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.