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Journal Club

Stuart Mather – 21 st October 2013. Journal Club. About the author – Ed Wright. BSc Virology (1999 – Edinburgh); PhD in Molecular Virology (2003 – Cambridge) MRC/UVRI Uganda Research Unit on AIDS (2004-2005) UCL – Postdoctoral Research Fellow in Robin Weiss lab – (2005-2011)

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Journal Club

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  1. Stuart Mather – 21st October 2013 Journal Club

  2. About the author – Ed Wright • BSc Virology (1999 – Edinburgh); PhD in Molecular Virology (2003 – Cambridge) • MRC/UVRI Uganda Research Unit on AIDS (2004-2005) • UCL – Postdoctoral Research Fellow in Robin Weiss lab – (2005-2011) • University of Westminster – Senior Lecturer & Principal Investigator at VPU Fitzrovia labs – (2011-present)

  3. Rabies • Viral infection of the brain and central nervous system – zoonotic (cross species; from animals to humans) • Transmitted in saliva – e.g. the bite of an infected dog http://asylumeclectica.com/asylum/malady/archives/rabies.htm http://dog-bitetreatment.com/category/rabies-in-dog-and-treatment • Symptoms – paraesthesia, malaise, fever, headache leading to acute pain, hyperactivity, excited/enraged behaviour, hydrophobia, paralysis and death • Symptomatic cases are nearly always fatal • Post-exposure prophylaxis (PEP) during virus incubation period can prevent illness – ≈15 million receive PEP annually

  4. Global burden of rabies • ≈55,000 deaths per year – 95% in Africa and Asia • Over 3 billion people worldwide at a realistic risk of transmitting rabies

  5. Rabies virus biology • Member of the Lyssavirus genus and Rhabdoviridae family • Enveloped, bullet-shaped virus • ≈ 120nm long and 75nm wide • Negative sense, single-stranded, linear RNA genome of ≈11kb, encoding for 5 proteins • Glycoprotein (G) = responsible for virus binding to cellular receptors (e.g. nAChR – acetylcholine receptor) and membrane fusion • G is also the main virus antigen http://www.nhs.uk/Conditions/rabies/Pages/introduction.aspx http://viralzone.expasy.org/all_by_species/22.html

  6. Lyssavirus genus Banyard AC et al Adv Virus Res. 2011;79:239-89. doi: 10.1016/B978-0-12-387040-7.00012-3. ‎

  7. Traditional rabies serology • Fluorescent Antibody Virus Neutralisation (FAVN) Test • Rapid Fluorescent Focus Inhibition Test (RFFIT) • Enzyme Linked Immunosorbent Assay (ELISA) Infection http://www.vet.k-state.edu/depts/dmp/service/rabies/favn.htm Neutralisation http://www.cdc.gov/rabies/specific_groups/doctors/serology.html

  8. Pseudotype viruses • ‘Chimeric’ viruses made up of a retroviral core (e.g. HIV), a heterologous envelope (e.g. rabies G) and encapsulating a quantifiable reporter gene (e.g. luciferase) • HIV - core • Rabies G – envelope • Luciferase – reporter • Non-infectious - Can be used instead of infectious virus in serological assays to determine neutralising antibody titres

  9. Pseudotype virus production Mather et al (2013) Future Virology 8(8); 745-755

  10. 4 main aims of the study: • Comparative serology using RABV pseudotype neutralisation assay against FAVN in a vaccination trial • Production of Mokola (MOKV), Duvenhage (DUVV) and Lagos bat (LBV) pseudotype viruses • Incorporation of lacZas a pseudotype reporter gene • Stability of pseudotype viruses

  11. Outline of rabies vaccination trial

  12. Pseudotype neutralisation assay (PNA) performance

  13. FAVN assay performance

  14. Correlation between PNA and FAVN

  15. Production of other lyssavirus pseudotypes

  16. Incorporation of lacZ reporter gene

  17. Stability of pseudotype viruses

  18. Summary • Rabies pseudotype neutralisation assays perform as well as FAVN for vaccine evaluation • MOKV, LBV and DUVV lyssavirus pseudotypes have been successfully produced • lacZ is a cheaper alternative to luciferase and GFP reporter genes • Rabies (CVS-11) pseudotypes are relatively stable after freeze-thawing and long term storage

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