Influenza viruses mutate frequently therefore, it is crucial to closely monitor circulating viruses to determine if they match the vaccine viruses and to update the vaccine if they don’t. Generally the hemagglutination inhibition (HI) assay is used to assess the antigenic properties of influenza viruses due to the simplicity and high throughput of the assay. For HI assays to be performed two essential elements are required; firstly influenza viruses must be able to be grown in tissue culture to sufficient titre and secondly the viruses must hemagglutinate red blood cells (RBC). However, recently many influenza A(H3N2) viruses have had very low or no HA titre using a variety of RBC, even though there was clear CPE and high influenza neuraminidase (NA) activity present, indicating significant levels of influenza virus.
Nine of these low-HA A(H3N2) viruses were further passaged up to six times in MDCK-SIAT cells. HA titres using guinea pig RBC increased for all isolates. To investigate whether this RBC binding was via HA or NA binding, the NA inhibitor oseltamivir was added to the hemagglutination assay. The HA titre of all 3C.2a viruses dropped dramatically upon addition of oseltamivir, but viruses from other HA phylogenetic clades 3C.3a and 3C.3b did not show reductions in HA titre. Sequencing of the HA genes from the passaged viruses showed there were no amino acid changes while NA gene sequencing showed mutations D151N, and some with T148I in later passages exclusively in the 3C.2a clade viruses; but these did not become a pure population.
This study did not identify changes in the HA gene of low/no agglutinating A(H3N2) viruses that could explain the increased HA titre. As a result these viruses will need to be continually assessed using virus neutralization assays instead of HI assays.