Objective To assess the replication efficiency of H6N6 avian influenza virus (AIV) with dual receptor binding properties in human respiratory cells and tissues in vitro, to conduct gene sequencing and analyze its genetic characterization, and to explore the potential of the virus crossing inter-species to infect humans.

Methods The receptor binding preference of chicken-originated H6N6 AIV to sialic acid receptors was detected by solid-phase direct binding assay. H6N6 AIV with dual receptor binding properties was selected, and its replication in human respiratory cells A549, BEAS-2B and human respiratory tissues was assessed. Sequencing of viral genomes, sequence alignment, and homology analysis were conducted to identify amino acid variations of all eight gene segments.

Results The H6N6 subtype AIV with dual receptor binding properties could effectively replicate in human respiratory cells A549 and BEAS-2B. Virus isolation and culture from isolated human bronchial and lung tissues were positive, and influenza virus nucleoprotein (NP) in human bronchial and lung tissues was also positive. Four strains of H6N6 subtype AIV with dual receptor binding properties originated from the Eurasian lineage and possessed the characterization of low pathogenicity AIV. H156K and S263K mutations in the HA receptor binding domain were found, suggesting that these two site mutations may be related to the dual receptor binding properties of H6N6 virus.

Conclusion Chicken-originated H6N6 subtype AIVs with dual receptorbinding properties can replicate effectively in the human respiratory tract without prior adaptation, indicating a potential risk of cross-species transmission to humans. These findings highlight a potential threat to human health and public health security.