Previously, the polymerase subunit PB2 has been shown to play an important role in host range restriction of avian influenza viruses in mammalian cells [37] [38] [39] . In influenza virus strains that circulate in humans, amino acid residue 627 in PB2 is a lysine, whereas in the majority of avian strains it is a conserved glutamic acid residue. The presence of glutamic acid at PB2 627 (avian-like) has been reported to account for the lower replication of avian influenza strains in mammalian cells and has been linked with reduced polymerase activity at lower temperature (33uC) in some cell systems [23, 24] . To assess the potential impact of this PB2 amino acid residue in restriction of avian influenza viruses at 32uC, we generated a recombinant A/Victoria/3/75 virus containing the PB2 K627E mutation and compared its growth with that of the isogenic wild-type virus in HAE at 32uC and 37uC. The K627E mutation resulted in restriction of the virus at both temperatures (Figure 4Ai ), and although titer at 32uC was 1.3 logs lower than at 37uC at 24 hrs pi, this difference was no greater than the differences in growth for wild-type virus at these temperatures (1.5 logs; Figure 4Ai ). Moreover, at the later time points analyzed, 48 and 72 hrs pi, the PB2 mutant did not show a significant difference in titer between the two temperatures. These data indicate that the K627E mutant virus was restricted for growth in HAE but that restriction was not temperature-dependent. Indeed, quantification of the numbers of infected cells identified by en face staining revealed that the K627E mutant virus infected a similar percentage of cells compared to wild-type virus at 24 hrs pi ( Figure 4Aii ) and that the mutant was capable of spread since new cells were infected by 48 hrs with similar kinetics to that of wildtype A/Victoria/3/75 at both 32uC and 37uC (Figure 4Aii ). Statistically, there was no difference between the wild-type and PB2 mutant viruses at either 32uC or 37uC at 48 hrs pi with respect to percent influenza virus-antigen positive epithelium. Together, these data suggest that the amino acid residue at PB2 627 influences viral fitness in HAE, but does not confer to a human virus the temperature-dependent phenotype of avian influenza virus infection in human ciliated airway epithelium.