The ability of isogenic A/England/195/09 recombinant viruses that differed only at residue 275 in the NA to infect and spread in MDCK or in differentiated HAE cells was assessed by Recombinant influenza viruses containing the NA from representative pandemic influenza A/England/195/09 were assessed for their ability to catabolize mono-and multivalent substrates. The MuNANA substrate was used to determine IC 50 values for two NA inhibitors and assess enzyme K m values. IC 50 and K m values are given as the mean of two determinations, and the SEM is indicated for K m values. For red cell elution, chicken erythrocytes were first mixed in equal volumes with 2-fold serially diluted virus and incubated on ice to determine haemagglutination titres. The microtitre plate was then moved to 378C to allow NA to cleave sialic acid residues and reverse haemagglutination. The geometric mean titre derived from duplicate wells shown is the last dilution at which an effect was seen. A lower desialylation titre indicates a reduced ability of the NA to cleave sialic acid and reverse haemagglutination.