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.