Background and aims: H9N2 subtype avian influenza virus (AIV) has low-pathogenicity but causes respiratory symptoms and drop in egg production in chicken with long-term virus shedding, resulting in great economic losses due to high mortality related to secondary infection with other pathogens. In recent years, H9N2 viruses have been posing a threat to public health, causing human infection in China. Compared to studies on other AIV subtypes, there are relatively few studies on the pathogenic mechanism of the H9N2 virus in mammals. H9N2 subtype AIV has been circulating worldwide in many avian species and transmitting with high efficiency in poultry. It can provide internal genes for other subtypes to produce new viruses, causing a pandemic risk. It is important to carry out long-term surveillance and pathogenic characteristics of the H9N2 virus. In this study, we conducted environmental surveillance of live poultry markets in Anhui province from 2013 to 2018, and 33 representative environmental isolates were selected and studied systematically.

Methods: The genomic RNA of Anhui H9N2 isolates was subjected to RT-PCR amplification followed by sequencing analysis.

Results: Thirty-three strains were isolated from the embryonated eggs of specific-pathogen-free chickens. Phylogenetic analysis indicated that h9.4.2.5-like H9N2 viruses were predominant during 2013-2018 and acquired multiple specific amino acid mutations that may have increased their affinity for mammals and enhanced their infectivity and transmissibility. Additionally, six internal genes of H9N2 clustered together with the novel human-lethal reassortant viruses, such as the low-pathogenicity H7N9, H10N8, and Anhui H5N6 viruses, and even HPAI H7N9.

Conclusion: Because H9N2 viruses may be the donors of internal genes that lead to the generation of novel reassortant viruses with enhanced pathogenicity in Anhui province, continuous environmental surveillance of live poultry markets, a key source of reassorted H9N2 and other avian influenza viruses, is of great importance.