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).