Compared to 37uC, viruses with a preference for binding to a2,3 SA, including Vic-226-228HA, were restricted for growth and spread in HAE at 32uC. Notably, the H5N1 strain examined in this study also maintains preference for a2,3 SA binding [45] ; thus, we may surmise that this characteristic of A/VN/1203/04 contributes to its attenuation observed in HAE. The contribution of a2,3 SA usage to replication of influenza viruses investigated by Hatta et al. in the upper respiratory tract of mice may have been masked in the mouse model (the 627 mutation in PB2 being more apparent) as mice express solely avian virus-like receptors (a2,3 SA) in their airways [46] . Restriction of a2,3 SA-binding viruses in HAE at 32uC was not due to a discrepancy in SA expression since HAE maintained at either 32uC or 37uC expressed similar levels of a2,6 and a2,3 SA (as detected by Sambucus nigra (SNA) and Maackia amurensis (MAA) lectin staining, respectively; data not shown).