1. Journal Club Stuart Mather – 21 st October 2013

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 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 http://dog-bitetreatment.com/category/rabies-in-dog-and-treatment http://asylumeclectica.com/asylum/malady/archives/rabies.htm

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) http://www.vet.k-state.edu/depts/dmp/service/rabies/favn.htm Infection Neutralisation http://www.cdc.gov/rabies/specific_gr oups/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 lacZ as 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