Discussion
Wild birds are the natural hosts of AIVs
(Globig et al., 2009), and recently with
the improvement to people’s ecological awareness, more and more data
have been obtained in the monitoring of avian influenza in wild birds.
It has shown that the H10-H12 subtypes could be isolated almost every
year worldwide and the number of these strains began to increase
gradually since 2000 (Figure 4a). Up to Apr 20, 2020, about 3000 H10-H12
HA sequences were available in GenBank database, and more than 80% of
them were from wild birds (Figure 4b). However, compared with the total
numbers, relatively few H10-H12 subtypes were isolated from wild birds
in China. There are about 50 H10-H12 subtype viruses were detected from
wild birds, and the majority of them were from Jiangxi and Hong Kong.
Especially for H12 subtype, only 1 strain could be found in the database
in China.
Shanghai is one of the most important overwintering and stopover site
for wild birds on the East Asian-Australian Migration route. Every year,
millions of migratory birds and hundreds of species pass through
Shanghai. Nanhui Dongtan wetland of Pudong and Jiuduansha Natural
Reservation Zone were two important wetlands for waterfowls in Shanghai.
Given the importance of H10-H12 subtypes in the influenza viral
ecosystem, it is essential that extensive surveillance of these rare
subtypes in wild birds in this area should be implemented to narrow this
knowledge gap.
It has shown that H11N9 virus could be transmitted directly from ducks
to humans (Gill et al., 2006), and the
recombination events could occur between H11 and H7 subtypes, such as
the H11 subtype can provide internal gene fragments for the highly
pathogenic H7 subtype (Shi et al., 2013).
H10 subtype is frequency isolated from wild birds and domestic poultry.
The NS1 gene of H10 AIVs mutates, leading to site substitution, which
increases virulence and pathogenicity of the virus to mammals
(El-Shesheny et al., 2018). The adaptation
of virus to the host usually involves the reassortment of gene fragments
of co-infected virus strains adaptive mutations in various virus genes
(Ince et al., 2013). The H10 was reported
to be associated with all possible NA subtypes, which contributed to the
diversity of the HA lineage (Wille et al.,
2018). In this study, we isolated 2 H10N4, 1 H11N9, 1 H11N2 and 1 H11Nx
strains during 2016-2019 surveillance. The HA-NA recombination of H10
and H11 subtypes showed in this manuscript might be frequently detected
in nature in wild birds. Interestingly, we identified 7 H12 subtype
strains in this study, including 3 NA combinations: H12N2, H12N5 and
H12N8. The H12 subtypes were relatively less than the other two subtypes
and they had might an NA bias for N2, not for N5
(Wille et al., 2018). Among these
positive samples, one strain was isolated from Gruiformes in
2016, while the remaining eleven strains were isolated fromAnseriformes , so Anseriformes should play an important
role in the maintenance of these rare subtypes
(Wille et al., 2018).
Phylogenetic analysis of these H10-H12 strains showed that a frequent
occurrence of reassortment could be identified among these subtypes. For
example, two H10N4 strains were recovered from the same site in
Jiuduansha Natural Reservation Zone in 2018, but parts of their internal
gene segments were belonged to the different sublineages (Figure S1),
suggesting that these two H10N4 viruses might share a different
ancestor. The similar evolution patterns also could be found in H12N2
and H12N5 strains (Figure S3), indicative of frequent reassortment
occurred among these subtypes in eastern China. Most of the eight gene
segments of these 12 strains were clustered into the Eurasian lineage,
and they shared high sequence identity with those isolated from wild
birds and domestic ducks in Japan, Korea, Bangladesh, Vietnam and China
where belong to the East Asian-Australasian Flyway route. In addition,
wild bird populations had been shown to be the main source of new
reassortment, rather than poultry (Lu et
al., 2014). Several evidences have showed that intercontinental
transfer of highly pathogenic avian influenza viruses by migratory birds
could be frequently occurred (Koehler et
al., 2008), such as H5N8 viruses, but it is rare for low pathogenic
avian influenza viruses via wild birds
(Krauss et al., 2007;
Winker et al., 2007). In this study, the
intercontinental reassortment of avian influenza viruses between
Eurasian and North American could be observed in two H12N2 strains
(NH112319-H12N2 and NH101807-H12N2) (Figure S3), thus the gene flow
between these sites were existed. In the same way, the study of AIV gene
segments showed in different regions and recombination between different
viral lineages, greatly increasing our understanding of the ecology of
virus in this fragile and pristine environment
(Hurt et al., 2016;
Olsen et al., 2006). It is therefore wild
birds play an essential role in the transmission of the influenza
viruses.
In this study, multiple strains of H10-H12 subtypes were isolated from
wild birds in Shanghai, the genetic analysis results of these strains
indicated that avian influenza viruses in wild birds were diverse which
may pose a threat to public health due to the frequent reassortment
between wild birds and poultry. Shanghai plays an important role in the
ecology of avian influenza virus in China and even the whole world, so
monitoring and studying the rare subtypes in this region is of great
help to improve our understanding of these virus subtypes. In future,
more avian influenza virus surveillance in the migratory flyways should
be strengthened, and which might provide a timely and effective method
for understand the infection situation of AIVs and the avian influenza
virus ecology in world.