1 INTRODUCTION
Since its first emergence in February 2013 in China, H7N9 avian influenza viruses (AIVs) have continued to circulate in mainland China and caused five epidemic waves of human cases during 2013-2017 (Kang et al., 2017; Liu et al., 2013; X. Wang et al., 2017). Till now, H7N9 AIVs have sickened 1568 individuals and caused 616 fatalities according to the WHO report (http://www.who.int/influenza/human_animal_interface). Early H7N9 isolates are all low pathogenic (LP) until late 2016, after circulation for a few years in live poultry markets of China, LP H7N9 AIVs evolved into a highly pathogenic (HP) form (Shi et al., 2018). HP H7N9 AIVs were characterized by insertion of polybasic amino acid residues at the HA cleavage site. This insertion alters the phenotype of H7N9 AIVs from low pathogenic to highly pathogenic, causing up to 100% mortality among infected chickens (Shi et al., 2017). Thus, H7N9 AIVs pose a tough challenge to both public health and poultry industry.
Phylogenetic analyses suggested that the HP H7N9 viruses are tri-reassortants of H7N3, H10N9, and H9N2 subtypes of AIV. These new reassortants carry several mammalian-adaption molecular markers, making the viruses cross the species barrier and bind to mammalian receptors with a high affinity in addition to a predominant binding preference for avian-like receptors (Q. Zhang et al., 2013). Compared to LP H7N9 AIVs, the HP isolates were more pathogenic and can cause more severe clinical symptoms in mice as well as in ferrets (Imai et al., 2017; Shi et al., 2018). Meanwhile, previous research has demonstrated that though airborne transmissibility of HP H7N9 viruses didn’t increase compared to LP isolates, some HP strains have obtained the traits associated with enhanced transmission in mammals, including a more extensive tissue tropism and a lower threshold pH for membrane fusion (X. Sun et al., 2019). All these characteristics of the HP H7N9 AIVs have made it an ongoing threat to humans and a potential risk to cause a next pandemic.
Vaccination is the most effective measure to control avian influenza. Given the risk of H7N9 AIVs to public health and poultry industry, a series of vaccination program have been implemented since 2017. An H5/H7 bivalent inactivated vaccine (H5 Re-8 and H7 Re-1) was introduced into the poultry industry in September 2017 following the emergence of HP H7N9 viruses in the fifth outbreak. In December 2018, the vaccine was updated by using the H5 Re-11, Re-12, and H7 Re-2 vaccine seed viruses. The vaccination program successfully reduced the prevalence of H7N9 viruses in poultry and humans (Wu et al., 2019). But imperfectly, H7N9 influenza virus has not been eradicated from poultry with continuous sporadic isolation of HP H7N9 strains. During our routine surveillance in poultry in 2020, we isolated two H7N9 subtype AIVs from breeder farms in northern China. These two chicken-origin H7N9 isolates, A/chicken/Northern China/BJ3426/2020(H7N9) and A/chicken/Northern China/SX1616/2020(H7N9) were both HP based on the sequence of the HA gene with a four-amino-acid (KRTA) insertion at position 339-342 in the cleavage site. To better understand their characteristics, we conducted sequencing and phylogenetic analysis, solid-phase direct binding assay, thermal stability assay, acid stability assay and animal experiments. Our results provide important insights into the persisting evolution of H7N9 AIVs, highlighting the importance of the surveillance in poultry.