4 DISCUSSION
In China, zoonotic influenza virus was dominated by H5N1 until 2013, the emergence of H7N9 changed this situation significantly. H7N9 influenza virus was first isolated from patients in Shanghai and Anhui Province (Gao et al., 2013). Since then, the virus has spread quickly and continued to circulate in mainland China. As a result, HP H7N9 AIVs emerged after four years evolution in nature, causing severe avian and human infections with high mortality. Countless numbers of poultry were culled due to H7N9 infections. Besides, epidemic waves of human infections occurred and 1568 cases were laboratory-confirmed with 616 deaths. Thus, H7N9 AIVs pose a huge threat to the poultry industry as well as human health.
The emergence of H7N9 AIVs has aroused widespread concerns, and in order to effectively control the epidemic situation, a large number of vaccine candidates have been generated with various vaccine platforms. H7N9-inactivated vaccine (H7-Re1) has been first introduced into the poultry industry in the fall of 2017 following the emergence of HP H7N9 AIVs during the fifth wave. Thereafter, H7-Re2 replaced H7-Re1 in December 2018. Surveillance data after the vaccination program indicated that the vaccine application has successfully prevented the spread of H7N9 between poultry and humans, and new outbreaks never emerged again (Shi et al., 2018; Wu et al., 2019). It is worth noting that although the prevalence of H7N9 influenza virus has declined dramatically, it hasn’t been truly eradicated from poultry with continuous sporadic isolation of HP H7N9 strains in chickens and zoo birds in northern China between 2018 and 2019 according to the CADC reports (http://www.cadc.net.cn/sites/MainSite/tzgg/sygb/).
In this study, two H7N9 subtype AIVs were isolated during our routine surveillance in poultry in 2020. They were from different breeder farms vaccinated with H7 Re-2 vaccine in northern China. Homologous analysis showed that these two chicken-origin H7N9 isolates, BJ3426 and SX1616 shared the highest nucleotide identities with HP H7N9 AIVs isolated in 2018 and 2019. Particularly, HA and NA genes showed the highest homologies with identical strain A/chicken/China/HD1/2019(H7N9), an antigenic variant of HP H7N9 AIV (Jiang et al., 2020), indicating they were also antigenic variants.
Phylogenetic analysis based on nucleotide sequences of the HA gene showed that these two viruses clustered into the HP H7N9 clade belonging to Yangtze River Delta lineage. Of note, they located in different small branches with H7 Re-2 vaccine, suggesting the antigenic drift of them. Analysis of internal genes showed that they were derived from dominant S genotype (G57-like) H9N2 AIVs, demonstrating that they were reassortants of H7N9 viruses with H9N2 isolates. According to the deduced amino acid sequence of HA, both strains possessed multiple consecutive basic amino acids (PEVPKRKRTAR↓GLF) insertion at the cleavage site, suggestive of high pathogenicity to chickens. This was further confirmed by animal experiment in SPF chickens, with all challenged chickens died within two days and the values of IVPI 2.92 (BJ3426) and 2.84 (SX1616), respectively.
Receptor-binding characteristic is a key factor affecting the interspecies transmission ability of AIVs (de Graaf & Fouchier, 2014; Ge & Wang, 2011), and HA protein plays an important role in determining the binding properties of viral receptors (Ni, Kondrashkina, & Wang, 2018; Van Poucke et al., 2015). Previous studies have demonstrated that mutations in HA protein receptor binding sites including T160A, G186V, Q226L and G228S contribute to virus-host specificity to human-type receptors (SAα-2,6Gal) (Dortmans et al., 2013; Gog et al., 2007). Except for G186V mutation, 226Q, 228G and 160T were conserved in both isolates, indicating the preference for avian-type (SAα-2,3Gal) receptors. This was consistent with the result of solid-phase direct binding assay showing 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.
Further analyzing the HA gene, we found that the V135T and A160T mutations made both strains obtain two extra potential glycosylation sites, which have been reported to contribute to the antigenic drift from the H7-Re2 vaccine (Yin et al., 2021). Besides, compared with H7 Re-2 vaccine, both strains have obtained F102V, R140K, S145P and N157D mutations located in or near antigenic epitopes, which were possible to change the antigenicity of these two isolates. Remarkably, the F102V and N157D mutations have never been reported before, to which more attentions should be paid.
Analyses of internal genes showed that the well-known molecular markers contributing to the increased virulence in mice such as PB2-E627K and D701N mutations didn’t appear in these two isolates. Recent research has reported that A286V and T437M mutations in NP could attenuate H7N9 viruses in mice (Ma et al., 2020), whereas both isolates retained 286A and 437T, indicating increased pathogenicity to mice. In mouse experiment, mice inoculated with both viruses only experienced a transient infection at the dose of 106.5EID50 on 4-5 days post infection and no deaths were observed during the two-week’s observation, indicating that these two H7N9 viruses were low virulent to mice. These results confirmed that the virulence of influenza virus in mice is a combined effect among different viral proteins (J. Gu et al., 2020; Ma et al., 2020). Besides, molecular makers associated with enhanced pathogenicity in mammals and interspecies transmission were detected in both isolates, posing a threat to public health.
Thermal and acid stabilities are important for influenza viruses to survive in the environment. In the present study, these two isolates exhibited relatively stable in high-temperature or acidic conditions, suggesting they were able to survive in disadvantageous environment. Thus, they showed a higher risk to infect poultry and humans.
In summary, our findings provide important insights into the evolution of HP H7N9 influenza viruses since the application of vaccines in poultry in China, and highlight the emergence of antigenic variants after 2019 (Jiang et al., 2020; J. Zhang et al., 2020). Vaccination is an effective measure to control the occurrence of H7N9 outbreaks, but when vaccines are used in the field, the potential emergence of novel variants that can evade vaccine-induced immunity should always be a concern. Therefore, continuous surveillance is necessary to check whether vaccination has induced changes in antigenicity. Furthermore, new antigen-matched vaccines and more comprehensive measures are urgently required to eliminate highly pathogenic H7N9 viruses.