WT and the 13 mutant viruses were therefore generated by transfection of cells with plasmids encoding the eight segments in cDNA form (27, 30) . Notwithstanding the reduced transcriptional activity associated with some of the non-synonymous changes in PB1, it was possible to rescue all of the mutants. Virus stocks were amplified in MDCK cells and titred as a first assessment of virus fitness (7, 30, 31) . For the mutants with non-synonymous changes in PB1, the endpoint titres showed a strong correlation with polymerase activity in the minireplicon system. The most deficient virus in this system (T32G) rescued only once out of four attempts in 293T and MDCK cells, where it showed a six log 10 growth defect relative to wild-type virus ( Figure 3C ). The A78T+G101T mutant was successfully rescued five times out of seven tested and reached endpoint titres of $1% of WT. All other mutants rescued on every attempt (between two and six times each). The G28A mutant, which had a 2-fold reduction in transcriptional activity, had a growth defect of $10-fold. Only the C74G virus grew similar levels as WT virus. However, not all viruses with unaltered PB1 coding sequences grew normally. Three of the mutant viruses with alterations around AUG1 (ACC, CCC and TCC), showed growth defects of between 8-and 20-fold relative to WT virus. In contrast, the T22A, T22G and T22C viruses grew normally, despite also having mutations to the upstream Kozak consensus of AUG1 that showed similar perturbations to segment 2 translation in the IVT system to the ACC, CCC and TCC changes. Similar relative results were also obtained when virus stocks were passaged in embryonated eggs, although here the T32G virus grew better, to $10 6 PFU/ml or 0.5% of the WT control (data not shown).