Introduction
H9N2 avian influenza virus (AIV) spread rapidly and infected more than
90% of chicken flocks since its breakout in Hebei province, China in
1998. It became one of the most important epidemics in poultry industry
in China (Gu,Xu,Wang, & Liu, 2017). Since then, vaccination strategy of
inactivated vaccine for control of H9N2 avian influenza had been
extensively executed, and worked well (Li et al., 2005). However, H9N2
virus is undergoing adaptive evolution under the vaccine immune
pressure. As a major antigen and receptor binding protein of H9N2 virus,
the haemagglutinin (HA) from the circulating field strains were
clustered into three lineages before 2007, A/Chicken/Beijing/1/94-like
(BJ/94-like), A/Quail/Hong Kong/G1/97-like (G1-like), and A/Duck/Hong
Kong/Y439/97-like (Y439/97-like) (Sun & Liu, 2015). In 2013, G57 was
emerged as the predominant genotype of H9N2 virus. A new genotype G118
was discovered in 2015 (Jin et al., 2020). With the evolution of H9N2
virus, the specific antibodies induced by inactivated vaccines could not
effectively block the attachment of HA of the circulating virus to the
target cells (Chambers,Kawaoka, & Webster, 1988). This resulted in the
decrease in the protection efficacy of the existing vaccines and
isolation of breakthrough H9N2 viruses in vaccinated chicken flocks with
high antibody titer (Li et al., 2019). Therefore, it is important to
monitor antigenic mutation of the HA from H9N2 virus.
Currently, over 30 antigenic sites of H9N2 virus have been reported,
most of which were mapped by monoclonal antibody (mAb) precisely
(Peacock et al., 2016; Jin et al., 2019; Kaverin et al., 2004; Okamatsu,
Sakoda, Kishida,Isoda, & Kida, 2008; Ping et al., 2008; Wan et al.,
2014; Zhu et al., 2015). The viral evolution could promote virus
escaping from the neutralization of antibody by adding N-linked
glycosylation (NLG) to shield the antigenic sites (An et al., 2019;
Cherry,Lipman,Nikolskaya, & Wolf, 2009), changing virus-antibody
binding property (Li et al., 2013), or altering receptor-binding
specificity (Wan & Perez, 2007; Peacock et al., 2017; Teng et al.,
2016; Yang et al., 2017).
We previously reported that the H9N2 vaccine representative strain
A/Chicken/Shanghai/F/1998 (F/98, H9N2), which belonged to BJ/94-like
lineage, occurred antigenic variation continually when passaged in
specific pathogen-free (SPF) chicken embryos or SPF chickens with or
without homologous vaccine antibodies (Jin et al., 2018; Su et al.,
2020). In this study, we generated recombinant F/98 viruses containing
single HA mutation from the passaged viruses occurring antigenic drift,
and identified the contribution of these HA mutations to the antigenic
variation using HI assay. We made a comprehensive analysis of the role
of the key mutation A198V in antigenic variation or immune escape by
cross-microneutralization (MN) assay and cross-protection in
vivo . Our results showed that A198V substitution caused the decreased
readouts of cross-MN titers significantly by enhancing the receptor
binding activity though it did not prevent Abs binding physically. In
addition, the strong receptor binding avidity increased the NA activity,
prevented viral release from cells, and slightly affected
cross-protection in vivo .