DISCUSSION
Though investigation of the prevalence of H9N2 virus in lower and middle income countries, either by poultry sero-surveys or by passive sampling, the virus was observed to be present at particularly high rates, especially in LPMs[10]. LPMs are not only a hub for poultry trading, but are also a major component of the disease transmission pathway[18], contributing to the spread of AIVs among poultry as well as facilitating zoonotic infections. The purpose of this study was to understand the evolution and molecular features of the H9N2 viruses, which were constantly circulating in LPMs in Anhui province between 2013 and 2018.
Phylogenetic analysis of the surface HA genes indicated that the lineage h9.4.2.5 was predominant in 2013–2018 in Anhui province. Although environmental strains belonging to h9.4.2.6 were not identified in this study, two strains isolated from chickens clustered into the 9.4.2.6 lineage, which is well established in southern China. The frequent live poultry trade aids the co-circulation of h9.4.2.5 and h9.4.2.6 viruses in Anhui. G1-like PB2 and M genes combined with the genetic backbone of F/98-like viruses was the predominant form of the six internal genes in the strains analyzed in this study. H9N2 subtype AIVs in China have evolved into diversified clusters and genotypes, according to previous research[9], and viruses isolated from the environment in this study were designated as genotype S (generally equivalent to genotype G57). The genotype S H9N2 AIVs were mainly prevalent in the Yangtze River Delta region in eastern China, and Anhui province is located within this area. Evidence has been reported that genotype S exhibits greater infectivity than the other genotypes, and has been predominant since 2010 across China, causing a significant threat to the poultry industry[2].
To control and eradicate both the H7N9 low and highly pathogenic viruses, the H5/H7 bivalent inactivated vaccine was used to protect chickens and ducks from infection. This intervention decreased the prevalence of H7N9 viruses in poultry and humans, confirming that the vaccination of poultry played an important role in preventing the spread of H7N9 virus. However, from our research, it is clear that all internal genes of the early H7N9, H5N6(Anhui) and H10N8 AIVs, including HPAI H7N9, were clustered into one group together with the strains analyzed in the present study. These strains isolated from the environment originated from LPMs in Anhui province, where HPAI H7N9 and H10N8 outbreaks have not occurred. H9N2 AIVs exhibited wide antigenic variability, donating internal genes to other AIVs[19]. Therefore, if immune escape H5 or H7 AIVs emerge, it is possible that highly pathogenic H7N9 or H5Nx AIVs may reappear. Moreover, high genetic compatibility was found between H9N2 and H1N1pdm(2009) viruses, and reassortment with H3N2 is possible, potentially leading to the gain of respiratory transmissibility[3]. Our study showed that the absence or presence of PNLGSs and key amino acid mutations in RBSs, such as Q226L, may affect receptor affinity[20]. The N166D mutation in the HA that appeared in the strains isolated in 2018 in this study, may contribute to the failure of H9N2 vaccination in the field[21]. In this study, the 95K, 224N and 242N residues in the M1 protein and 21G in the M2 protein may all increase AIV infectivity[22]. Taken together, our findings indicate that most viral isolates from the environment in Anhui province possessed the ability to increase the affinity for mammals, and enhance infectivity and transmission.
In summary, we systematically analyzed H9N2 environmental isolates from Anhui province from 2013 to 2018. Phylogenetic analysis indicated that h9.4.2.5-like H9N2 viruses were predominant during 2013–2018. Additionally, these viruses had undergone dramatic reassortment and evolution, resulting in wide ranging genetic diversity. The acquired characteristics of increased affinity for mammals and enhanced infectivity and transmission among viral isolates are worthy of close attention.