A modified plaque assay method for accurate analysis of infectivity of influenza viruses with uncleaved hemagglutinin

A modified plaque assay method for evaluation of infectivity of influenza viruses is described. The modification consists of a double agar overlay, trypsin being included in the second layer added 24 hours after infection. The modified method allows one to determine accurately the infectivity titer of influenza viruses possessing uncleaved hemagglutinin, and to compare the infectivity of viruses possessing cleaved or uncleaved forms of hemagglutinin. The standard method in which trypsin is included into agar overlay just after infection artificially increases the infectivity titer of viruses with uncleaved hemagglutinin and therefore is not suitable for these purposes.