A simple screening assay for receptor switching of avian influenza viruses

Background: Adaptation of the receptor-binding preference from α2,3- to α2,6-linked sialic acid is an essential step for an avian influenza virus to transmit efficiently in human population and become a pandemic virus. The currently available assays for receptor-binding preference are complex and not widely available. Objectives: A simple high-throughput screening assay will facilitate early detection of a potential pandemic virus, which is crucial for the prevention and control of the possible pandemic. We wanted to develop a simple assay to differentiate influenza viruses with α2,3- or α2,6-linked receptor-binding preference. Study design: The assay employs a specific sialidase (from Salmonella thyphimurium) that can eliminate α2,3-linked sialic acid from red blood cells. A reduction of hemagglutination titer indicates α2,3-linked receptor preference in this assay. Results: Using a panel of H5N1 avian influenza isolates and H1/H3 human influenza isolates, as well as mutated H5 reverse genetics virus, the assay could accurately differentiate the viruses according to their receptor-binding preference. Furthermore, the assay was sufficiently sensitive to detect a minor variant with α2,6-linkage-specificity in a background of α2,3-linkage-specific virus. Conclusions: We have developed a simple screening assay capable of detecting avian influenza viruses that have switched their receptor-binding preference.