Protocol S1 Supplementary analyses. Schema analysis on the HIV envelope gene. Found at: doi:10.1371/journal.ppat.1000418.s001 (0.02 MB DOC)  Avian Influenza Virus Glycoproteins Restrict Virus Replication and Spread through Human Airway Epithelium at Temperatures of the Proximal Airways Transmission of avian influenza viruses from bird to human is a rare event even though avian influenza viruses infect the ciliated epithelium of human airways in vitro and ex vivo. Using an in vitro model of human ciliated airway epithelium (HAE), we demonstrate that while human and avian influenza viruses efficiently infect at temperatures of the human distal airways (37°C), avian, but not human, influenza viruses are restricted for infection at the cooler temperatures of the human proximal airways (32°C). These data support the hypothesis that avian influenza viruses, ordinarily adapted to the temperature of the avian enteric tract (40°C), rarely infect humans, in part due to differences in host airway regional temperatures. Previously, a critical residue at position 627 in the avian influenza virus polymerase subunit, PB2, was identified as conferring temperature-dependency in mammalian cells. Here, we use reverse genetics to show that avianization of residue 627 attenuates a human virus, but does not account for the different infection between 32°C and 37°C. To determine the mechanism of temperature restriction of avian influenza viruses in HAE at 32°C, we generated recombinant human influenza viruses in either the A/Victoria/3/75 (H3N2) or A/PR/8/34 (H1N1) genetic background that contained avian or avian-like glycoproteins. Two of these viruses, A/Victoria/3/75 with L226Q and S228G mutations in hemagglutinin (HA) and neuraminidase (NA) from A/Chick/Italy/1347/99 and A/PR/8/34 containing the H7 and N1 from A/Chick/Italy/1347/99, exhibited temperature restriction approaching that of wholly avian influenza viruses. These data suggest that influenza viruses bearing avian or avian-like surface glycoproteins have a reduced capacity to establish productive infection at the temperature of the human proximal airways. This temperature restriction may limit zoonotic transmission of avian influenza viruses and suggests that adaptation of avian influenza viruses to efficient infection at 32°C may represent a critical evolutionary step enabling human-to-human transmission. Influenza viruses circulating in the human population are predominately type A and B, with type A being more common [1] . All influenza type A viruses originate from aquatic birds and successful introduction of these avian viruses into the human population, by either direct adaptation or reassortment with already circulating human viruses, has led to influenza pandemics of historical significance (reviewed in [2] [3] [4] 5] ). Still, documented evidence of transmission of avian influenza viruses directly from birds to humans is rare, partly because species barriers restrict avian influenza virus infection of the epithelial cells of the human respiratory tract, the primary site of influenza virus infection and spread.