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VirologyL1 – Virus structure and taxonomy List A diseases:  Transmissible diseases that have the potential for very serious and rapid spread, irrespective of national borders. They have particularly serious socio-economic or public health consequences and are of major importance in the internationa...

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Virology L1 – Virus structure and taxonomy List A diseases:  Transmissible diseases that have the potential for very serious and rapid spread, irrespective of national borders. They have particularly serious socio-economic or public health consequences and are of major importance in the international trade of animals and animal products  14/15 List A diseases are viral. Emerging infectious diseases:  Avian flu  Dengue  Ebola and Marburg hemorrhagic fevers  Nipah virus encephalitis  Hendra virus disease  Hantavirus pulmonary syndrome  Monkeypox  SARS  HIV  West Nile virus  Hepatitis viruses B and C  Human herpesviruses 6 and 8  Parvovirus B19. - Includes new diseases and existing diseases that are rapidly increasing in incidence or geographic range. - ~60-70% of emerging infectious diseases are zoonotic. LO: Explain the basic properties of viruses, and how these are different from the properties of other micro-organisms. LO: Describe the relative size and structural components of viruses compared with other micro-organisms. LO: Be able to explain the basis for classifying viruses and the benefits of doing so.

Virus definition  Viruses are small, obligate intracellular parasites. Size: - 17-300nm - RBC > bacteria > virus - Veterinary viruses vary in size. o Double stranded DNA (dsDNA) > single stranded DNA (ssDNA) Viruses are ubiquitous - There are viruses of plants, animals, fungi and bacteria. Obligate intracellular lifestyle - No free-living viruses - Viruses lack the complex energy-generating and biosynthetic ‘machinery’ necessary for independent existence. - In order to replicate themselves, viruses ‘highjack’ the cellular machinery of the host. The information stored in the virus genetic material allows it to take over the machinery of the cell, converting it to a factory to make copies of itself. - Phases of viral existence: o Transmission Phase: variably fragile o Replication phase: subversion of hose cellular machinery to produce copes of themselves.  In their simplest form, viruses consist of nucleic acid and protein. Nucleic acid - RNA or DNA (never both) - Single stranded or double stranded

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RNA: positive (same as mRNA) or negative (complementary to mRNA) Monopartite (single molecule of nucleic acid) or multipartite (segmented) Haploid – one copy of the gene (Exception: retrovirdae) Often infectious

Protein - 1 to >100 proteins - Structural o Used to construct components of the virus o Protection of viral nucleic acid o Attachment and entry o Important in host immune response - Non-structural (enzymes) o Nucleic acid and protein metabolism o Shutdown of host cell function o Subdiversion of cellular machinery - Other components: o Glycoproteins: most often as peplomers (spikes) extending outwards from the envelope. o Lipids: Viral envelope (derived from cell membranes). Infectious agents simpler than viruses  Viroids: infectious RNA  Prions: infectious protein Viral structure Virion = complete virus particle  Capsid (protein coat + Viral nucleic acid = nucleocapsid  ± lipid envelope (host derived) ± matrix protein between nucleocapsid and envelope (packing material)   Capsomers: morphologic “building blocks” Capsid symmetry - Icosahedral (cubic) symmetry - Helical symmetry - Virions self-assemble from protein subunits.

Envelope - A lipid bilayer formed when a virus particle buds through cellular membranes - Host derived has Peplomers (spikes) Virus derived…? - Enveloped virions are often pleomorphic as the envelope is not riigid. Classification of viruses

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Informal classification = Subspecies, Strains, Variants o Diagnostics, vaccine development L2 – Virus replication and cultivation of viruses

LO: Explain what happens during each of the 7 stages of viral replication Step 1 Attachment: Virus attaches to the host cell  Adsorption: non-specific binding of viral particle to the cell membrane – random initial virus-cell contact  Attachment: specific binding of viral glycoproteins to cell surface receptors o Reversible o Followed immediately by penetration – irreversible. o Virus surface protein (ligand) is the key. Cell surface molecule (receptor) is the lock.  Cell surface molecule is crucial for fundamental cellular functions – highly conserved.  Many different cellular molecules are used as receptors by different viruses, they are mainly cell surface glycoproteins or carbohydrate residues present on glycoproteins or glycolipids, components of the extracellular matrix and receptors involved in cell signaling and activation.  E.g. ICAM-1 on leukocytes and endothelial cells – Rhinoviruses. CD4 on macrophages and T cells – HIV. Sialic acid – orthmyxoviruses (flu) and paramyxoviruses (New castle disease virus. - Specificity of attachment o A virus can only infect the cells which have the correct receptor. o Provides the basis for a virus’s host range o Limits viruses to only infecting certain cell types within their hosts. Step 2 Penetration: Virus enters the cell – crosses the plasma membrane  Modes of transport: o Receptor-mediated endocytosis (adenovirus, orthmoyxoviruses) o Fusion with plasma membrane (herpesvirus) o Direct penetration of a genome (parvovirus)  Irreversible process. Step 3 Uncoating: Viral genetic material (nucleic acid) is released inside the cell.  Viral DNA or RNA has to be released from the surrounding capsid  Complete or incomplete  Various mechanisms and various sites used by different viruses o At the cell surface o Within a cytoplasm o At a nuclear pore o Within a nucleus  Uncoating may be targeted by drugs e.g. amantadine for influenza viruses. Step 4 Synthesis: Large amount of viral proteins and viral nucleic acid is produced inside the host cell.  In a normal cell cycle: transcription occurs in the nucleus (DNA  RNA), and translation occurs in the cytoplasm (mRNA  protein)  The synthesis of all viruses involves transcription or generation of positive mRNAs from their genomes and the use of host ribosomes to translate the viral mRNA into viral proteins. Step 5 Assembly: viral nucleic acid is packed into viral protein coat to form large number of viral particles. Step 6 Maturation: The viral particles undergo structural changes in the protein coat until they become infectious. Step 7 Release: All the infectious virions are released from the host cell via bursting (cell lysis) or budding of the host cell.

Replication cycle:

LO: Provide examples on how this knowledge can be exploited in the design of antiviral drugs.

LO: Explain how viruses are cultivated in the laboratory Cultivation of viruses  Viruses only replicate in living cells o Experimental animals o Avian embryos (eggs) o In vitro cell culture o In vitro organ culture Experimental animals - Still used for virus isolation (infrequently). When cell culture methods have not been developed e.g. wobbly possum disease.

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Are still used in virus research. o Animal models for viral pathogenesis and host immune responses. o Vaccine testing o Production of reagents.

Avian embryos - Bacteriologically sterile - Immunologically incompetent - Still used for isolation of certain viruses o Influenza virus o ILT o Poxviruses Organ cultures - Tracheal rings - Embryonic intestine - Nerve tissue cultures Cell culture - Cell culture medium needs to take into consideration nutrients, growth factors and +/- antibiotics - Types of cell cultures: o Primary cell cultures  Prepared directly from tissues  Often more susceptible to viral infection  Difficult to establish, time consuming, expensive o Secondary cell cultures  Primary cells passaged in vitro for a limited number of passages o Continuous cell lines  Immortal cell lines  Easy to work with  Less susceptible to viral infection  99% done this way o Organ cultures  Specialized techniques hardly used. LO: Explain how one can detect viral growth in cell culture Recognizing virus growth in cell culture  Cytopathic effects (CPE)  Haemadsorption/haemagglutination  Immuno-staining (IF, IHC)  Polymerase chain reaction (PCR) NEED TO LISTEN TO RECORDING TO FINISH THIS LECTURE

L3 – Viral pathogenesis (mechanisms of disease development) LO: Describe the main steps in the course of viral infection and their relationship to disease, viral spread and persistence.

1. Entry Routes: Skin

Respiratory tract

Breach in integrity. E.g. Abrasions or bites.

Protective mechanisms include, mucus blanket produced by goblet Minor abrasions cells; ciliated  Papillomaviru epithelium; alveolar ses macrophages. MOST  Herpesviruses COMMON ROUTE OF ENTRY  Pox viruses Arthropod bite  Bluetongue virus  Equine encephalitidi es virus  West Nile virus Bite of vertebrate  FIV  Rabies Iatrogenic

Localised infections  Rhinoviruses  Mammalian influenza viruses  Adenoviruses Systemic infections  Canine distemper virus  Newcastle disease virus  Foot-andmouth disease

Alimentary tract

Urogenital tract

Protective mechanisms include, Mucus + IgA; acid and bile; proteolytic enzymes

Small tears or abrasions during sexual activity

Acid-stable nonenveloped viruses  Rotaviruses  Caliciviruses  Enteroviruses Acid-labile enveloped viruses  Coronaviruses  Pestiviruses  Avian influenza virus

Iatrogenic (AI, reproductive exam)

Conjunctiva Herpesvirus Canine adenovirus 1 and 2

Herpesviruses Equine arteritis virus Papillomaviruse s

LO: Define ‘incubation period’ and ‘period of infectivity’

2. Incubation and dissemination 

Incubation period: the time from virus entry (infection) to onset of observable clinical signs. o SHORT – when clinical signs are due to viral replication at the site of entry (2-5 days) o LONGER – when virus must spread before causing disease (1-2 weeks) o There are many other virus and host-associated reasons for variation.

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Dissemination: Spread from the site of initial infection. o Local spread on epithelial surfaces:  Direct cell-to-cell  Aided by mechanical action (e.g. peristaltic)  E.g. influenza, rotaviruses, papillomaviruses, poxviruses. o Via blood stream:  Subepithelial invasion

Placenta Damage to the placenta Pass through placenta to a fetus Reproductive losses: Abortion Neonatal death Persistently infected animals.

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Direct inoculation (arboviruses) Viraemia (presence of virus in the blood)  Cell-associated – canine distemper, Rinderpest  Free in plasma – avian inclusion body hepatitis (adenovirus) Via nerves:  Uncommon, often fatal.  Relatively slow spread  Axonal or via Schwann cells  May use olfactory nerve endings  E.g. Rabies, Aujeszky’s disease, herpes B, Borna disease virsuses.

3. Disease (not always) LO: Explain how viruses cause disease. LO: Describe virus-induced changes at the level of the cell Viruses’ effects upon cells  Cell death: apoptosis vs necrosis o Cytopathic viruses o Removal of virus infected cells by the immune system.  Loss of ‘specialised’ functions – viruses that bud rather than lyse cells  Cell fusion  Cell transformation (neoplasm)  No detectable adverse effects (non-pathogenic viruses) Disease results when:  A sufficient number of essential, non-replaceable cells are killed: o Motor neurons in Taflan disease, hepatocytes in CAdV-1 infection.  Rate of cell destruction exceeds max cell regeneration rate, resulting in organ failure o Feline panleukopenia – loss of PBL and all dividing cells.  Circulatory disturbances due to infection of endothelium (or DIC) occur  thrombosis, haemorrhage, effusions, ischaemia.  Epithelial damage and/or immunosuppression allow secondary invasions by other pathogens o Parainfluenza 3, BVD, FIV  Widespread inflammation – canine distemper.  Interference with normal morphogenesis in the foetus o Pestiviruses  Formation of immune complexes (immunopathology) o FIP 

Disease results from an interaction between the host and virus

LO: Define the term virulence Virulence: The extent to which a virus causes disease in a specified host.  Some influenza viruses cause deadly HPAI in chickens, but no disease at all in ducks  For many viruses, virulence varies by strain, not just by species

4. Spread to other animals

Period of infectivity: Period during which an infected individual can infect others. May begin before the end of the incubation period (e.g. FMD). Can be short (days) or long (weeks to months). Chronically infected animals (‘carriers’) may shed the virus in the absence of clinical signs. Continuous or intermittent shedding is possible.

5. Spread to other animals Mechanisms of virus shedding:  From the respiratory tract: coughing, sneezing, eating drinking  From the oropharynx and intestinal tract: Diarrhoea (Rotavirus), saliva – licking, grooming, biting (Rabies, FeLV, FIV.  From the skin: Through direct contact and small abrasions (Papillomaviruses), Large quantities of virus in vesicles (FMD virus)  From the urinary tract: Hantaviruses  Genital tract: EAV  Milk: CAE  Blood and tissues: Arboviruses, iatrogenic transmission  Infection without shedding: via germplasm, via consumption of contaminated tissues: hog cholera, ASF.

6. Recovery OR death OR persistent infection   

Death Recovery Persistent infection o Latent infection o Chronic infection o Slow infection

LO: Describe mechanisms of viral persistence. Mechanisms of persistent infection  Cell-associated infections – herpesviruses (latency), retroviruses (integration of the genome)  Persist in an immunologically ‘privileged’ or inaccessible site – neural ganglia (herpesvirus)  Induce immunological tolerance in the host – BVD  Induce immunodeficiency in the host – Lentiviruses (FIV), herpesviruses, IBD  Continuing antigenic change – lentiviruses (FIV)  Non-immunogenic agents – prions. LO: Explain in detail pathogenesis of EHV-1 infection.

L4 – Diagnosis of viral infection LO: Explain in general, diagnostic approaches for detection of viral infections with examples of tests used Laboratory confirmation is needed:  For specific diagnosis of zoonotic diseases  When a specific diagnosis is required to optimize clinical management (e.g. diarrhoea in a large group of valuable young animals) or help with future management decisions  When it is necessary to certify animals free of particular infections e.g. in pre-export testing.  To prevent transmission of pathogens, e.g. during AI, embryo transfer, blood transfusions.  To facilitate disease eradication programmes. Diagnostic tests:  Non-specific o Haematology/biochemistry/cytology – on blood, urine, CSF, body cavity fluids. o Post mortem examination – gross findings and histopathology  Specific o Detection of specific antigen – antigens, nucleic acid, cultivation o Detection of specific antibodies – in blood, CSF etc. Inclusion bodies  Presence of inclusion bodies is very suggestive of viral infection.  Intracytoplasmic (e.g. rabiesvirus)  Intranuclear (e.g. herpesvirus)  Both (e.g. paramyxoviruses)  Inclusion bodies sometimes represent “packed” virus particles in an almost ‘crystalline array. Approaches to diagnostic tests: Detection of whole pathogen – isolation/growth of pathogens:  Specify what you are looking for  Different viruses have different requirements for in-vitro culture  Fresh samples are best  Keep the pathogens alive – temperature, transport media, reliable courier etc.  It may take up to 3 passages (3 weeks) for the virus to show CPE (cytopathic effects)

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Success is highly variable o Timing of sampling o Transport (cold chain) o Availability of susceptible cells Not all viruses cause a cytopathic effect – may be difficult to recognize viral growth. For successful virus isolation, samples need to be: o Collected at the early stages of disease o Collected into a viral transport medium – bacterial swabs are not suitable. o Maintain cold (not frozen) – repeated freeze-thawing should be avoided. o Transported to the lab ASAP o Processed ASAP or stored at -80˚C – appropriate cells need to be available.

Detection of whole pathogen – electron microscopy  Direct observation if >106 particles/ml  Rapid  Applicable to some enteric, skin and mucous membrane infections.  Submit faeces, scabs, vesicular fluids, scrapings (unfixed)  Antibodies may be added before imaging to improve sensitivity/specificity – immunoelectron microscopy (agglutinate virions).  Limitations: o Expensive equipment o Expensive maintenance o Requires an experienced observer o Sensitivity is low. Detection of pathogens components – nucleic acids  Traditional PCR  Real-time PCR  RT – PCR  In-situ PCR  In-situ hybridization (ISH) LO: Explain the principles behind various tests used for detection of viruses/viral antigens/antiviral antibodies LO: Explain what factors that should be taken into account when selecting an appropriate diagnostic test and be able to apply this knowledge to specific diagnostic situations. LO: Explain the limitation of testing a single serum sample for the presence of antiviral antibodies. What information can you get from such testing. LO: Explain the pros and cons of virus isolation from clinical samples. LO: Explains the pros and cons of molecular detection (PCR) of viral nucleic acids. Herpesvirus Describe characteristic features typical to all herpesviruses. • A large and diverse family of dsDNA viruses capsid 100 nm, genome 125 to 235 kbp; encode >100 proteins  Relatively fragile in the environment  Spread via mucosal contact or by droplet  Replicate in the cell nucleus producing visible eosinophilic, intranuclear inclusion bodies  Typically host-specific  Establish latency in their hosts  Cause a range of diseases: o Respiratory disease

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Foetal death/abortion ulcerative skin lesions nervous system diseases lymphoproliferative diseases (e.g., lymphoid malignancies, ‘glandular fever’)

Explain viral latency and provide examples of its clinical significance. Viral latency: Viral genome is present but only a few (if any) genes are transcribed and there is NO production of infectious virus.  Herpesviruses characteristically establish latency after primary infection.  Sites of latency vary between herpesviruses, typically lymphoid or neural tissues  Virus is periodically reactivated and shed from latently-infected animals.  This recrudescence occurs even in the presence of circulating antiviral antibodies.  Recurrent episodes are usually milder than the primary episode, may be sub-clinical. Reactivation from latency:  Reactivation from latent infection (=recrudescence) is generally associated with stressors; e.g.,: o Crowding o Exhaustion o Other diseases o Transportation o Exposure to excessive UV light o Exposure to extreme cold or heat  During latency period the viruses are invisible to the immune system, no production of infectious virus.  Active infection: cell lysis with release of millions of progeny viruses. Immune response:  An immune response has little effect on latent virus i.e. it does not present recrudescence nor recrudescence nor clears the virus from latently infected animals  Humoral immune responses are directed at viral glycoproteins – high Ab levels not always protective.  Cellular immune responses possibly more important. Explain some other mechanisms of viral survival/persistence in nature. Describe briefly range of clinical presentations that herpesviruses can cause. Illustrate your answer with examples. Bovine herpes virus 1  Resp disease – infectious bovine rhinotracheitis  Genital disease – infectious pustular vaginitis  Keratoconjunctivitis  Abortion  Gastrointestinal disease in calves  Clinical signs: o Fever, depression, inappetence o Profuse nasal discharge (serous to mucopurulent) o Hyperaemia and ulcers on nasal mucosa o Early marked rhinitis and conjunctivitis o Gastroenteritis (particularly in calves – often fatal) o Occasion...