The use of equine influenza pseudotypes for serological screening

Standard assays used for influenza serology present certain practical issues, such as inter-laboratory variability, complex protocols and necessity for handling certain virus strains in high biological containment facilities. In an attempt to address this, avian and human influenza HA pseudotyped retroviruses have been successfully employed in antibody neutralisation assays. In this study we have generated equine influenza pseudotyped lentiviruses for serological screening. This was achieved by co-transfection of HEK293T cells with plasmids expressing the haemagglutinin (HA) protein of different H3N8 subtype equine influenza virus strains, HIV gag-pol and firefly luciferase reporter genes and harvesting virus from supernatant. To produce infective pseudotype particles it was also necessary to co-transfect a plasmid encoding the TMPRSS2 endoprotease to cleave HA. High titre pseudotype virus (PV) was then used in PV antibody neutralisation assays (PVNAs) to successfully distinguish between vaccinated and non-vaccinated equines. The sera were also screened by single radial haemolysis (SRH) assay. There was a 65% statistical correlation between the results of the two assays, with the PVNA assay appearing more slightly more sensitive. Future work will extend the testing of the PVNA with a larger cohort of serum samples to assess sensitivity/specificity, inter/intra-laboratory variability and to define a protective titre.