4th Year MPharm SRP: Introduction to Pseudotype Viruses Stuart Mather Project Leaders: Dr Nigel Temperton and Dr Simon Scott

Influenza virus Member of the Orthomyxoviridae family of viruses 3 genera = Influenza A, B & C Seasonal influenza - WHO estimate = 1 billion cases, 3-5 million severe illnesses, 300-500k deaths Huge financial implications – treatment and vaccination costs, loss in workforce productivity Certain subtypes responsible for pandemic influenza outbreaks – e.g. Spanish flu (H1N1) – death toll of approx. 50 million in 10 months http://www.nhs.uk/Conditions/vaccinations/Pages/flu-influenza-vaccine.aspx National Museum of Health and Medicine, Armed Forces Institute of Pathology, Washington, DC, USA (NCP1603).

Influenza virion RNA genome – eight segments, single stranded, negative sense HA = responsible for binding of virus to sialic acid cellular receptor NA = cleaves sialic acid to release progeny virus during egress 17 subtypes of HA and 10 subtypes of NA: http://en.wikipedia.org/wiki/Influenza

Influenza A Virus Transmission Taken from ‘Fields Virology’ (Eds Knipe & Howley)

Antigenic drift Accumulation of random mutations in epitopes of viral antigens ‘Evolution’ of HA so that existing antibodies less efficient at neutralising drift variants Cause of recurrent influenza epidemics – necessary to repeatedly vaccinate http://www.virology.ws http://www.rapidreferenceinfluenza.com

Antigenic shift Introduction of new or evolved influenza subtype into the human population Direct avian to human transmission Reintroduction of historic virus subtype Genetic reassortment Cause of devastating global influenza pandemics - population has totally naïve immunity to the novel virus

Pseudotype viruses are a solution! Serological assays Serology – study of plasma serum – identification of antibodies PRNT – ‘gold standard’ for measuring virus-neutralising antibody (VNAb) response Required to use infectious virus Low-throughput – takes ~6 days to produce assay results Other assays, such as HI, SRH and ELISA are employed Do not measure neutralising antibody response Issues with low sensitivity and specificity in some cases Need for a sensitive and specific method for measure VNAb response without handling live virus Pseudotype viruses are a solution!

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

Cloning of HA gene - plasmids http://www.addgene.org/plasmid_protocols/subcloning/

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

Reporter gene flexibility Variety of reporters can be incorporated into the pseudotype platform Ability to ‘cost-customise’ the system Temperton and Wright (2009) Encyclopedia of Life Sciences