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.