New Reassortant Clade 220.127.116.11b Avian Influenza A(H5N6) Virus in Wild Birds, South Korea, 2017-18.
During December 2017-January 2018 in South Korea, we isolated 6 H5N6 HPAIVs from 231 fecal samples of wild birds collected from the banks of the Cheongmi-cheon River (37[degrees]06'56.9"N, 127[degrees]25'18.3"E) and 34 from 222 fecal samples collected from the banks of the Gokgyo-cheon River (36[degrees]45'12.3'N, 127[degrees]07'12.7'E) (online Technical Appendix 1, https://wwwnc.cdc.gov/EID/article/24/10/180461-Techapp1.pdf). These wild bird habitats are wintering sites of migratory waterfowl, including mallard (Anas platyrhynchos), spot-billed duck (Anas poecilorhyncha), Mandarin duck (Aix galericulata), and common teal (Anas crecca). The Gokgyo-cheon River is a major habitat site for Mandarin ducks, and numerous HPAIVs were detected in fecal samples from Mandarin ducks during 2011, 2015, and 2016 (9). We identified avian influenza virus-positive fecal samples from 38 Mandarin ducks and 2 mallards, based on DNA barcoding technique (10). We performed full-length genome sequencing and comparative phylogenetic analysis on 19 of the 40 isolates (online Technical Appendix 1; online Technical Appendix 2, https://wwwnc.cdc.gov/EID/ article/24/10/18-0461-Techapp2.xlsx).
All H5N6 isolates shared high nucleotide sequence identities in all 8 gene segments (99.58%-100%) and were identified as HPAIVs based on the presence of multiple basic amino acids at the HA proteolytic cleavage site (PLREKRRKR/G). Searches of the GISAID (https:// www.gisaid.org) and BLAST (https://blast.ncbi.nlm.nih. gov/Blast.cgi) databases indicated that all 8 genomes had the highest nucleotide identity with A/Great_Blackbacked_Gull/Netherlands/1/2017 (Netherlands/1) clade 18.104.22.168 subgroup B H5N6 strain from December 2017 (99.17%-99.79%), rather than subgroup B H5N6 viruses from Japan and Taiwan collected during December 2017 (97.18%-99.27%).
In phylogenetic analysis, we identified 2 genotypes of subgroup B H5N6 viruses (online Technical Appendix 1 Figures 1, 2): genotypes B.N6.1 and B.N6.2. The genotype B.N6.1 viruses were identified from South Korea, Japan, Taiwan, Greece, and the Netherlands (Netherlands/1 strain), and the genotype B.N6.2 viruses were detected from England, Germany, and the Netherlands. For genotype B.N6.1, all genes except NA clustered with H5N8 HPAIV of previously reported genotypes, H5N8-NL cluster I in the Netherlands (6), Ger-11-16 in Germany (5), and Duck/Poland/82a/16-like in Italy (4). The NA gene clustered with LPAIVs circulating in wild birds in Eurasia and separated into 2 clusters, suggesting the potential for >2 independent reassortment events between H5N8 virus and unidentified wild bird origin N6 segments. Consistent clustering of South Korea isolates with the Netherlands/1 strain in maximum-likelihood (ML) phylogenies for each gene supported by high ML bootstrap values (86-100) suggests their close relationship. The genotype B.N6.2 viruses had different polymerase basic 2 (PB2) and polymerase acidic (PA) genes from genotype B.N6.1. The polymerase basic 2 gene probably originated from other LPAIVs, and a polymerase acidic gene originated from H5N8-NL cluster II genotype (6,7). The phylogenetic network and ML analysis suggest that H5N6 viruses have evolved from subgroup B H5N8 viruses into 3 independent pathways, detected in Greece, Europe/South Korea, and Japan/Taiwan (online Technical Appendix 1, Figures 2,3).
The time of most recent common ancestry (tMRCA) for each gene of genotype B.N6.1 H5N6 viruses isolated during winter 2017-18 in Eurasia, except for the NA gene, ranged from January 2016 to October 2016, suggesting that genotype B.N6.1 viruses diverged during the previous year. The tMRCA of the NA gene was September 2015 (95% highest posterior density August 2014-August 2016). The tMRCA of the NA gene has wide 95% highest posterior density range because only a few recent N6 genes of LPAIVs were available in databases for analysis. The tMRCA for each gene of H5N6 HPAIVs identified in South Korea ranged from July through September 2017, suggesting that ancestors of these viruses emerged among wild birds during or after summer 2017, possibly at the breeding and molting sites in the Palearctic region (Table; online Technical Appendix Figure 4). Detection of H5N6 HPAIV from fecal samples of wild birds in South Korea during the 2017-18 wintering season and our phylogenetic analysis suggest that the viruses had moved through wild birds during the fall migration season.
On the basis of our data and migratory pattern of birds, we estimate that H5N6 viruses possibly descended from H5N8 viruses circulating during 2016-17, reaching breeding regions of wild birds during early 2017, followed by dissemination into Europe and East Asia during the fall migration. Enhanced surveillance in wild birds is needed for early detection of new introductions of HPAIV and to trace the transmission route of HPAIV.
This work was funded by Konkuk University in 2015.
Mr. Kwon is a PhD candidate at Konkuk University, Seoul, South Korea. His primary research interest is the epidemiology of HPAIVs in wild birds. Ms. Jeong is a PhD candidate at Konkuk University, Seoul. Her primary research interest is the epidemiology of viruses in wild birds.
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Address for correspondence: Chang-Seon Song, Avian Disease Laboratory, College of Veterinary Medicine, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul, 143-701, South Korea; email: email@example.com
Jung-Hoon Kwon,  Sol Jeong,  Dong-Hun Lee, David E. Swayne, Yu-jin Kim, Sun-hak Lee, Jin-Yong Noh, Tseren-Ochir Erdene-Ochir, Jei-Hyun Jeong, Chang-Seon Song
Author affiliations: Konkuk University, Seoul, South Korea (J.-H. Kwon, S. Jeong, Y.-J. Kim, S.-H. Lee, J.-Y. Noh, T.-O. Erdene-Ochir, J.-H. Jeong, C.-S. Song); US Department of Agriculture, Athens, Georgia, USA (D.-H. Lee, D.E. Swayne)
 These authors contributed equally to this article.
Table. Time to most recent common ancestor for each gene segment of genotype B.N6.1 influenza A(H5N6) viruses isolated in South Korea, December 2017-January 2018 * South Korea isolates, node 1 Gene Mean 95% HPD PB2 2017 Sep 2017 Jul-Oct PB1 2017 Sep 2017 Jul-Oct PA 2017 Sep 2017 Aug-Oct HA 2017 Sep 2017 Jul-Oct NP 2017 Jul 2017 Apr-Sep NA 2017 Jul 2017 Mar-Sep M 2017 Aug 2017 May-Oct NS 2017 Jul 2017 Apr-Oct South Korea and Europe isolates, node 2 Gene Mean 95% HPD PB2 2017 May 2016 Dec-2017 Sep PB1 2017 May 2017 Feb-Aug PA 2017 Jul 2017 Apr-Sep HA 2017 May 2017 Feb-Jul NP 2017 Mar 2016 Nov-2017 Jun NA 2017 Feb 2016 Jul-2017 Jul M 2017 May 2017 Jan-Aug NS 2017 Mar 2016 Nov-2017 Jun South Korea, Europe, Japan, Taiwan, and Greece isolates, node 3 Gene Mean 95% HPD PB2 2016 Mar 2015 Oct-2016 Jun PB1 2016 Jul 2016 May-Aug PA 2016 Oct 2016 Jul-Dec HA 2016 Mar 2015 Dec-2016 May NP 2016 Jan 2015 Jul-2016 May NA 2015 Sep 2014 Aug-2016 Aug M 2016 Mar 2016 Jan-May NS 2016 Feb 2015 Oct-2016 Jun * Nodes of the temporally structured maximum clade credibility phylogenetic tree (online Technical Appendix Figure 4, https://wwwnc.cdc.gov/EID/article/24/10/18-0461-Techapp1.pdf). HA, hemagglutinin; HPD, highest posterior density; M, matrix; NA, neuraminidase; NP, nucleoprotein; NS, nonstructural; PA, polymerase acidic; PB, polymerase basic.
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|Title Annotation:||RESEARCH LETTERS|
|Author:||Kwon, Jung-Hoon; Jeong, Sol; Lee, Dong-Hun; Swayne, David E.; Kim, Yu-jin; Lee, Sun-hak; Noh, Jin-Yo|
|Publication:||Emerging Infectious Diseases|
|Date:||Oct 1, 2018|
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