Sequence and phylogenetic analysis of H11 subtypes
In the three strains of the H11 subtype, two 2016 strains (PD112440-H11N9 and PD112452-H11Nx) were isolated from Eurasian coot (Fulica atra ) and Common teal (Anas crecca ), respectively, and the other 2017 strain (NH101834-H11N2) was isolated from Eurasian wigeon (Anas penelope ) at Nanhui Dongtan wetland of Pudong. Except for the NA gene of PD112452-H11Nx, the whole genome sequences of the three strains were obtained. The two 2016 isolates (PD112440-H11N9 and PD112452-H11Nx) were almost genetically identical and the six gene nucleotide sequences identity were 99.1% to 100%, whereas their PA genes shared 93.7% identity; accordingly, their closest relative gene segments were the same, and showed in Table 2. Except for NA gene, the 2017 isolate NH101834-H11N2 shared a relative low nucleotide identity (91.0%-97.6%) with the two 2016 isolates in other 7 gene segments and had the most similarity with the duck and wild bird viruses circulating in Vietnam, Japan, Korea, South Africa and China (Table 2).
In the phylogenetic tree, HA genes of the three strains were clustered in the Eurasian Lineage, and grouped into 2 sublineages. The two 2016 strains grouped together in a small sublineage, and more closely related to H11N9 virus circulating in ducks in Ibaraki (A/duck/Ibaraki/99/2016). The 2017 isolate NH101834-H11N2 was grouped in another sublineage and showed a close relationship with the H11N3 and H11N9 strains circulating in ducks and mandarin ducks in Bangladesh and South Korea (Figure 2). Phylogenetic analyses of the NA genes showed that 2016 strain PD112440-H11N9 was closely related to H11N9 virus circulating in ducks in Japan, and 2017 strain NH101834-H11N2 showed a close relationship with the H6N2 and H5N2 viruses circulating in wild ducks in Korea and China (Figure 2). Phylogenetic relationships among the six internal genes of the three strains revealed the topologic structure were similar to the HA gene tree, that is the two 2016 strains closely clustered together in the same sublineage, and the 2017 strain clustered into another sister sublineage. They were all genetically closed to those of viruses isolated from domestic ducks or wild birds circulating in Japan, Korea, Bangladesh, Mongolia, Vietnam, Cambodia and China (Figure S2).
Sequence and phylogenetic analysis of H12 subtypes
To better understand the evolutionary relationship of these H12 subtypes influenza viruses with other influenza viruses, we performed phylogenetic analysis. Of the seven H12 subtypes, four were of the H12N2 subtype, two were of the H12N5 subtype and one was of the H12N8 subtype. Five strains were isolated from Common teal (Anas crecca ) and the other two strains were from Mallard (Anas platyrhynchos ) at Jiuduansha Natural Reservation Zone and Nanhui Dongtan wetland of Pudong during 2017-2019. The whole genome sequences of these H12 strains were sequenced and analyzed. The homological analysis showed that they shared a low similarity in nucleotide sequence identity among the eight gene segments, such as 77.9% to 99.9% for the HA gene, 51.0% to 98.8% for the NA gene, 83.7% to 96.9% for PB2 gene, 87.9% to 99.0% for PB1 gene, 92.1% to 99.6% for PA gene, 92.1% to 98.9% for NP gene and 95.0% to 98.7% for M gene, 70.0% to 99.7% for the NS gene, indicated that these H12 subtypes were highly divergent. BLAST (https://www.ncbi.nlm.nih.gov/blast/) search showed that the HA, PB2 and PB1 genes of NH112319-H12N2, and PB1 gene of NH101807-H12N2 shared the highest sequence identity with A/Mallard/Alaska/AH0029066S.1.A/2016 (H12N5) and A/mallard/California/3070/2012 (H11N2) viruses, respectively, which were circulating in North American sublineage. The remaining of other gene segments of these H12 subtypes shared >97% identity with those AIVs were from wild birds or ducks along the East Asian-Australasian Flyway route, such as in Japan, Korea, Mongolia, Bangladesh, Vietnam, Netherlands and China (Table 2).
Phylogenetic analysis of HA genes showed that the seven HA genes of these H12 subtypes belonged to two sublineages: NH112319-H12N2 was clustered into the North American lineage, while the other six strains clustered into the Eurasian lineages (Figure 3). Two H12N2 strains (NH102615-H12N2 and NH110165-H12N2) were highly similarity and closely clustered together, and grouped with another H12N2 virus (NH101807-H12N2) and other duck viruses from Japan into a small sublineage, and then with two H12N5 strains formed a sister sublineage. The H12N8 strain (JDS110203-H12N8) shared high homology with A/mallard/Novosibirsk region/964k/2018 (H12N5) and formed another clade in the Eurasian lineages.
Phylogenetic analysis showed that NA gene of these H12 strains were all clustered into the Eurasian lineage. Likewise, two H12N2 strains of NH102615-H12N2 and NH110165-H12N2 were closely clustered together, and then grouped with the other two H12N2 strains and other Southeast Asian viruses, such as Vietnamese and Indian, in two small clades (Figure 3). The N5 and N8 genes of these H12 subtypes grouped with H6N5, H12N5 and H3N8 viruses circulating in Mongolia, China and Japan, respectively (Figure 3).
Phylogenetic analysis of the internal genes showed that PB2 and PB1 genes of NH112319-H12N2, and PB1 gene of NH101807-H12N2 were clustered into the North American lineage, and in contrast the other internal genes all belonged to the Eurasian lineage and most of them formed a relatively unique clade with other strains isolated form migratory waterfowls, domestic aquatic birds and chickens in Japan, Korea, Mongolia, Bangladesh, Vietnam, and China (Figure S3). The results indicated that two H12N2 strains (NH112319-H12N2 and NH101807-H12N2) were generated through genetic reassortment between viruses belonging to the Eurasian and North American virus lineage.