To characterize the temperature dependency of avian vs. human influenza viruses in a relevant model of the target cell types of the human airways, we utilized an in vitro model of human ciliated airway epithelium (HAE). This model closely mimics the morphological and physiological features of the human airway epithelium in vivo and has been previously used to investigate infection by diverse respiratory viruses [26] [27] [28] [29] [30] . In humans, ciliated airway epithelium is present throughout the airways, extending from the nasal cavity and large proximal airways into the distal bronchiolar airway regions. Previously, we have shown that both human and avian influenza viruses replicate well in HAE and that human and avian influenza virus cell tropism correlates with the respective distribution of the specific sialic acid linkages [13] . However, these previous studies were conducted at 37uC, reflecting conditions encountered in the distal airways [13] . Others have also utilized these airway cell systems to characterize influenza virus replication of wild-type and recombinant viruses at 35uC [14, 31, 32] . In the present study, we utilize the HAE model, in combination with influenza virus reverse genetics, to investigate the influence of temperature on human and avian influenza virus infection, replication and spread. We demonstrate that, compared to human influenza viruses, avian influenza viruses are severely restricted for infection of human airway epithelium at the temperature of the human proximal airways. Then, using different strategies to 'avianize' human influenza viruses, we show that the temperature restriction of avian viruses is closely associated with the avian HA and NA glycoproteins.