1 INTRODUCTION
Since December 2019, the global outbreak of coronavirus disease 2019
(COVID-19) has seriously threatened human health and public health
security. We are currently making every effort to prevent and control
the COVID-19; however, it is impossible to ignore the harm the influenza
virus can inflict on humans (F. Wu et al., 2020). In the past two
decades, highly pathogenic avian influenza viruses H5N1, H7N9, and H5N6
continuously crossed the species barrier from poultry to humans (Claas
et al., 1998; Gao et al., 2013; Shen et al., 2016). Low-pathogenic H6
subtype influenza virus was also isolated from a patient diagnosed of
lower respiratory tract infection in 2013, and a highly close H6 virus
was subsequently isolated from a dog in 2014 (Wei et al., 2013),
indicating H6 subtype virus could crossed the species barriers to
mammals as well thus posing a potential threat to human health.
H6 subtype influenza A virus was first isolated from turkey in 1963
(Lupiani & Reddy, 2009). Since then, H6 subtype AIV has widely spread
around the world. It is mainly divided into North American and Eurasian
lineages, where the Eurasian lineage can be further divided into Group I
(ST339-like), Group II (ST2853-like), Group III (HN573-like) (Bahl,
Vijaykrishna, Holmes, Smith, & Guan, 2009; Wallensten et al., 2005; zu
Dohna, Li, Cardona, Miller, & Carpenter, 2009), and W312-like minor
poultry group (Chin et al., 2002). In China’s live poultry market, H6
viruses (including H6N1, H6N2, H6N5, H6N6, H6N8, etc.) have been
transmitted to poultry and prevalent for a long time (Huang et al.,
2012; Zhao et al., 2011). From 2000 to 2005, ST339-like Group I H6N2 was
the most frequently detected H6 subtype in live poultry markets in
southern China. However, since the emergence of the H6N6 virus in 2005,
the ST2853-like Group II H6N6 has gradually replaced the H6N2 subtype.
Similar group replacement scenario has also been observed in eastern
China but generally 2-4 years later than southern China, possibly due to
the induction from there. More importantly, the H6N6 virus transmitted
from waterfowl to land poultry is causing an endemic disease in poultry
(H. Wu et al., 2016; Zhao et al., 2011; Zou et al., 2016).
The specific recognition and binding of hemagglutinin (HA) to sialic
acid (SA) receptors on the host cells play a crucial role in determining
the host range of influenza virus. Most AIV binds to avian-like
receptors SAα-2,3 galactose(Gal), while the human influenza virus
recognizes human-like receptors SAα-2,6Gal. For an effective human to
human transmission, AIV must first acquire the ability to bind to the
human-like receptor. Zou et al (Zou et al., 2016) found that 4
out of the 14 H6N6 AIV strains acquired the ability to recognize and
bind to the human-like receptor. Wang et al (G. Wang et al.,
2014) isolated 70 H6N6 viruses from the live poultry market in southern
China from 2008 to 2011 and found that 48 strains (68.6%) preferential
bind to a human-like receptor. Bi et al (Bi et al., 2020) found that
some H6N2 and H6N6 strains isolated in live poultry markets during
2016–2019 in China preferential bind to a human-like receptor. The
molecular epidemiological investigation found that reassortment between
H6 subtype and non-H6 subtype (especially H5N6 virus prevalent in
Southern China) may easily occur (Li et al., 2019). Regarding the cases
of human infection by H5N6 virus that occurred in 2014, it was found
that H5N6 virus was generated by reassortment of H6N6, H7N9/H9N2, and H5
viruses (Bi et al., 2016), which indicated that H6N6 acted as the
progenitor of H5N6, a novel avian influenza virus infecting human.
Meanwhile, some H7N9 viruses infecting humans have changed their NP and
NS gene fragments by reassorting with H6N6 and H5N6 virus genes (Jin et
al., 2017), which indicated that AIV H6N6 is currently in the dynamic
status of evolution and is prone to reassorting with other subtypes of
viruses, thus increasing the genetic diversity of the viruses. In 2011,
swine-originated H6N6 virus A/SW/GD/K6/2010 (GDK6) was isolated in
Southern China (Zhang et al., 2011). Gene sequencing analysis indicated
that the virus originated from domestic ducks, and HA belongs to Group
II virus of H6 Eurasian lineages. Animal experiments showed that the
virus has limited transmissibility between ferrets (Sun et al., 2017),
suggesting that the H6N6 virus could pass the interspecific barriers and
infect humans. The seroepidemiological investigation found that the
serum of exposed people from turkey farms in the United States was
positive for H6 (Kayali G, 2010), and Li et al detected H6
antibody in the serum of 15,689 exposed people, resulting in a positive
rate of 0.4% (Xin et al., 2015). Therefore, the possibility for humans
to get infected by the H6N6 virus is increasing, while the risk for
virus epidemic also rises.
H6N6 subtype AIV is widely prevalent in wild waterfowl and poultry in
Eurasia, and its host range had gradually expanded to mammals, such as
swine. Some viruses have even acquired the ability to recognize and bind
to human-like receptor SAα-2, 6 Gal (G. Wang et al., 2014; Zhang et al.,
2011; Zou et al., 2016). Following the continuous evolution of the
virus, it remains unclear how readily it can develop the ability to
cross interspecific barriers, thus affecting humans. In this study, the
H6N6 subtype of avian influenza viruses isolated from chickens were
sequenced and phylogenetically analysed, and the receptor-binding
preference of the virus was analyzed. Then, the mice in vivo and
human lung tissue in vitr o were inoculated with viruses to
observe replication in mice and human lung tissue, as well as the
molecular characteristics of the virus that infects mice and replicates
in humans. Furthermore, we evaluated the potential of H6N6 viruses to
infect humans and the key molecules that support binding to human-like
SAα-2,6Gal preference and replication in humans.