Abstract
In recent decades, multiple novel reassortant avian influenza viruses (AIVs) carrying internal genes derived from H9N2 viruses have emerged in poultry in China and have repeatedly caused human infections, highlighting the pivotal role of the H9N2 internal genes cassette in facilitating cross-species transmission. In this study, five H6N1 and one H6N2 AIVs were isolated from chickens and Houdan chickens exhibiting respiratory symptoms. Genetic analyses revealed that the heamagglutinin (HA) genes of these H6 AIVs belonged to the ST339-like lineage and originated from H6 AIVs circulating in domestic geese in China, whereas the neuraminidase (NA) genes were derived from H9N2 or H5N1 AIVs, respectively. Notably, all six internal genes of these H6 isolates were derived from the G57 genotype H9N2 virus. In vitro studies demonstrated that these H6 AIVs replicated effectively in both avian and mammalian cells. The H6N2 isolate displayed dual receptor binding affinity for both avian-like and human-like receptors, whereas the five H6N1 viruses showed no detectable receptor binding affinity under the assay conditions in vitro. Reverse-genetics reassortant assay further revealed that the H6N1 HA itself possesses intrinsic dual receptor binding affinity. Furthermore, these H6 isolates exhibited high virulence in mice, and induced obvious respiratory symptoms in chickens, with efficient transmission among birds. Collectively, our findings demonstrate that H6 AIVs carrying the G57 H9N2 internal gene constellation are actively circulating in chicken populations and pose a substantial threat to poultry health, underscoring their potential risk to public health and the need for continued surveillance at the poultry-human interface.