The inability of avian influenza viruses to replicate efficiently at cooler temperatures has been linked to the viral polymerase subunit, PB2 [23, 24] . In the present study, mutating position 627 in a human virus PB2 to an avian virus conserved residue resulted in growth restriction at both 32uC and 37uC, suggesting that this residue is important for general viral fitness in HAE, but is not responsible for the differences in infection seen at 32uC vs. 37uC. Two recent reports also found that viruses with 627E in PB2 were attenuated regardless of temperature in human bronchial epithelial cells and MDCK cells, respectively, although in other cell systems including human small airway epithelial cells, a temperature specific effect was found [24, 42] . It should be emphasized that those studies were performed in non-differentiated epithelial cells unlike our studies that use human differentiated airway epithelial cells. We and others have previously shown that differentiated airway epithelial cell models enable discrimination of attenuated phenotypes of respiratory virus infection whereas non-differentiated cells do not [26, 27, 43] . In addition, we also show using HAE, that the H5N1 strain A/VN/1203/04, which possesses a lysine at position 627 (human adaptation), is still restricted for growth at 32uC, albeit less so than avian influenza viruses that have never infected humans. The attenuation in HAE of this H5N1 isolate which possesses a ''human'' amino acid at residue 627 in PB2 suggests other residues in the polymerase subunit or other viral proteins altogether are involved in temperature sensitivity of avian influenza viruses.