Avian influenza viruses are attenuated at temperatures below 37uC and cold sensitivity of avian viral RNA replication in cell lines was linked to the presence of a glutamic acid at amino acid 627 in the avian virus polymerase subunit, PB2, instead of a lysine in the human virus PB2 [23] . Lysine substitution at residue 627 of H5N1 viruses improved virus replication in mice [24] . In addition to PB2, work utilizing human-avian reassortant viruses in MDCK cells provided initial evidence that avian glycoproteins, HA and neuraminidase (NA), may mediate temperature-dependent effects on viral growth [25] . To our knowledge, other viral genes have not been well characterized, nor the HA and NA further evaluated, in their contribution to temperature sensitivity of avian influenza viruses.