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.