Virology Flashcards

Question

Give examples of viral diagnostic techniques based on identifying a virus's **antigenicity**, and describe their advantages & disadvantages.

Answer 1

Detecting virus using antibodies in **immunoassays**; can use **monoclonal** antibody specific for one antigenic epitope or **polyclonal** antibodies, ie., a mix of antibodies to detect several epitopes **Advantages**: fast, don't require live or infectious virus, specific **Disadvantages**: no amplification, relatively low sensitivity **Types:** **Antigen ELISA** - plate covered with antibody in sandwich in capture assay to detect antigen **Immunohisto/cytochemistry (tissue or cells)** - viral antigen in cells is detected by specific antibody, which carries fluorescent label (immunofluorescence assay) or enzyme label that reacts with colour substrate (immunoperoxidase assay) **Immunochromatography (fluids)** - eg. Rapid ImmunoMigration Witness FeLV test for finding p27 antigen in blood sample, uses antigen-specific antibody labeled with colloidal gold

Answer 2

**Neutralisation assay: **serum sample mixed with virus ➔ antibodies in sample bind to virus ➔ mixture added to cells ➔ if antibodies present, virus won’t infect cells ➔ if no antibodies, viruses affect cells, which show cytopathic effects such as Negri inclusion bodies **Haemagglutination inhibition assay: **based on some viruses' ability to agglutinate RBCs, esp influenza virus. Either a big blob of blood in the well or a “button” or “dot” of blood in the case of no reaction ie., presence of antibody (positive result for antibody response to virus) **Antibody ELISA** - opposite of antigen ELISA; plate is coated with antigen ie. whole virus or purified virus protein ➔ antibody in serum sample binds to antigen ➔ second antibody that recognises IgG is added ➔ second antibody carries enzyme label that changes colour substrate when bound

Answer 3

This involves identifying a virus based on detection of viral nucleic acid. There are two techniques: ## Footnote **1. PCR - polymerase chain reaction:** - amplification of viral sample - if not a DNA virus but RNA, then RNA needs to be converted to cDNA for PCR analysis - PCR is based on ability of Taq heat-stable polymerase to amplify DNA, by replicating from short primers specific for virus sequence; Taq can survive repeated cycles of heating to 94 C - generation of amplicon (viral sequence) of predicted size indicates presence of virus nucleic acid in sample; ie., produces large number of copies that are easy to detect - separated on agarose gel using electrophoresis - with electrophoresis must have right positive control **2. Real-time quantitative PCR** **- fluorescence-tagged:** - labelled DNA provides quantitative measure of amt of viral DNA in sample, eg measure of viral load; no need for electrophoresis **Advantages of both**: very sensitive, can use v small amt of starting material; doesn’t require live virus; specific as primers designed for target DNA **Disadvantages**: very sensitive, danger of contamination & false positives

Answer 4

1. Interference with Interferon (IFN) of the innate immune system 2. Hiding out in immune-privileged sites such as CNS, eye & gonads 3. Syncytium formation 4. Transformation 5. Viral production of antigen decoys 6. Antigenic DRIFT 7. Antigenic SHIFT 8. Interfering with MHC Class I Pathway of antigen presentation 9. Avoiding detection by NK cells 10. Superantigens

Answer 5

Some viruses have evolved to circumvent Type I IFNs (α, β, Ω): - virus produces early proteins that interfere with the signalling cascade produced by the infected cell’s toll-like receptors. This viral interference means the cell can’t produce IFN & its viral-resistance genes are NOT switched on - virus’s early proteins could BLOCK IFN-receptors, so the cell can’t respond to IFN & its viral-resistance genes are NOT switched on

Answer 6

Pestiviruses

Answer 7

These sensitive areas could be irreparably damaged by lymphocytes’ inflammatory & destructive immune processes; the BBB, for example, protects not only from infection but also from lymphocytes. Some viruses, such as Rhabdoviruses, deliberately migrate to CNS, gonads & eyes via neurons, for example, to evade immune detection

Answer 8

Viruses produce *fusion protein*, which enables cells to form syncytium ie., fusion of cells, which faciliates continguous spread of virus from cell to cell without having to exit into the ECF, where they’re susceptible to neutralisation by antibodies. ## Footnote NB microscopically, viral syncytia appear as a clump of cells with many nuclei

Answer 9

The clue is in the name: Bovine Respiratory Syncytial Virus (BRSV)

Answer 10

Oncogenic (cancer-causing) retroviruses integrate their DNA into host genome of infected cell ➔ Cell undergoes malignant transformation & proliferates out of control ➔ Viral genome, with oncogenes, replicated with each cell division ## Footnote Potential risk for xenotransplantation as endogenous retrovirus sequences could become activated to create pathogen

Answer 11

FeLV - Feline Leukemia Virus In FeLV-induced thymic lymphoma, the thymus becomes enlarged due to infection

Answer 12

1. Virus causes infected cell to secrete decoy antigen produced under direction of viral genome ➔ soluble antigen then complexes most of the virus-neutralising antibodies in the ECF ➔ virus can bind to target cell without interference. OR 2. Some viruses express decoy antigens on surface that are much more immunogenic (more attractive to antibodies, maybe bigger, for example) than the protein used for attachment ➔ antibodies bind to these decoy antigens ➔ virus’s functional antigens enable it to bind to target receptor

Answer 13

Ebola virus ## Footnote - produces decoy antigen similar to its structural protein but soluble, secreted by infected cell to saturate antibody binding sites.

Answer 14

Virus genes are subject to mutation, so virus-neutralising antibodies specific for defined epitopes of viral antigen can no longer recognise the virus if epitope changes. This is a *slow process* of natural mutation. Natural mutation ➔ protein sequence altered ➔ antibody doesn’t recognise epitope/antigen ➔ virus evades immune system

Answer 15

1. Feline calicivirus (FCV) strains ## Footnote 2. Feline panleukopenia virus (FPV) - mutations enabled FPV spike antigen to bind to transferrin receptor on canine enterocytes

Answer 16

Whole genes such as RNA segments are swapped between viral strains in a secondary host, such as a pig, to radically change antigens expressed - virus acquires novel antigenic gene that host has never seen before This is a rapid process.

Answer 17

Influenza viruses: the HA & NA genes have swapped between humans and animals.

Answer 18

They interfere in three ways with MHC Class I Pathway of antigen presentation to CD8+ Killer T-cells: 1. Viral proteins block TAP transporters that convey degraded antigen peptide from cytoplasm to MHC 1 molecule in ER for processing and surface presentation 2. Viral proteins cause MHC 1 to be retained in ER 3. Viral proteins cause dislocation of MHC into cytoplasm, where it gets degraded by proteosomes * NB: This impairment of MHC I also ↑ susceptibility of virus to innate killing response mediated by NATURAL KILLER NK cells, which target cells that show deficient/defective MHC production*

Answer 19

Virus causes infected cell to produce “fake” MHC molecules that “fool” NK cells into “thinking” the cell is ok ➔ virus avoids NK killing

Answer 20

Some retroviruses produce “superantigens” that “glue” MHC and T-cell receptors together non-specifically, ie., even if they don’t recognise the peptide ➔ polyclonal instead of antigen-specific T-cell response stimulated ➔ every T-cell activated in presence of infection, so T-cells are not effective against the viral pathogen

Answer 21

**Strategy**: *Direct immunosuppression* - targeted infection & destruction of cells of the immune system, esp. T-helper cells, macrophages & dendritic cells **Example**: *Feline Leukemia Virus (FeLV)* - oncogenic retrovirus causes neoplasia (lymphoma), myelosuppression (anaemia - stem cells in bone marrow infected) and immunosuppression

Answer 22

Lentiviruses use both Direct & Indirect Immunosuppression ## Footnote **Direct**: Targeted infection & destruction of cells of the immune system, esp. T-helper cells, macrophages & dendritic cells **Example**:* Feline Immunodeficiency Virus (FIV)* - the fighting kind; lymphopenia develops so WBC count low; CD4 T-cell numbers reach critically low level & immunosuppression symptoms become clinically evident **Indirect**: Subversion of cytokine responses; viruses produce proteins that bind to cytokines, acting as antagonists that neutralise cytokine reaction; viruses produce FAKE cytokines, acting as agonists **Example**: *Epstein-Barr virus (glandular fever) in humans & Orf virus in sheep* - produce FAKE interleukin-10 (IL-10) that causes infected host cell to “think” there’s no infection, so immune response is very weak.

Answer 23

**Strategy**: Tolerance **Example**: *Bovine Viral Diarrhoea Virus (BVDV)* - Transplacental spread of NON-CYTOPATHIC BIOTYPE of virus from dam to foetus ➔ antigen present in primary lymphoid tissues during lymphocyte development of foetal bone marrow & thymus ➔ *clonal deletion of virus-antigen-reactive lymphocyte*s, as if it was self-antigen ➔ calf becomes immunotolerant to virus NB: non-cytopathic biotype is the one that causes persistent infection, only mild diarrhoea. When calf is infected with cytopathic biotype, it develops mucosal disease & dies.

Answer 24

**Strategy**: Latency - virus enters quiescent/dormant stage of development undergoing no replication - Acute-phase stage of infection with replication and cell destruction can occur in one cell type but latency can occur in another cell type eg., acute phase in epithelial cells & latency in neurons or lymphocytes - Recrudescence (reactivation) to become fulminant again ➔ further shedding ➔ clinical disease can recur ➔ triggered by stress, meds or lowered immunity **Example**: *Feline Herpesvirus-1 (FHV)* - Cats often shed virus after pregnancy as recrudescence often occurs after parturition ➔ kittens infected ➔ kittens show signs of “cat flu” & develop latent infection ➔ persistently infected **Example**: *Equine herpesviruses (EHV-1, EHV-4)* - Virus moves into circulation via cell-associated viraemia, ie., lymphocytes become Trojan horse for virus ➔ virus moves to sites of secondary replication eg., pregnant uterus or CNS ➔ abortion or neurological disease

Answer 25

Antigenic DRIFT. Small numbers of mutations in three amino-acid residues of Feline Panleukopenia Virus (FPLV) CAPSID GENE allowed it to infect dogs & spread within new host order.

Answer 26

Antigenic SHIFT. Virus acquires different H type from different species than original host as result of re-assortment of viral segments Pigs can act as mixing vessels (receptor for avian+human influenza viruses) H1N1 - Spanish flu: avian virus might have jumped to humans H2N2 - Asian flu: reassortment of H1N1 with human strain & acquired 3 new genes H3N2 - Hong Kong flu: reassortment of human & avian flu viruses H1N1 - re-emerging: possibly released by lab H5 & H7 can become highly pathogenic 16 “H” types carry specific haemagglutinin (HA) antigens ie., spike envelope proteins 9 “N” types carry specific neuraminidase (NA)

Answer 27

An influenza virus that undergoes antigenic drift in the genes for its haemagluttinin spike proteins could cause it to bind differently to sialic acid on different host cell types and even different species’ cells. If the sialic acid is long and thin in structure due to α-2,3-link with galactose, then the AVIAN influenza virus's HA will bind, but if the sialic acid is slightly wider because it’s formed an α-2,6 linkage with galactose, then this kinked shape is recognised by HUMAN influenza virus's HA. If the cleavage sites are changed, even by just two amino-acid residues, then the influenza viruses could bind instead to different species’ receptors. Ie., Kinked α-2,6 linkage sialic acid receptors don’t usually bind to avian influenza virus but deep in our lungs humans have receptors for α-2,3-linkage that is recognised by H5N1 avian influenza.

Answer 28

An arbovirus is an arthropod-born virus that replicates in the arthropod vector as well as in the host. A virus that doesn't replicate in the arthropod vector but merely uses it for mechanical transmission of infection is not a true arbovirus.

Answer 29

A & B Leporivirus that causes myxomatosis in rabbits is a pox virus that targets epithelial cells of the skin & replicates in host-cell cytoplasm. As a pox virus, it is very large & carries its own replication proteins, thus doesn't need to enter the nucleus to replicate. Also, it uses insects and mosquitoes only as mechanical vectors, but doesn't replicate in them, so it isn't an arbovirus.

Answer 30

1. Flaviviridae - Flavivirus 2. Bunyaviridae - Bunyavirus 3. Reoviridae - Orbivirus 4. Asfarviridae - Asfivirus 5. Togaviridae - Alphavirus

Answer 31

Most arbovirus diseases that affect humans are caused Flaviviridae flavivirus arboviruses. Positive-sense single-stranded RNA viruses, enveloped. West Nile Virus Japanese Enchephalitus

Answer 32

An arbovirus in the Flaviviridae family, genus Flavivirus. **Epidemiology**: Endemic in Africa, Asia, Australia, Middle East, Europe and US **Vectors**: Mosquitoes mainly of genus *Culex* **Hosts**: Mosquitos & Birds (Humans, horses, bats, chipmunks, skunks, squirrels, rabbits, llamas are dead-end hosts that can carry low viraemia only ie., not infectious) **Transmission**: Bird-Mosquito-Bird Inoculation by mosquito → viraemia → mosquito vector takes blood meal from infected bird → infects other birds or mammals → targets cells of CNS in mammals **Clinical**: Neurotropic in mammalian hosts Case fatality of horses with clinical signs ~33% but high proportion of infected animals show no clinical signs Humans: \<1% cases result in serous illness (encephalitis, meningitis, death) **Control**: No commercial vaccine though DNA vaccine recently produced; Mosquito control

Answer 33

A tick-born arbovirus of the Flaviviridae family & Flavivirus genus that affects sheep in upland UK. **Vector**: *Ixodes ricinus* (sheep tick) * *Hosts**: Mainly sheep, but also red grouse - Cattle, horses, deer & man are dead-end hosts that end up with only low-level viraemia, subclinical not infectious. In rare cases causes severe neurological symptoms eg. meningoencephalitis **Transmission:** Tick takes blood meal in sheep ➔ virus replicates in lymphoid tissue ➔ transient viraemia of 1-5 days ➔ immune system eliminates from non-neuronal tissue but virus attacks CNS cells **Clinical:** Sheep - encephalomyelitis, ataxia, leaping gait, paralysis, but not all develop clinical disease; Red grouse - high mortality, up to 70% **Control**: Tick-control through acaricide treatment of domestic sheep 3x a year and regular vaccination against virus 2x in first year (young lambs borne to vaccinated ewes are protected by MDA) if possible/affordable.

Answer 34

The flavirus is present on hills and moors & prevalent in spring, but they could increase in numbers with climate change, which could extend the springtime period and/or expand the region of infection from hills to other areas. ## Footnote Sheep can also develop immunity to the 3x-yearly acaricide treatment and vaccine.

Answer 35

Blue Tongue (Blue Tongue Virus, BTV) African Horse Sickness (AHSV)

Answer 36

BTV is an arbovirus of the Reoviridae family & Orbivirus genus. It affects sheep & cattle, but sheep have much more visible clinical signs. **Epidemiology**: Endemic in warmer climates but from 1998-2004 there were outbreaks of diff. serotypes; emergency vaccination in 2008 brought it under control but we're seeing more now. **Vector**: *Culicoides* spp biting midges **Hosts**: Culicoides midges, domestic sheep / cattle / wild ruminants **Transmission**: Midges inoculate hosts with BTV ➔ replicates in monocytes, macrophages, lymphocytes, endothelial cells in lymphoid tissue, skin, lungs & elsewhere ➔ prolonged but transient viraemia ➔ damage to endothelial cells / blood vessels in target tissues ➔ ulcers & haemorrhage in oral cavity, upper GIT, pulmonary artery, oedema **Clinical**: **Sheep** - focally extensive reddening & ulceration of lower lip; vascular leakage (oedema) & haemmorhages around head & face; extensive erosion & shedding of mucosa on tongue; fever; necrosis of skeletal & cardiac muscle; erosion & ulceration of oral cavity, oesophagus & forestomachs; congestion; bloody nasal discharge **Cattle - **similar to sheep but in general show much less sign of disease **Control**: Attentuated virus vaccines (OVP); Inactivated virus vaccines are safest but drawbacks include multiple doses & limited number of serotypes avail.

Answer 37

AHSV is a mosquito-borne arbovirus of the family Reoviridae & genus Orbivirus, which affects equids. **Epidemiology**: Endemic to central tropical regions of Africa; Seasonal (late summer/autumn) and an epizootic cyclical incidence, associated with drought followed by heavy rain; Northern expansion of vector field to Mediterranean threatens Europe **Vector**: *Culicoides* spp. biting midges (C. imicola mainly) **Hosts**: Equids - horses, zebra, donkeys, mules **Transmission**: Mosquito inoculation with AHSV ➔ replicates in blood cells 7-14 days ➔ Cell-associated viraemia (RBCs, WBCs) 4-8 days, up to 21 days in horses ➔ vasculitis, ↑ vascular permeability **Clinical**: **Cardiac form: **swelling of head, neck, chest; mild fever, swelling above eye **Pulmonary form:** most severe with pulmonary oedema & 95% mortality Mortality rate in horses is 70-95%, mules around 50%, and donkeys around 10%; infection in zebra and African donkeys is subclinical **Control**: vector control, live-attenuated vaccine

Answer 38

Schmallenberg (genus Bunyavirus) Rift Valley Fever (genus Phlebovirus) Crimea-Congo Haemhorragic Fever (genus Nairovirus) We mainly need to know about Schmallenberg.

Answer 39

Schmallenberg virus is an arbovirus in the family Bunyaviridae, genus Bunyavirus. It affects sheep and cattle in Europe and the UK. **Vector**: Culicoides spp biting midges **Transmission**: Midge inoculates host with SBV → infects immune cells → inflammation, viraemia (4-6 days) → targets neurons / CNS **Clinical:** arthrogryposis in neonate sheep & calves especially Predominant CNS lesions or conditions were cerebellar and cerebral hypoplasia, hydranencephaly, porencephaly, hydrocephalus, and micromyelia **Control**: vaccine

Answer 40

ASFV is a tick-borne arbovirus of the Asfarviridae family, genus Asfivirus. Don't mix this up with African Horse Sickness Virus, which is an Orbivirus in the Reoviridae family. **Epidemiology**: Enzootic in most countries of sub-Saharan Africa; eradicated in Europe except for Sardinia **Vector**: Soft ticks, genus *Ornithodoros* **Hosts**: Domestic pigs **Transmission**: Soft tick takes blood meal & inoculates pig with ASFV ➔ virus replicates in perinuclear region of cytoplasm of macrophages, monocytes (doesn’t need nucleus machinery) ➔ incubation 3-15 days ➔ viraemia ➔ haemorrhage **Clinical**: Acute - Lethal haemorrhagic disease Reddening (white pigs) – tips of ears, tail, distal extremities, ventral chest, abdomen Anorexia, listlessness, cyanosis, incoordination within 24–48 hours before death, ↑ pulse & respiratory rate,V&D Death within 6–13 days, up to 20 days Lots of haemhorrages everywhere Survivors become carriers In domestic swine, mortality ~ 100% **Control**: No vaccine or treatment - import controls & careful waste disposal in free countries - limit pig movementto avoid contact with ticks - rapid slaughter & disinfection during outbreaks

Answer 41

Equine encephalitis (genus Alphavirus) We didn't really go over this in the course but it was highlighted by Noad as something we need to know.

Answer 42

Both are arboviruses. African Swine Fever Virus is in the family Asfarviridae, genus Asfivirus. Vector is the soft tick (genus Ornithodoros), host is the pig. Clinically it causes haemhorragic disease and death or carrier state. The virus replicates in the cytoplasm of host cells, in the pernuclear area, not the nucleus. Mainly sub-Saharan, eradicated in Europe except for Sardinia. African Horse Sickness Virus is in the family Reoviridae, genus Orbivirus. Vector is biting midges (*Culicoides*, esp. *C. imicola*), host is equids. Clinically there's the milder cardiac form that causes swelling about the head, neck & face; and the more severe pulmonary form, which results in pulmonary oedema & death in about a majority of infected horses. The virus causes cell-associated viraemia in WBCs, & significantly increases vascular permeability.

Answer 43

The “prion” theory, also known as the “protein only” theory, proposes that a normal cellular protein, named PrPc, is converted spontaneously into or comes into contact with a pre-existing abnormal isoform, PrPSc, which goes on to convert other normal isoforms into self-aggregates, fibrils and plaques. These accumulations of PrPSc interfere with normal neurological function, resulting in disease of the CNS. **NORMAL**: The normal PrPc protein is a cellular protein that is highly expressed on neurones. PrPc is a continuously produced and degraded, ie., a “recycling protein”, with a half-life of ~ 24 hours. It is also protease sensitive, which means it can be degraded by proteases. Secondary and tertiary structure of normal PrPc contain many α-helices, which don’t easily form compact aggregates, plaques or amyloid fibrils, as in tertiary form they form right-angles to each other. **ABNORMAL**: The disease isoform, PrPSc, like the normal isoform, are highly expressed in neurones, so when they aggregate, they interfere with neuronal function, specifically synaptic function. PrPSc is also relatively resistant to protease, making it difficult to degrade using low levels of protease. Secondary & tertiary structure of PrPSc contain a lot of β-sheets, which are basically parallel structures that easily aggregate to form amyloid fibrils and plaques. The isoform also facilitates conversion of the normal protein into the diseased form, and is transmissible through ingestion of the diseased protein. Other misfolding-protein neurodegenerative diseases include: * *Alzheimers** (amyloid plaques in the brain) * *Dementia with Lewy bodies (**α-synuclein) * *Parkinson’s** (α-synuclein)

Answer 44

Transmission of scrapie occurs via the alimentary tract. Dissemination of prions into the environment (soil) can occur from infectious placenta or amniotic fluid and possibly environmental contamination by saliva or excrement. The protein enters animal, mainly sheep, through intestines or cuts in the skin. It’s frequently transmitted in family lines, so some form of maternal transmission may occur at a pre- or postnatal stage. Prions stimulate cytokine production from astrocytes (astrocytosis) & microglia (brain macrophages) but there is NO infiltration of neutrophils, T-cells or B cells ➔ no adaptive immune response ➔ no IgG antibodies (so can’t detect by antibody ELISA unless in PrP knockout mice) ➔ replication requires active immune system ➔ replicates within follicular dendritic cells ➔ travels from GALT (Peyer’s patches) & gut to damage CNS / neuronal synapses, which is irreversible

Answer 45

Cattle became exposed to the abnormal prions through the feeding of ruminant-derived protein within feedstuffs such as meat-and-bone meal (MBM). The details of pathogenesis are unknown, but studies indicate that after oral exposure the agent replicates in the Peyer’s patches of the ileum followed by migration, via peripheral nerves, to the CNS. No evidence of horizontal transmission or environmental factor.

Answer 46

Prion disease is now more often detected by 96-microwell ELISA method. Prion disease is also detected by Western Blot identification.

Answer 47

Four steps: 1/ Run an extract of cells or tissue suspected of BSE infection through gel electrophoresis, which separates out proteins by 3D structure or length of their polypeptides, using molecular weight or electric charge. 2/ Gel-separated proteins then transferred onto a membrane, usually nitrocellulose. 3/ Proteins on membrane stained with antibodies specific to the target protein. There are three forms of the PrPc molecule: it is the same PrP protein glycosylated three different ways. Antibody can detect the differences, although it’s difficult to produce an antibody that selectively binds to PrPSc. Antibody to PrP can be produced by: - Immunising an animal with PrP from a different species. - Add a T-cell epitope to PrP protein so it will be recognised by T-cell as foreign - Immunise PrP-knockout mice (mice with no PrP proteins) with PrP to make antibody 4/ Split the sample into two and protease-digest one sample. The one that’s left over because it’s resistant is the infective PrPSc protein.