In our initial experiments, spread of avian influenza viruses from cell to cell at 32uC was compromised in cultures inoculated at low MOI, suggesting a potential role for the envelope glycoproteins, HA and NA, in mediating temperature restriction. Previous work by Kaverin and colleagues also demonstrated temperature Figure 5 . Cell tropism of human, avian and avianized viruses in HAE. Representative cross-sections of inoculated HAE, fixed 24 hrs pi, were probed for viral antigen (NP; green) and a2acetylated tubulin, a marker for ciliated cells (red). Notably, the staining pattern for wild-type A/Victoria/3/ 75 was identical to that of PR8+Vic HA/NA. Arrows mark ciliated cells infected with either wild-type A/Victoria/3/75 or PR8+Vic HA/NA; arrow-head denotes non-ciliated cells infected by these viruses. These data indicate that viruses with Victoria glycoproteins were able to infect both cell types previously shown to express a2,6 SA [13] . Viral antigen was detected only in ciliated cells in cultures inoculated with Vic-226-228HA (in the Victoria background with either endogenous N2 or avian N1 or PR8+Chick HA/NA). Scale bar equals 20 mm. doi:10.1371/journal.ppat.1000424.g005 effects on growth of human-avian reassortant viruses containing avian glycoproteins [25] , although this work was performed in non-polarized MDCK cells and did not investigate additional correlates of infection such as spread and CPE. In our study, we generated recombinant influenza viruses based on the A/Victoria/ 3/75 or A/PR/8/34 genetic backbone that were engineered to contain avian-like and/or avian glycoproteins and characterized infection in HAE. Kinetic studies showed that although human influenza viruses that possessed avian or avian-like surface glycoproteins were modestly restricted compared to wild-type viruses at 37uC, these mutant viruses were able spread like wildtype viruses throughout HAE at this temperature. Wide-spread infection throughout HAE was even observed for viruses in which their endogenous HA was replaced or mutated to preferentially bind a2,3 SA, restricting tropism to ciliated cells. Efficient replication of Vic-226-228HA at 37uC in our studies corroborates previous work by Matrosovich and colleagues in which little effect of HA-specificity 'switching' on replication was noted unless a very low MOI (0.00004) was used for inoculation [44] . In contrast, Wan and Perez described more profound differences in replication in HAE at 37uC with recombinant viruses that differed only in their receptor specificity [31] . However, it should be noted that their recombinant viruses were based on an H9N2 avian strain that yielded relatively low titers, and their initial infections were performed at 35uC before incubating at 37uC [31] .