique, Western blotting, is widely used to determine the presence and size of a protein in a biologic sample.
Southern is the last name of the scientist who first blotted DNA from a separating gel to a membrane, a technique since called Southern blotting. By analogy, Northern blotting was applied to the technique of transferring RNA from a gel to a membrane, and Western blotting was applied to protein transfer.

271 Western Blotting In Western blotting a protein mixture is electrophoretically separated on a polyacrylamide slab gel in the presence of sodium dodecyl sulfate [SDS], a dissociating agent. The final positions of different proteins in the gel are a function of their molecular size.

The protein bands are transferred from the separating SDS-PAGE to a support membrane [eg, nitrocellulose membrane] by capillary action [blotting] or by electrophoresis, such that the membrane acquires a replica of the array of separated proteins present in the gel. SDS is displaced from the protein during the transfer process and native antigenic determinants are often regained as the protein refolds.

The individual protein bands are identified by flooding the nitrocellulose membrane with radiolabeled polyclonal or monoclonal antibody specific for the protein of interest. The Ag-Ab complexes that form are visualized by autoradiography. Antibody probes labeled with enzymes that generate chemiluminescent signals and leave images on photographic film have been used.

Clinical Uses The Western blot is used to determine whether a positive result in a screening immunologic test is a true-positive or a false-positive result. For example the test is used to confirm a positive ELISA test for HIV infection or Lyme disease; to detect feline immunodeficiency virus antibodies, antibodies to Sarcocystis neurona [equine protozoal myeloencephalitis, EPM], etc.

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The HIV virus is dissociated into its constituent proteins by treatment with SDS and the proteins separated by SDS-PAGE. The separated proteins are transferred to a nitrocellulose sheet and reacted with the serum from the patient. Anti-HIV antibodies in the serum bind to the various HIV proteins [primarily gp41 and p24] and are detected using horseradish peroxidase-linked anti-human immunoglobulin, which produces a visible color change when the enzyme substrate is added.