Recombinant PR8 viruses were produced by transfection of plasmids into 293T cells in suspension as previously described (7, 30) . Rescued viruses were passaged once in MDCK cells at an input MOI of 0.001, and where indicated, once in 11-day-old embryonated eggs using an inoculum of 1000 PFU. Virus titres were determined by plaque assay on MDCK cells (30) , and the presence of the desired mutations in segment 2 were confirmed by sequencing. Multiple independent rescues were performed (minimum twice, mostly 3-6 times) to ensure that a given phenotype did not result from adventitious mutations elsewhere in the virus genome. Virus infections of MDCK cells were performed at an MOI of 3-5 in serum free media for 30 min at 37 C, after which cells were overlaid with serum-containing media. Haemagglutination (HA) assays were performed as previously described (30) .