3 RESULTS
3.1 Virus isolation and identification
Two H7N9 AIVs were isolated from clinical samples of layer chickens
during our routine surveillance in poultry in 2020. They were from
different breeder farms
vaccinated with
H7 Re-2 vaccine in northern China.
These two chicken-origin H7N9 isolates were named
A/chicken/Northern
China/BJ3426/2020(H7N9)
[BJ3426] and A/chicken/Northern
China/SX1616/2020(H7N9)
[SX1616], respectively. Full
genomes of these two H7N9 AIVs have been sequenced and deposited in the
Global Initiative on Sharing Avian Influenza Data (GISAID) under the
accession numbers EPI 1921163-EPI 1921178.
3.2Molecular andphylogenetic
analysis
Firstly, homologous analysis was conducted at the nucleotide level. As
shown in Table 1, all eight genes of
these two viruses shared 97.5%-99.3% nucleotide identity, of which NS
gene presented the lowest, while PB1
and
NP showed the highest homologies.
When compared with sequences from GISAID, they shared the highest
nucleotide identities with the same HP H7N9 AIVs isolated in 2018 and
2019. Specially, HA and NA genes, PA and NP genes showed the highest
homologies with identical strains A/chicken/China/HD1/2019(H7N9) and
A/chicken/Hebei/S1177/2019(H7N9), respectively, while PB2, PB1, M and NS
genes shared highest nucleotide identities with different viruses.
Subsequently, in order to infer genetic evolution of these two isolates,
phylogenetic trees of all the eight genes were constructed with
reference sequences available in the GenBank database. The HA gene-based
phylogenetic analysis revealed that these two strains clustered to the
HP H7N9 clade belonging to Yangtze River Delta lineage.
They were closely related to
previously reported antigenic variant HD1 (Jiang et al., 2020), while
located in different small branches with H7 Re-2 vaccine, suggesting the
differences in their antigenicity. The NA gene of these two strains was
in the RD5-like (H10N9) branch and their internal genes were closely
related to the dominant S genotype (G57-like) of the H9N2 subtype (M.
Gu, Xu, Wang, & Liu, 2017; Pu et al., 2015) (Figure 1).
We further analyzed several key molecular characteristics of
these two strains. As shown in Table
2, the HA protein of both strains
had a four-amino-acid (KRTA) insertion at position 339-342 and an I335V
mutation in the cleavage site to make the motif
PEVPKRKRTAR↓GLF, indicating highly
pathogenic to chickens. A160T mutation as well as 226Q and 228G in HA
suggested the preference for avian-like receptors, whereas 186V
indicated a binding specificity for human-like receptors (Shi et al.,
2017; Yang et al., 2017). The V135T and
A160T mutations in HA made
both strains obtain two extra
potential glycosylation sites, which contributed to the antigenic drift
from the H7-Re2 vaccine (Yin et al., 2021).
Besides, compared with H7 Re-2
vaccine, both strains have acquired F102V, R140K, S145P and N157D
mutations in HA protein, which may change the
antigenicity of
these two isolates.
Remarkably, the F102V and N157D
mutations have never been reported before. PB2-E627K or D701N mutation
was considered to increase the virulence of H7N9 viruses in mammals (Shi
et al., 2017), while in our study, neither was found in any of these two
isolates. I368V mutation in PB1, which can enhance the pathogenicity to
ferrets (Tscherne & Garcia-Sastre, 2011), was found in both isolates.
PA-K356R, a molecular maker associated with the bird-to-human
transmissibility of the H7N9 viruses, was also detected in both strains
(Xu et al., 2013). It has been reported that A286V and T437M mutations
in NP could attenuate H7N9 viruses in mice, whereas these two isolates
retained 286A and 437T, indicating increased pathogenicity to mice (Ma
et al., 2020).
3.3Receptor-binding
properties of these two isolates
Receptor-binding property is an important basis to evaluate the
interspecific transmission ability of AIVs (H. Sun et al., 2016; Xiong
et al., 2013). To investigate the receptor-binding properties of these
two isolates, we conducted solid-phase direct binding assay. Meanwhile,
HD/05(H5N1) and CA04(H1N1) were used as controls with absolute
preference for avian-type (SAα-2,3Gal) and human-type (SAα-2,6Gal)
receptors, respectively. The results showed that both strains had
dual-receptor characteristics, whereas bound to the α-2,
3-sialylglycopolymer with high affinity and to the α-2,
6-sialylglycopolymer with low affinity (Figure 2).