Virology LOs.pdf

VM 7535 Veterinary Virology Learning Objectives Ch. 1 - Introduction to Viruses Learning Objectives 1. List six important ways that viruses impact domestic animals, humans,...

VM 7535 Veterinary Virology Learning Objectives Ch. 1 - Introduction to Viruses Learning Objectives 1. List six important ways that viruses impact domestic animals, humans, and science. a. Viruses cause significant diseases in animals b. Viruses cause agriculturally and economically important exotic diseases c. Viruses cause important zoonotic diseases (public health importance) d. Viruses induce cancers in animals e. Viruses are responsible for several newly recognized (emerging) diseases f. Viruses are invaluable for studying basic biological processes and are useful as vectors 2. Describe four general aspects of viral biology that make viruses so formidable. a. Virus transmission is very efficient and occurs in many ways i. Often reach high titers, infected animals excrete large amounts of virus ii. Some are very stable in the environment iii. Use a wide variety of routes for transmission b. They (especially RNA viruses) are highly prone to genomic mutations i. Results in development of new virus strains that cause significant disease in their usual hosts or acquire the ability to infect new hosts (“species-jumping”) c. They successfully evade host defenses (immune evasion) by a number of mechanisms i. Latency, antigenic variation, destruction/suppression of immune system ii. By evading the immune system, they are able to replicate, cause disease, and often persist and transmit to other hosts iii. Important Q for vets: where and how do viruses persist? Why? Persisting viruses are the source of the next infection d. Unlike bacteria, viruses are NOT susceptible to common antibiotics i. Safe and effective antiviral drugs are limited and primarily used for treating certain human viral infections 3. List the five major primary sources of virus that affect animals, and for each source identify one way to mitigate transmission of virus to an uninfected host. Primary infection - reinfection → isolation of infected host Zoonotic transmission → decrease interspecies interaction Environmental infection → sanitation, preventative measures, appropriate decontamination Persistently infected host → antiviral drugs if available, supportive care and isolation Vector-borne transmission → decrease interspecies interaction, preventative measures 4. List and be able to diagram the 2-3 main structural components of viruses. 5. Identify how viruses differ from bacteria in their multiplication, genomic make up, protein synthesis, energy production, autonomous growth and antibiotic susceptibility. Lack metabolic machinery to generate energy → obligate intracellular parasites Do not replicate by binary fission Transmit genetic material from one host to another and use host machinery to replicate  6. Have an understanding/perspective on the size of viruses and how they are visualized. Viruses are considered the smallest microorganisms known to infect humans and animals They are several orders of magnitude smaller than the smallest bacterium The sizes of viruses are commonly expressed in nanometer (nm) Individual virus particles can only be seen by electron microscopy Ch. 2 - Virus Structure and Classification Learning Objectives 1. Recognize the diversity of viral genomes: nucleic acid type, structure, polarity. Viral genomes come in many types: - RNA or DNA (but not both) - Linear or circular - Single-stranded or double-stranded - Single molecule or may come in segments - Polarity: +, -, or +/- (ambisense) - Range in size from 2kb to >200kb - Haploid (except retroviruses – diploid) **Regardless of genome type – transcription of +mRNA from the viral genome must occur! 2. Outline the differences between a viral capsid and envelope with regards to their contents and how they are derived. Viral capsid Envelope A protective protein shell in which the viral A lipid membrane that surrounds the capsid  nucleic acid is packaged - Composed of a lipid bilayer (host cell - Encoded by viral genome origin) and associated glycoproteins - Composed of a variable # of identical (viral origin) protein subunits (capsomeres) - Enveloped vs non-enveloped/naked - Each capsomere is comprised of viruses multiple protein subunits (protomers) - Lipid bilayer portion acquired by - Protomers → capsomeres → capsid budding through a host cell - Formed by self-assembly of membrane - usually plasma protomers and capsomeres membrane - Can be icosahedral or helical - Glycoproteins - carbohydrate moieties - added by cellular machinery by virus (aka “spikes”) 3. Identify five ways/conditions that inactivate enveloped viruses and explain how some can be exploited to control infections in veterinary medicine. In general, enveloped viruses are NOT stable in environment; more sensitive to: - Heat - Oxidation - Freezing/thawing - Desiccation - Detergents High temps are very effective for inactivation (surface protein denaturation) → helpful for sanitation protocols Sensitivity to detergents → specific cleaning solutions can be used 4. Explain why capsid proteins are optimal targets for serological testing and vaccine development. Capsid proteins are usually conserved, and contain “group-specific antigens” common to variants of a particular virus. They elicit robust immune responses (both humoral and cell-mediated) Capsid proteins are the basis for many serodiagnostic tests (ELISA, immunofluorescence, etc.) and used as targets for vaccine development. 5. Identify the common functions of capsid and envelope proteins for naked and enveloped viruses. Functions of capsids: - Protects viral genome in extracellular environment; - Mediates binding of the virus (if non-enveloped) to host cells to facilitate entry;  - Interacts with viral and cellular proteins to promote uncoating after entry into the cell and packaging of the viral genome during assembly of virus particle; - Interacts with cellular proteins/factors during exit from the cell Functions of envelope: - Carries glycoprotein molecules that are important in virus interaction with the cell, including binding to cellular receptors and fusion with the cell membrane for entry into cell; - In addition to entry, virus envelope glycoproteins facilitate exit from host cell; - In some viruses envelope proteins play a role in uncoating of viruses; - Contain protective (neutralizing) antigens that elicit antibody response 6. Define "serotype." Explain how new viral serotypes may arise, what limits the emergence of novel serotypes, and the potential consequences of novel serotype emergence. Serotype (viral serotype): a set of viruses that can be distinguished from other viruses in the same species on the basis of antigenic properties - important for understanding neutralization properties Ch. 3 - Viral Replication Learning Objectives (Part 1) 1. List the key steps of viral replication and briefly describe what occurs at each step. Step 1: Virus Attachment - Virus replication begin with binding/attachment of the virion to the host cell - Viral attachment protein (VAP) on capsid or envelope, AKA ligand, on virus → virus receptor, on the cell (receptor already present, the virus is just able to utilize it) - Attachment is always a receptor-ligand mediated event - The cell receptor may be protein, carbohydrate, or lipid - Cells without appropriate receptors are NOT susceptible - Appropriate receptor is a primary determinant of host cell specificity (but not the only determinant) Step 2: Penetration - Following successful ligand-receptor binding, the virus gains access into the cytoplasm by passing through a cellular membrane (e..g, plasma and endosomal membranes) - Non-enveloped: direct penetration - Enveloped: membrane fusion - Receptor-mediated endocytosis - For viruses that do not penetrate at the plasma membrane – RME - Used by both enveloped and non-enveloped viruses - Provides environment (i.e., low pH) that promotes fusion and/or uncoating  - Facilitates intracellular trafficking – a “free ride through the cytoskeleton” - Penetration: non-enveloped virus - No envelope = no fusion - Membrane puncture: virion creates pore in membrane, genome (not capsid) released into cytosol - Direct penetration: the capsid/genome passes through 1. Perforation: no major lysis of the membrane 2. Lysis: dissolution of the membrane - Penetration: enveloped virion - Enveloped virions → membrane fusion – using viral fusion proteins - Different protein than VAP or distinct domain - Fuses viral with cellular membrane → capsid/genome into the cytoplasm - Depending on virus, fusion occurs at either plasma or endosomal vesicle membrane Step 3: Uncoating - One of the least understood steps in virus replication - Removal of capsid, liberation of the viral genome - Uncoating can occur simultaneously with, or independent of, penetration - Aided by viral or cellular factors, and/or drop in pH - Uncoating can occur at plasma membrane, within endosomes, or at nuclear membrane Step 4: Biosynthesis - Primary requirements for the virus at this step: - Synthesize mRNAs recognized by cell translation machinery → 1. Proteins for replication and components of the virion structure - Replicate viral genome → 2. New genomes for packaging into virions - Incredible diversity in how different viruses/virus families achieve these two goals - In all cases by exploiting the biology of the host cell - Clinically relevant features of biosynthesis: - Diversity of virus genome structures - Replication and expression of DNA viruses - Replication and expression of RNA viruses - Processing of viral mRNAs and proteins Step 5: Assembly - Goal: virus proteins and genomic nucleic acid must co-localize and assemble into a functional virus particle - Formation and assembly of capsid (protomers and capsomeres) - Selective packaging of nucleic acid genome and other essential viral components - Acquisition of an envelope (depending on virus) - Virion assembly - “Signals” in the relevant amino acid sequences determine interaction - Movement often involves the cellular microtubule apparatus and other transport systems (vesicles) - Site of assembly often where diagnostic inclusion bodies can be seen - RNA viruses – cytoplasm  - DNA viruses – nucleus Step 6: Release - Non-enveloped viruses - Generally, requires cell death (cell lysis, “lytic infection”) - The cell dies → membranes degrade → virions released - Enveloped viruses - Do not require cell lysis - Assemble adjacent to cell membrane and mature by budding - Process of budding is not necessarily lethal to cell - Non-infectious until the virion acquires an envelope 2. Be able to align the steps of the viral replication cycle with steps of the viral growth curve.  3. Describe what the one step growth curve tells us about a virus and why the eclipse stage is named as such. The one-step growth curve is a classic study of viral replication 1. Inoculate cultured mammalian cells with virus at a high multiplicity of infection (MOI – ratio between virus particles and number of cells) 2. At varying times post-inoculation, collect culture supernatant (liquid that sits on top, contains growth medium for cells) and cell lysates 3. Determine amount of virus produced by titration on fresh cells 4. Plot amount of virus over time A one-step growth curve tells us how time influences infectious virus units per cell as the virus undergoes a replication cycle. The “eclipse period” occurs when the virus is uncoating, and synthesizing its genome and proteins, within a host cell. The infectious virus is not detectable at this time. What the growth curve tells us: 1. Virus production does NOT occur in a linear fashion (more like a stepwise fashion) 2. Only virus growth has eclipse phase 3. How fast the virus replicates 4. How many virions are produced per infected cell 4. Given a viral particle and a cell be able to identify the locations of a viral ligand and viral receptor. Be able to describe how they influence both host and tissue tropism. A viral ligand or viral attachment protein (VAP) is located on the virus, as either a part of the capsid or a part of the envelope. It interacts with a virus receptor on the host cell. The receptor is already present, but the virus is able to utilize it to attach/bind. Viral tropism: the ability of different viruses to infect different cellular types ultimately to produce a successful infection This is influenced by the type of receptor a viral ligand can bind to The appropriate receptor is a primary determinant of host cell specificity (but not the only determinant). Virus ligand-host receptor interactions is a key point of virus vulnerability → one of the best ways to defend against viruses is to prevent (neutralize!) infection by blocking entry into cells Antibody → block binding of virion to its receptor → neutralize infectivity Distribution of virus receptors is an important factor in defining the tissues affected (and thus the pathogenesis of the disease) **Change in receptor usage can result in host range expansion and emerging disease** 5. Explain two potentially serious consequences that could occur if a virus alters viral receptor usage. Give one example where this has happened in veterinary medicine. If a virus alters viral receptor usage… 1. It may be able to expand its host range (cat → dogs and cats) 2. It may be able to expand its tissue range (skin → visceral organ) ?? One example: the story of Canine Parvovirus - In the 1920s, FPV caused a recognized disease in domestic cats; probable interspecies transmission between FPV and related viruses of feral carnivores - 1976-1978 – mutation in FPV capsid protein gave the virus the ability to bind to canine TfR (transferrin receptor; iron!) → 2 new diseases emerged in dogs: myocarditis and enteritis - 1978-1980 – new canine parvovirus (CPV-2) spreads worldwide; CPV-2 can infect dogs, but no longer infects cats - 1984-1990 – Additional mutations in CPV-2 reinstates the ability to infect cats: CPV-2a replaces CPV-2 throughout the world 6. Identify the steps in the viral replication cycle that are most vulnerable to interference by antibodies. Step 1: Virus Attachment - Antibody → block binding of the virion to its receptor → neutralize infectivity - Virus ligand-host receptor interactions = key point of virus vulnerability Step 2: Penetration - Fusion proteins are targets for host neutralizing antibody response – can prevent fusion and genome release Step 4: Biosynthesis - Some viruses (like Herpesviruses) make their own DNA polymerase to copy DNA 7. Describe how viral fusion proteins and distribution of cellular proteases affect tissue tropism and virulence between low- and high-pathogenic avian influenza viruses. During Step 2 (Penetration), - Fusion proteins  - Fusion proteins contain hydrophobic domains (a.k.a. Fusion peptides) which insert into the cell membrane - In native conformation of the envelope glycoprotein – fusion peptide is hidden - Stimulus causes conformational changes, position the fusion peptide for insertion into cellular membrane → active - Variety of mechanisms - Activation by cellular protease - Envelope glycoproteins often translated as precursor polyprotein - Made from host cell but encoded by viral genome - Cleavable by cellular proteases → rearrangement of the protein - Additional conformational changes usually required for full activation (for influenza and others, pH change) - Clinical relevance of fusion proteins - Targets for host neutralizing antibody response - Targets for antiviral drugs - Leads to ‘syncytial cell’ formation by fusing cells (diagnostic) - Can be important determinant of tissue tropism, host range, and virulence - Specificity of proteolytic cleavage site is an important virulence determinant - Protease cleavage site and distribution of cellular proteases can affect tissue distribution and pathogenicity Low pathogenic avian influenza (LPAI) vs highly pathogenic avian influenza (HPAI): - Protease usage dictates the disease severity - In LPAI, proteases are localized to the respiratory and intestinal organs → mild disease - In HPAI, there are ubiquitous proteases, protease is in different tissues → lethal disease Learning Objectives (Part 2) 1. Identify the single reason why replication of all viruses must result in the production of positive (+) sense mRNA, whether they possess a RNA or DNA genome. +mRNA is read by the host ribosome for the production of viral proteins. 2. Explain two strategies that RNA viruses employ to produce positive sense mRNA. RNA viruses use virus-encoded enzymes for replication of RNA by one of two unique pathways: 1. RNA-dependent RNA synthesis [RNA-dependent RNA polymerase/RdRp] a. RNA → RdRp → +mRNA 2. RNA-dependent DNA synthesis (reverse transcription) [RNA-dependent DNA polymerase/RdDp; reverse transcriptase/RT] a. RNA → RdDp/RT → DNA → +mRNA b. retroviruses 3. Identify the major reason why RNA viruses are more prone to developing genomic mutations. RNA viruses are more prone to developing genomic mutations because they do not have a proof-reading mechanism, which results in a higher error/mutation rate during genome replication. That facilitates genetic variation and adaptation/immune evasion. 4. Describe why it is advantageous for a virus to have a segmented genome. It is advantageous for a virus to have a segmented genome as viruses with differing segmented genomes can swap genome segments and create new viruses. 5. Explain why viral encoded proteins important in viral replication are excellent targets for developing antiviral therapeutics and give examples of two viral targets. Viral encoded proteins important in viral replication are great targets for developing antiviral therapeutics as they are virus-specific, so therapeutics would not target the host machinery. Additionally, these therapeutics affect the viral replication cycle and are thus important in treating viral infections. Example 1: RdRp, RT, and DNA viral polymerases are virus-unique enzymes and are excellent targets for antiviral drugs. Example 2: Viral proteases are good targets for antiviral drugs – they are needed for the final processing of a viral polyprotein during biosynthesis. Ch. 4 - Virus-Cell Interactions Learning Objectives 1. Explain the difference between susceptible and permissive cells and identify what is necessary for productive viral infection. Infection: the process by which a virus introduces its genome into the cell Susceptible (cell): has correct receptors for the virus to attach - Determined by presence of receptors - Does not imply production/release of infectious virus Permissive (cell): steps of virus life-cycle beyond attachment - The host cell environment supports complete replication of a virus - Depends on internal biochemistry, not presence of receptors – does not imply susceptibility Productive infection: production of progeny virions occurs following an infection Susceptible + Permissive = Productive Get new infectious viral particles Abortive infection: production of progeny virions does not occur following an infection Susceptible + NOT Permissive = Abortive Do not get new viral particles 2. Describe how CPE (cytopathic effect), cellular metabolism, specialized functions, and viral productivity are different between cytolytic, productive persistent, and latent persistent infections. Cytolytic infections include cytopathic effects and cell death - Cytolytic viruses – productive infection in susceptible and permissive cells - Resulting CPE can be observed in light microscopy - The nature of the CPE may be characteristic of a particular virus (aid in diagnosis) 3. Define CPE and explain what the presence of CPE tells you about a sample submitted to a diagnostic lab. CPE = cytopathic effects; refers to virus-induced cellular changes visible by light microscopy - Cytoskeletal disruption - Syncytia - Necrosis - Apoptosis - Lysis - Cell rounding commonly seen prior to lysis - Lysis – strategy for release - Occurs late – after virions assembled - Inclusions Cytolytic infections include cytopathic effects and cell death - Cytolytic viruses – productive infection in susceptible and permissive cells - Resulting CPE can be observed in light microscopy - The nature of the CPE may be characteristic of a particular virus (aid in diagnosis) 4. Recognize that some CPE are specific for groups of viruses. Syncytial cell formation: lentivirus, coronavirus, paramyxovirus, herpesvirus Inclusion bodies: adenovirus, herpesvirus, Rabies virus 5. Define viral latency and describe how it may influence which diagnostic test you choose to detect infection. Viral latency: virus is present but not replicating Considering a latent virus is non-productive, there will be no observable effects on cells. 6. Describe three ways that DNA viruses promote tumor development. Mechanisms of Transformation by DNA viruses DNA viruses produce oncoproteins that: 1. Inactivate tumor suppressor gene proteins 2. Bind growth factor receptors and activate signaling pathways 3. Produce oncoproteins that drive or limit transcription (transcription factors) 7. Using the appropriate terminology, explain the four ways that retroviruses cause tumors. Transformation by Retroviruses As a consequence of integration, retroviruses can: 1. Acquire host genes – move them to other cells (transduction) 2. Activate cellular genes (Cis-activation) – virus promoter (LTR) activates nearby cellular genes (insertional activation) 3. Activate cellular genes (Trans-activation) – virus gene product acts as transcription factor to induce cell transformation 4. Activation of signal transduction – virus protein stimulates cellular pathways to trigger cell growth 8. List three features of retroviral replication/biology that lends to their ability to cause neoplasia. Transformation by retroviruses results from key features of retrovirus replication: - Reverse transcription of (+) ssRNA into dsDNA - Integration of dsDNA into host cell chromosome  - Transcriptional regulatory sequences in the viral long terminal repeat (LTR) → viral promoter Ch. 5 - Viral Pathogenesis Learning Objectives - Part 1 1. Explain the difference between pathogenicity and virulence. Pathogenicity: ability of the virus to cause disease (YES or NO; qualitative) – property of virus and host Virulence: relative measure (quantitative) of pathogenicity – property of the virus only - How severe is the disease? 2. The majority of infections in a population do not result in disease. Describe two main management practices that you can implement to maintain a larger underwater portion of the iceberg (keep disease inapparent in most infections). Management strategies that keep the underwater portion of the iceberg larger: - Vaccination and herd immunity - Nutrition - Good biosecurity - Hygiene - Decrease stress / overcrowding 3. We discussed that as a veterinarian you should be concerned about genetic changes in a virus because they can lead to expansion of tissue tropism, host range, or potential loss of vaccine immunity. Additionally, viral genetic changes can lead to a change in virulence. Describe how a more virulent virus would shift the iceberg. A more virulent virus would shift the iceberg, as more animals would have more severe disease and consequently, experience more severe clinical signs. 4. List the three principal factors that determine the outcome of viral infection and the severity of disease. What factors at the individual host level can influence disease outcome? Major factors that influence the outcome of infections: - Genetic features of virus - Exposure DOSE of virus (if high → overwhelm immune system, increases viral burden) - Host factors - Age (i.e. very old, very young) - Immune status - Concomitant infections (can increase likelihood of secondary infections)  - Nutritional status (helps w/ normal function of immune system) - Genetics 5. Describe how DEED influences the risk of viral transmission. DEED Dose → higher dose, harder to overcome Envelope or not → no envelope = more stable Environment → temperature of medium / transmission; humidity, wind Distance → must work harder if further distance 6. List and describe the major anatomic portals that viruses use to enter and exit a host. Portals of Entry – Horizontal Transmission Mucosal Surfaces - Dividing, living cells - Access to submucosa - Dissemination via lymphatics Sites of Entry Barriers Respiratory Mucociliary clearance Digestive Acid, bile, proteases, Urogenital Peristalsis Conjunctival Rapid turnover Skin Viruses that enter via skin do so via abrasions and penetrations - Injuries, cuts, scrapes - Mechanical insults - Arthropod vectors Iatrogenic transmission – veterinary/animal husbandry practices - EIAV transmitted via contaminated needles, twitches, etc. (i.e. blood) - Bovine papillomavirus, BLV – ear tagging, tattooing, dehorning Portals of Entry – Vertical Transmission - Transmission from dam to embryo/fetus - Usually refers to transmission prior to birth - In germ line of fertilized eggs - Across the placenta (a few are able) - During passage through the birth canal - Perinatal transmission – colostrum Consequences of vertical virus transmission: - Abortion (Herpesvirus) - Congenital disease (Bluetongue virus) - Persistent infection (Bovine Viral Diarrhea Virus)  - Neoplasia (Retroviruses) 7. Describe the major differences between transient and persistent infections regarding duration of infection, infectious period, and whether clearance of virus occurs. Transient vs Persistent Infections Transient Infections Persistent Infections Virus eliminated from the host Virus not cleared by immune system Usually short duration (days to weeks) Virus avoids/interferes with the immune response Self-limiting, controlled by immune response Productive or latent Variable disease – from subclinical to progressive A non-equilibrium process – virus and host never Always source of virus! come into balance Equilibrium between virus and host – balance - Minimal tissue damage when in equilibrium - Viral replication generally restricted (except immune tolerance!) - Virus infection generally non-cytopathic 8. List the five mechanisms that viruses use to persist in an infected host. Mechanisms of viral persistence: - Evade adaptive immune response - Immune tolerance - Infect tissues with reduced immune surveillance - Infection of immune system cells - Restricted viral gene expression/latency 9. Describe immunological tolerance as a mechanism of viral persistence, and identify what laboratory test you would use to diagnose persistent viral infection in an animal that is immunologically tolerant to said virus. Example: BVDV – Bovine Viral Diarrhea Virus - Infection of fetus with non-cytopathic BVDV during the first 120 days of pregnancy induces tolerance and persistent infection - Virus continually replicates – viral antigens are recognized as “self” Lab test to diagnose persistent viral infection: viral titer?? 10. Briefly describe how the skin and brain are inherently different in supporting viral persistence by reduced immunosurveillance. Tissues with reduced immune surveillance have a high threshold of immune activation (it takes a lot to activate)  - e.g. skin, glands - Papillomaviruses - Why? → because tissues are always being exposed to antigens! Tissues devoid of immune activation (immune-privileged sites) - e.g. the brain - e.g. infection of the cerebral cortex (equine) by Borna disease virus 11. Describe tactics used by viruses to evade and subdue the adaptive immune response Evasion of adaptive immune response: 1. Virus induces ineffective antibody a. Animal makes antibody, but against wrong antigen (a non-neutralizing Ab) i. ex: African swine fever, retroviruses (FIV, HIV-1) 2. Change neutralizing antigens a. “Antigenic variation” by some viruses – i.e. EIAV (Equine infectious anemia virus) i. Changes surface proteins so Ab cannot bind ii. This is how EIAV remains persistent!!** 3. Interfere with “antigen presentation” a. Block steps in pathways of antigen presentation by MHC Class I and II i. Proteins never make it to the cell surface for presentation! 12. Describe how restricted viral gene expression aids in viral persistence. The absence of (or minimal) viral proteins allows the virus to hide from immune system recognition. Examples: Herpesviruses, Lentiviruses, and other retroviruses - Slow, persistent, progressive infections - Restricted expression for long periods Example: Papillomaviruses - Early (cell cycle regulation) vs late (structural) gene expression 13. Describe how the results of a diagnostic test for antibody (serology) would be different between viruses that use immunologic tolerance and those that use latency (restricted gene expression) as mechanisms of persistence. What about when testing for viral antigen (protein)? A diagnostic test for antibody would be different between viruses that use immunologic tolerance and those that use latency (restricted gene expression): - Those that use immunologic tolerance – virus recognized as “self” so no antibodies made  - Those that use latency/restricted gene expression – few antibodies as infections are slow and there is little gene expression and subsequently, little protein (antigen) production by the virus When testing for viral antigen: - There would be a high viral titer / increased amounts of viral antigen for viruses that cause immunological tolerance - There would be a low viral titer / low amount of antigen for viruses that use latency Learning Objectives - Part 2 14. Outline how localized and systemic viral infections differ in sites of replication, spreading, incubation period (onset of clinical signs), shedding, and severity of disease. Localized vs Systemic Viral Infections Localized Systemic Sites of replication Virus replicates at or near the site of entry Virus spreads from site of entry Replication at sites of entry in the respiratory and alimentary tracts (mucosal) or skin Spreading Spread by infection of neighboring cells Lymphatic and hematogenous (cell-to-cell) dissemination Virus may enter lymphatic system, but does not replicate well Incubation period / Usually have a short incubation period (days Occurs after secondary (peak) onset of clinical to weeks) viremia – ~8-9 days signs post-infection Onset of fever, seeding of target organs 1-2 weeks for clinical signs to appear Shedding Entry, replication, lesion formation, and Sites of entry, the ‘target’ shedding occur in, and from, the same organ organ(s), and the site of shedding may or may not be the Shedding into lumen – NO VIREMIA same Maximal shedding before peak clinical 10-12 days, replication occurs in disease target organ, and virus shedding occurs – followed by clinical disease, signs, and transmission Severity of disease Faster time to recovery Takes longer to clear Does not imply mild disease Can be more severe  Examples Canine papillomavirus Feline infectious peritonitis (FIP) Calf – rotavirus infection (severe!) Canine Distemper 15. Given an infection scenario that includes characteristics listed in the previous objective, be able to identify whether an infection is localized or systemic. 16. Describe the difference between primary and secondary viremia and identify why secondary viremia is more diagnostically useful. Primary viremia is often subclinical or undetectable, and occurs after approximately 4 days post-infection. Secondary (peak) viremia involves high enough / detectable amounts of viral titers and usually coincides with onset of clinical signs, after approximately 8-9 days post-infection. Secondary viremia is more diagnostically useful because there is a higher amount of virus circulating in the blood. 17. List the 4 key factors that govern whether a virus remains localized or spreads systemically. Key factors that govern whether a virus infection remains localized or spreads systemically: 1. Directional release 2. Availability of susceptible/permissive cells in deeper tissues 3. Macrophage/monocyte susceptibility and permissiveness 4. Temperature range of the virus 18. Briefly explain why macrophage susceptibility plays an important role in viral infection outcomes. Macrophages are present in all body compartments. The outcome of the virus-macrophage encounter is often important in systemic spread of the virus. The macrophage may ingest and kill the virus, or become infected - If infected, since they are mobile cells, they facilitate spread to many tissues! 19. Be able to determine whether a viral infection is likely to be localized or systemic when given the directional release of virus particles. Directional Release - From the apical surface → luminal release – back where you started - Localized infection - From the basolateral surface → move away from luminal defenses and gain access to underlying tissues - Systemic infection  20. Briefly explain how a change in body core temperature (hypothermia) may influence the anatomic site of viral replication in an infected host. Many viruses replicate more efficiently at lower temperatures → infection will remain localized to cooler areas (i.e. respiratory/oral mucosa, skin). Hypothermia may influence the anatomic site of viral replication by allowing the virus to replicate and spread into deeper areas. Example: Feline herpesvirus – pneumonia can occur in neonatal hypothermic kittens as the virus is able to replicate in the lungs due to cooler body temperatures 21. List the two major factors that contribute to the termination of an active viral infection and identify the difference in relative importance of these factors between localized and systemic infections. Resolution of Infection 1. Depletion of susceptible cells a. More important for localized infections 2. The immune response a. Innate immune responses very important for localized infections b. Adaptive immune responses important in recovery from systemic infections c. Abs prevent infection d. CTLs kill infected cells 22. Explain the timing differences in peak viral replication, immune responses, clinical signs, and when diagnostic samples should be collected between localized and systemic infections. Timing Differences: Localized vs Systemic Infections Localized Systemic Peak viral replication Around days 3-4 as maximal Around days 8-9 as central virus shedding occurs depots amplify virus and secondary viremia occurs Immune responses Innate immune response kicks Adaptive immune response in ~ day 2 as viral replication kicks in ~ day 9 or 10 as virus reaches peak shedding occurs in target organ and virus replication is at peak Clinical signs Begin at around day 2 when After secondary viremia, innate immune response kicks anywhere between 8-12 days in, other signs around day 4 when inflammatory response ramping up When diagnostic samples During peak viral replication During secondary (peak) viremia should be collected stage – around days 2-4 – around 8-9 days PI Ch. 6 – Host Response to Viral Infections Learning Objectives 1. Describe the three main host defense systems that animals possess to defend against viral infection. Host defenses against viral infections Portals of entry 1) Physical and biochemical barriers at body surfaces a) Repels many pathogens b) Skin, mucociliary clearance, acid, bile, proteases, natural inhibitors in fluids Following infection 2) Innate immune response a) Generalist immune response b) Rapid activation as soon as pathogen is recognized c) Induces the adaptive immune response 3) Adaptive immune response a) Antigen (pathogen)-specific b) Takes days to weeks to develop c) Result in memory of the antigen 2. The host defense systems can be considered 1st, 2nd and 3rd lines of defense based on their roles in protecting the host. List the order in which they exert their protective effects and understand the relationship between the latter two systems, clinical signs of disease, and diagnostic tests. Physical and biochemical barriers attempt to prevent infection at portals of entry → Innate immune response → Adaptive immune response 3. Describe how the innate immune system detects viral infections and how this detection system leads to stimulation of other components of the immune system. The innate immune response detects viruses by sensor molecules; pattern recognition receptors (PRRs) recognize pathogen associated molecular patterns (PAMPs) associated with viral infection. The binding of PRRs to respective PAMPs initiates cell signaling cascade → type I IFNs and proinflammatory cytokines induced. This detection leads to the induction of type I IFNs and cytokines; then there is an influx of NK and phagocytic cells; finally, there is induction of the adaptive immune response.  4. Explain the role of type I interferons in antiviral immunity. Type I interferons (IFNs) are a family of cytokines produced by almost all cells in response to viral infection - IFN-alpha and IFN-beta - Rapid response – within 3-4 hours after infection - Stimulated by viral PAMPs (ex: dsRNA) - Secreted by the virus-infected cells - Protects other cells against viral infection - Induce antiviral response in neighboring cells through action of IFN-stimulated genes (ISGs) IFNs are a very important innate defense mechanism, and help slow down the infection in early stages. They do NOT protect virus-infected cells; they induce antiviral effects in neighboring, UNINFECTED cells. IFNs are usually host-specific. 5. Know what causes fever during viral infection and how the febrile response benefits the host in the context of viral infections. Proinflammatory cytokines are released by infected cells, and exert direct and indirect antiviral activity: - Fever (all are potent pyrogens) - Inflammation - Induction of adaptive immune responses **They contribute to the subjective feeling of malaise (“flu-like” symptoms) The febrile response benefits the host in the context of viral infections: - Inhibits / slows viral replication - Increases rate of many defense mechanisms - Dilation of blood vessels - Increased blood flow to affected areas - Increased rate of inflammatory response **Only use anti-pyretics like NSAIDs if fever dangerously high; if low-grade, do not 6. Briefly describe the role of NK cells and phagocytes in the host response to viral infection. NK cells and phagocytes… - Are not virus antigen-specific, and are relatively fast (2-3 days) to respond. - They kill virus-infected cells (NKs), phagocytose/degrade viruses (macs, neutrophils)  - Elaborate vasoactive substances that increase the speed and intensity of the immune response - Secrete cytokines important in induction of adaptive immune response NK cells must be activated by activating ligands to initiate cell killing (regulated – apoptosis); otherwise will favor inhibition and self-tolerance. 7. Describe how the humoral and cell-mediated immune responses work together to address viral infections. What aspect of a viral infection does each arm of the adaptive immune response address? The adaptive immune response takes days to weeks to develop in naive animals. Very specific response - triggered by antigen-receptor interactions Effector cells = B and T lymphocytes - B lymphocyte → antibody production - Helper T lymphocyte → cytokines → B cell and CTL activation - Cytotoxic T lymphocyte → killing of virus-infected cells Viruses exist as extracellular virions, yet replicate and assemble within a cell → susceptible to both antibody and cell-mediated immunity → viruses differ with respect to which type of immunity is most effective Humoral immune response - Extracellular virus: antibodies → block infection Cell-mediated immune response - Intracellular virus: CMI → kill infected cells 8. List and briefly describe the three main types of antibody-mediated host defense mechanisms to viral infection. Neutralization: - Neutralizing antibodies specifically bind to virions and prevent attachment, penetration, uncoating - Does not involve cells or cellular immunity - Not all antibodies are neutralizers - Neutralizing Abs detected by virus neutralization test - Test determines if produced Ab do neutralize virus - Complement activation can cause lysis of free virus or infected cells Opsonization and Phagocytosis: - Facilitates uptake/intracellular destruction of viruses - Antibody-virus complexes – internalized by phagocytic cells - Occurs via interaction between Fc receptors on macrophages and neutrophils - Not neutralization because it requires cell!  - Downside of opsonization: some viruses that replicate in macrophages (e.g. FIPV) can use this mechanism to enter cells Antibody-dependent cell-mediated cytotoxicity (ADCC): - Mechanism for killing cells bound by specific Abs to cell surface viral antigens (i.e. glycoproteins) - Effector cells: NK cells, macrophages, neutrophils - Presumably far less important than CTL-mediated killing of infected cells 9. Describe the mechanisms of virus neutralization by antibodies (not involving cells). Mechanisms of virus neutralization: - Steric hindrance – antibody binds and prevents interaction and attachment - Blocks ligand-receptor interaction - Inhibition of penetration/uncoating – prevent conformational changes that expose fusion peptide; stabilization of capsid - Aggregation - fewer infectious particles – less circulating virus able to infect - Antibody + complement → lysis of viral envelope 10. Explain how cytotoxic T cells help clear viral infections and the role played by helper T cells. CTLs are essential for control of most virus infections - Viruses replicate intracellularly - Viral proteins are processed through the endogenous pathway - Viral peptides are presented with MHC class I molecules on cell surface - CTLs recognize viral antigens presented by MHC Class I - Activated CTL releases perforin and granzymes – kills virus-infected cell by inducing apoptosis Helper T cells help activate CTLs 11. List and briefly describe the strategies that viruses use to evade the innate and adaptive immune responses. Viral strategies to evade the immune response: - Escape by antigenic variation - Prevention of apoptosis of infected cells - Escape by latency/restricted gene expression - Modulation of MHC class I expression - Inhibition of antigen presentation - Destruction of immune cells - Cytokine targeting - Inhibition/interference of interferon response pathway  12. Explain how the immune response to viruses can contribute to the pathogenesis of viral diseases such as FIP. Products of the immune response itself can cause disease in several viral infections. Pathogenesis of FIP: Feline enterocoronavirus (FECV) → mild/subclinical enteritis Feline enterocoronavirus → mutation → Feline infectious peritonitis virus (FIPV) → replication in macrophages → immune complex formation in blood vessels → vasculitis → clinical forms of disease (effusive - wet; non-effusive - dry) Effusive form: fluid accumulation due to vasculitis, resulting in fluid leaking from blood into the abdomen Example of immune complex-mediated tissue injury Ch. 7 – Prevention and Treatment of Viral Infections Learning Objectives 1. Know the main goals of vaccination. Goals of vaccination: - Prevent or reduce disease (not necessarily infection!) - Reduce transmission 2. Describe how vaccination fits in with other methods of controlling viral disease. Vaccines help to prevent and control viral diseases in a population, and assist with preventing transmission, along with quarantine and disinfection. 3. Describe the ideal characteristics of an attenuated vaccine virus strain and the methods used for developing attenuated virus strains. An attenuated vaccine is the most common form of live virus vaccines Goal: reduced virulence but still induce protective immunity Use naturally occurring avirulent strains - Vaccine virus may be from the same host species or different species Experimentally attenuated strains - Goal is to select for a virus that: - Has reduced virulence - Retains protective antigens - Replicates sufficiently well to stimulate immune response - Empirically derived mutants - Apply “selective pressure” – i.e. passage in cell culture or foreign host - Induction of mutations through growth in mutagenic chemicals or radiation - Test for reduced virulence in host species or closely related species - Temperature-sensitive (ts) mutants - Select for mutants with altered temperature preference - Virus ‘forced’ to adapt to lower temperature by repeated in vitro passage - Loses its ability to replicate at normal body temperatures - Genetically engineered mutants - Based on a rational approach - Requires knowledge of which genes control virulence - Introduce targeted mutations in virulence genes by: - Modification (deletion, site-directed mutagenesis) - Gene deletion (referred to as “Marker” or “DIVA” vaccines) - DIVA = Differentiating Infected from Vaccinated Animals  4. List the types of viral gene product vaccines available and know how these products differ from whole virus vaccines. Virus proteins / gene products = subcomponents of virus (vs whole virus) - Protein subunit - VLP – viral proteins assembled in a virus-like particle - DNA – purified DNA that codes for a protective antigen (viral protein gene) - RNA – mRNA message that codes for protective antigen (viral protein gene) 5. Define a DIVA vaccine and describe the advantages it confers over standard attenuated vaccines. DIVA vaccines have gene deletions/markers which help to differentiate infected from vaccinated animals. A naturally infected animal will have antibodies to a specific gene product, whereas a vaccinated animal will not have antibodies for that gene product (because the gene was deleted). 6. Know the reason why live virus and inactivated vaccines generally should not be mixed. Live virus and inactivated vaccines generally should not be mixed as they could recombine to form a virulent virus?? 7. Understand the advantages and disadvantages of replicating vs. non-replicating vaccines in terms of effectiveness, safety, and stability. Replicating vaccines: - Effective at inducing protective immunity - Ability to revert to virulent strain Non-replicating vaccines: - Less effective at inducing protective immunity - No ability for reversion 8. Describe the barriers to producing effective antiviral drugs (especially for veterinary use). - Drugs must be both safe and effective - Safety - Viruses are obligate intracellular pathogens (OIPs) → use cell machinery or encode related proteins - Compounds interfering with virus growth often have adverse effects - Very difficult to make a drug that inhibits viral replication without toxicity to the host! - Specificity of viral agent - Target specificity is narrow - few “broad-acting” antivirals  - So need etiologic diagnosis (need to know exact virus causing infection) - Treatment generally must be early to be effective - Many viruses have maximal replication before overt disease - Most useful if given shortly after infection - In vitro replication - Some viruses do not replicate in cell culture - difficult to assess efficacy - Cost - Pharmaceutical companies - expense vs profit potential - Owners - cost-prohibitive (i.e. rimantadine would be ~$600/day for a horse) - Cost of Hepatitis C treatment (cure) in people - $84,000 for 12 weeks 9. Be able to describe the general modes of action for antiviral drugs that target different stages of the virus life cycle. Direct-acting antiviral drugs target stages in the virus replication cycle. - Uncoating - Interfere with uncoating - Blocks M2 ion channel → blocks uncoating - Resistance an issue - Amantadine/Rimantadine for influenza - Replication / Transcription (viral polymerases) - Nucleoside analogs (like Acyclovir) - Similar to guanosine for example - Highly effective against herpes simplex viruses (human) - Less effective against varicella zoster and many veterinary alpha-herpesviruses - Acyclovir is a pro-drug - no anti-viral activity until converted to active TP form, which is a DNA chain terminator - First phosphorylation is carried out by viral kinase - Highly toxic in infected cells but no antiviral activity in uninfected cells - Acyclovir has low toxicity in non-infected cells - Nucleotide analog (Cidofovir) - Selectivity based on preferential use by viral DNA polymerases - Nephrotoxicity - best for topical application - Endonuclease inhibitors (Xofluza) - Endonuclease inhibitor - targets polymerase acidic (PA) endonuclease, which is part of the viral nuclease - Viral endonuclease needed for genome replication (cap snatching mechanism) - Single treatment - Release - Tamiflu = Neuraminidase (NA) inhibitor  - Influenza virus HA binds to sialic acid on surface of cells - Release requires cleavage of sialic acid by NA - NA inhibitors prevent virus release - Viral protein cleavage / protein synthesis - Paxlovid = protease inhibitor - targets viral 3CL protease - 3CL protease responsible for processing viral polyprotein - Combined with ritonavir, affects drug degradation → can affect other meds P is on; hard to treat with this drug, esp if taking other meds