Results
A total of 234 HA gene sequences of AIVs gathered in water samples were reported from nine countries between 2003 – 2020. No AIV sequences were reported from Africa, Oceania, or South America. Most of the AIV sequences were obtained from China (47.44%, n=111) and Bangladesh (23.93%, n=56), mainly from two subtypes: LPAI H9N2 and HPAI H5N1. The H10N7 was the only subtype described in Europe. Most subtypes (H3N2, H4N8, H4N9, H5N1, H5N6, H5N8, H6N2, H6N6, H7N9, H9N2, H10N8, H11N2, H11N9, and H12N7) were reported only by Asian countries.
The highest AIV diversity was observed in China with twelve subtypes followed by Japan with six subtypes. H3N8 and H4N6 subtypes were described in both Asian and American countries. H4N9, H7N9, H10N8, H11N2, and H12N7 subtypes were exclusively reported in China. Similarly, H4N2, H4N8, and H6N6 subtypes were solely detected in Mexico, Japan, and Vietnam, respectively. Likewise, H1N1, H5N2, and H7N2 subtypes were only isolated in the United States (Figure 1).
The AIV sequences were detected in water samples from live bird markets (64.10%, n=150), wild bird habitats (19.23%, n=45), poultry farms (12.82%, n=30), and unknown sampling site (3.85%, n=9). AIV sequences from live bird markets were mainly reported by Asian countries (98.67%, n=148), especially China (54.66%, n=82) and Bangladesh (37.33%, n=56). The United States was the only country on the American continent that reported AIV sequences from a live bird market (1.33%, n=2) (Figure 2A).
Likewise, AIV sequences from wild bird habitats and poultry farms were detected in both Asia and America. Most of the AIV sequences from wild bird habitats were reported in China (46.67%, n=21) and Japan (35.56%, n=16) (Figure 2B), especially from the Dongting Lake (Hunan) and the Izumi Plain (Kagoshima), respectively. However, IAV sequences were also identified in water samples from the Qinghai lake in China and from a Kodai pond in a Japanese zoo. In the American continent, the IAV sequences were reported in Mexico, the United States, and Canada (Figure 2B). In particular, IAV sequences were detected from the Agassiz National Wildlife Refuge and the Thief Lake Wildlife Management Area in Minnesota, United States, filtered wetland water in Alaska, United States, a wetland in Central Mexico, and a wild bird habitat in Alberta, Canada.
The IAV sequences from poultry farms were mainly reported in the United States (66.67%, n=20) (Figure 2C), corresponding to water samples from barns of turkey flocks in Minnesota and Nebraska. One IAV sequence belonged to a free-range farm in the Netherlands. All the IAV sequences (H3N2, H3N8, H4N9, H11N2, and H12N7) detected from poultry farms in China (20%, n=6) were located close to aquatic environments (i.e., a domestic duck and goose farm close to a river in Hunan; as well as duck farms in the Dongting Lake region, China). On the other hand, nine AIV sequences (H5N1, H5N6, H6N6, H7N9, and H11N9) from four Asian countries did not specify the sampling site (Figure 2D).
The highest subtype diversity was found in water samples from wild bird habitats with eleven subtypes (H1N1, H3N8, H4N2, H4N6, H4N8, H5N1, H5H6, H5N8, H6N2, H9N2, and H10N8). The wild bird habitats in Japan represented the highest subtype diversity with six subtypes (H3N8, H4N6, H4N8, H5N6, H5N8, and H6N2), two of them have also been reported from North American wild bird habitats (H3N8 and H4N6) (Figure 2B).
In the context of live bird markets, seven subtypes (H3N2, H5N1, H5N6, H6N2, H7N9, H9N2, and H11N9) were reported in Southeast Asia, and one subtype (H7N2) was detected in the United States (Figure 2A). Similarly, in poultry farms, six subtypes (H3N2, H3N8, H4N9, H5N1, H11N2, and H12N7) were reported in Southeast Asia, one subtype (H10N7) in the Netherlands, and one subtype (H5N2) in the United States (Figure 2C). China evidenced the highest subtype diversity in both live bird markets and poultry farms.
The live bird market samples were obtained from poultry cage water (27.33%, n=41), sewage (21.33%, n=32), and contaminated drinking water troughs (14.67%, n=22). The greatest subtype diversity was found in water from drinking troughs with six subtypes (H3N2, H5N1, H5N6, H6N2, H9N2, and H11N9). However, a high percentage of the live bird market samples did not report the water source (36.67%, n=55) (Figure 3A).
Likewise, the poultry farm samples were obtained principally from turkey barn water (66.67%, n=20) and duck’s environmental water with fecal matter (16.67%, n=5); and to a lesser extent from sewage, carcass wash water, and hand-wash water (3.33%, n=1 each one). The greatest subtype diversity was found in environmental water of duck farms with five subtypes: H3N2, H3N8, H4N9, H11N2, and H12N7. Only the poultry farm along a river in China and the free-range farm in the Netherlands did not specify the water source (Figure 3B).
The circular phylogenetic tree based on the analysis of the HA sequences detected in water samples showed that the H1 subtype was closely related to the H6 subtype, and the H12 to the H9 subtype (Figure 4). Likewise, the H7 subtype had close relation with the H10 subtype, and these were genetically close to the H3 and the H4 subtype.
The H3N2 HA sequence (Accession Nº: LC041327) detected from a live bird market in Vietnam shared 95.88% of nucleotide sequence identity with the H3N2 HA sequence (Accession Nº: CY146748) from a duck farm in China. Moreover, the H3N8 HA sequence (Accession Nº: CY146756) isolated from a duck farm in the Dongting Lake region was grouped in a clade with H3N8 HA sequences from the Izumi plain in Japan. Meanwhile, H4 subtype evidenced two geographically separated clades, the first containing H4N6, H4N8, and H4N9 HA sequences from Asia, and the second H4N2 and H4N6 HA sequences from America. In particular, the H4N9 HA sequence (Accession Nº: CY146732) isolated from a duck farm close to the Dongting Lake in China was related to AIVs detected in the Izumi plain, Japan with 97.99-98.05% nucleotide sequence identity.
All the H5N1 HA sequences were grouped together, except for one H5N1 HA sequence (Accession N°: KX247930) detected in a live bird market in China, which was related to H5N6 HA sequences from a Kodai pond and the Izumi plain in Japan, as well as, from a live bird market in China with 95.95-96.34% nucleotide sequence identity. Similarly, all the H5N8 HA sequences detected in wild bird habitats were grouped in the same clade, except the H5N8 HA sequence (Accession Nº EPI553362) detected in the Izumi plain in Japan which was phylogenetically related to the H5N2 HA sequences detected in turkey barns from the United States with 97.95 - 98.30% nucleotide sequence identity.
The phylogenetic analysis also showed that all the H9N2 HA sequences were grouped together in two clusters by country (China and Bangladesh) and were closely related to the H12N7 HA sequence (Accession Nº: CY146764) detected from a duck farm located in the Dongting Lake region in China. Finally, H11N9 HA sequences from live bird markets and the H11N2 HA sequence from a duck farm close to a wetland in China were clustered in the same clade with 95.58-95.70% nucleotide sequence identity.