We again compared virus replication and spread of the recombinant viruses to that of wild-type A/Victoria/3/75 at the two temperatures. As stated above, replication measured for the wild-type virus was slightly compromised at lower temperature, noticeable at 24 hrs pi. Restriction at this time point was also observed during infection of HAE with Vic-226-228HA, as it had been for the PB2 mutant virus. Specifically, a 2.5 log decrease in virus growth was determined for Vic-226-228HA at 32uC compared to 37uC at the 24 hr time point (Figure 4Bi ). However, unlike the PB2 mutant virus, the difference between replication at 32uC and 37uC for Vic 226-228HA was also significant at the 48 hour time point. Moreover, this mutant virus with avian viruslike sialic acid usage spread less efficiently than wild-type at 32uC so that by 48 hrs pi the number of virus antigen-positive cells was significantly different (Figure 4Bii ). In contrast, at 37uC, Vic-226-228HA infected similar numbers of cells as the wild-type virus by 48 hrs; indeed, the mutant virus was able to spread significantly more efficiently at the higher temperature (Figure 4Bii) .