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