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4 th Year MPharm SRP: Introduction to Pseudotype Viruses

4 th 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.

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4 th Year MPharm SRP: Introduction to Pseudotype Viruses

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  1. 4th Year MPharm SRP: Introduction to Pseudotype Viruses Stuart Mather Project Leaders: Dr Nigel Temperton and Dr Simon Scott

  2. Influenza virus • Member of the Orthomyxoviridaefamily 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).

  3. 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

  4. Influenza A Virus Transmission Taken from ‘Fields Virology’ (EdsKnipe & Howley)

  5. 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.rapidreferenceinfluenza.com http://www.virology.ws

  6. 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

  7. 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!

  8. 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

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

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

  11. 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

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