One potential caveat of the recombinant viruses with avianized HA and/or NA utilized in our previous analysis was that they contained HA and NA pairs that had not co-evolved. To eliminate the possibility that the restriction we observed with these recombinant viruses was due to an imbalance between the activities of the surface glycoproteins that were not evolutionarily optimized, we next generated reassorted influenza viruses on a common genetic background, possessing human or avian glycoproteins with co-evolved pairings. This was achieved using human recombinant A/PR/8/34 (H1N1) in which the wild-type H1 and N1 glycoproteins were replaced by the H3 and N2 glycoprotein pair from A/Victoria/3/75 (generating PR8+Vic HA/NA) or the H7 and N1 glycoprotein pair from A/Chick/ Italy/1347/99 (generating PR8+Chick HA/NA, previously termed RD3) [41] . Since we and others have shown differential cell-type tropism between human and avian influenza virus in HAE [13, 14] , we next determined if avianizing the human virus HA by mutation or substitution (in the presence or absence of an avian NA) recapitulated the cell-type tropism exhibited by wholly avian influenza viruses in HAE. As shown by immunofluorescent detection in histological sections of infected HAE, PR8 containing A/Victoria/3/75 glycoproteins infected both ciliated and nonciliated cells in HAE with a tropism similar to wild-type A/ Victoria/3/75 ( Figure 5 ). In contrast, A/Victoria/3/75 with two avian-like amino acid substitutions in HA and PR8+Chick HA/ NA only infected ciliated cells, a tropism that was mirrored by wholly avian virus [13, 14] . These data clearly show that the ciliated cell tropism of avian influenza viruses is dictated by properties of the viral glycoproteins. These results correlate with the known increased sialic acid binding preference of avian HA for a2,3-linked SA, and to the presence of a2,3-linked SA on ciliated cells in HAE [8, 13, 14] .