Development of lentiviral pseudotypes for surveillance studies on animal influenza viruses

Pseudotyped viruses (PVs) provide a safe, flexible platform for fundamental virological studies and antibody screening assays. Generation of influenza PVs involves co-transfection of producer cells with plasmids encoding the necessary viral components. The pseudotype virus neutralisation assay (PVNA) is a sensitive technique to measure protective antibody responses which cause neutralisation of virus particles. Many traditional methodologies, e.g. Haemagglutination Inhibition (HI) and Single Radial Haemolysis (SRH), detect only surface glycoprotein binding antibodies whereas the PVNA quantifies infectivity-neutralising responses.

Haemagglutinin (HA) and neuraminidase (NA) are the two major surface glycoproteins embedded within the membrane of an influenza virion. HA is responsible for virion attachment and entry into a host cell and NA is essential for viral egress and thus spread of infection. Two enzymes crucial for the infectivity of influenza viruses are; HA-cleaving cellular proteases and the NA itself. Optimisation of both enzymes in PV production is necessary to increase the titre of PVs. Producing high titre PVs is important as this permits minimal quantities to be used in PVNAs, and repeat experiments can be carried out using the same batch of virus, minimising intra-study variability.

Optimising PVs and employing them in a novel situation, such as an equine influenza vaccine efficacy trial, has been carried out with promising results. We have demonstrated that data obtained from the PVNA correlates well with the traditional SRH assay and consequently there is the potential for more widespread adoption in research and commercial settings. Furthermore, PVs have been manipulated to assess how single amino acid changes with equine influenza virus can affect the neutralisation efficacy of sera generated by vaccination.

Novel PVs, such as those derived from canine and phocine (seal) influenza strains have also been produced and provide a new platform for sero-surveillance of these viruses particularly in the case of wild or feral animals, due to issues with obtaining samples from wild animals during acute infection.

Overall, PVs have been demonstrated as useful and readily-manipulated tools for studying antibody responses against equine, canine and phocine influenza viruses.