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 type A viruses are endemic in aquatic birds but can cross the species barrier to infect the human respiratory tract. While transmission from birds to humans is rare, the introduction of novel avian influenza viruses into immunologically naïve human populations has significant pandemic potential. Avian influenza viruses are adapted for growth at 40°C, the temperature of the avian enteric tract. However, the human proximal airways, the likely site of initial inoculation by influenza viruses, are maintained at a cooler temperature (32°C), suggesting that zoonotic transmission may be limited by temperature differences between the two hosts. Using an in vitro model of human ciliated airway epithelium, we show that avian influenza viruses grow well at 37°C, a temperature reflective of distal airways, but are restricted for infection at 32°C. A panel of genetically manipulated human influenza viruses possessing avian or avian-like surface glycoproteins were also restricted at 32°C, but not 37°C, suggesting that avian virus glycoproteins are not adapted for efficient infection at the temperature of the proximal airways. Thus, avian influenza virus infection is restricted in the human proximal airways due to the cooler temperature of this region, thus limiting the likelihood of zoonotic and subsequent human-to-human transmission of these viruses.