Active surveillance of avian influenza virus (AIV) in wetlands and lakes is important for exploring the gene pool in wild birds. Through active surveillance from 2015 through 2019, 10,900 samples from wild birds in central China were collected, and 89 AIVs were isolated, including 2 subtypes of highly pathogenic AIV and 12 of low-pathogenic AIV; H9N2 and H6Ny were the dominant subtypes. Phylogenetic analysis of the isolates demonstrated that extensive intersubtype reassortments and frequent intercontinental gene exchange occurred in AIVs. AIV gene segments persistently circulated in several migration seasons, but interseasonal persistence of the whole genome was rare. The whole genomes of one H6N6 and polymerase basic 2 (PB2), polymerase acidic (PA), hemagglutinin (HA), neuraminidase (NA), M, and nonstructural (NS) genes of one H9N2 virus were found to be of poultry origin, suggesting a spillover of AIVs from poultry to wild birds. Importantly, one H9N2 virus only bound to human-type receptor, and one H1N1, four H6, and seven H9N2 viruses possessed dual receptor-binding capacity. Nineteen of 20 representative viruses tested could replicate in the lungs of mice without preadaptation, which poses a clear threat of infection in humans. Together, our study highlights the need for intensive AIV surveillance. IMPORTANCE Influenza virus surveillance in wild birds plays an important role in the early recognition and control of the virus. However, the AIV gene pool in wild birds in central China along the East Asian-Australasian flyway has not been well studied. Here, we conducted a 5-year AIV active surveillance in this region. Our data revealed the long-term circulation and prevalence of AIVs in wild birds in central China, and we observed that intercontinental gene exchange of AIVs is more frequent and continuous than previously thought. Spillover events from poultry to wild bird were observed in H6 and H9 viruses. In addition, in 20 representative viruses, 12 viruses could bind human-type receptors, and 19 viruses could replicate in mice without preadaption. Our work highlights the potential threat of wild bird AIVs to public health.
Keywords: avian influenza virus; pathogenic; reassortment; viral surveillance; wild birds.