influenza a virus infections in birds account for important inputs into the evolutionary porcine - human complex of this prominent anthropozoonotic pathogen . among influenza
a viruses , which broadly exhibit 17 ha and 9 na antigenic subtypes , only three haemagglutinin ( ha ) subtypes ( h1 , h2 , and h3 ) and two neuraminidase ( na ) subtypes ( n1 and n2 ) have circulated widely in swine and human populations since the 20th century .
viruses from waterfowl reassorted with existing human and/or porcine influenza viruses to generate the 1957 , 1968 , and 2009 ( novel swine - origin influenza a ( h1n1 ) virus investigation team , 2009 ) pandemic influenza viruses and may expectably play a similar role in the creation of future pandemic viruses .
in addition , on multiple occasions , it has been evident that avian influenza viruses ( aivs ) , chiefly the subtypes h5n1 , h7n7 , and h9n2 , directly transmitted from birds to humans [ 3 , 4 ] .
avian - originated h1n1 , h3n2 , h5n1 , and h9n2 viruses have been recovered from pigs in asia , europe , and canada [ 57 ] .
pigs have been postulated , hence , to be the ultimate  mixing vessels  for mammalian influenza viruses and aivs and can play an important role in the genetic reassortment of influenza viruses .
therefore , detection and characterization of aivs , especially those subtypes having the potential to transmit to mammals , including pigs and humans , are significant .
avian h7n7 and h3n8 strains are contracted and circulated by horses , as well , and an equine h3n8 virus apparently was involved in the formation of the pandemic h3n2 virus , and perhaps the h3n8 pandemic virus of 1889 .  
the genome of influenza a viruses consists of eight different segments of single - stranded negative - sense rna .
most gene segments encode for one protein ; whereas the gene segments 2 ( polymerase basic1pb1 ) , 7 ( matrix  m ) , and 8 ( nonstructural  ns gene ) , each encodes one additional protein from alternatively spliced mrna . among the proteins , 9 are structural , while the rest two , namely pb1-f2 and ns1 , are nonstructural .
aquatic birds are the primary reservoir of type a influenza viruses and are classified into various antigenic subtypes , based on their two surface glycoproteins , the ha , and the na .
so far , seventeen ha ( h1h17 ) and nine na subtypes ( n1n9 ) have been identified from avian species , representing the entire pool of influenza a viruses known today .
although largely shaped by the ha , the virulence of influenza a viruses is polygenic in nature .
h5 and h7 influenza viruses with multiple basic amino acids nearby to the cleavage site of the ha glycoprotein exhibit a wide range of tissue tropism and lead to systemic disease in chickens with fatality .
it has been demonstrated that the amino acids at positions 627 and 701 of the polymerase basic 2 ( pb2 ) protein influence the outcome of infection in mice [ 12 , 13 ] .
several studies have reported that the ns1 protein is also associated with the virulence of influenza viruses [ 14 , 15 ] .
the glutamic acid at position 92 of ns1 of h5n1 influenza virus confers virulence and resistance to antiviral cytokines in pigs . in spite of those well - known
, meaningful functions of the ns gene concerning virulence and escape from host cytokine response , the degree of variation in the ns gene pool of aivs in their natural reservoirs , particularly in the northern pintail ( anas acuta ) ( thereafter referred to as pintail ( s ) ) ducks , a globally major influenza host wintering in japan , is poorly studied . in this paper
, we focused on genetic analysis of h1n1 , h1n2 , and h1n3 strains of aivs that we isolated from apparently healthy migratory pintails wintering in japan [ 17 , 18 ] . also , we analyzed the ns genes of h5 and h7 subtypes , in addition to the above - mentioned aivs .
moreover , polymerase acid ( pa ) , nucleoprotein ( np ) , m , and ns genes of h5n9 strain , an uncommon antigenic combination which we isolate for the first time in asia , were further looked into in the present study .
viruses used in this study ( table 1 ) were of low pathogenicity and isolated in embryonated chicken eggs ( ece ) from fecal materials of migratory , apparently healthy pintails wintering in japan [ 17 , 18 ] .
working stocks of viruses were prepared by 3rd passage in ece , and allantoic fluid was harvested at 3 days after inoculation ( dpi ) and stored at 80c .
total viral rna was extracted from infected allantoic fluid using the isogen - ls ( nippongene , tokyo , japan ) , in accordance with the manufacturer 's instructions .
reverse transcription was carried out with the uni12 primer ( 5-agc aaa agc aag g-3 ) and mmlv reverse transcriptase ( geneamp rna pcr kit , applied biosystems , tokyo , japan ) , followed by full length amplification of each gene segment , as described earlier .
the pcr product was purified using qiaquick gel extraction kit ( qiagen , valencia , ca ) , according to the manufacturer 's instructions .
the pcr product was sequenced by dideoxy chain terminating method , using dye terminator cycle sequencing fs ready reaction kit ( perkin - elmer applied biosystems , japan ) .
sequencing was carried out with the same primers used to amplify the gene and continued with subsequently designed sequence - specific primers .
nucleotide sequences were determined using an automated dna sequencer ( abi 310 dna sequencer , applied biosystems , foster city , ca ) , edited and assembled with genetyx - mac ( version 10.0 ; software development corp . , tokyo , japan ) .
multiple sequence alignments and processing were performed with the molecular evolutionary genetics analysis ( mega ) version 4.1.0 software , with an engine based on the clustalw algorithm .
the phylogenetic analyses were performed using neighbor joining tree inference analysis , with the 1000 bootstrap replications , to assign confidence levels to branches .
nucleotide sequences of entire open reading frame ( orf ) of each gene segment ( except pb1 gene of a / northern pintail / aomori/1130/08 h1n3 ( aomori/1130/08 h1n3 ) ) were used in the phylogenetic analysis .
the pb1 gene of aomori/1130/08 h1n3 was partially 1350 base pair ( 1 - 1350  bp ) sequenced and used in the phylogenetic analysis .
furthermore , the ha gene of a / northern pintail / akita/1364/08 h1n2 ( akita/1364/08 h1n2 ) was sequenced partially 765  bp ( 760 - 1524 ) and was not included in the phylogenetic analysis .
the nucleotide sequences generated in this study have been deposited in genbank and are available under accession number ab546149ab546193 .
as a part of longitudinal virological studies , we have isolated various subtypes of aivs from fecal materials of migratory , apparently healthy pintails wintering in tohoku district , japan [ 17 , 18 ] . of these , h1n1 , h1n2 , and h1n3 viruses were characterized genetically . for phylogenetic relationships of ns gene , the latter from viruses bearing h1 , h5 , and h7 ha was sequenced and compared with viruses from genbank . the sequenced ha genes ( n = 6 ) of h1 subtype with different na subtype combinations ( n1 , n2 , and n3 ) isolated from pintails during 2007 - 2008 were closely related to each other ( figure 1(a ) ; table 2 ) .
the nucleotide and amino acid sequence identities were found to range from 96.5 to 100% and 98.0 to 100% , respectively .
phylogenetically , the viruses were clustered with the viruses from eurasian countries , mostly with viruses from china , namely a / duck / hebei/843/05 h1n2 and a / wild duck / jx/12416/05 h1n1 ( > 97% nucleotide homology ) ( figure 1(a ) ) .
the ha genes of sequenced viruses were grouped into the h1.1.2 ( subtype h1 ) .
viruses of the present study had a sister group relationship with the japanese h1 viruses including a / pintail / shimane/324/98 h1n9 isolated earlier . north american strains , and swine , human seasonal and pandemic h1 strains integrated sharply in different branches .
it means that although bearing the same antigenic subtype ( h1n1 ) and obtained during 2007 - 2008 , our isolates were unrelated to the porcine - derived 2009 pandemic and seasonal flu strains .
like ha genes , the nucleotide homologies of the na genes of the isolates with each other were 96.899.8% and 99.799.9% for n1 ( n = 3 ) and n2 ( n = 3 ) , respectively , for each subtype . on the other hand ,
the amino acid sequence identities were found to range from 97.4 to 99.8% ( n1 ) and 99.499.8% ( n2 ) .
a duck - originated japanese strain obtained 2 years earlier , a / duck / tsukuba/67/05 h1n1 ( tsukuba/67/05 h1n1 ) was found most closely related to one of the isolates  a / northern pintail / aomori/422/07 h1n1 ( aomori/422/07 h1n1 ) ( > 98% homology at both nucleotide and amino acid sequences)of the present study , while two other viruses have maximum homology to a mongolian strain , a / duck / mongolia/116/02 h1n1 ( mongolia/116/02 h1n1 ) , isolated in 2002 ( > 98% homology at both nucleotide and amino acid sequences ) . for n2 nas , a chinese strain , namely a / goose / gui yang/3799/05 h5n2 (
gui yang/3799/05 h5n2 ) isolated from goose in 2005 was found rather closely related ( > 98% nucleotide and amino acid identity ) .
the n3 na gene of aomori/1130/08 h1n3 has > 99% nucleotide and amino acid homology to a duck strain , namely a / duck / niigata/514/06 h5n3 ( niigata/514/06 h5n3 ) isolated in 2006 in japan ( table 3 ) .
principally , the na genes of the sequenced viruses were clustered with viruses of eurasian origin ( figures 1(b ) and 1(c ) ) .
na gene of aomori/422/07 h1n1 strain branched separately from other strains isolated by us in 2008 ( figure 1(b ) ) , the n2 na genes of which were all integrated into a single branch represented by a h5n2 strain , gui yang/3799/05 , which was isolated in china in 2005 ( figure 1(c ) ) .
like h1 ha genes , the n1 na genes were distinctly separated from pandemic strains ( figure 1(b ) ) .
a remarkably diverse variation in the nucleotide sequence identities ( 50100% ) was found among the ns genes of the sequenced viruses ( table 4 ) .
when analyzed phylogenetically , they were distinctly branched into two branches , allele a and b , and clustered with eurasian origin viruses ( figure 1(d ) and table 4 ) .
nucleotide sequence identities of ns genes within allele a and within allele b were 97.7100% and 94.4100% , respectively .
however , the divergence between the two alleles was approximately 50% , regardless of their isolation time and geographical location .
the majority of the viruses of the present study were branched with allele a viruses represented with / by a / mallard / sweden/2724/06 h5n1 and a / duck / hong kong / y439/97 h9n2 ( hong kong / y439/97 h9n2 ) .
in contrast , a / northern pintail / aomori/372/08 h7n7 ( aomori/372/08 h7n7 ) , aomori/385/08 h5n3 , and a / northern pintail / miyagi/674/08 h7n7 ( miyagi/674/08 h7n7 ) viruses were clustered with allele b viruses represented with / by a / goose / guangdong/1/96 h5n1 ( guangdong/1/96 h5n1 ) . as for allele a , two pairs 
h5n2 and akita/1355/07 h5n2 , alongside with akita/1353/08 h1n2 and akita/1364/08 h1n2 viruses  were found 100% homologous , while in allele b aomori/372/08 h7n7 and miyagi/674/08 h7n7 viruses were found to be entirely identical .
significantly , the ns1 genes of influenza viruses in general have been divided into two alleles : a and b .
regardless of some exceptions , all influenza viruses circulating in mammalian species and many viruses from avian species comprise the allele a , while allele b is found within only aivs .
the ns1 gene of the majority of highly pathogenic avian influenza ( hpai ) h5n1 viruses isolated from humans since 1997 is affiliated with allele a. in contrast , the ns1 gene of the guangdong/1/96 virus , which was the source of the initial hpai h5n1/1997 ha gene , belonged to allele b .
the ns1 genes of viruses of the present study  all lpai  belong either to allele a or allele b. curiously , it was pointed out that some strains of the hpai h5n1 virus were preserved genetically unchanged from 1997 up to 2005 . in that connection , perhaps , it is of note that the ns gene of some viruses , for example aomori/372/08 h7n7 and miyagi/674/08 h7n7 of this study have only < 3% nucleotide disparity from that of a russian strain , namely a / duck / chabarovsk/1610/72 h3n8 ( chabarovsk/1610/72 h3n8 ) , isolated in 1972 .
the ns gene of one of the viruses we had isolated from pintail ducks , namely akita/714/06 h5n2 , has 98.3% nucleotide homology to the same russian isolate chabarovsk/1610/72 h3n8 .
it means that the ongoing mutation rate during about 35 years was only about 1.7% , which is markedly less than the expected one .
this may imply that throughout about a third of that 35 years period the ns gene has been somehow conserved , considering that the estimated ns gene evolutionary rates for avian , classic swine , and equine lineages are 0.841.27 nucleotide changes per site per year , and even higher , within both wild and domestic avian host species .
basically , the two reasons thought to possibly account for unexplained genetic conservation in influenza a viruses are cross contamination , or interference with vaccine strains .
the strain chabarovsk/1610/72 or related strains are not held in our laboratory , and interference with a vaccine strain seems unlikely , in that case .
these , together with the location of chabarovsk being close to the regular migration route of pintails between japan and siberia , have to further be looked into .  
prevalence of allele a and b viruses in their natural hosts has been reported to vary spatially , as well .
generally , allele b viruses were reported to be less common in their natural hosts than allele a. out of 11 ns genes sequenced in this study , 72.7% ( 8/11 ) were classified as allele a , and 27.3% ( 3/11 ) as allele b. in asia , the prevalence of allele b viruses in all avian species has been reported to be only 15% , which is markedly lower than found in this study .
however , the prevalence rate of allele b viruses in north american free flying birds has been documented to be 30% , similar to that observed in pintails in our study , but higher than found in northern european mallards ( 13% ; 6 out of 45 ) .
most pintails which winter in japan originate from eastern russia [ 33 , 34 ] . besides
, pintails marked in north america have been recovered during winter in japan and vice versa [ 35 , 36 ] .
moreover , some pintails migrate from north america to eastern russia , where they could come into contact with birds that migrate from six continents , including asian wintering sites .
furthermore , from the recent satellite telemetry data , provided by the united states geological survey ( usgs ) and the us fish and wildlife service , it is evident that pintails marked during winter in japan move to alaska through russia and return to japan , following the same route ( alaska science center , movements of northern pintail ducks and whooper swans marked with satellite transmitters in japan http://alaska.usgs.gov/science/biology/avian_influenza/pintail_movements_virus.php .
thus , inter- and intra continental exchange of genes and genomes of aiv could occur through congregation of north american and asian migrants at shared summer habitats in eastern russia , or by means of pintails that migrate between north american nesting grounds and japanese wintering grounds ( assessment of virus movement across continents : using northern pintails ( anas acuta ) as a test .
also the number of samples may possibly have effect on the results found in this study .
the np genes of the sequenced viruses have 90.499.9% nucleotide homologies among each other , regardless of their temporal and spatial differences in isolation ( table 5 ) .
broadly , viruses isolated from eastern hemisphere have high homologies to the np genes of the viruses of this study ( 97.2 to 99.5% nucleotide identity ) .
it is noteworthy that the np gene of the majority of viruses ( 75% of 8 viruses ) investigated in this study had a high homology to a porcine strain , namely a / swine / korea / c12/08 h5n2 ( korea / c12/08 h5n2 ) isolated in 2008 , suggesting that the np genes of these viruses and the korea isolate might have a common ancestral origin .
similar to np gene of h1 viruses , the ha genes of some h5n2 viruses isolated by us during 2006 - 2007 were found to be closely related to this korean virus [ 19 , 26 ] . in conjunction ,
biogeographically and phylogenetically , other related strains , namely a / garganey / san jiang/160/06 h5n2 , a / baikal teal / hongze/14/05 h11n9 , and a / duck / hong kong / mps180/03 h4n6 originated from china , and one strain , a / mallard / hokkaido/24/09 h5n1 , from japan . for np
gene viruses from both eurasian countries and north america were clustered with the viruses of the present study ( figure 1(e ) ) .
the nucleotide sequence disparity between viruses of this study and north american viruses clustered together ranged from 1.4 to 9.3% .
especially , and not by chance , in all likelihood , aomori/422/07 h1n1 has 98.6% nucleotide identity to the strain a / pintail / alaska/1/07 h3n8 , meaning isolated from the same host in the same year . in that case ,
viruses of north american lineage integrated distinctly from those of eurasian lineage , suggesting , in light of the mentioned high np gene homology , intercontinental gene exchange through reassortment , rather than whole genome intercontinental conveyance .
the ha amino acid sequences deduced from nucleotide sequences analysis revealed that all the viruses contain seven potential n - linked glycosylation sites ( 5 in ha1 and 2 in ha2 ) throughout the ha molecule . except for aomori/422/07 h1n1 , the amino acid motif of the ha0 cleavage site of all the sequenced h1 viruses was psiqsr*glf ( * cleavage point ) , which is common in all avian , porcine , and human viruses .
h1n1 contains a rare amino acid motif , psvqsr*glf . among the h1 avian strains isolated globally and sequences reported in genbank during 19802009 , only one strain , namely a / mallard / new york/170/82 h1n2 has identical ( psvqsr*glf ) amino acids .
however , aomori/422/07 h1n1 solely clustered with the eurasian viruses ( figure 1(a ) ) , implying , ostensibly that it has no relation with the mallard - originated new york strain and evolves individually .
still , the extreme rareness of the mentioned motif might possibly be supportive of genetic recombination event that perhaps took place within collocated ducks from north america and asia .
the ha molecules of all of the sequenced h1 viruses contain residues gln ( q ) and gly ( g ) at positions 240 and 242 ( h1 numbering ) , respectively , which indicate the avian receptor specificity .
on the other hand , the amino acid sequence analysis of nas revealed that all the viruses contain full length na protein with seven potential n - linked glycosylation sites in n1 and n2 known to be conserved in wild ducks . in all na molecules ,
a residue his ( h ) was found at position 274 ( n2 numbering ) , thereby indicating sensitivity to oseltamivir or zamamivir .
besides , typical catalytic sites , framework sites as well as other specific regions were found completely conserved as described previously .
residue position 198 in n3 na contains asn ( n ) instead of asp ( d ) and is also shown to be conserved .
the n1 and n2 na of the sequenced viruses possessed e119 and r292 amino acid residues , indicating that the commonly mutated residues in na inhibitor - resistant viruses were not present .
deletion of several amino acids in the ns1 gene has been observed more frequently in aivs in recent years , a feature of possible adaptation of these viruses to poultry [ 24 , 43 ] .
the viruses sequenced in our study contained full length ns genes , indicating basically wild bird strains .
nevertheless , all isolates possessed residue ala ( a ) position 149 , which is important for replication of viruses in chickens .
also , residue asp ( d ) instead of glu ( e ) was found at position 92 of the ns1 , which is reported to be involved in modulation of cytokine response , and has been associated with the high virulence exhibited by hpai h5n1 viruses in pigs .
all the sequenced viruses contain a pdz ( postsynaptic density , psd-95 ; discs large , dlg ; zonula occludens-1 , zo-1 ) domain ligand at the c terminus of ns1 ( esev - cooh ) , which plays an important role in many key signaling pathways of viral replication .
when compared to pandemic and seasonal influenza viruses , the ha genes ' nucleotide identity of viruses of the present study was found to range from 67.1 to 76.4% , while amino acid sequence homology was found 81.892.1% ( table 6 ) . in difference , for na gene , a somewhat higher nucleotide and amino acid identity was found ( 79.386.5% nucleotide and 84.992.5% amino acid sequence identities ) .
not only the ha and na genes but also other internal genes of pandemic and seasonal flu strains were clustered distinctly from those of our isolates , suggesting no relation between these viruses .
basically , genomic analyses of the last four pandemic strains showed that the genes contributed by avian strains are those that encode for the polymerases and surface antigens , as follows:1918pb1 and pa ; 1957pb1 , ha , and na ; 1968pb1 and ha ; 2009pb2 and pa .
this means that among the presently prevailing pandemic and seasonal strains , only the seasonal h3n2 has past affinity to avian ha , when it originally formed as a then pandemic strain in 1968 , thereafter considerably drifting genetically for already 43 years .
independent drift probably took place during that period of time within the precursor avian h3 ha gene in bird populations .
in conclusion , our genetic analysis suggests that the sequenced viruses were to an appreciable degree characteristic of the pintail populations we sampled , which typically winter in japan .
except in one strain , the ha0 cleavage site of h1 was found as usually found in all avian , porcine , and human viruses .
the residues that compose the catalytic and framework sites of the na enzyme were completely conserved in the studied viruses .
the np gene of the majority of the strains sequenced in this study ( 5/8 ) was related to that of the porcine strain korea / c12/08 h5n2 .
thus , it may be presumed that our isolates evolved through reassortment process during the cocirculation of these strains .
our findings clearly demonstrate that two distinct gene pools , corresponding to both ns allele a and b , were present within the pintail populations wintering in japan .
it is noteworthy that some strains contain ns gene highly related to a duck isolate obtained in russian in 1972 , about 35 years prior to our isolate , indicating significant gene conservation .
it is also concluded that although bearing the same antigenic subtype ( h1n1 ) and obtained during 2007 - 2008 , our isolates were unrelated to 2009 pandemic and seasonal flu strains .
 the pandemic influenza virus strains of 1918 ( h1n1 ) , 1957 ( h2n2 ) , 1968 ( h3n2 ) , and 2009 ( h1n1 ) have genes related to avian influenza viruses ( aivs ) . 
 the nonstructural ( ns ) gene of aivs plays a significant role in host - viral interaction . 
 however , little is known about the degree of diversity of this gene in northern pintail ( anas acuta ) ducks wintering in japan . 
 this study describes characteristics of pintail - originated h1n1 , h1n2 , h1n3 , h5n2 , h5n3 , h5n9 , and h7n7 viruses . 
 most of the viruses were revealed to be avian strains and not related to pandemic and seasonal flu strains . 
 nevertheless , the np genes of 62.5% ( 5/8 ) viruses were found closely related to a a / swine / korea / c12/08 , indicating exchange of genetic material and ongoing mammalian - linked evolution of aivs . besides 
 , all the viruses , except aomori/422/07 h1n1 , contain psiqsrglf motif usually found in avian , porcine , and human h1 strains . 
 the aomori/422/07 h1n1 has a psvqsrglf motif identical to a north american strain . 
 this findings linked to an important intercontinental , asian - american biogeographical interface . 
 phylogenetically all the viruses were clustered in eurasian lineage . 
 cocirculation of allele a and b ( ns gene ) viruses was evident in the study implying the existence of a wide reservoir of influenza a viruses in pintail wintering in japan . 