ViroSpot microneutralization assay for antigenic characterization of human influenza viruses

The hemagglutination inhibition (HI) assay has been used for the antigenic characterization of influenza viruses for decades. However, the majority of recent seasonal influenza A viruses of the H3N2 subtype has lost the capacity to agglutinate erythrocytes of various species. The hemagglutination (HA) activity of other A(H3N2) strains is generally sensitive to the action of the neuraminidase inhibitor oseltamivir, which indicates that the neuraminidase and not the hemagglutinin is responsible for the HA activity. These findings complicate the antigenic characterization and selection of A(H3N2) vaccine strains, calling for alternative antigenic characterization assays. Here we describe the development and use of the ViroSpot microneutralization (MN) assay as a reliable and robust alternative for the HI assay. Serum neutralization of influenza A(H3N2) reference virus strains and epidemic isolates was determined by automated readout of immunostained cell monolayers, in a format designed to minimize the influence of infectious virus doses on serum neutralization titers. Neutralization of infection was largely independent from rates of viral replication and cell-to-cell transmission, facilitating the comparison of different virus isolates. Other advantages of the ViroSpot MN assay include its relative insensitivity to variation in test dose of infectious virus, automated capture and analyses of residual infection patterns, and compatibility with standardized large scale analyses. Using this assay, a number of epidemic influenza A(H3N2) strains that failed to agglutinate erythrocytes, were readily characterized antigenically.