Establishment of an H6N2 Influenza Virus Lineage inDomestic Ducks in Southern China�†
K. Huang,1,2‡ J. Bahl,1,2‡§ X. H. Fan,2 D. Vijaykrishna,1,2§ C. L. Cheung,1,2 R. J. Webby,3R. G. Webster,3 H. Chen,1,2 Gavin J. D. Smith,1,2§ J. S. M. Peiris,2 and Y. Guan1,2*
International Institute of Infection and Immunity, Shantou University Medical College, Shantou 515031, Guangdong, China1;State Key Laboratory of Emerging Infectious Diseases and Department of Microbiology, Li Ka Shing Faculty of Medicine,
The University of Hong Kong, 21 Sassoon Road, Pok Fu Lam, Hong Kong SAR, China2; and Division ofVirology, Department of Infectious Diseases, St. Jude Children’s Research Hospital,
332 North Lauderdale Street, Memphis, Tennessee 38105-27943
Received 4 February 2010/Accepted 30 April 2010
Multiple reassortment events between different subtypes of endemic avian influenza viruses have increasedthe genomic diversity of influenza viruses circulating in poultry in southern China. Gene exchange from thenatural gene pool to poultry has contributed to this increase in genetic diversity. However, the role of domesticducks as an interface between the natural gene pool and terrestrial poultry in the influenza virus ecosystem hasnot been fully characterized. Here we phylogenetically and antigenically analyzed 170 H6 viruses isolated fromdomestic ducks from 2000 to 2005 in southern China, which contains the largest population of domestic ducksin the world. Three distinct hemagglutinin lineages were identified. Group I contained the majority of isolateswith a single internal gene complex and was endemic in domestic ducks in Guangdong from the late 1990sonward. Group II was derived from reassortment events in which the surface genes of group I viruses werereplaced with novel H6 and N2 genes. Group III represented H6 viruses that undergo frequent reassortmentwith multiple virus subtypes from the natural gene pool. Surprisingly, H6 viruses endemic in domestic ducksand terrestrial poultry seldom reassort, but gene exchanges between viruses from domestic ducks and migra-tory ducks occurred throughout the surveillance period. These findings suggest that domestic ducks insouthern China mediate the interaction of viruses between different gene pools and facilitate the generation ofnovel influenza virus variants circulating in poultry.
Southern China has long been considered a hypotheticalepicenter for facilitating the emergence of pandemic influenzaviruses (13, 14). Systematic influenza surveillance has shownthat the ecology of influenza virus in southern China has be-come increasingly complex in the last 2 decades (18). Multiplesubtypes of influenza viruses have emerged, causing repeatedpoultry outbreaks, human infection, and dissemination tomany countries in Eurasia that has given rise to a persistentpandemic threat. Epidemiological and genetic analyses re-vealed that three subtypes of influenza viruses, including highlypathogenic avian influenza (HPAI) H5N1 virus and low-patho-genic H9N2 and H6N1 subtypes, cocirculated within a varietyof poultry hosts in this region. These viruses have also under-gone extensive reassortment either between different endemicvirus subtypes or with viruses from the influenza virus naturalreservoir (3, 5, 23).
In the last decade, major research efforts have focused onunderstanding the emergence and development of the aboveviruses (1, 3, 8, 17, 22, 23). As approximately 60% of the world
population of domestic ducks (approximately 600 millionbirds) are farmed in southern China (7), this represents thelargest aquatic bird reservoir for influenza viruses in the world.Yet many subtypes of influenza viruses isolated from this pop-ulation have not been systematically studied. Thus, the mech-anisms for virus emergence and interaction in this populationremain to be determined.
Aquatic birds are known to host a high diversity of avianinfluenza A viruses (AIV) (6, 19). All domestic ducks, exceptMuscovy ducks, are derived from mallards, and these hostsrepresent an interface between the aquatic bird natural AIVreservoir and the domestic poultry population (1, 17). Regulartransmission of AIV between the aquatic reservoir and thedomestic birds in China has resulted in increased genetic di-versity of all sublineages known to circulate in domestic birds(5, 17). However, the movement and interaction of influenzavirus between migratory and domestic ducks have not beenfully explored. As H6 subtype viruses are among the mostfrequently encountered in our surveillance, in both wild anddomestic aquatic birds, we have analyzed the evolutionarygenomics of this subtype to elucidate the role that domesticducks play in the influenza virus ecology of this region.
Genetic characterization of 170 H6 subtype representativeviruses isolated from 2000 to 2005 in southern China revealedgenetically distinct H6N2 hemagglutinin (HA) lineages in do-mestic ducks. Our results show that these viruses reassortmuch less frequently than do other influenza A viruses, i.e.,they maintain a stable genome constellation through time andspace and have been endemic in domestic ducks in Guangdong
* Corresponding author. Mailing address: State Key Laboratory ofEmerging Infectious Diseases, Li Ka Shing Faculty of Medicine, TheUniversity of Hong Kong, 21 Sassoon Road, Pok Fu Lam, Hong KongSAR, China. Phone: 852 2819-9830. Fax: 852 2817-1958. E-mail: [email protected].
† Supplemental material for this article may be found at http://jvi.asm.org/.
‡ These authors contributed equally to this work.§ Present address: Duke-NUS Graduate Medical School, Singapore.� Published ahead of print on 12 May 2010.
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since the late 1990s. Our findings also showed that the replace-ment of the surface genes occurred and resulted in the estab-lishment of a new H6N2 virus lineage that largely maintainedthe same internal gene complex. The epidemiological and eco-logical behavior of H6 viruses in domestic ducks highlightsproperties of the influenza virus ecosystem in southern Chinathat have not been observed before. In particular, our resultsshow for the first time that a unique nonreassortant lineage hasbecome endemic in domestic ducks in the region. Our findingsalso demonstrate that gene exchange between the domesticduck and migratory duck gene pools occurs frequently but thatthere is only limited gene flow between H6 viruses endemic interrestrial hosts and domestic ducks. Furthermore, we foundthat the sources for some gene segments currently circulatingin the dominant H5N1 variants were likely H6 viruses intro-duced from migratory ducks. The findings of our study suggestthat domestic ducks in southern China have a dual role in theinfluenza virus ecosystem—as a component of the natural res-ervoir along with wild aquatic birds but also as a major poultrytype that harbors endemic virus lineages.
MATERIALS AND METHODS
Surveillance and virus isolation. Avian influenza virus surveillance was con-ducted in live-poultry markets in six provinces of southern China from July 2000to December 2005. Cloacal and tracheal swabs were taken from apparentlyhealthy poultry if possible; otherwise, fecal swabs were collected. Sampling wasconducted weekly. The surveillance program was initiated in Guangdong prov-ince (Shantou) from July 2000 and then extended to Hunan and Jiangxi in 2002,followed by Guangxi and Fujian in 2004 and Guizhou in 2005. In Jiangxi,sampling was carried out on sentinel duck farms located on Poyang Lake andfrom 2004 on was extended to capture migratory ducks at the lake (2). In allother sites, poultry types sampled include chicken, goose, duck, and severalminor poultry species. Occasionally mallard-like birds that could be distinguishedfrom domestic ducks at the live-poultry markets were labeled as wild duck toindicate this distinction. It is unclear whether these mallard-like birds were bredin captivity or were captured migratory ducks. Virus isolation and subtype iden-tification were conducted using standard protocols as previously described (11).One hundred seventy H6 viruses were randomly and proportionally selectedfrom the positive candidates based on surveillance region, sampling occasion,and isolation rate as representatives for further analyses.
Antigenic analysis. Antigenic characteristics of the representative H6 influ-enza viruses were compared by hemagglutination inhibition (HI) assay withpostinfection chicken antisera raised against chicken/Hong Kong/17/1977 (Ck/HK/17/77, H6N1), laughing gull/Delaware/4/1994 (LG/DE/4/94, H6N8), teal/Hong Kong/W312/1997 (Te/HK/W312/97, H6N1), quail/Hong Kong/1721-20/1999 (Qa/HK/1721-20/99, H6N1), quail/Hong Kong/YU1564/2000 (Qa/HK/YU1564/00, H6N?), and duck/Jiangxi/227/2003 (Dk/JX/227/03, H6N1) that weregenerated in our laboratory.
Phylogenetic and molecular analyses. RNA extraction, cDNA synthesis, andPCR were carried out as previously described (21). Sequencing was performedfor representative viruses by using a BigDye Terminator v3.1 cycle sequencing kiton an ABI 3730 genetic analyzer (Applied Biosystems) according to the manu-facturer’s instructions. DNA sequences were compiled and edited by usingLasergene 8.0 (DNAstar, Madison, WI).
Multiple sequence alignment of representative H6 viruses was performedtogether with alignment of sequences downloaded from GenBank using BioEdit7.0 (9). Full-length gene sequences for each segment from the first start codonwere used for phylogenetic analyses. Phylogenetic analysis was based on thefollowing nucleotides: PB2, 1 to 2280; PB1, 1 to 2274; PA, 1 to 2151; HA, 1 to1704; NP, 1 to 1494; NA, 1 to 1410; M, 1 to 982; and NS, 1 to 844. The programMrModeltest 2.2 was used to determine the appropriate DNA substitutionmodel and rate heterogeneity (12). The generated model was used in all subse-quent analyses. Neighbor-joining and maximum-likelihood trees were con-structed using PAUP* 4b10 (16) and Garli 0.96, respectively (24). BayesianMarkov chain Monte Carlo (MCMC) analysis of two independent replicates of2 million generations with six chains and sampling every 200 generations wasconducted with MrBayes 3.1 (10). Estimates of the phylogenies were calculatedby performing 1,000 neighbor-joining bootstrap replicates, and Bayesian poste-
rior probabilities were calculated from the consensus of 18,000 trees after exclu-sion of the first 2,000 trees as burn-in.
Nucleotide sequence accession numbers. The nucleotide sequences obtainedin the present study are available from GenBank under accession numbersHM144388 to HM145740.
RESULTS
Epidemiology of H6 influenza viruses from aquatic poultry.A total of 153,063 swabs were collected from apparentlyhealthy poultry in southern China from 2000 to 2005. Sixteenpercent of the total influenza virus isolates (1,325/8,325) wereof H6 subtype with 413 isolated from minor poultry (par-tridges, quails, etc.) and 1 from chicken. The remaining ma-jority were isolated from domestic ducks and geese (911/1,325). Isolation rates in winter and spring were generallyhigher than those in summer and autumn, although H6 virusescould be identified year-round. H6N2 was the most prevalentsubtype among all H6 viruses from aquatic birds, followed byH6N1 and others, such as H6N5 or H6N6. We found only 2%positive isolates from tracheal swabs in aquatic poultry (16/911), and the remaining 98% of viruses were isolated fromcloacal or fecal swabs, suggesting that those H6 viruses repli-cate primarily in the gut.
Phylogenic analysis of surface genes. A total of 170 H6viruses (about 20%) isolated from aquatic birds (ducks, n �165; geese, n � 5) were selected to represent positive samplingoccasions for sequence analysis. At least one virus was se-quenced from each positive sampling occasion. For those sam-ples having more than two isolates, viruses were proportionallysequenced.
Phylogenetic analysis of the H6 hemagglutinin (HA) geneclassified the duck isolates into three major lineages or cladescomprising isolates from both wild and domestic aquatic birds.The first group (group I), represented by Dk/ST/339/00(ST339-like), comprised those viruses isolated exclusively fromGuangdong (Shantou) and Hong Kong. This lineage formed amonophyletic group with a common ancestor isolated in HongKong in 1998 (Dk/HK/1037-1/98) (Fig. 1a). Within this groupthe majority of the isolates were H6N2 (n � 81) or 51% of totalH6 viruses tested. The other neuraminidase (NA) subtypecombinations were much less frequently encountered, and nowild bird or sentinel duck isolates phylogenetically groupedwith these taxa. This indicated that these viruses represented astable H6 lineage in domestic ducks from this region.
The second group (group II), represented by WDk/ST/2853/03 (ST2853-like), had both H6N2 and H6N6 viruses thatwere isolated from Guangdong and Fujian (Fig. 1a). This cladecirculated in domestic ducks from 2002 to 2005 and cocircu-lated with group I in at least Guangdong province. Thirty-twopercent of the H6 isolates sequenced clustered into this clade.This clade had a sister relationship with those H6 subtypeviruses, represented by teal/HK/W312/97 (W312-like), estab-lished in terrestrial minor poultry in southern China since atleast 1997 (4). Only two isolates from this study grouped withpreviously described H6 viruses from terrestrial poultry (WDk/ST/867/2002 and Dk/ST/1275/2004), suggesting that those es-tablished terrestrial bird viruses were rarely transmitted backto aquatic birds.
The third group (group III), represented by Dk/HN/573/02(or HN573-like), was composed of those viruses isolated from
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inland regions such as Jiangxi and Hunan (Fig. 1a), and itsmembers were phylogenetically related to H6 viruses isolatedfrom wild or migratory birds and included some viruses iso-lated from poultry disease outbreaks from different countries(Fig. 1a). Viruses of this clade obviously belong to the Eurasiangene pool and included the biggest diversity of NA subtypecombinations, including N1, N2, N5, and N8 (Fig. 1b). Mostimportantly, all isolates from the sentinel duck farm in Jiangxiand from domestic ducks in Guangxi and Hunan, but only asingle isolate from Shantou, belong to this group. This suggeststhat within Guangxi, Hunan, and Jiangxi there is a greaterdegree of interaction between migratory and domestic ducksthan there is for lineages established in Guangdong province.It has been noted that those H6 viruses introduced into NorthAmerica (represented by Mall/MN/Sg-00776/08 H6N2) alsobelong to this lineage.
The majority of the H6 subtypes encountered in our surveil-lance were H6N2 (79%). Other H6 subtypes, including H6N1(8 isolates), were rarely encountered. Phylogenetic analysis ofthe N2-NA gene tree showed that it could be delineated intothree major lineages (group I, group II, and group III) (Fig.1b). Group I formed a strongly supported monophyletic cladeprimarily comprised of aquatic isolates from Shantou (ST339-like). However, two isolates from Hunan (Dk/HN/908/2005and Dk/HN/989/2005) were also nested within this clade. Sim-ilarly to the HA phylogeny, this clade was not associated withany wild aquatic bird and sentinel duck isolates, a finding thatconfirmed a distinct lineage unique to domestic ducks. GroupII was closely affiliated with aquatic bird isolates from neigh-boring countries and with different HA subtype combination.Isolates of group III (HN573-like) included those from senti-nel and migratory ducks in Jiangxi and domestic ducks fromHunan and Guangxi as well as those from Eurasian countriesand represent a natural gene pool and transmission betweenthe wild reservoir and domestic ducks. Only one N2 gene fromWDk/ST/867/02 clustered with terrestrial H6/H9N2-like vi-ruses that have become endemic in this region since the early1990s (Fig. 1b).
Phylogenetic analyses of internal genes. In general, for allinternal gene trees the majority of aquatic source virusesformed a monophyletic group identified as group I, whichincluded the ST339-like viruses (Fig. 2 and 3). Also, in allinternal gene trees, gene segments circulating in the naturalreservoir could also be identified and classified as group III.However, unlike the surface proteins, group II viruses rarelyformed a monophyletic clade in the internal gene phylogenies.In some clades H6 viruses clustered with HPAI H5N1 reassor-tant viruses. However, in all trees terrestrial and aquatic H6isolates were distinct and rarely mixed.
Phylogenetic analyses of the PB2 gene demonstrated that sixlineages cocirculated in Eurasia. H6 isolates tested were incor-porated into each of those lineages (Fig. 2a). The majority ofH6 viruses from aquatic sources were monophyletic (group I)and formed a sister relationship with W312-like terrestrial H6viruses. It was noted that the representative viruses for groupI and group II were all clustered together in this lineage. Theremaining H6 viruses joined two clades that contained bothwild and domestic duck isolates and belonged to the naturalgene pool corresponding to those group III viruses in the HAphylogenetic tree. The second clade comprised a mix of do-mestic and wild birds, including H5N2 isolated from migratoryducks (MDk/HK/MP206/05) in Hong Kong. Notably, two H6isolates grouped in this clade, with Dk/HN/491/05 falling intothe basal portion of the lineage leading to the dominant H5N1variants (represented by Ck/HK/YU22/02 [genotype Z] andMD/JX/2136/05 [genotype V]) (5, 17). The remaining lineagesbelonged to those H5N1 or H9N2 lineages established in thisregion during the past 2 decades.
Similar phylogenies were observed for the PB1 and PAgenes. In the PB1 gene tree, the majority of characterized H6viruses clustered together and formed the group I lineage,while viruses isolated from Jiangxi, Hunan, and Guangxi joinedthe Eurasian gene pool lineage. It has been noted that about 10H6 viruses isolated from wild duck (WDk/ST/2395/01) in Shan-tou formed another independent lineage with a sister-grouprelationship to the Gs/Guangdong/1/96-like highly pathogenicH5N1 virus lineage (Fig. 2b). None of the aquatic lineagesformed a strong phylogenetic relationship with terrestriallyderived H6 viruses. The group I virus lineage had a sisterrelationship with Ck/Beijing/1/94-like H9N2 virus lineage withstrong bootstrap and Bayesian support, suggesting that thesetwo lineages might be derived from a common ancestor re-cently (Fig. 2b). Phylogenetic analysis of the PA gene segmentshowed that group I viruses were sisters to established terres-trial H6N1 (W312-like) and H9N2 (G1-like, CK/Bei-like) vi-ruses (Fig. 2c). The remaining H6 viruses incorporated intodifferent lineages of the Eurasian influenza virus gene pool.
Phylogenetic analyses of the NP, M, and NS genes all con-firmed the establishment of an H6 virus lineage in domesticducks in southern China which occupied the main body ofviruses tested and covered the whole sampling period. Virusesof this lineage were detected from birds in Shantou and Fujian.H6 viruses from Hunan, Guangxi, and Jiangxi, isolated fromeither domestic, sentinel, or migratory ducks, were clusteredinto different lineages that belonged to the Eurasian influenzavirus gene pool, suggesting multiple transmissions or gene mix-ing between migratory waterfowl and domestic aquatic birds(Fig. 3). Interestingly, the NP gene phylogeny showed H6
FIG. 1. Phylogenetic relationships of the H6 HA (a) and N2 NA (b) genes of representative H6 influenza viruses. The phylogenetic trees weregenerated by the maximum-likelihood method using Garli (version 0.96). Numbers above and below branches indicate Bayesian posteriorprobabilities and neighbor-joining (NJ) bootstrap values, using MrBayes 3.1 and PAUP* 4b10 programs, respectively. Analysis was based on codingnucleotides 1 to 1704 and 1 to 1410 of the H6 HA and N2 NA gene segments, respectively. The H6 HA and N2 NA trees are both rooted toturkey/Canada/63 (H6N2). Viruses highlighted in blue are those characterized in this study. Viruses highlighted in green were those isolated fromminor poultry, and the ones in red are reference strains. Abbreviations: Ck, chicken; Ck/Bei, chicken/Beijing/1/94-like; Cu, chukar; Dk, duck; FJ,Fujian; G1, Qa/HK/G1/97-like; GD, Guangdong; Gf, guinea fowl; Gs, goose; Gs/GD, goose/Guangdong/1/96-like; GX, Guangxi; HK, Hong Kong;HN, Hunan; JX, Jiangxi; Mall, mallard; MDk, migratory duck; Pa, partridge; Ph, pheasant; Qa, quail; SAU, southern Australia; ST, Shantou; TW,Taiwan; Ty, turkey; WDk, wild duck; W312, teal/HK/W312-like. The length of the scale bar corresponds to 0.01 nucleotide substitution per site.
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FIG. 2. Phylogenetic relationships of the PB2 (a), PB1 (b), and PA (c) polymerase genes of representative H6 influenza viruses. Analysis was based on theindicated nucleotides: PB2, 1 to 2280; PB1, 1 to 2274; and PA, 1 to 2151. The PB2, PB1, and PA trees were rooted to equine/Prague/1/56 (H7N7), pintailduck/Alberta/628/79, and Ann Arbor/6/60, respectively. Viruses characterized in this study are highlighted in blue. Viruses shown in green were those isolatedfrom minor poultry, while viruses in red represent prototype viruses. Abbreviations are the same as those defined in the legend to Fig. 1. The length of the scalebar corresponds to 0.01 nucleotide substitution per site.
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FIG. 3. Phylogenetic relationships of nucleoprotein (NP) (a), matrix (M) (b), and nonstructural (NS) protein (c) genes of representative H6influenza viruses. Analysis was based on the indicated nucleotides: NP, 1 to 1494; M, 1 to 982; and NS, 1 to 844. The NP, M, and NS trees are rootedto equine/Prague/1/56 (H7N7). Viruses characterized in this study are highlighted in blue. Viruses highlighted in green were those isolated fromminor poultry, while viruses in red represent prototype viruses. Abbreviations are the same as the ones listed in the legend to Fig. 1. The lengthof the scale bar corresponds to 0.01 nucleotide substitution per site.
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aquatic isolates with a sister or basal relationship to each of themajor reassortments leading to the emergence of novel H5N1and H9N2 genotypes (Fig. 3a). The M gene showed a stronghost restriction phylogenetic signal among all established lin-eages, as the current H6 lineage from domestic duck andH9N2 and H5N1 lineages as well as the gene pool lineage wereeasily distinguishable in the tree (Fig. 3b). The NS gene phy-logeny revealed that multiple genetic lineages from both alleleA and allele B cocirculate in the H6 subtype gene pool fromaquatic market birds (Fig. 3c).
Gene interactions and reassortment events. In summary,phylogenetic analyses of each of the eight gene segments ofthose H6 viruses revealed that group I viruses with ST339-likegene constellation were persistently prevalent in domesticducks from 2000 to 2004 (Table 1). Even though a few reas-sortant group I viruses with internal gene segments from thegene pool were observed, these novel viruses were sporadicallyencountered and not established in domestic ducks. The groupII viruses were reassortant group I-like viruses with new sur-face genes introduced as early as 2002. From 2002 to 2005,group I and group II viruses cocirculated in the field and groupII viruses became established gradually in the domestic duckhosts from 2005 onward (see Table S1 in the supplementalmaterial). Further, in 2005 a large number of group II virusesincorporated a new NP gene from the gene pool. The group IIIviruses analyzed were representative of the influenza virusgene pool in this region which clustered with other subtypes ofinfluenza viruses in all internal gene trees (Table 1). Among alltested viruses, only two isolates belonged to the H6N1/N2 viruslineage of established terrestrial H6 viruses. The majorities ofreassortants, or novel genome constellations, were transientand resulted from gene exchange between group I and group IIor the gene pool (Table S1). These findings showed that geneinteraction between the influenza virus gene pool and thoseestablished virus lineages in domestic ducks occurred through-out the surveillance period.
Antigenic analysis. To understand the antigenic propertiesof group I, group II, and group III H6 viruses, representativesfrom each of those three H6 lineages were tested by HI usinga panel of reference antisera (Table 2). None of the identifiedgroups reacted well with anti-Teal/HK/W312/97 or anti-Qa/
HK/1564/00, representative viruses of H6 viruses established interrestrial poultry, whereas all groups (group I, group II, andgroup III) reacted well with anti-Dk/ST/5540/01, representa-tive of group I viruses. Group II viruses were moderately re-active with anti-Dk/ST/14966/01, representative of group IIviruses, whereas group I and group III viruses showed low tomoderate reactivity compared to the homologous titer. GroupIII viruses reacted well with anti-Dk/JX/227/03 viruses, butgroup I and group II were only moderately reactive. Thissuggests that even though these lineages are phylogeneticallydistinct, they are similar in their antigenic properties.
DISCUSSION
It has generally been accepted that ducks and shorebirds arethe natural reservoirs of influenza A viruses (1, 6, 19). How-ever, the role of domestic ducks (compared to migratoryducks) in influenza virus ecology, particularly the interactionand prevalence of different subtypes of influenza viruses be-tween migratory and domestic ducks, has not been fully de-fined. In this study, phylogenetic analysis of viruses isolated insouthern China revealed that multiple lineages of H6 subtypeviruses cocirculated in the domestic duck population. Our find-ings demonstrated that the majority of H6 duck isolates be-longed to a single H6N2 virus lineage with a single gene con-stellation (group I), distinct from the natural gene pool, thathad become endemic. Our results also revealed the emergenceof a novel H6 lineage (group II) as well as the circulation ofviruses from the Eurasian natural gene pool (group III) in thishost in southern China.
Influenza A viruses belonging to the natural gene pool, rep-resented by viruses from diverse geographical origins, havebeen frequently detected from domestic ducks in inland prov-inces. These viruses cannot be distinguished from those virusesdetected directly from migratory ducks or sentinel ducks in oursurveillance program. This suggests that domestic ducks in thisregion form a significant part of the natural influenza virusreservoir.
As one of the major poultry types, domestic ducks in Chinahave the largest population size in the world (7). H6N2 viruswas one of the most frequently detected subtypes in our sur-
TABLE 1. Gene constellation of H6 subtype influenza viruses isolated from southern Chinaa
VirusGene origin
Yr(s) of detection No. ofvirusesHA NA PB2 PB1 PA NP M NS
Dk/ST/339/00 Gr I Gr I Gr I Gr I Gr I Gr I Gr I Gr I 2000–2004 65Dk/ST/1840/00 Gr I Gr I Gr I Gr I Gr I GP Gr I Gr I 2000, 2002, 2003 3Dk/ST/2167/01 Gr I Gr I Gr I GP Gr I Gr I Gr I Gr I 2001, 2003 4Dk/ST/4534/01 Gr I Gr I GP Gr I Gr I GP Gr I GP 2001 1Dk/ST/3656/02 Gr I Gr I Gr I Gr I Gr I GP Gr I GP 2002 1Dk/ST/1838/03 Gr I Gr I GP GP Gr I GP Gr I Gr I 2003 1WDk/ST/1494/02 Gr II Gr I Gr I Gr I Gr I Gr I Gr I Gr I 2002, 2004, 2005 4WDk/ST/2853/03 Gr II Gr II Gr I Gr I Gr I Gr I Gr I Gr I 2003–2005 21Dk/ST/2472/05 Gr II Gr I Gr I Gr I Gr I GP Gr I Gr I 2005 14WDk/ST/180/05 Gr II Gr II Gr I Gr I Gr I GP Gr I Gr I 2005 2Dk/FJ/12371/05 Gr II Gr II GP Gr I Gr I Gr I Gr I Gr I 2005 1Dk/ST/83/00 Gr III Gr II N Am GP GP Gs/GD GP GP 2000 1Dk/HN/573/02 Gr III Gr III GP GP GP GP GP GP 2002, 2005 3MDk/JX/12147/05 Gr III Gr III GP GP GP GP GP GP 2005 1
a Abbreviations: Gr I, group I; Gr II, group II; Gr III, group III; GP, gene pool; N Am, North America lineage; Gs/GD, goose/Guangdong/1/96-like.
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veillance program. Phylogenetic analyses revealed that mostH6N2 viruses isolated in Guangdong and Fujian had the samegene constellation, especially their internal gene complex. Thelong-term presence of this stable genotype indicates that virusbehavior in domestic ducks is similar to that seen in terrestrialpoultry species in the region (3). Domestic ducks thereforeappear to act as part of the natural reservoir along with wildaquatic birds but also as a major poultry type that harborsendemic virus lineages. Even though H6 viruses were alsodetected from mallards in North America over many years,those viruses were characterized by frequent reassortment anda stable virus genotype of this kind was not recognized (1, 6).
Reassortment or gene exchange between the established H6virus lineage and the natural gene pool occurred sporadicallythroughout the surveillance period (see Table S1 in the sup-plemental material), but few reassortants were persistent orprevalent. It is likely that those transient reassortant viruseshave reduced viral fitness or that there may be a fitness advan-tage for those established internal gene complexes. This is verydifferent from highly pathogenic H5N1 variants that cocircu-late and also show genotypic replacement over relatively shortperiods of time, e.g., 3 years (5, 8, 17, 20).
The lack of reassortment in the aquatic bird H6 lineage isinteresting given that H5N1, H9N2, and terrestrial birdH6N1/N2 subtype viruses, which are known to readily reassortwith each other, have cocirculated in the poultry populationsunder surveillance (8, 22, 23). Interestingly, two H6 duck iso-lates from the gene pool (group III) appear to have donatedgenes for the generation of the dominant H5N1 variants (ge-notypes Z and V). So, it is not the established H6N2 viruslineage but the viruses from the influenza virus gene pool indomestic ducks that contributed to the genetic diversity ofH5N1 and H9N2 viruses (5, 22, 23).
Although the W312-like H6N1/N2 viruses have been en-demic in minor terrestrial poultry in southern China for over10 years, their transmission to domestic ducks is very limited.Interspecies transmission of H6 virus from domestic ducks to
terrestrial poultry was also not recognized. These results high-light the host restriction that is prevalent between domesticducks and other terrestrial poultry. However, our previousstudies showed that domestic duck was the major host in whichH5N1 influenza viruses were endemic and that reassortmentoccurred frequently in this host with subsequent transmissionto terrestrial poultry (15). The mechanism responsible for theheterogeneity in virus behavior in precisely the same ecosystemremains unclear.
In this study our data show that virus or virus gene intro-duction from migratory duck to domestic duck occurredthroughout the surveillance period. Even though interspeciestransmissions of H6 subtype virus from domestic ducks toterrestrial poultry were not common, virus or gene introduc-tion from domestic ducks to other types of poultry of othervirus subtypes occurred frequently, especially in the case ofhighly pathogenic H5N1 virus. Thus, domestic ducks could betreated as intermediate hosts between the “real gene pool”from migratory ducks and those terrestrial poultry in the wholeinfluenza virus ecosystem. Further investigation and surveil-lance are required to understand the role of the domestic duckpopulation in facilitating virus interaction and the generationof genetic diversity.
ACKNOWLEDGMENTS
This study was supported by the Li Ka Shing Foundation, the Na-tional Institutes of Health (NIAID contract HHSN266200700005C),and the Area of Excellence Scheme of the University Grants Commit-tee (grant AoE/M-12/06) of the Hong Kong SAR Government.G.J.D.S. is supported by a career development award under NIAIDcontract HHSN266200700005C.
REFERENCES
1. Bahl, J., D. Vijaykrishna, E. C. Holmes, G. J. D. Smith, and Y. Guan. 2009.Gene flow and competitive exclusion of avian influenza A virus. Virology390:289–297.
2. Chen, H., G. J. D. Smith, S. Y. Zhang, K. Qin, J. Wang, K. S. Li, R. G.Webster, J. S. M. Peiris, and Y. Guan. 2005. Avian flu: H5N1 virus outbreakin migratory waterfowl. Nature 436:191–192.
TABLE 2. Antigenic analysis of H6 subtype of influenza viruses isolated from domestic ducks in southern Chinaa
a Abbreviations: DE4, laughing gull/Delaware/4/90; W312, teal/HK/W312/97; HK1564, Qa/HK/Yu1564/00; HK17, Ck/HK/17/77; ST5540, Dk/ST/5540/01; ST14966,Dk/ST/14966/05, JX227, Dk/JX/227/03; �, HI titer of �1:40.
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