We next generated a recombinant influenza virus containing both the 226-228HA and Chick N1 and tested infection and growth in HAE at 32uC and 37uC in comparison to wild-type A/Victoria/3/ 75. At 24 hrs pi, the double glycoprotein-altered virus exhibited similar restriction as observed for the other viruses. Nonetheless, an overall evaluation of the double glycoprotein-altered virus suggested that as infection proceeded, this virus was profoundly restricted at 32uC compared to 37uC (Figure 4Di ), exhibiting .2 log reduction in titer at 48 hrs. Notably, titers for the wild-type virus differed by less than 0.5 logs between temperatures at this time point. Furthermore, the double glycoprotein-altered virus was still significantly restricted at 72 hrs pi when titers at 32uC were compared to those at 37uC. The level of restriction observed for the double mutant was greater than that observed for either virus containing each of these mutations/ substitutions individually. Moreover, analysis of viral antigen positive cells at 72 hrs by en face staining of infected HAE indicated compromised spread of Victoria (226-228HA)+Chick N1 which was more severe at 32uC than 37uC (Figure 4Dii) .