Human influenza viruses with avian-like glycoproteins display restricted replication and spread at 32uC in HAE Our initial phenotype indicated that A/Dk/Eng/62 was restricted in its ability to spread from cell to cell within the epithelium at 32uC (Figure 2A ). Several events in the viral life cycle that are critical for spread, including release of progeny virions from previously infected cells and attachment and entry into new target cells, are mediated by influenza virus glycoproteins. Thus, we hypothesized that glycoprotein function could be responsible for the restricted infection of HAE by avian influenza viruses at the lower temperature of 32uC. To test whether HA and/or NA contributed to the restricted phenotype of avian influenza viruses at 32uC, we used reverse genetics to generate mutant viruses genetically altered to confer avian virus-like glycoprotein specificities on the A/Victoria/3/75 background. First, mutations in HA previously shown to switch sialic acid usage from a2,6 to a2,3 linkages (L226Q, S228G) [40] were introduced to generate the Vic-226-228HA virus. Second, we generated a reassortant virus in which the Victoria NA was replaced by that of the avian virus A/ Chick/Italy/1347/99 to generate Vic+Chick N1.