Volume 27, Number 4—April 2021
Highly Pathogenic Avian Influenza Clade 188.8.131.52 Subtype H5N6 Viruses Isolated from Wild Whooper Swans, Mongolia, 2020
We identified clade 184.108.40.206 highly pathogenic avian influenza A(H5N6) viruses from whooper swans (Cygnus cygnus) found dead in Mongolia. The identification of these infections in wild birds in this area is of concern because of the potential for virus dissemination during fall migration.
Highly pathogenic avian influenza (HPAI) H5Nx viruses have been continuous threat to poultry and public health since the detection of A/Goose/Guangdong/1/1996(H5N1) (Gs/GD) in 1996 in Guangdong Province, China. The Gs/GD-lineage has evolved into 10 genetically distinct clades (0–9) and subclades (1). A novel clade 220.127.116.11 of H5Nx viruses bearing multiple neuraminidase subtypes, including N2, N5, N6, and N8, has been identified in China since 2008 (2), and the H5 genes have been phylogenetically differentiated into 4 subgroups (A–D) (3). Clade 18.104.22.168 H5N6 viruses have been causing worldwide epizootics in poultry and wild birds, and human cases have also been reported since 2014 (4). In this study, we report the identification and genetic analysis of 2 HPAI clade 22.214.171.124 H5N6 viruses isolated from whooper swan carcasses in central Mongolia during April 2020.
Wild whooper swan (Cygnus cygnus) carcasses were found in April 2020 on the banks of 2 small ponds (48°25′39.8′′N, 102°36′09.6′′E, and 47°56′22.0′′N, 102°32′54.0′′E) around the Orkhon River located nearby Khunt Lake (Bulgan Province, 48°25′59.8′′N, 102°34′51.1′′E) and Doitiin Tsgaaan Lake (Arkhangal Province, 47°34′20.1′′N, 102°31′45.4′′E) (Appendix 1 Figure 1). These lakes are a major stopover site of migratory wild birds in central Mongolia between their breeding sites in the north and the wintering sites in the south and are also major breeding and molting habitats of wild bird species, including whooper swan. The lakes were the outbreak sites of HPAI H5N1 in wild birds in 2005, 2006, and 2009 (5). Two HPAI viruses, A/Whooper swan/Mongolia/24/2020(H5N6) and A/Whooper swan/Mongolia/25/2020(H5N6), referred to as MN-H5N6/2020 viruses in this article, were isolated from 2 brain tissue samples of whooper swan carcasses. We conducted whole-genome sequencing (6) and phylogenetic analysis on the isolates (Appendix 1). The nucleotide sequences have been deposited in GenBank (accession nos. MT872354–69).
The 2 MN-H5N6/2020 viruses shared high nucleotide similarity (99.65%–100%) across all 8 gene segments. Polybasic amino acid motif on the cleavage site of hemagglutinin (HA) genes (PLRERRRKR/G) suggested that the MN-H5N6/2020 viruses are HPAI. BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and GISAID (https://platform.gisaid.org) searches showed that the MN-H5N6/2020 viruses share high nucleotide identity (99.4%–99.9%) across all 8 gene segments with the HPAI H5N6 viruses, referred to as Xinjiang-H5N6/2020 viruses in this article, isolated from wild swans (mute swans [Cygnus olor] and whooper swans) during January 2020 in Xinjiang Province, China (Table 1) (7), which is located ≈4,800 km southwest of the isolation sites of the MN-H5N6/2020 viruses. These results suggested that the MN-H5N6/2020 viruses might have been introduced through the Central Asian flyway to central Mongolia most likely during early spring migration in 2020.
In the maximum-likelihood phylogenetic trees, all 8 gene segments of the MN-H5N6/2020 were closely clustered with the sequences of the Xinjiang-H5N6/2020 viruses and the H5N6 viruses of clade 126.96.36.199 group C isolated during 2016–2019 in China, Vietnam, and Russia, including human isolates (Appendix 1 Figure 2). The phylogenetic relationship and high nucleotide identity indicated that the MN-H5N6/2020 viruses possess the identical genome constellation with the Xinjiang-H5N6/2020 viruses (7). The time of most recent common ancestor for each gene of the MN-H5N6/2020 viruses and the Xinjiang-H5N6/2020 viruses ranged from May to December 2019, suggesting that the MN-H5N6/2020 viruses and the Xinjiang-H5N6/2020 viruses had diverged from a common ancestor most likely during the second half of the previous year (Table 2; Appendix 1 Figure 3). The time of most recent common ancestor for each gene of the MN-H5N6/2020 viruses ranged from January through March 2020. These data and understanding of waterfowl migration patterns suggest that H5N6 viruses were maintained among wild birds during fall and winter 2019 and reached Mongolia by late winter, most likely carried by long-distance flights of infected migrating wild birds during spring migration. A previous satellite-tracking study of whooper swans between northern China and Mongolia showed that the most stable period for the wintering population of whooper swans in the Sanmenxia Reservoir area was from late December to early January (8). For spring migration, departure dates of the wintering population of whooper swans ranged from February 17 to March 27, and arrival dates at the breeding sites in Mongolia ranged from February 27 to May 23. This spring bird migration pattern coincided with the timing and direction of H5N6 virus transmission between Xinjiang and Mongolia.
The MN-H5N6/2020 and the Xinjiang-H5N6/2020 viruses had mutations associated with increased HA receptor binding affinity to human-like receptor (α-2,6 sialic acid), including D94N, S133A, S154N, and T156A (H5 numbering) (9) (Appendix 2 Table 2), although they maintained the amino acids related to the binding tropism to avian-like (α-2,3 sialic acid) receptors (222Q and 224G). Unlike 7 H5N6 human isolates of clade 188.8.131.52 group C, the MN-H5N6/2020 and the Xinjiang-H5N6/2020 viruses had amino acid substitution at HA position 188 (H5 numbering), from threonin to isoleucine, which is known to enhance receptor binding affinity to human-like receptor (10). In the maximum-likelihood phylogenetic tree and maximum clade credibility tree of polymerase basic 2 gene, the closest isolates of the MN-H5N6/2020 and Xinjiang-H5N6/2020 viruses were human H5N6 isolates from China (Table 2; Appendix 1 Figure 2, 3).
Wild migratory birds have played an important role in disseminating and maintaining of Gs/GD lineage HPAI H5Nx viruses, as observed in the epizootics of H5N1 clade 2.2 during 2005–2006 (11), H5N1 clade 2.3.2 in 2009 (12), clade 184.108.40.206c in 2015 (13), and clade 220.127.116.11 since 2014 (14). During widespread dissemination of the HPAIV clade 2.2 during 2005–2006 and clade 2.3.2 in 2009, these viruses were also detected from wild birds at Doitiin Tsgaaan Lake and Khunt Lake, highlighting that these areas are useful locations for monitoring of HPAI in wild birds as a pathway for the spread of HPAI during migration of waterfowl. Identifying 2 H5N6 HPAI viruses in wild waterfowl in this area signifies the potential for wide spread of this clade 18.104.22.168 H5N6 viruses during the 2020 fall migration.
Since the first report of a human infection with HPAI clade 22.214.171.124 H5N6 virus in Sichuan Province, China, in April 2014 (4), a total of 24 cases had been reported from China as of August 2020 (15). The mammalian host-specific markers found in the MN-H5N6/2020 viruses suggest that these viruses are potentially infectious for mammals. Considering the possibility of future dispersal of the H5N6 HPAI viruses through wild birds and the presence of mammalian host-specific genetic markers, enhanced active surveillance in wild birds, poultry, and mammals is needed to monitor spread and understand the potential for zoonotic infection.
Ms. Jeong is a PhD candidate at Konkuk University, Seoul, South Korea; her primary research interests include the molecular epidemiology and host–pathogen interaction of avian influenza viruses. Ms. Nyamsuren is a researcher and MSc student at Mongolian University of Life Sciences, Ulaanbaatar, Mongolia; her primary research interests include the molecular epidemiology and diagnosis of transboundary animal diseases.
We would like to thank the State Central Veterinary Laboratory of Mongolia for providing Biosafety Level 3 facilities and technical support.
This research was supported by the Bio and Medical Technology Development Program of the National Research Foundation, funded by the government of South Korea (grant no. NRF-2018M3A9H4056535).
- WHO/OIE/FAO H5N1 Evolution Working Group. Toward a unified nomenclature system for highly pathogenic avian influenza virus (H5N1). Emerg Infect Dis. 2008;14:
- Gu M, Liu W, Cao Y, Peng D, Wang X, Wan H, et al. Novel reassortant highly pathogenic avian influenza (H5N5) viruses in domestic ducks, China. Emerg Infect Dis. 2011;17:1060–3.
- Lee DH, Bahl J, Torchetti MK, Killian ML, Ip HS, DeLiberto TJ, et al. Highly pathogenic avian influenza viruses and generation of novel reassortants, United States, 2014–2015. Emerg Infect Dis. 2016;22:1283–5.
- Pan M, Gao R, Lv Q, Huang S, Zhou Z, Yang L, et al. Human infection with a novel, highly pathogenic avian influenza A (H5N6) virus: Virological and clinical findings. J Infect. 2016;72:52–9.
- Gilbert M, Jambal L, Karesh WB, Fine A, Shiilegdamba E, Dulam P, et al. Highly pathogenic avian influenza virus among wild birds in Mongolia. PLoS One. 2012;7:
- Lee DH. Complete genome sequencing of influenza A viruses using next-generation sequencing. Methods Mol Biol. 2020;2123:69–79.
- Li Y, Li M, Li Y, Tian J, Bai X, Yang C, et al. Outbreaks of highly pathogenic avian influenza (H5N6) virus subclade 126.96.36.199h in swans, Xinjiang, Western China, 2020. Emerg Infect Dis. 2020;26:2956–60.
- Li S, Meng W, Liu D, Yang Q, Chen L, Dai Q, et al. Migratory whooper swans Cygnus cygnus transmit H5N1 virus between China and Mongolia: combination evidence from satellite tracking and phylogenetics analysis. Sci Rep. 2018;8:7049.
- Suttie A, Deng YM, Greenhill AR, Dussart P, Horwood PF, Karlsson EA. Inventory of molecular markers affecting biological characteristics of avian influenza A viruses. Virus Genes. 2019;55:739–68.
- Yang ZY, Wei CJ, Kong WP, Wu L, Xu L, Smith DF, et al. Immunization by avian H5 influenza hemagglutinin mutants with altered receptor binding specificity. Science. 2007;317:825–8.
- Chen H, Li Y, Li Z, Shi J, Shinya K, Deng G, et al. Properties and dissemination of H5N1 viruses isolated during an influenza outbreak in migratory waterfowl in western China. J Virol. 2006;80:5976–83.
- Li Y, Liu L, Zhang Y, Duan Z, Tian G, Zeng X, et al. New avian influenza virus (H5N1) in wild birds, Qinghai, China. Emerg Infect Dis. 2011;17:265–7.
- Bi Y, Chen J, Zhang Z, Li M, Cai T, Sharshov K, et al. Highly pathogenic avian influenza H5N1 Clade 188.8.131.52c virus in migratory birds, 2014-2015. Virol Sin. 2016;31:300–5.
- Global Consortium for H5N8 and Related Influenza Viruses. Role for migratory wild birds in the global spread of avian influenza H5N8. Science. 2016;354:213–7.
- World Health Organization. Avian influenza weekly update number 754. 2020 [cited 2020 Aug 19]. https://www.who.int/docs/default-source/wpro—documents/emergency/surveillance/avian-influenza/ai-20200814.pdf?sfvrsn=30d65594_68
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Original Publication Date: March 09, 2021
1These authors contributed equally to this article.