Discussion
Avian influenza viruses have posed a significant threat to the global
poultry industry, in addition to public health. Due to the wide host
range, some H9N2 AIVs not only circulate in poultry and wild bird
population, but have also been detected in mammals, and which elevates
the risk of transmission to humans. Another concern is the extensive
genetic reassortment with other influenza serotypes, with H9N2 AIV
considered to be the most common and destructive LPAIV subtype. While
vaccination is an effective method to control the spread of influenza
viruses, the efficacy of the H9N2 AIV vaccine has been challenged by the
perpetual antigenic drift. Our previous research demonstrated that H9N2
field isolates have undergone antigenic drift to evolve into distinct
antigenic groups, which also resulted in significant antigenic
differences with the commercial vaccines. Unfortunately, the key amino
acids associated with those antigenic drift events remain elusive. Two
discrete antigenic sites “H9-A” and “H9-B” (or group I and II) which
include at least 46 amino acid sites have been identified (T. Peacock et
al., 2016). We also analyzed the evolution of these 46 aa in isolates
collected over the most recent five years and found that most of these
sites were completely conserved among circulating H9N2 viruses. To
identify which single mutations of these viruses are driving the
antigenic drift, 6 high-frequency mutations including those at aa sites
164, 168, 171, 198, 200 and 201 were screened by comparing amino acid
alignments of the H9N2 AIVs isolated from China in 2014-2019. It should
be noted that 5 of these (164,168,198,200 and 201) are located both
in the antigenic sites and RBS region (Kaverin et al., 2004). Three of
them (164, 168 and 201) were under positive selection (data not shown)
pressure (Su et al., 2020). Therefore, these sites were likely
responsible for the significant antigenic drift observed in recent
years.
A variety of mechanisms, namely changes to epitope structure,
acquisition of additional glycosylation sites and modulation of
receptor-binding avidity, can contribute to both actual and apparent
antigenic change (Abe et al., 2004; Das et al., 2011; Hensley et al.,
2009). In the present study, 2 sites containing a high-frequency of
mutations were identified that contribute to the viral antigenic drift.
The A168N and D201G substitutions resulted in significant antigenic
changes.
The HA protein of influenza is a highly glycosylated. N-linked
glycosylation of HA has been reported to contribute to immune escape and
virulence of influenza (Gao et al., 2021), and obtaining new
glycosylation sites is also an important mechanism of viral antigenic
drift. None of the mutations characterized here introduced novel
glycosylation sites into the virus. In response to escape under
neutralizing antibody pressure of the virus, influenza A virus could
evolve by regulating HA receptor avidity via amino acid substitutions in
the HA1 globular head domain (Das et al., 2011), many of which
simultaneously alter the antigenicity (Hensley et al., 2009; Wu &
Wilson, 2020). Some studies have shown that receptor avidity may be a
more important factor than antigenicity in avoiding neutralizing
antibodies (Crowe, 2012). Here, the effects of two mutations at
antigenic site II of residue 201 (D201G and D201A) on antigenicity were
significantly altered, in which the glycine substitution had a
significant effect both on antigenicity and immune escape, while alanine
had no effect. In addition, a serine substitution at residue 201 can
also lead to antibody escape (Wan et al., 2014). It was observed that
the change of surface structure was a result of the D201G substitution
(G: hydrophobic, D: hydrophilic). This in turn may be due to the change
of hydrophilicity, thus leading to the change of receptor avidity and
antigenicity of the virus. It is important to note that changes in
receptor avidity were not validated at residue 201 in this study. Of
note, 201G mainly exists in predominant subclade C (Figure 1), which may
result in immune escape in chicken flocks in the future. The other sites
that may cause a similar phenomenon are at residues 201 and 168, which
are both in the antigenic epitopes and RBS region. It was observed that
asparagine (N, 37.1%) and alanine (A, 35.7%) accounted for the largest
proportion at residue 168 in 2019-2020, which was significantly
different from the major site (aspartic acid, D, 52.9%, and A, 33.0%)
in 2014 by amino acid comparisons (Table S1). The substitution of those
three amino acids can affect the antigenicity of H9N2 viruses (T.
Peacock et al., 2016).
Comprised of a weak genetic basis of preferences for alternative avian
receptors and human-like receptors, residue 198 may be a multifunctional
site which could function to modulate polyclonal antisera binding and
receptor-binding avidity at the same time (T. P. Peacock et al., 2020).
In the present study, T198A was capable of immune escape in a reciprocal
HI assay using polyclonal antibodies, while escape was not observed in a
microneutralization assay. One possible explanation for this result is
that the HI receptor-binding avidity and neutralization antibody
epitopes are different. Thus, the molecular basis requires an in depth
analysis, which is beyond the scope of this report.
In conclusion, key amino acid substitutions that may drive antigenic
drift of H9N2-AIV in a recent five year periods were identified. Two
predominant substitutions, including A168N and D201G were demonstrated
to significantly affect antigenicity, but did not change growth
characteristics and cell tropism. It is worth noting that the D201G
substitution not only changed the antigenicity, but also produced an
immune escape variant of the parental virus. The data presented here
provide a reference for the prediction of the evolutionary direction of
H9N2-AIV, and the development of effective vaccines.