on october 28 , 2016 , we isolated an h5n6 subtype hpaiv from 1 of 391 fecal samples collected from wild birds in gokgyo - cheon , south korea ( 364512.3n , 1270712.7e ) . gokgyo - cheon is a wild bird habitat for wintering of migratory waterfowl , including mallard ( anas platyrhynchos ) , spot - billed duck ( anas poecilorhyncha ) , mandarin duck ( aix galericulata ) , and common teal ( anas crecca ) . the species of the positive fecal sample was identified as mandarin duck on the basis of dna barcoding technique as described ( 9 ) . full - length genome sequencing and phylogenetic analysis were conducted to trace the origin of a / mandarin_duck / korea / k16 - 187 - 3/2016(h5n6 ) virus , hereafter referred to as md / kr/2016 . we entered genome sequences in the gisaid ( global initiative on sharing all influenza data ) epiflu database ( https://www.gisaid.org ) under accession nos . the isolate was identified as an hpaiv on the basis of multiple basic amino acids at the ha proteolytic cleavage site ( plrerrrkr / g ) . gisaid blast ( https://blast.ncbi.nlm.nih.gov/blast.cgi ) searches indicated that h5 and n6 genes had high nucleotide identity in ha ( 99.17% ) and na ( 99.24% ) with a / great_egret / hong_kong/00032/2016 ( h5n6 ) ( table 1 ) . internal gene segments , except the polymerase basic 1 ( pb1 ) gene , had high nucleotide identity with other h5n6 subtypes isolated in guangdong and jiangxi , china ( pb2 , 99.09% ; polymerase acidic , 98.96% ; nucleoprotein , 99.16% ; matrix , 98.98% ; and nonstructural protein [ ns ] , 98.31% ) . however , the pb1 gene had high nucleotide identity ( 97.01% ) with h4 low pathogenicity avian influenza viruses ( lpaivs ) . * gisaid , global initiative on sharing all influenza data ( http://www.gisaid.org ) ; ha , hemagglutinin ; mp , matrix ; na , neuraminidase ; np , nucleoprotein ; ns , nonstructural protein ; pa , polymerase acidic ; pb1 , polymerase basic 1 ; pb2 , polymerase basic 2 . in previous phylogenetic analyses , the ha gene of clade 2.3.4.4 viruses was divided into 4 distinct subgroups ( technical appendix 1 figure 1 ) ( 10 ) . group intercontinental a ( ica ) contains h5n8 subtype virus and its reassortant viruses identified in china , south korea , japan , taiwan , canada , the united states , and countries in europe during 20132016 . group b contains h5n8 subtype viruses identified in china and south korea during 20132014 , and in russia in late 2016 . group c contains h5n1 and h5n6 subtype viruses identified in china , vietnam , laos , and hong kong , including isolates from humans in guangdong , yunnan , and hunnan provinces , china . group d contains h5n6 subtype viruses identified in china and vietnam , including an isolate from a human in sichuan province , china . the ha gene of md / kr/2016 virus belonged to group c and clustered with h5n6 subtype viruses isolated from humans , cats , and the environment in guangdong during 20142015 and a migratory aquatic bird in hong kong during january 2016 ( a / great_egret / hong_kong/00032/2016 [ h5n6 ] ) ( technical appendix 1 figure 1 ) . a previous study reported that a / environment / guangdong / gz693/2015 ( h5n6 ) , hereafter referred to as gz693/2015(h5n6 ) , is a 7:1 gene reassortant virus between h5n6 hpaiv and lpaivs found in southern china ( 7 ) . md / kr/2016 clustered with gz693/2015(h5n6 ) virus for all 8 genes ( technical appendix 1 figure 2 ) . in particular , the ha , na , pb2 , polymerase acidic , nucleoprotein , matrix , and ns protein genes clustered with gz693/2015(h5n6 ) and other clade 2.3.4.4 group c h5n6 viruses . the pb1 gene clustered with gz693/2015(h5n6 ) ( nucleotide identity 92.79% ) and lpaivs , such as h3n2 and h4n2 subtype viruses , from southern china . phylogenetic analysis and blast search collectively suggest that md / kr/2016 virus had an identical genotype to gz693/2015(h5n6 ) . most of clade 2.3.4.4 group c viruses have leucine or serine at position 129 ( h5 numbering ) in ha protein . however , md / kr/2016 had a single amino acid deletion at position 129 ( table 2 ) , as did a / great_egret / hong_kong/00032/2016 ( h5n6 ) . this deletion at position 129 and phylogenetic network analysis suggested that md / kr/2016 is closely related to h5n6 subtypes isolated from wild birds in hong kong in 2016 ( technical appendix 1 figure 3 ) . md / kr/2016 contained the mutation associated with increased virulence in mammals and mammalian transmissibility ( s123p and t156a mutations in the ha gene ; p42s and d92e mutation , and elongated c - terminus with pdz binding motif in ns gene ) . however , this isolate lacked the q226l and g228s mutations in ha , which have been associated with increased binding to human - type receptor ( -2,6linked sialic acid ) and lacked q591k , e627k and d701n mutations in pb2 , which have been associated with enhanced pathogenicity and adaptation to mammalian hosts ( 11 ) . all of the 9 h5n6 subtype human isolates of group c lacked the q226l and g228s mutations in ha , but 5 viruses contained the e627k mutation in pb2 ( table 2 ) , suggesting that some purported mammalian adaptation amino acid substitutions were not necessary for sporadic virus infection of h5n6 hpaiv in humans . * del , deletion ; ha , hemagglutinin ; na , neuraminidase ; ns , nonstructural protein ; pb2 , polymerase basic 2 ; pdz , pdz binding motif . s123p , s133a , t156a , q222l , and g224s mutations in ha have been associated with increased binding to human - like receptor ( -26 sialic acid ) . q591k , e627k , and d701n mutations have been associated with improved replication of avian influenza virus in mammals . 42s , 8084 deletion , and esev pdz binding motif have been associated with increased virulence in mice . wild aquatic birds have been suspected to play a key role in dissemination of hpaivs to various regions , as seen with clade 2.2 h5n1 hpaiv in 2005 , clade 2.3.2.1 h5n1 hpaiv in 2009 , and clade 2.3.4.4 h5n8 hpaiv in 2014 ( 4,12 ) . some populations of mandarin ducks are year - round residents in south korea and japan ; others populations migrate between russia and eastern asia ( 13 ) . in south korea , hpaiv was detected from mandarin duck samples in 2010 ( h5n1 ) and 2014 ( h5n8 ) ( 14,15 ) and again in 2016 during this study , suggesting that mandarin ducks are a major host species for clade 2.3.4.4 h5 hpaiv and can disseminate the virus throughout south korea and into other countries . detection of the h5n6 hpaiv clade 2.3.4.4 in a migratory bird species in south korea ; reports of h5n6 outbreaks in poultry from china , laos , and vietnam ; and diagnosis of lethal human cases of highly homologous h5n6 viruses in china raise a concern over the potential for broad geographic dissemination of zoonotic h5n6 hpaiv by wild birds outside eastern asia . additional information on reassortant clade 2.3.4.4 avian influenza a(h5n6 ) virus in a wild mandarin duck , south korea , 2016 . influenza virus isolates used in the study of reassortant clade 2.3.4.4 avian influenza a(h5n6 ) virus in a wild mandarin duck , south korea , 2016 .
a reassortant clade 2.3.4.4 avian influenza a(h5n6 ) virus was isolated from a fecal sample of a mandarin duck ( aix galericulata ) in south korea during october 2016 . 
 this virus was genetically similar to h5n6 subtype virus isolates from china , vietnam , laos , and hong kong , including human isolates .