Retrovirus

Retrovirus

1. Replication

   • 1. Virus attachment to plasma membrane
     • Adhesion to cell surface by specific (protein) receptor
     • Membrane fusion and release of nucleocapsid
   • 2. Reverse transcriptase synthesises cDNA within the capsid
     • → Initial reverse transcriptase – envelope removed, so ions and nucleases can get in
     • Retrovirus nucleocapsid contains almost everything it needs to reverse transcribe its RNA
     • Reverse transcription can take place in vitro, if virions are permeableised by mild detergents and the media contains a source of metal cations (Mg, Mn, Zn) and neucleotides
     • DNA synthesis takes place inside the virus capsid, the virus does not induce interferon and is refractory to its effects.
     • Retrovirus integrates into the host genome without de novo expression of antigen making it invisible to the immune system
   • 3. Several copies of virus cDNA integrated into host genome at “random” loci
     • → Can only insert where genome is unwound
   • 4. Synthesis of mRNA by host mRNA polymerase under control of strong virus promotor
     • → Viral RNA – viral proteins made by ribosomes
   • 5. Synthesis of new virus proteins
   • 6. Synthesis of new genome by host RNA polymerase
     • → +ve ss RNA – capped and poly-A tail (as made in mammalian cell), therefore looks different to all other viral and +ve ss RNA
   • 7. Assembly and budding of new virus particles
   • Schematic of reverse transcriptase
     • 50-100 copies per virus
   • copies RNA into DNA (RNA used as a template)
     • Can use RNA or DNA as a template, always producing DNA
     • RNA/DNA runs through the centre of the template
     • RNA is degraded at the RNAse H active site
       • Produces ss DNA and bits of RNA attached
   • Reverse transcriptase has both RNA dependent DNA polymerase and DNA dependent DNA polymerase activity.
     • By a complex series of steps in makes a complete double stranded DNA copy of the virus genome
     • Copies and degrades +ve sense RNA
     • Restart at the other end and run through it again – end up with a complete copy
       • Template switching copies -ve sense DNA to make duplex
     • ds DNA produced, containing all genetic information in original RNA
   • Classes of retroviruses
     • Replication-competent retroviral genome
       • Forms a Provirus ds DNA incorporated into the host genome
         • Doesn’t produce new virus, just sits in host genome
       • Strong viral Enhancer promoters
         • (may promote a nearby c-onc)
       • Cis-activating
     • Replication-defective rapidly transforming
       • (insertion of v-onc interfering with virus replication require helper virus)
         • V-onc is therefore interfering with replication
       • Double genome (means two pro-virus per cell)
     • Endogenous Retro Virus:
       • A complete copy of a retroviral genome (pro-virus) without promotors transmitted in the germ line from parent to progeny present in every cell. (and can exist in every member of some species).
       • Triangles indicate repeating sequences at either end
       • These proviruses are under the control of cellular regulatory genes and are normally silent
         • They usually do not contain v-onc (no ‘on’ gene).
       • Not replication defective and can be induced by a variety of external stimuli (irradiation, mutagen, immune stimulus, retrovirus)
         • Virus in genome that is silent can be activated by another virus particle with activation sequence
     • Replication-competent rapidly transforming
       • (Rous Sarcoma virus)
         • V-onc has been inserted and will not affect replication
       • Gene with v-onc causes tumour, therefore v-onc was the first oncogene to be found to cause tumours
       • Combination of defective and competent
         • Will replicate very well in a single host
         • Will not pass on well to a new host

2. Characteristics

   • Genome
     • 2 copies of +ve sense single stranded RNA
     • Structure of retrovirus Genome (cf.mRNA)
       • Only diploid virus
         • (2 identical strands +ve sense RNA poly A tail and capped)
       • Only +ve RNA not to act directly as an mRNA immediately after infection
       • U3, R, U5 – strong promoters for translation process
       • Only viral RNA to be synthesised and processed by host ENZYMES
     • Only viral RNA genome to be synthesised and processed by host ENZYMES
     • GAG = group associated antigen
       • Core protein in hexagonal area
     • U3, U5 – untranslated protein
     • Pol – reverse transcriptase, etc.
     • Env – peplomers etc.
     • Oncgenes
       • Originally defined in retroviruses
       • Viral genes that promote tumour development
         • → ‘cancer causing genes’
       • e.g. v-myc, v-mos, v-fos, v-src (>60)
         • Written in italics when associated with a virus
         • Myc = myelocytoma
         • Fos = feline osteosytoma
       • Many were found to have cellular equivalents
         • e.g. c-myc, c-mos, c-fos
         • Non-Italics - belong to host
         • C = cellular
       • V-Oncogens encode a range of proteins that affect growth promoting cell signalling pathways. (cell growth = virus growth)
         • E.g. growth factor, translation factor
         • Viral promotion of replication – if near cell growth genes, get a tumour
       • v-onc differ from host genes
         • They do not have introns
           • Can be pushed out very quickly – made by reverse transcriptase of host RNA
         • Are expressed out of their normal cellular context.
           • Under the control of strong viral promotors.
         • Have a high frequency of mutations. (generated during reverse transcription)
         • The strong promotors of a retrovirus inserted close to a cellular oncogene may induce overexpression of the cellular oncogene (Cys-acting).
       • Promoter acts on same strand of DNA, at lots of different points
   • Structure
   • Family: Retroviridae
     • Genus Alpha retrovirus:
       • Avian leukosis Viruses
       • Rous sarcoma virus
     • Genus Beta retrovirus:
       • Pulmonary adenomatosis of sheep (Jaagsiekte)
       • Several primate viruses
     • Genus Gamma retrovirus:
       • Feline leukemia virus
       • Feline sarcoma virus
     • Genus Delta retrovirus:
       • Bovine Leukemia virus
       • HTLV 1 & 2
       • Simian T lymphotrophic virus
     • Genus Lentivirus:
       • HIV
       • FIV
       • BIV
       • Maedi/Visna
       • Caprine arthritis-encephalitis virus
       • Equine infectious anaemia
     • Genus Spumavirus:
       • Bovine
       • Feline
       • Human Foamy virus (problem in cell culture)

3. Alpharetrovirus (avian)

   • There are a large number of avian retroviruses which affect many species of birds including poultry.
   • All chickens contain Endogenous Retro Viruses rarely expressed not pathogenic.
     • Passed on in egg – present in fertilised ovum
   • Avian leukosis virus : exogenous viruses, replication competent (no v-onc, cis acting slow transforming) they cause disease sporadically in birds >13 weeks
     • Infect one to another
   • One egg with retrovirus – will shed viral particle throughout life. Other chickens infected and virus gets oncogene – get tumour development

4. Feline leukaemia virus FeLV

   • Infection occurs in high density cat populations via respiratory, grooming or bites
   • Infection of adult cat
     • Immune response controls virus infection.
     • ± Latent provirus infection of lymphocytes
       • Will ‘turn off’, go and sit in lymph node. Virus is in genome and it is in a ‘shut down’ cell
     • Can’t get rid of virus totally by immune response, but can shut the disease down
   • Infection of pregnant queen or new-born kittens
     • 50% Immune response - control virus infection, latent infection of lymphocytes
     • 50% No immune response – persistent virus infection, immuno-tolerant, productive infection of lymphocytes
       • Will likely develop tumours
         • Likely to trigger an oncogenic effect
       • Source of infection for other cats
     • Infection at a late stage of pregnancy – still shedding at partuition
     • More common in feral cat populations
   • Cells infected with FeLV express Feline Oncovirus Cell Membrane Antigen (env gene product of ERV FeLVC)
     • Immune competent cats
       • Self-limiting infection associated with antibodies to viral antigens and FOCMA, remain non viraemic.
       • Immune response to exogenous and endogenous infection
     • Immune tolerant cats
       • Persistent infection with viraemia, no antibodies to virus antigens or FOCMA.
       • Infectious to other cats and develop disease.
   • FeLVA is a slow transforming (cis-acting) virus
     • Wide range of diseases can be caused by this virus
     • Depending on cell type/ oncogene involved
     • Lymphosarcomas 30% of feline tumours; thymic, muticentric (T-cell), alimentary (B-cell)
     • Scattered around – spleen, lymph nodes
     • Myeloproliferative: erythromyelosis (red cell precursor), granulocytic leukemia (neutrophil precursor), myelofibrosis (fibrobla sts) lymphocytic leukaemia
   • Diagnosis of vraemic immuno-tolerant cats depends on detection of P27virus antigen in blood or serum
     • In practice test kits are available online – lots of virus in circulation (+ve test)
   • Abortive – can miss the virus particle in blood – but doesn’t mean it isn’t there
   • Provirus DNA – virus RNA as well (virus going around in blood)
   • Anti FOCMA antibodies – antibodies to an oncogenic antigen
   • Prevention
     • Testing and removal of infected animal from Catteries
   • Vaccines
     • Cell culture derivedgp70, FOCM Aantigen
     • (Leucocell2/quantum cat FeLV)
     • E. coli expressed Subunit vaccine (Leucogen)
     • Recombinant canary pox virus
     • Non core vaccine
       • Not a high risk for the majority of cats.
         • Often used to keep under control at breathing level

5. Equine infectious anaemia (swamp fever)

   • Targets Monocyte macrophages.
   • Frequently asymptomatic (avirulent strains)
     • More virus than disease
   • Virulent strains (acute viraemia, thrombocytopaenia and anaemia)
   • Cyclical pathology (immune escape mutations)
   • Partial recovery with time
   • Notifiable in UK
   • Antibodies in blood (Coggins test)
   • Chinese attenuated vaccine
     • Gives a +ve coggins test result
   • Need to carry out serological tests to prove clearance of disease
   • May be important for import/export certificate

6. PCR test for pro-viral DNA in lymphocytes

   • Amplification of LTR region (avoids endogenous retrovirus false positive results)
   • PCR able to detect 1 copy of viral DNA, but this requires an infected lymphocyte to be in the initial sample, so false –ve results do occur
     • Promoters at either end are lost in endogenous so look for these in exogenous
     • Not every lymphocyte is infected – lung cells are initial target

7. Lentivirus infections

   • Feline Immunodeficiency Virus
     • 3-6% of healthy cats positive for antibodies 30% of sick cats
       • Disease burden associated with virus (indirect association with other pathologies)
     • A widespread and common virus of cats, which was first identified in 1987.
     • Related to HIV and similar in pathogenesis
     • Takes a long time for infection to develop
     • High incidence of FIV and FeLV tend to go together
   • Lifelong infection: virus is present in blood and saliva
     • Cats become infected usually by fighting, but can be trans-placental or at parturition (viraemic mother)
     • Env binds to receptors CD134 and CXCR4
       • Not many cells have CD134
       • CXCR4 is a chemokine receptor (lots of cells have it) – T helper cells
     • Virus infects activated feline CD4+ T-cells
     • Acute phase I (2 months) → viraema, infection spreads to more CD4+ cells.
     • Fever, malaise and lymphadenopathy
   • Initial immune response, antibody production, and cytotoxic CD8 T-cells which reduce the number of infected CD4+ cells with apparent clinical recovery.
     • Phase II. may last several years
     • Mutations of the virus allow it to escape from CD8+ T-cell control
     • CD4+ T-cells containing provirus cannot be eliminated
     • Gradual decline of CD4+ T-cells follow recurrent cycles of expansion, immune elimination, mutation and escape of virus infected cells.
   • Central role of CD4+ T-helper cells in immune responses and progressive drop in CD4+
     • Reduced IL2 production
       • Major CD4+ growth factor
     • Reduced antibody production,
     • Reduced blastogenesis
     • Reduced Cell Mediated Immunity
     • Phase III: onset of symptoms due to immunosuppression associated with opportunistic infections
     • High frequency of fevers, gingivitis and mouth ulcers.
       • Common presenting signs
   • Strong association with FeLV infection, and B-cell lymphomas
     • Unsure of which causes the other, as occur at the same time
     • Neurological symptoms can also occur in rare cases were infection of astrocytes and microglial cells predominates
   • Diagnosis
     • Depends of the detection of antibodies to virus
   • Treatment
     • Azidothymidine, which inhibits reverse transcriptase is partially effective.
     • Several experimental vaccines but none effective virus persists in the presence of antibody and cytotoxic response.

8. Maedi/Visna in sheep

   • (Similar pathogenesis to FIV)
   • Maedi, infection of Monocytes and monocyte precursors, new virus is produced when monocytes become activated
   • Lifelong infection with late onset of disease symptoms, chronic inflammation/fibrosis induced by infected monocytes
   • Symptoms of breathlessness, consolidation of lungs and enlarged bronchial lymph nodes, fibrosis and hyperplasia of alveolar septae with monocyte infiltration
   • Visna chronic demyelinating encephalitis with monocyte in filtration caused by a distinct subtype of virus. (Rare)
   • Virus persists in the presence of antibody and cellular immune response possibly due to constant mutation and generation of escape variants.
   • Transmission from mother to lambs in colostrum milk.
   • Transmission of virus by aerosol from symptomatic sheep or from blood by biting insects.
   • Control by eradication
     • Removal of symptomatic sheep
     • Removal of seropositive sheep
     • Removal of PCR +ve sheep
     • This hasn’t been engaged with with too much enthusiasm – would need to remove a lot of sheep to get the problem under control

9. Caprine Arthritis/encephalitis

   • Similar virus to maedi/visna, but lesions occur as a proliferative synovitis with lymphocyte plasma cell and monocyte infiltration.
   • Spread in colostrum and milk. (kids develop neurological symptoms)
   • Several small ruminant lentivirus have been identified and sub-typed according to serology and host range.
   • Some are found mainly in sheep (Maedi/visna) others are mainly goat (Caprine arthritis)
   • Some occur equally in sheep and goats.
   • They vary in their pathogenicity.

10. Ovine pulmonary adenomatosis (Jaagsiekte)

    • Worldwide disease of sheep (except Australasia)
    • Both sheep and goats contain several (27) related endogenous retroviruses (JSRV) in their genome
    • Develop tumours in the lungs
    • Neoplastic transformation of type II alveolar cells and non-cilliated bronchial epithelial cells
    • Nodular adenomas/adenocarcinomas secrete large amounts of surfactant containing fluid.
    • Metastasis to L.N.
    • The disease is spread by the respiratory route, disease incidence can be as high as 20% following introduction of virus.
    • But more commonly 1-5% of sheep in endemic flocks become affected.
    • Clinical signs include breathlessness, lung sounds, large amount of pulmonary fluid only seen in some cases
    • Tip sheep up on front legs, and fluid will pour out of their nose
    • The JSRV genome does not contain a conventional oncogene.
      • JSRV env protein binds to Hyaluronidase 2 (Hyal2)
        • A hyaluronidase enzyme on the surface of many types of cell
        • Including type 2 pneumocytes.
      • JSRV env protein has transforming properties when transfected into a wide range of cells through binding to Phosphatidylinositol 3-kinases (a cell signalling pathway).
        • Only exogenous protein affects the signalling pathway
        • enJSRV env protein does not bind PI3_Kinase and lacks transforming properties.
        • But enJSRV Env is expressed in the placenta and foetus where it has an important function in the development of the multinucleated trophoblast cells essential for implantation.
          • Nearly identical, but without env gene
        • Cells normally stop growing when they touch each other (e.g. skin), these will keep going
      • Affected sheep have no antibody response to JSRV env protein as the expression of endogenous retrovirus causes the sheep to be immuno-tolerant to the jaagseike virus.
        • Not all infected sheep develop lesions
        • In infected flocks many more sheep have JSRV in peripheral blood lymphocytes than have lung lesions (despite lack of antibody response).
          • Endogenous proteins block replication of exogenous
      • Initial infection of a cells by JSRV is much more efficient in dividing cells.
        • Type 2 pneumocytes are actively dividing in lambs in which JSRV is more likely to establish lung infection leading to the development of lung lesions.
        • In Older sheep intercurrent disease with lung damage leads to pneumocyte division as part of the healing process which increases the risk of tumour development
        • Co-expression of mutant GAG proteins from some endogenous retroviruses disrupts assembly and exit of mature wild type virus; hence act to limit JSRV spread within the lung and prevent development of tumours.
        • Susceptible when healing
        • ↑ risk of replication, develop tumour
        • Virus is a trigger/amplifier of effects – usually another factor that triggers formation of a tumour

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