In conjunction with the HA, the sialidase activity of NA is crucial for successful virus penetration of mucus layers for initial infection and subsequent release of progeny virions from infected cells [47, 48] . This is especially critical both in vivo and in HAE models in which the luminal epithelial cell surface is robust with glycoconjugates displaying abundant terminal sialic acid moieties that may act as false receptors for influenza viruses [49] . Using standard laboratory assays that employ small monovalent soluble substrates for cleavage by NA (MUNANA), we were not able to demonstrate any temperature-dependent loss of NA activity associated with either human or avian virus (data not shown). However, the ability of the avian virus NA to cleave biologically relevant substrates present in HAE may be compromised at 32uC vs. 37uC restricting both initial infection and subsequent spread of the virus throughout the epithelium. This is supported by our data which demonstrate restricted growth and spread of reassortant viruses containing avian virus NA, including Vic+Chick N1 and PR8+Chick HA/NA in HAE at 32uC.