Introduction
Evolutionary changes in Influenza A(H3N2) viruses in recent years have made it difficult to detect antigenic change using haemagglutination inhibition (HI) assays, due to their loss of ability to agglutinate red blood cells from avian or mammalian sources. Here we describe the use of a virus microneutralization assay called the Focus Reduction Assay (FRA) to test the antigenic characteristics of currently circulating human A(H3N2) viruses.
Methods
Ferret post-infection antisera generated against a number of genetically distinct haemaglutinin clades of circulating H3N2 viruses were serially diluted and incubated with a test viruses diluted at a pre-determined virus titre. The serum-virus mixes were then added to MDCK-SIAT seeded 96 well plates, incubated for 90 minutes, and an Avicel overlay was added to restrict virus spread. Plates were incubated overnight at 350C, 5%CO2, and were then formalin fixed. Immunostaining was performed using a primary mouse anti-Influenza A NP antibody followed by a secondary goat anti-mouse IgG HRP conjugate, which was detected by True Blue substrate. Spots of infected cells were scanned and analysed by the CTL immunospot analyser.
Results and Conclusions
Using the Focus Reduction Assay has allowed the rapid antigenic characterisation of many human A(H3N2) viruses isolated at the WHO Influenza Centre that were not able to be characterised by the traditional HI method. This has provided valuable data for influenza virus surveillance, and will help in the selection of suitable H3N2 candidate vaccine viruses for influenza virus vaccine updates. The assay is more sensitive than the HI assay, making it a more robust assay for comparing antigenic differences amongst circulating A(H3N2) isolates. As 2017 has been a very severe influenza season in Australia, dominated by H3N2 viruses, this assay is now an important adjunct to our suite of viral analysis tools.