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During the routine surveillance, we isolated nine H4N6 subtype avian influenza viruses (AIVs) in Jiangsu Province, China, in March 2023. Phylogenetic analysis revealed that nine H4N6 viruses belonged to the Eurasian lineage and underwent complex genetic recombination among Asian countries during their evolution. It is particularly noteworthy that the PB2 and PB1 genes of our representative virus were descended from clade 2.3.4.4b H5 high-pathogenic AIVs in Japan. Mutations of D3V and D622G in PB1, N66S in PB1-F2, N30D, I43M, and T215A in M1, and P42S and I106M in NS1 were observed in nine isolates, which may increase the pathogenicity of the viruses in mice. The receptor binding analysis showed that the tested H4N6 virus could bind to both avian-type and human-type receptors. Vitro infection kinetics revealed that the representative virus could efficiently replicate in mammalian cells, including MDCK and 293T cells. Pathogenicity tests in mice indicated that the representative virus could replicate in nasal turbinates and lungs without prior adaptation. Our data reveal the potential public health issues represented by H4N6 viruses from wild birds and highlight the need to strengthen routine surveillance of wild birds. [ABSTRACT FROM AUTHOR]