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 Orthomyxoviridae family of viruses
3 genera = Influenza A, B & C
Seasonal influenza - WHO estimate = 1 billion cases, 3-5 million severe illnesses, k 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
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:

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

6. 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

7. 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

8. 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!

9. 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

10. Cloning of HA gene - plasmids

11. Pseudotype virus production
Mather et al (2013) Future Virology 8(8);

12. 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