Introduction
Chickens natural infected with the H9N2 subtype of low pathogenic avian influenza (LPAI) exhibited mild respiratory signs and decreased egg production. Co-infection with other pathogenic microorganisms will aggravate the clinical signs. Although the H9N2 subtype of avian influenza virus (H9N2-AIV) is of low pathogenicity to birds, the actual threat lies in its broad host range. The virus not only infects birds, but has been reported to jump species to infect humans and other mammals. More serious H9N2-AIV frequently donates gene segments to facilitate the generation of novel reassortants, causing epidemics or even pandemics in poultry (Gerloff et al., 2014). An analysis of the hemagglutinin (HA) gene sequence database of the National Center for Biotechnology Information (NCBI) in 2016 revealed that <90% of the H9N2-AIV isolates came from Asia, of which 78% originated in China (Li et al., 2017). Another dataset showed that the AIV positivity rate was 12.73% between 2016 and 2019 in China, of which H9N2 accounted for 72.75% of cases (Bi et al., 2020). This shows that H9N2-AIV has become the dominant AIV in recent years within China, which seriously threatens the public health of humans as well as the livestock and poultry industries.
Vaccination of poultry is a key element of disease control in endemic countries. Influenza A virus mutates rapidly, resulting in antigenic drift and poor year-to-year vaccine efficacy. Commercial vaccine strains of H9N2 in China, including A/chicken/Shandong/6/96 (SD696), A/chicken/Guangdong/SS/94 (SS) and A/chicken/Shandong/F/98 (F98), were all isolated prior to 2000. Previously, we demonstrated that H9N2 virus isolated from 2013 to 2016 in China underwent antigenic drift to evolve into distinct antigenic groups, and accumulated significant antigenic differences compared with the commercial vaccines (Xia, Cui, et al., 2017). A growing body of research supports these observations (Sun & Liu, 2015). The identification of antigenic sites for monitoring of variants for the development effective vaccines is crucial. More than 46 HA amino acid antigenic sites were identified in H9N2-AIV (T. P. Peacock et al., 2017; Song et al., 2020; Su et al., 2020; Wan et al., 2014; Zhu et al., 2015). Some of those positions are multifunctional, such as the D200N substitution, which also increases replication in chicken embryo fibroblast cells and embryonated chicken eggs (Song et al., 2020). It was reported that N166D also affects the pathogenicity (Jin et al., 2019; T. P. Peacock et al., 2017), and the 220 loop deletion could arise in the field due to immune selection pressure, which also reduces HA stability (T. P. Peacock et al., 2017). However, it is still unknown which single substitution of recent isolates is responsible for the observed antigenic drift.
Although the epitopes of H9N2-AIV are not well characterized, it is known that not all substitutions affect viral antigenicity (T. P. Peacock, Harvey, Sadeyen, Reeve, & Iqbal, 2018). For example, mutations in H3N2 and H1N1 viruses near the receptor binding site (RBS) determine major antigenic changes, but are also affected by mutations to adjacent sites as well (Koel et al., 2013; Lewis et al., 2014; Santos et al., 2019). Interestingly, our previous analyses established a link between high-frequency substitutions and those key antigenic sites of H3N2 viruses (Xia et al., 2020). Therefore, the high-frequency mutation sites near the H9N2-AIV HA RBS protein may be related to the key amino acid sites producing antigenic variation. In this study, we aimed to demonstrate that the single high-frequency mutation site near the RBS could drive antigenic drift of H9N2-AIV circulating in 3 recent years in China.