Similarly, the reassorted virus Vic+Chick N1 displayed a 2 log decrease in viral titer in HAE at 32uC compared to 37uC at 24 hrs pi. Although this difference was not appreciably greater than the difference in titer between temperatures for either wild-type virus or the PB2 mutant, Vic+Chick N1, unlike wild-type A/Victoria/3/75 and Vic 627PB2, maintained the ,2-log difference in growth at 48 hrs pi (Figure 4Ci ), suggesting this virus was more restricted at the cooler temperature. Quantification of numbers of infected cells illustrated that, like Vic-226-228HA, Vic+Chick N1 was restricted for spread at 32uC which was significant at 48 hrs, but was capable of spread similar to wild-type A/Victoria/3/75 at 37uC (Figure 4Cii ). Together these data suggest that avianizing either the HA or NA glycoprotein of an otherwise human influenza virus limits spread and subsequent infection at 32uC compared to 37uC.