Tropism and innate host responses of influenza A/H5N6 virus: an analysis of ex vivo and in vitro cultures of the human respiratory tract.

Since their first isolation in 2013, influenza A/H5N6 viruses have spread amongst poultry across multiple provinces in China and to Laos, Vietnam and Myanmar. So far, there have been 14 human H5N6 infections with 10 fatalities.We investigated the tropism, replication competence and cytokine induction of one human and two avian H5N6 isolates in ex vivo and in vitro cultures derived from the human respiratory tract. Virus tropism and replication were studied in ex vivo cultures of human nasopharynx, bronchus and lung. Induction of cytokines and chemokines was measured in vitro in virus-infected primary human alveolar epithelial cells.Human H5N6 virus replicated more efficiently than highly pathogenic avian influenza (HPAI) H5N1 virus and as efficiently as H1N1pdm in ex vivo human bronchus and lung and was also able to replicate in ex vivo cultures of human nasopharynx. Avian H5N6 viruses replicated less efficiently than H1N1pdm in human bronchial tissues and to similar titres as HPAI H5N1 in the lung. While the human H5N6 virus had affinity for avian-like receptors, the two avian isolates had binding affinity for both avian- and human-like receptors. All three H5N6 viruses were less potent inducers of pro-inflammatory cytokines compared with H5N1 virus.Human H5N6 virus appears better adapted to infect the human airways than H5N1 virus and may pose a significant public health threat.

Transmission of H7N9 Influenza Viruses with a Polymorphism at PB2 Residue 627 in Chickens and Ferrets.

UNLABELLED: Poultry exposure is a major risk factor for human H7N9 zoonotic infections, for which the mode of transmission remains unclear. We studied the transmission of genetically related poultry and human H7N9 influenza viruses differing by four amino acids, including the host determinant PB2 residue 627. A/Silkie chicken/HK/1772/2014 (SCk1772) and A/HK/3263/14 (HK3263) replicated to comparable titers in chickens, with superior oropharyngeal over cloacal shedding; both viruses transmitted efficiently among chickens via direct contact but inefficiently via the airborne route. Interspecies transmission via the airborne route was observed for ferrets exposed to the SCk1772- or HK3263-infected chickens, while low numbers of copies of influenza viral genome were detected in the air, predominantly at particle sizes larger than 4 µm. In ferrets, the human isolate HK3263 replicated to higher titers and transmitted more efficiently via direct contact than SCk1772. We monitored "intrahost" and "interhost" adaptive changes at PB2 residue 627 during infection and transmission of the Sck1772 that carried E627 and HK3263 that carried V/K/E polymorphism at 60%, 20%, and 20%, respectively. For SCk1772, positive selection for K627 over E627 was observed in ferrets during the chicken-to-ferret or ferret-to-ferret transmission. For HK3263 that contained V/K/E polymorphism, mixed V627 and E627 genotypes were transmitted among chickens while either V627 or K627 was transmitted to ferrets with a narrow transmission bottleneck. Overall, our results suggest direct contact as the main mode for H7N9 transmission and identify the PB2-V627 genotype with uncompromised fitness and transmissibility in both avian and mammalian species. IMPORTANCE: We studied the modes of H7N9 transmission, as this information is crucial for developing effective control measures for prevention. Using chicken (SCk1772) and human (HK3263) H7N9 isolates that differed by four amino acids, including the host determinant PB2 residue 627, we observed that both viruses transmitted efficiently among chickens via direct contact but inefficiently via the airborne route. Chicken-to-ferret transmission via the airborne route was observed, along with the detection of viral genome in the air at low copy numbers. In ferrets, HK3263 transmitted more efficiently than SCk1772 via direct contact. During the transmission of SCk1772 that contained E and HK3263 that contained V/K/E polymorphism at PB2 residue 627, positive selections of E627 and K627 were observed in chickens and ferrets, respectively. In addition, PB2-V627 was transmitted and stably maintained in both avian and mammalian species. Our results support applying intervention strategies that minimize direct and indirect contact at the poultry markets during epidemics.