Establishment and Lineage Replacement of H6 ... - Journal of Virology

Establishment and Lineage Replacement of H6 Influenza Viruses in Domestic Ducks in Southern China Kai Huang,a,b Huachen Zhu,a,b Xiaohui Fan,b Jia Wang,a,b Chung-Lam Cheung,b Lian Duan,a,b Wenshan Hong,a Yongmei Liu,b Lifeng Li,b David K. Smith,a,b Honglin Chen,a,b Robert G. Webster,c Richard J. Webby,c Malik Peiris,b and Yi Guana,b International Institute of Infection and Immunity, Shantou University Medical College, Shantou, Guangdong, Chinaa; State Key Laboratory of Emerging Infectious Diseases, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, Chinab; and Division of Virology, Department of Infectious Diseases, Saint Jude Children’s Research Hospital, Memphis, Tennessee, USAc

Domestic ducks in southern China act as an important reservoir for influenza viruses and have also facilitated the establishment of multiple H6 influenza virus lineages. To understand the continuing evolution of these established lineages, 297 H6 viruses isolated from domestic ducks during 2006 and 2007 were genetically and antigenically analyzed. Phylogenetic analyses showed that group II duck H6 viruses had replaced the previously predominant group I lineage and extended their geographic distribution from coastal to inland regions. Group II H6 virus showed that the genesis and development of multiple types of deletions in the neuraminidase (NA) stalk region could occur in the influenza viruses from domestic ducks. A gradual replacement of the N2 NA subtype with N6 was observed. Significant antigenic changes occurred within group II H6 viruses so that they became antigenically distinguishable from group I and gene pool viruses. Gene exchange between group II H6 viruses and the established H5N1, H9N2, or H6N1 virus lineages in poultry in the region was very limited. These findings suggest that domestic ducks can facilitate significant genetic and antigenic changes in viruses established in this host and highlight gaps in our knowledge of influenza virus ecology and even the evolutionary behavior of this virus family in its aquatic avian reservoirs.

A

quatic birds are accepted as the natural reservoirs of influenza A viruses, and these viruses have been introduced to other animals, shaping the current ecology of influenza viruses (17). Alteration of the influenza virus ecosystem by the emergence of novel host species or marked changes in the size and structure of host populations can impact the behavior of virus evolution. The establishment of multiple influenza virus subtypes (H5N1, H6N1, and H9N2) in the poultry of southern China provides the best example of this (4, 5, 20). Domestic ducks in China have substantially increased in numbers over the last 2 decades such that now 75% of the domestic ducks in the world are bred in China (15). Three phylogenetic groups or lineages of the H6 subtype of influenza viruses were prevalent in domestic ducks in southern China from 2000 to 2005 (9). Two lineages were specifically established in these ducks, while the third represented viruses from the gene pool of Eurasian avian influenza viruses (9). Thus, H6 viruses in domestic ducks in southern China are part of both the gene pool and specific viral lineages. An H6N1 virus (W312-like) has been endemic in this region in terrestrial poultry since the late 1990s (4), but it is not yet clear whether the H6 viruses established more recently in ducks would further spread to terrestrial poultry. The Asian highly pathogenic H5N1 lineages and two H9N2 (G1- and Ck/Bei-like) lineages, which are still endemic in southern China in poultry, are considered to be pandemic threats (19). Novel reassortant variants from these virus lineages have continually emerged and reemerged in the region (4, 5, 20). Interaction between these endemic virus lineages and other viruses from domestic ducks or other aquatic or shore birds has not been well defined. Whether multiple established H6 duck influenza viruses would promote such interactions or gene exchange is still unknown. Continuation of influenza surveillance from 2006 to 2007 suggests that the H6 subtype was still one of the most prevalent influ-

June 2012 Volume 86 Number 11

enza virus subtypes in domestic ducks in southern China. Genetic analyses of 297 H6 viruses isolated during this period suggest that group II H6 viruses have become predominant in the field and replaced the previously dominant group I viruses. Group II viruses have also spread to neighboring inland provinces and have occasionally been transmitted to swine (21). Antigenic analyses showed significant changes in the group II H6 viruses over time. Molecular characterization also revealed the emergence and development of multiple types of deletions in the stalk regions from different neuraminidases (NAs) of group II H6 viruses. These findings suggest that the established H6 duck viruses from southern China are not in evolutionary stasis but have undergone significant genetic and antigenic changes. MATERIALS AND METHODS Surveillance and virus isolation. Influenza virus surveillance of live poultry was conducted in seven provinces of southern China (Guangdong, Guangxi, Guizhou, Fujian, Hunan, Jiangxi, and Yunnan) as previously described (4, 9). Sampling was conducted weekly or with a 10-day interval from apparently healthy birds at live-poultry markets in the sampling sites. To avoid contamination and expand representation, no more than two ducks were sampled from each cage. Paired swabs from trachea and cloaca were taken from individual birds where possible. If this was not practical, either cloacal or fresh fecal swabs were collected. Swabs were kept in a cool box and shipped to the laboratory within 2 h. Virus isolation

Received 22 September 2011 Accepted 7 March 2012 Published ahead of print 21 March 2012 Address correspondence to Huachen Zhu, [email protected], or Yi Guan, [email protected]. Supplemental material for this article may be found at http://jvi.asm.org/. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.06389-11

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was conducted using 9- to 11-day embryonated chicken eggs, and virus subtypes were determined by hemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests as described previously (4, 9). Antigenic analysis. Antigenic changes of H6 viruses were analyzed by HI test with a panel of reference ferret antisera prepared for the present study. Two adult influenza virus-free male ferrets were intravenously and intranasally inoculated with 106 50% egg infection doses (EID50s) of different reference viruses to produce antisera. The ferret antisera produced were anti-Duck/Shantou/2195/2003 (Dk/ST/2195/03) (group I) and antiDk/ST/2853/03, anti-Duck/Fujian/1695/2005 (Dk/FJ/1695/05), and antiDuck/Guangxi/183/2007 (Dk/GX/183/07) (group II) which cover the group I and II viruses at several time points. All antisera were treated with receptor-destroying enzyme (RDE; Denka Seiken Co. Ltd., Tokyo, Japan) to remove nonspecific inhibitors, and tests for HI started at a 1:40 dilution. Viruses tested against the antisera were from all three groups and covered a range of isolation times. Viruses and virus sequencing. At least one virus from each positive sampling occasion was sequenced. When two to eight H6 isolates were obtained, a second virus was randomly selected for sequencing. If more than eight H6 viruses were isolated, a further virus was randomly selected from each subsequent group (or part thereof) of eight isolates. Collectively, 297 viruses were used for genetic analyses. RNA extraction, cDNA synthesis, and PCR were carried out as described previously (4, 9). Sequencing was performed using a BigDye Terminator, version 3.1, cycle sequencing kit on an ABI 3730 genetic analyzer (Applied Biosystems) following the manufacturer’s instructions. DNA sequences were compiled and edited using Lasergene, version 8.0 (DNASTAR, Madison, WI). Phylogenetic analyses. Phylogenetic analyses were based on the following coding sequences (nucleotides): PB2, 1 to 2280; PB1, 1 to 2274; PA, 1 to 2151; hemagglutinin (HA), 1 to 1701; nucleoprotein (NP), 1 to 1497; NA, 1 to 1410; M, 1 to 982; and NS, 1 to 844. Multiple sequence alignments were compiled using Clustal W (3). Maximum-likelihood (ML) phylogenies of each gene segment were inferred using the GTR⫹I⫹⌫4 model (general time-reversible with invariant sites and 4 gamma distributed heterogeneous substitution rates) in PhyML (version 3.0) (8). Robustness of the ML topology was evaluated with the approximate likelihood ratio test (aLRT), and the result is presented as the branch support (1). The group I, II, and III lineages were originally assigned based on clear phylogenetic subclades detected with strong bootstrap support (⬎70%) and Bayesian posterior probability (⬎0.9) in our previous work (9). Viruses from these lineages were included in the trees constructed here. Nucleotide sequence accession numbers. The nucleotide sequences obtained in the present study are available from GenBank under accession numbers CY109223 to CY110734.

RESULTS

Virus isolation and prevalence. From January 2006 to December 2007, a total of 56,055 duck swabs were sampled at live-poultry markets or duck farms from seven provinces of southern China. Of the 7,937 influenza A viruses isolated during this period, 1,849 were of the H6 subtype, giving an isolation rate of 3.3% for H6 viruses from the swab samples (Table 1). The majority of the H6 viruses (about 95%) were detected in domestic ducks from Shantou (eastern Guangdong province) and Fujian province, the southeastern coastal regions. Viruses could be detected yearround but with a higher prevalence during winter and were mainly isolated from fecal or cloacal swabs, which is consistent with our previous study (9). Most of the H6 viruses (95%) had an N2 or N6 NA gene but N1, N5, N8, and N9 NA genes were also observed. As part of our overall surveillance program from 2000 to 2007, H6 viruses in chickens and minor poultry were also assessed (4, 9). Ten H6 isolates from 70,475 chicken swabs, an isolation rate similar to that seen in chickens in eastern China (22), and 401 H6 isolates from 15,252 minor poultry swabs were obtained (4, 9).

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TABLE 1 Prevalence of H6 influenza virus in domestic ducks in southern China and viruses sequenced

Location

No. of samples

No. of isolates

No. of H6 isolates

No. of H6 isolates sequenced (%)

Guangdong Hunan Jiangxi Guangxi Fujian Yunan Guizhou

9,536 5,255 16,596 5,030 10,024 4,820 4,794

1,388 452 850 921 2,394 537 1,455

851 4 16 20 911 2 45

127 (15) 4 (100) 8 (50) 12 (60) 129 (14) 2 (100) 15 (33)

Total

56,055

7,937

1,849

297 (16)

Antigenic analysis. A panel of ferret antisera against four viruses, one from group I and three isolated in different years from group II, were prepared and used to conduct antigenic characterization. Selected H6 influenza viruses from the three groups were characterized by HI assays. Group I and gene pool (group III) viruses had moderate to high reactivity to anti-ST/2195/03 (group I) and anti-ST/2853/03 (early group II) but no reactivity to antiFJ/1695/05 and anti-GX/183/07. Most group II viruses were reactive to anti-GX/183/07 while about half were reactive to anti-FJ/ 1695/05, but only a few reacted to anti-ST/2853/03 (early group II) or anti-ST/2195/03 (group I) (Table 2). This suggests that the antigenicity of group II viruses altered after the group became established. Phylogenetic analyses of H6 duck viruses. A total of 297 viruses have been sequenced (16% of isolates), of which 153 had the full-length genome sequenced, and for the remaining 144 only the HA and NA genes were sequenced. All sequence data acquired in our previous study on duck H6 viruses isolated from 2000 to 2005 in the same region (9) and from publicly available data sets were also included in the analyses. Surface genes. Phylogenetic analysis of the complete set of H6 HA genes showed that duck H6 isolates from southern China clustered into the three major groups (I, II, and gene pool/III) as previously described (9). Of the 297 H6 HA genes sequenced in this work, no viruses from group I, which was the predominant group during 2000 to 2005 (9), were identified. Most (93%) of the viruses from 2006 to 2007 belonged to group II. The remaining viruses belonged to the gene pool (group III) (Fig. 1A). Group II H6 viruses, which were observed only in the coastal regions of Guangdong and Fujian from 2000 to 2005 (9), have also been isolated in ducks from inland regions, such as Jiangxi, Hunan, and Guizhou, since 2006. One group II H6N6 virus has also been isolated from swine in Guangdong (21). A large proportion of H6 viruses from 2006 to 2007 had an N6 NA (63%) rather than an N2 NA (32%) gene, which was predominant during 2000 to 2005 (9), and N1, N5, N8, and N9 subtypes made up the remaining NA genes, with only N8 being found in group II viruses. Phylogenetic analysis of the N2 genes showed that they also formed three distinct lineages, group i, ii, and gene pool (group iii) (Fig. 1B). The group II H6 viruses incorporating both group i and ii N2 NAs continue to prevail in the field and were also found in the inland regions (Fig. 1B) although their numbers are decreasing relative to N6 NA. Almost all N6 NA genes of group II H6 duck viruses from this study clustered to-

Journal of Virology

Lineage Replacement of H6 Inﬂuenza Viruses in Ducks

TABLE 2 Antigenic analysis of H6 viruses from 2006-2007 in southern China HI titer with the indicated antiserumb Group I antiserum, ST/2195/03

Group II antiserum

Isolate group and virusa Group I Dk/ST/2195/03 Dk/ST/339/00 Gs/GX/4442/06 Dk/ST/5540/01

640 320 320 640

640 1,280 1,280 2,560

⬍ ⬍ ⬍ 80

⬍ ⬍ ⬍ ⬍

Group II WDk/ST/2853/03 Dk/FJ/1695/05 Ck/GX/183/07 Dk/ST/8/06 Dk/ST/4636/06 Dk/ST/14167/06 Dk/FJ/9062/06 Gs/GY/1142/07 Dk/GX/908/07 Dk/JX/4981/07 Gs/GX/1305/07 Dk/GX/1330/07 Pa/ST/238/07 Dk/ST/168/07 Dk/FJ/1804/07

640 320 ⬍ ⬍ ⬍ ⬍ 80 ⬍ ⬍ ⬍ ⬍ ⬍ ⬍ ⬍ 80

2,560 640 ⬍ ⬍ ⬍ ⬍ 80 ⬍ ⬍ ⬍ ⬍ ⬍ ⬍ ⬍ ⬍

⬍ 2,560 640 320 ⬍ ⬍ 320 320 320 640 ⬍ ⬍ ⬍ ⬍ ⬍

⬍ 1,280 2,560 1,280 640 80 1,280 1,280 1,280 1,280 320 160 160 80 ⬍

Group III Dk/HN/573/02 Dk/JX/227/03 WDk/JX/10668/05 Dk/GY/2773/06 Dk/YN/3136/06 Dk/GX/2736/06

160 160 80 160 80 320

160 160 80 640 160 640

⬍ ⬍ ⬍ ⬍ ⬍ ⬍

⬍ ⬍ ⬍ ⬍ ⬍ ⬍

ST/2853/03 FJ/1695/05

GX/183/07

a The four viruses used to construct the panel of antisera and their respective HI titers are underlined. Virus abbreviations are listed in the legend of Fig. 1. b ⬍, HI titer of ⬍40 (serum dilution of 1:40).

gether to form a major lineage (Fig. 1C, ST192-like). The N2 NA appears to be being replaced by an N6 NA, which has also established a lineage along with the group II H6 viruses. Internal genes. Generally, the internal genes of the group II viruses isolated from 2006 to 2007 clustered together to form a large clade, which was named the group I/II lineage in our previous report (9). Although remotely related in some of the genes (NP and PB1), none of these H6 duck viruses obtained their internal genes directly from the contemporary H9N2 (G1-like and Ck/Bei-like), H6N1 (W312-like), and highly pathogenic H5N1 lineages that are endemic in the region in poultry (Fig. 2 and 3; also data not shown). Viruses with all six group I/II-like internal genes were predominantly detected in coastal provinces, i.e., Guangdong and Fujian (Fig. 2 and 3 and Table 3; see also Table S1 in the supplemental material). Some H6 isolates from Shantou (Guangdong) reassorted with an NP gene from the gene pool while the remaining genes belonged to the group I/II lineage (Fig. 1 to 3 and Table 3; see also Table S1). However, the majority of group II H6 viruses detected in the inland provinces (Jiangxi, Hunan, Guizhou, Guangxi, and Yunnan) had most or all internal genes from the


gene pool (Fig. 2 and 3 and Table 3; see also Table S1). The occurrence of several gene exchanges between migratory waterfowl and domestic ducks would account for these observations. Exchanges between the gene pool and the group I/II internal genes occurred more frequently for the NP, M, and NS genes, with a few involving the polymerase genes (Table 3; see also Table S1). Molecular characterization. Almost all H6 viruses isolated from domestic ducks had the amino acid sequence PQIETR2G at the HA cleavage site (indicated by the down arrow) which is also commonly observed in H6 viruses from terrestrial poultry. All H6 duck viruses except for one had residues 226Q and 228G (H3 numbering of the mature protein) at the HA receptor-binding pocket. This suggests that the binding preference of these viruses favors ␣-2,3 receptors. All duck H6 viruses tested had 627E in PB2. Overall, HA nucleotide sequence difference levels were ⬍7% within groups I, II, and III and ⬎10% between groups. NA deletion. Among the 467 duck H6 viruses (isolated from 2000 to 2007 in southern China) and sequenced here and in our previous report (9), 60 of the N2 NA and 5 of the N6 NA genes isolated from 2003 to 2007 had a deletion in the NA stalk region (Table 4). No deletion was found in any other subtype of NA. All viruses with an NA deletion belonged to the H6 group II viruses. Deletions in N2 formed four groups: (i) a 19-amino-acid deletion of residues 63 to 81 (N2-⌬19) in 25 viruses, (ii) an 18-amino-acid deletion of residues 43 to 60 (N2-⌬18) in one virus, (iii) a 10amino-acid deletion of residues 66 to 75 (N2-⌬10a) in eight viruses, and (iv) a 10-amino-acid deletion of residues 69 to 78 (N2⌬10b) in 26 viruses. In the N6 NA gene four groups of deletion were observed: (i) an 11-amino-acid deletion of residues 53 to 63 (N6-⌬11a) in two viruses, (ii) an 11-amino-acid deletion of residues 59 to 69 (N6-⌬11b) in one virus, (iii) an 11-amino-acid deletion of residues 60 to 70 (N6-⌬11c) in one virus, and (iv) a 12-amino-acid deletion of residues 59 to 70 (N6-⌬12) in one virus (Table 4; see also Table S1 in the supplemental material). Deletions in the NA stalk region might develop as the NA subtype becomes predominant in the field. For the N2 NA, no NA deletions were observed in the first 3 years (2000 to 2002). The number of deletions detected increased during 2005 to 2006 when the N2 subtype was most common and then declined as the number of N2 viruses decreased in the field (Table 4). DISCUSSION

Even though wild aquatic birds have long been recognized as the primary hosts or natural reservoirs of the influenza A virus, the evolutionary behaviors of this virus family in domestic ducks have not been fully defined. Our previous studies demonstrated that domestic ducks in southern China actually act as intermediate hosts between the gene pool from migratory ducks and terrestrial poultry in the influenza virus ecosystem of this region (4, 9). This study shows, as did our previous observation, that domestic ducks could also facilitate the establishment of H6 virus lineages and also support the endemicity of the viruses within this host. Genetic analyses of 297 sequenced H6 duck viruses isolated during 2006 and 2007 from southern China revealed lineage replacement between the two previously established H6 virus lineages. Group II H6 viruses became predominant in the field, and their NA genes also gradually changed from N2 to N6. Group II H6 viruses also underwent significant antigenic changes within the group and became distinguishable from H6 viruses from group I and the gene pool.

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FIG 1 Phylogenetic relationships of the H6 HA (A), N2 NA (B), and N6 NA (C) genes of selected H6 influenza viruses from ducks in southern China. The phylogenetic trees were generated by the maximum-likelihood method using PhyML (version 3.0). Numbers below branches indicate approximate likelihood ratio test (aLRT) branch support. The H6 HA tree is rooted to Turkey/Canada/1965 (H6N2), N2 NA is rooted to Turkey/England/1969 (H3N2), and N6 is rooted to Duck/England/1/1956 (H11N6). Viruses highlighted in blue are those characterized in this study; those in red are reference strains. Virus subtypes other than H6 in the NA trees are indicated. Abbreviations are as follows: Aq, aquatic bird; Aus, Australia; Bei, Beijing; CA, California; Chn, China; Ck, Chicken; Cu, chukar; Deu, Germany; Dk, duck; E_Chn, Eastern China; FJ, Fujian; Fra, France; GD, Guangdong; Gs, goose; Gs/GD, Goose/Guangdong/1/96 (H5N1); GX, Guangxi; GZ, Guizhou; HK, Hong Kong; HN, Hunan; Ita, Italy; Jpn, Japan; JX, Jiangxi; Kor, South Korea; Md, mallard; MD, Maryland; MDk, migratory duck; MN, Minnesota; Mng, Mongolia; Nld, Netherlands; NSW, New South Wales, Australia; Pa, partridge; Ph, pheasant; Qa, quail; Rus, Russian Federation; Sb_Dk, spot-billed duck; SCk, Silky chicken; Sgp, Singapore; ST, Shantou, Guangdong; Sts, Sharp-tailed_sandpiper; Sw, swine; Swe, Sweden; TW, Taiwan; Ty, turkey; Vnm, Vietnam; WDk, wild duck; YN, Yunnan; Zaf, South Africa; Zmb, Zambia.

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FIG 2 Phylogenetic relationships of the H6 PB2, PB1, and PA genes of selected H6 influenza viruses from ducks in southern China. The PB2 tree was rooted to Equine/Prague/1/56 (H7N7), the PB1 tree was rooted to Pintail Duck/ALB/219/77, and the PA tree was rooted to Equine/London/1416/73 (H7N7). Highlighting and abbreviations are as described in the legend of Fig. 1, with viruses of subtypes other than H6 indicated.

It has been proposed that influenza A viruses in their natural reservoir (wild aquatic birds) are in a state of evolutionary stasis; i.e., the viruses show limited nonsynonymous nucleotide substitutions over the long term (17). The findings presented here suggest that viruses in domestic ducks may behave significantly differently from those in their wild counterparts. The establishment of group II H6 viruses in domestic ducks did cause significant genetic and antigenic changes. As this phenomenon was observed only in the H6 duck viruses in southern China, it is still unknown


whether similar evolution also occurs in other virus subtypes from aquatic birds (both wild and domestic) in different regions. This enigma about the evolution of influenza viruses in aquatic birds needs to be further explored. Phylogenetic analyses of influenza viruses from wild birds in North America demonstrated a remarkably high rate of genome reassortment forming transient “genome constellations” with no clear patterns (7). In the study here, the dominant group II H6 viruses had a relatively fixed internal gene complex. This was pre-

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FIG 3 Phylogenetic relationships of the NP, M, and NS genes of selected H6 influenza viruses from ducks in southern China. All trees were rooted to Equine/Prague/1/56 (H7N7). Highlighting and abbreviations are as described in the legend of Fig. 1, with viruses of subtypes other than H6 indicated.

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TABLE 3 Gene constellation of H6 viruses isolated from ducks in southern China Area and location (no. of viruses)a

Lineage of gene segmentb HA

NAc

PB2

PB1

PA

NP

M

NS

Coastal region ST (3) FJ (4) FJ (1) FJ (54), ST (3) FJ (1) ST (19), FJ (1) ST (27) FJ (1) FJ (2) FJ(1) FJ (1) ST (1) ST (1) ST (1) ST (1)

II II II II II II II II II II II II II II GP

2-i 2-ii 2-GP 6 6 6 2-i 2-ii 6 6 6 6 2-i 8-GP 2-GP

I/II I/II I/II I/II GP I/II I/II I/II I/II I/II I/II GP GP GP GP

I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II GP I/II GP GP

I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II I/II GP GP GP

I/II I/II I/II I/II I/II GP GP GP GP GP GP I/II GP GP GP

I/II I/II I/II I/II I/II I/II I/II I/II I/II GP GP I/II I/II GP GP

I/II I/II I/II I/II I/II I/II I/II I/II GP-B GP-B I/II I/II I/II GP GP

Inland region GX (2) GX (4), GZ (2) GZ (1) GZ (1) GZ (1), GX (1) HN (1) GX (1), HN (1), YN (1) JX (1), GZ (1) GX (1) GZ (3) YN (1) GZ (1) HN (1) JX (3) JX (1) GX (1) GX (1)

II GP GP GP GP GP GP GP GP II II II II II II II II

8-GP 2-GP 1-GP 5-GP 8-GP 9-GP 2-GP 1-GP 1-GP 2-i 6-GP 8-GP 8-GP 2-i 2-ii 2-i 6

GP GP GP GP GP GP GP GP GP GP GP GP I/II I/II GP I/II I/II

GP GP GP GP GP GP GP GP GP GP GP GP GP GP I/II I/II I/II

GP GP GP GP GP GP GP GP GP GP GP GP GP GP I/II I/II I/II

GP GP GP GP GP GP GP GP GP GP GP GP GP GP GP I/II I/II

GP GP GP GP GP GP GP GP I/II I/II I/II I/II I/II I/II I/II I/II I/II

GP GP GP GP GP GP GP-B GP-B GP I/II I/II I/II I/II I/II I/II I/II I/II

a

FJ, Fujian; GX, Guangxi; GZ, Guizhou; HN, Hunan; JX, Jiangxi; ST, Shantou; YN, Yunnan. GP, gene pool; GP-B, gene pool allele B; I, group I; II, group II; I/II, groups I and II. c The numbers indicate the NA subtype and lineage on the NA trees. 6, N6 lineage incorporated with the group II H6 viruses, represented by Wild duck/Shantou/192/2004 (ST192-like). b

viously suggested to occur only when interspecies transmission of an avian virus to an aberrant host causes the evolution of a distinct genomic configuration (7). Although domestic ducks are aquatic birds, it is possible that agricultural breeding and population management practices may have changed the viruses to behave as in an aberrant host. In the field, even though the H6 viruses were cocirculating with Asian highly pathogenic H5N1 viruses over a long period (4), reassortment between H6 and the endemic H5N1 viruses was not identified in this study. Similarly, reassortment events between the H6 viruses and other lineages established in terrestrial poultry were also not observed. Whether the established H6 virus had a relative fitness advantage in the host is still unknown. Probably, such reassortment would reduce virus fitness so that preservation of this lineage is favored. Formation of stable lineages within an aquatic avian host was first observed in this and our previous study (9). As there is no obvious difference of virus subtype distribution between domestic and migratory ducks (6), this might be a consequence of agricul-


tural practices, such as much higher population densities of domestic ducks within limited areas, different population age structures, and artificial seasonality for breeding, that are not present in wild bird populations or of other ecosystem factors. Whether the establishment of a virus lineage in domestic duck becomes the first step in, or favors, interspecies transmission from aquatic birds to terrestrial poultry is unknown. What we observed in our surveillance is that the two established H6 virus lineages from 2000 to 2007 had not been introduced to terrestrial poultry. All but five of the over 100 H6 viruses isolated and sequenced in this period from chickens and other terrestrial poultry were W312-like (4). Interspecies transmission and subsequent establishment of H6 viruses in terrestrial poultry occurred in southern China (4), Taiwan (10), and California (16, 18) before. Phylogenetic analyses demonstrated that the HA genes of these viruses were of aquatic avian sources and are of lineages different from the lineages of the duck viruses isolated here. However, as the H6 viruses in aquatic birds prior to the transmission events are not available, it cannot be known if they were established or not. H6

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TABLE 4 Development of NA deletion at the stalk region N2 NA deletion profile

N6 NA deletion profile

Year of isolation

N2-⌬19 (63–81)

N2-⌬10a (66–75)

N2-⌬10b (69–78)

N2-⌬18 (43–60)

No. of NA genes sequenced (no. of NA genes with stalk deletion)

2000 2001 2002 2003 2004 2005 2006 2007

0 0 0 3 3 17 2 0

0 0 0 0 0 1 2 5

0 0 0 0 0 7 19 0

0 0 0 0 0 1 0 0

7 27 28 17 (3) 11 (3) 41 (26) 75 (23) 21(5)

0 0 0 0 0 1 1 0

0 0 0 0 0 0 0 1

0 0 0 0 0 0 0 1

0 0 0 0 0 0 1 0

0 1 0 0 3 8 (1) 98 (2) 89 (2)

Total

25

8

26

1

227 (60)

2

1

1

1

199 (5)

No. of viruses with the indicated deletiona

a

N6-⌬11a (53–63)

N6-⌬11b (59–69)

N6-⌬11c (60–70)

N6-⌬12 (59–70)

No. of NA genes sequenced (no. of NA genes with stalk deletion)

No. of viruses with the indicated deletiona

Amino acid positions of the deletions are given in parentheses.

viruses are rarely found in chickens in this region and eastern China (22). Molecular analyses of the group II H6 duck viruses revealed the emergence and development of multiple types of deletion in the stalk region of different NA subtypes. Previously, a deletion in the NA stalk region was mainly observed in highly pathogenic H5 or H7 viruses and viruses established in terrestrial poultry (2, 12). The occurrence of deletions in the NA stalk region could influence the replication, host range, and, in some cases, the virulence of a virus (13, 14) and was considered a molecular mark of adaptation to terrestrial poultry (2, 14). One of the NA deletion types identified here (N2-⌬19, which has a deletion of amino acid residues 63 to 81) has been previously found in other NA sequences (in two viruses, A/chicken/France/03426/2003 [H5N2] and A/duck/ Kingmen/E322/04 [H6N2]) (11), and N2-⌬10a was observed in one chicken sample (9). However, the emergence and development of NA deletions in the stalk region of the group II H6 viruses has not yet been associated with any evidence of lineage establishment in terrestrial poultry but demonstrates that this kind of deletion could systematically occur in viruses of domestic aquatic birds. Even though the N2 NA genes had acquired different types of deletions in the stalk region, they were gradually replaced in the field by N6 NA genes, which mostly did not have deletions in their stalk regions. Thus, the stalk deletion does not appear to provide an HA-NA compatibility advantage, and the NA may not be key to the establishment of the H6 viruses in ducks. The NA replacement may be decided by an advantage of the subtype itself in the host. Whether these NA deletions would enable the H6 viruses to be more easily introduced into terrestrial poultry, and even mammals, needs to be further investigated. Genetic and antigenic analyses of the H6 duck viruses from southern China provided novel insights into the evolution of the influenza virus in aquatic birds. It is necessary to further analyze other subtypes of influenza viruses from this host. The findings would directly benefit the understanding of the entire ecology of influenza viruses. ACKNOWLEDGMENTS This study was supported by the Li Ka Shing Foundation, the National Institutes of Health (NIAID contract HHSN266200700005C), and the

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Area of Excellence Scheme of the University Grants Committee (grant AoE/M-12/06) of the Hong Kong SAR Government.

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