Seven RNA segments of laboratory-adapted strain [A/Puerto Rico/8/34 (H1N1)] were combined with the NA of a pH1N1 strain [A/England/195/09 (pH1N1)] to rescue a pair of isogenic viruses that differed only at position 275 (wild-type PR8+E195 and mutant PR8+E195 H275Y ). Similarly a pair of isogenic viruses based on the whole genome of A/England/195/09 were rescued according to published methods. 11 The sialidase enzyme properties of both wild-type NA and the mutant H275Y NA were assessed using a fluorescent substrate [2 ′ -(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid (MuNANA)] as previously described. 12 Enzyme kinetic experiments were performed on a standardized amount of each virus for which the NA metabolized 10 nmol substrate/h. A chicken erythrocyte elution assay, where NA is required to cleave sialic acid from multivalent cell surface moieties of the red blood cell, was established. Equal haemagglutination titres of each virus were diluted in 2-fold steps and mixed with PBS containing 1% chicken red blood cells in a V-bottomed microtitre plate and left at 48C for 1 h to determine the haemagglutination endpoint titre. The plate was then incubated for 2 h at 378C. The last dilution of virus at which haemagglutination was lost was defined as the desialylation endpoint titre.