Complex Reassortment of Multiple Subtypes of Avian Influenza Viruses in Domestic Ducks at the Dongting Lake Region of China

Guohua Deng; Dan Tan; Jianzhong Shi; Pengfei Cui; Yongping Jiang; Liling Liu; Guobin Tian; Yoshihiro Kawaoka; Chengjun Li; Hualan Chen

doi:10.1128/JVI.00776-13

. 2013 Sep;87(17):9452–9462. doi: 10.1128/JVI.00776-13

Complex Reassortment of Multiple Subtypes of Avian Influenza Viruses in Domestic Ducks at the Dongting Lake Region of China

Guohua Deng ^a, Dan Tan ^a, Jianzhong Shi ^a, Pengfei Cui ^a, Yongping Jiang ^a, Liling Liu ^a, Guobin Tian ^a, Yoshihiro Kawaoka ^b, Chengjun Li ^a,^✉, Hualan Chen ^a,^✉

PMCID: PMC3754128 PMID: 23804642

Abstract

To gain insight into the ecology of avian influenza viruses (AIV), we conducted active influenza virus surveillance in domestic ducks on farms located on the flyway of migratory birds in the Dongting Lake region of Hunan Province, China, from winter 2011 until spring 2012. Specimens comprising 3,030 duck swab samples and 1,010 environmental samples were collected from 101 duck farms. We isolated AIV of various HA subtypes, including H3, H4, H5, H6, H9, H10, H11, and H12. We sequenced the entire coding sequences of the genomes of 28 representative isolates constituting 13 specific subtypes. When the phylogenetic relationships among these isolates were examined, we observed that extensive reassortment events had occurred. Among the 28 Dongting Lake viruses, 21 genotypes involving the six internal genes were identified. Furthermore, we identified viruses or viral genes introduced from other countries, viral gene segments of unknown origin, and a novel HA/NA combination. Our findings emphasize the importance of farmed domestic ducks in the Dongting Lake region to the genesis and evolution of AIV and highlight the need for continued surveillance of domestic ducks in this region.

INTRODUCTION

Wild aquatic birds are the natural reservoir of all subtypes of avian influenza viruses (AIV), from H1 to H16 and N1 to N9 (1, 2), with the exception of the recently identified H17N10 influenza virus, which was isolated from bats (3). Wild aquatic birds serve as carriers and transmitters of AIV, expanding the geographic distribution of influenza viruses. In the United States, the active surveillance for AIV in wild aquatic birds initiated in 2006 has revealed that hemagglutinin (HA) gene sequences representing the H4, H8, H10, H11, and H12 subtypes are well established in North America, with no evidence of intercontinental exchange (4). Intercontinental exchange of AIV between the North American and Eurasian lineages has occurred, however, for other subtypes of lowly pathogenic AIV (H1, H2, H3, H6, H9, H13, and H16) over the past few decades (4).

The replication of most AIV is usually restricted to the intestinal duct of wild aquatic birds, resulting in no observable clinical signs (2). However, certain H5 and H7 strains may cause obvious symptoms and death (2); a prime example is the Qinghai Lake H5N1 influenza viruses. In 2005, thousands of migratory aquatic birds died in the Qinghai Lake area due to H5N1 virus infection (5, 6). The H5N1 viruses of the Qinghai Lake lineage subsequently spread to Europe and Africa (7–14). This wide dissemination of the Qinghai Lake H5N1 viruses exacerbated the global prevalence of avian H5N1 influenza viruses and greatly contributed to the increase in human cases of H5N1 influenza virus infection (15, 16). As of 4 June 2013, 630 laboratory-confirmed human cases of H5N1 influenza had been reported to the WHO, among which 375 cases were fatal (http://www.who.int).

Domestic ducks are positioned at the interface between wild aquatic birds and terrestrial poultry and are known to play an important role in the ecology of AIV (17–20). Domestic duck farming in China has grown substantially during the last 3 decades. Currently, more than 75% of the world's domestic duck population is bred in China (21). Various subtypes of AIV have been isolated from ducks, including the two accepted public health threats, i.e., H5N1 and H9N2 viruses (22–27). Domestic ducks can carry highly pathogenic H5N1 influenza virus yet remain healthy (22, 28, 29); however, in some cases, the virus can kill them (30–32). In Hong Kong in the 1970s and 1980s, H9 influenza viruses were detected only in apparently healthy ducks (33). In the 1990s, the H9N2 virus expanded its host range and became endemic in land-based domestic poultry in Asia (27, 34, 35). Recently, multiple subtypes of AIV were isolated from domestic ducks in the live poultry markets of southern China (17, 18). However, direct evidence of the cocirculation of multiple subtypes of AIV in domestic duck farms has not been reported.

Domestic duck farming in southern China occurs in high-density settings but in a free-range manner. There are no biosafety measures in place on these duck farms. This farming style creates an environment in which migratory birds and domestic ducks are in close contact, sharing water, food, and habitat. As a result, AIV of both sources coexist in duck farms, facilitating interspecies transmission and viral gene reassortment. The Dongting Lake region in Hunan Province, China, is on the East Asian-Australasian Flyway of migratory birds. Many different species of wild aquatic birds migrate there every year for breeding, wintering, and summering (36). The duck farms in this region are poorly built on the lake shore. The flock sizes range from dozens (backyard farms) to thousands of ducks among different farms. The ducks stay in the lake for most of the time during the day, sharing food, water, and habitat with the wild aquatic birds. Therefore, the Dongting Lake region offers a unique opportunity to observe the evolution of AIV and to gain a better understanding of the ecology of AIV in this region, which will provide invaluable insights into the general epidemic situation of AIV. In this study, we conducted active surveillance of AIV on domestic duck farms in the Dongting Lake region from winter 2011 to spring 2012. We report the isolation of multiple subtypes of AIV, including H5N1 viruses, from specimens collected from healthy domestic ducks and from the environment of duck farms in four counties of the Dongting Lake region. Our results emphasize the importance of duck farms to the ecology of AIV and draw attention to the need for control of H5N1 virus infection in ducks.

MATERIALS AND METHODS

Sample collection and virus isolation.

Active avian influenza virus surveillance was conducted over two periods in the Dongting Lake region of Hunan Province, China. From 9 November to 13 November 2011, 1,620 swab samples (a cloacal swab and tracheal swab from the same duck were put into the same sample collection tube and counted as one sample) and 540 environmental samples (water and soil with deposited fecal matter) were collected from 54 duck farms in Yuanjiang City, Xiangyin County, and Hanshou County, in the Dongting Lake region. Between 23 March and 25 March 2012, 1,410 swab samples and 470 environmental samples were collected from 47 duck farms in Yuanjiang City, Huarong County, and Hanshou County. All of the swab samples were collected from apparently healthy ducks. The samples were placed in a transport medium that consisted of phosphate-buffered saline (PBS) containing penicillin (2,000 U/ml) and streptomycin (2,000 μg/ml). The samples were immediately frozen with dry ice and kept on dry ice during transfer to the laboratory. All of the individual samples were inoculated into 10-day-old embryonated chicken eggs for 48 h at 37°C. The allantoic fluid was collected and tested for HA activity with 0.5% chicken red blood cells. Allantoic fluid that was positive for HA activity was aliquoted and stored at −80°C. All procedures for sample collection and virus isolation were conducted by experienced scientists. To avoid cross-contamination during egg inoculation and allantoic fluid collection, strict sterile techniques were implemented. When the procedure with one batch of eggs (fewer than 30 eggs) was completed in a biosafety cabinet (BSC), the cabinet was thoroughly disinfected with 70% ethanol and UV for 30 min before the next use. All of the instruments used during the procedure, such as forceps, scissors, and an egg perforator, were autoclaved before use and were disinfected with 70% ethanol after use with each egg. In addition, each specimen was divided into three aliquots, and only one aliquot was used for the virus isolation procedure. In the case of possible cross-contamination, one of the backup aliquots of the original specimen could be used to repeat the virus isolation. All virus isolation procedures were conducted in a biosafety level 3 (BSL-3) facility approved for such work by the Ministry of Agriculture, China.

HA subtyping of isolates.

When the HA assay was positive, hemagglutination inhibition (HI) assays were performed to determine the HA subtype of the isolated AIV and to exclude Newcastle disease virus (NDV), another avian virus frequently isolated from avian species. The HI assay was performed by using a panel of reference antisera against the 16 HA subtypes of AIV that was generated in specific-pathogen-free (SPF) chickens in our laboratory. NA subtypes were determined for representative isolates by means of direct sequencing (see below).

Genetic and phylogenetic analyses.

A total of 28 avian influenza virus isolates representing all of the 8 HA subtypes identified in this study were randomly selected for genetic and phylogenetic analyses. Viral RNA (vRNA) was extracted from virus-infected allantoic fluid by use of a QIAmp viral RNA minikit (Qiagen). cDNAs were synthesized from vRNAs by reverse transcription (RT) with the Uni12 primer and were amplified by PCR with primers complementary to the conserved promoter and noncoding regions of each gene segment (37). The coding sequences for the entire genomes of the 28 viruses were sequenced on an Applied Biosystems DNA analyzer at the Harbin Veterinary Research Institute. The primer sequences for RT-PCR and sequencing are available upon request. The nucleotide sequences were edited using the Seqman module of the DNAStar package. Phylogenetic analysis was performed using the ClustalX 1.81 software package, implementing the neighbor-joining method. Both North American and Eurasian lineages of the HA and NA genes were included in the phylogenetic trees (with the exception of H5 HA and N1 NA) and constituted the outline of the phylogenetic trees. Compared with the North American lineage, more genes of the Eurasian lineage were included. Regarding the internal genes, we selected virus isolates from China or other Asian countries for construction of the phylogenetic trees. When a virus was isolated in a tree, its closest relatives were identified by using the BLAST program of NCBI and were included in the tree to best reflect the phylogenetic relationship. We defined a “group” in the phylogenetic trees only when the genes of Dongting Lake viruses were present in that group. The definition of “group” was based on the phylogenetic relationship of the gene sequences. In general, the Dongting Lake viruses in the phylogenetic tree shared <∼96% sequence identity among different groups. The tree topology was evaluated by 1,000 bootstrap analyses and is shown by using TreeView 1.6.6 (38). The regions of nucleotide sequence used for the phylogenetic analysis were as follows: H3 HA, nucleotides (nt) 60 to 1720; H4 HA, nt 20 to 1704; H5N1 HA, nt 62 to 1696; H5 HA (low-pathogenicity avian influenza virus [LPAIV]), nt 115 to 1720; H6 HA, nt 18 to 1721; H9 HA, nt 46 to 1572; H10 HA, nt 32 to 1686; H11 HA, nt 33 to 1730; H12 HA, nt 63 to 1698; N1 NA, nt 21 to 1136; N2 NA, nt 38 to 1396; N3 NA, nt 19 to 1413; N6 NA, nt 19 to 1415; N7 NA, nt 44 to 1305; N8 NA, nt 35 to 1422; N9 NA, nt 28 to 1419; PB2, nt 55 to 2289; PB1, nt 35 to 2233; PA, nt 25 to 2168; NP, nt 46 to 1510; M, nt 58 to 985; and NS, nt 27 to 854.

Nucleotide sequence accession numbers.

The nucleotide sequences of the 28 viruses determined in this study have been deposited in GenBank under accession numbers CY146545 to CY146768.

RESULTS

Geographic location of sampling sites.

The active surveillance of AIV was conducted in Yuanjiang City, Xiangyin County, Huarong County, and Hanshou County, in the region of Dongting Lake, the third largest lake in China (Fig. 1). Dongting Lake is a national natural reserve and is one of the most important wetlands worldwide. It is located on the East Asian-Australasian Flyway of migratory birds. More than 200 species of birds breed there, including 153 species of migratory birds, 40 species of resident birds, and 25 species of passing migrants (36). A large population of domestic ducks breeds in the Dongting Lake region. Many of the duck farms are small-scale or backyard-style farms, with open environments and no biosafety measures. This mode of duck farming creates an environment in which the domestic ducks and wild aquatic birds live together, sharing water, food, and habitat.

Fig 1 — Geographical location of the Dongting Lake region under surveillance. The avian influenza viruses isolated in winter 2011 are shown by blue “stars,” whereas those isolated in spring 2012 are shown by red “stars”.

Avian influenza virus samples were collected over two periods: 9 to 13 November 2011 and 23 to 25 March 2012. A total of 3,030 swab samples collected from apparently healthy ducks and 1,010 environmental samples collected from duck farms were tested for the presence of AIV. Strikingly, we observed that 8 HA subtypes of AIV were circulating in the duck farms in the Dongting Lake region (Table 1; see Table S1 in the supplemental material). In total, 130 influenza viruses were isolated from the 4,040 samples collected. Among them, 107 influenza viruses were isolated from the 3,030 swab samples (isolation rate = 3.5%), and 23 viruses were isolated from the 1,010 environmental samples (isolation rate = 2.3%). Of the 130 viruses isolated, the H4 subtype was most dominant, and the H11 and H3 subtypes were the second and third most dominant subtypes, respectively.

Table 1.

Isolation of AIV from duck farms in Dongting Lake region of Hunan Province, China, between winter 2011 and spring 2012

Sampling time	No. of duck farms sampled	Total no. of samples from ducks	Total no. of samples from duck farm environment	No. of influenza virus isolates of HA subtype									No. of viruses isolated from:		Total no. of viruses
Sampling time	No. of duck farms sampled	Total no. of samples from ducks	Total no. of samples from duck farm environment	H3	H4	H5N1	H5N2	H6	H9	H10	H11	H12	Ducks	Environment	Total no. of viruses
Winter 2011	54	1,620	540	2	0	4	3	0	2	0	9	1	17	4	21
Spring 2012	47	1,410	470	21	66	2	0	2	0	2	16	0	90	19	109
Total	101	3,030	1,010	23	66	6	3	2	2	2	25	1	107	23	130

Open in a new tab

Genetic and phylogenetic analyses of HA genes.

To better understand the evolutionary relationship of AIV in the Dongting Lake region, we sequenced the entire coding sequences of the genomes of 28 of the isolates—23 from ducks and 5 from duck farm environments—representing all 8 of the HA subtypes we identified. This panel of viruses consisted of 13 different subtypes, including H3N2, H3N8, H4N2, H4N6, H4N9, H5N1, H5N2, H6N6, H9N2, H10N3, H11N2, H11N9, and H12N7 (Table 2).

Table 2.

List of Dongting Lake avian influenza viruses characterized genetically and phylogenetically in this study

Virus name	Subtype	Sampling date (day-mo-yr)
A/environment/Hunan/S4304/2011	H3N2	13-Nov-2011
A/environment/Hunan/S4350/2011	H3N8	13-Nov-2011
A/duck/Hunan/S1256/2012	H3N8	23-Mar-2012
A/duck/Hunan/S1824/2012	H3N8	24-Mar-2012
A/duck/Hunan/S11313/2012	H4N2	25-Mar-2012
A/duck/Hunan/S11090/2012	H4N6	25-Mar-2012
A/duck/Hunan/S11200/2012	H4N6	25-Mar-2012
A/duck/Hunan/S11893/2012	H4N6	25-Mar-2012
A/duck/Hunan/S11547/2012	H4N9	24-Mar-2012
A/duck/Hunan/S11643/2012	H4N9	25-Mar-2012
A/environment/Hunan/S11511/2012	H4N9	24-Mar-2012
A/duck/Hunan/S4030/2011	H5N1	09-Nov-2011
A/duck/Hunan/S4150/2011	H5N1	11-Nov-2011
A/duck/Hunan/S4220/2011	H5N1	13-Nov-2011
A/duck/Hunan/S4234/2011	H5N1	13-Nov-2011
A/duck/Hunan/S4101/2011	H5N2	11-Nov-2011
A/duck/Hunan/S4120/2011	H5N2	11-Nov-2011
A/duck/Hunan/S4124/2011	H5N2	11-Nov-2011
A/duck/Hunan/S1661/2012	H6N6	24-Mar-2012
A/duck/Hunan/S4111/2011	H9N2	11-Nov-2011
A/duck/Hunan/S11205/2012	H10N3	25-Mar-2012
A/environment/Hunan/S1798/2012	H11N2	24-Mar-2012
A/duck/Hunan/S4013/2011	H11N9	09-Nov-2011
A/duck/Hunan/S4137/2011	H11N9	11-Nov-2011
A/duck/Hunan/S4443/2011	H11N9	13-Nov-2011
A/duck/Hunan/S4474/2011	H11N9	13-Nov-2011
A/duck/Hunan/S1607/2012	H11N9	24-Mar-2012
A/environment/Hunan/S4484/2011	H12N7	13-Nov-2011

Open in a new tab

The four H5N1 viruses were all isolated from apparently healthy ducks. They possessed the stretch of multibasic amino acids (PQIERRRRKR/GL for DK/Hunan/S4234/11 and PQRERRRKR/GL for DK/Hunan/S4030/11, DK/Hunan/S4150/11, and DK/Hunan/S4220/11) at the HA cleavage site that is the signature of highly pathogenic H5N1 influenza viruses (39–41). Phylogenetic analysis of the four HA genes revealed that they all clustered in clade 2.3.2 (Fig. 2), represented by the human isolates Hubei/1/10 and Guangxi/1/09. Clade 2.3.2 viruses were originally identified from wild birds (42, 43) but have also been reported in domestic poultry, such as chickens and ducks (44–46). This clade contains recent H5N1 isolates and is located at the extremity of the phylogenetic tree, indicating active virus evolution.

The topology of the H3 avian influenza virus HA gene was separated into the North American lineage and the Eurasian lineage, as described previously (Fig. 2; see Fig. S1A in the supplemental material) (47). The HA genes of the four H3 viruses in this study, i.e., DK/Hunan/S1256/12 (H3N8), Duck/Hunan/S1824/12 (H3N8), EN/Hunan/S4304/11 (H3N2), and EN/Hunan/S4350/11 (H3N8), were all located in the Eurasian lineage but shared less than 92.9% identity and belonged to four distinct groups. The EN/Hunan/S4304/11-like HA gene had not been identified previously; in GenBank, DK/Siberia/100/01 (H3N8) contained the closest H3 HA gene to that of EN/Hunan/S4304/11, with an identity of only 93.4%.

The phylogenetic tree of the H4 HA gene was divided into the North American lineage and the Eurasian lineage (Fig. 2; see Fig. S1B) (48). All 7 of the Dongting Lake H4 HA genes in this study, including genes from 1 H4N2 virus, 3 H4N6 viruses, and 3 H4N9 viruses, clustered together in the phylogenetic tree. They shared 95.6% to 100% identity at the nucleotide level, indicating a common ancestor, although it is uncertain whether they were introduced into the Dongting Lake region from a single source or multiple sources.

The HA genes of the three H5N2 viruses in this study, i.e., DK/Hunan/S4101/11, DK/Hunan/S4120/11, and DK/Hunan/S4124/11, shared 99.9% identity. Two HA genes of H5N2 viruses in GenBank, namely, WB/Korea/L60-2/08 and EWC/Vietnam/8/07, were closely related to these three Dongting Lake H5N2 virus genes (Fig. 2; see Fig. S1C), with 98.1% to 98.9% nucleotide sequence identities. We speculate that they could be the ancestors of the HA genes of the three Dongting Lake H5N2 viruses. The other H5 HA genes in GenBank shared less than 80.6% identity with the Dongting Lake H5N2 viruses. Thus, the HA genes of the three Dongting Lake H5N2 viruses formed a distinct lineage (termed the WB/Korea/L60-2/08 lineage) with the H5N2 viruses isolated from other Asian countries.

Previously, Huang et al. reported that H6 influenza viruses were prevalent in domestic ducks in southern China (Fig. 2; see Fig. S1D) (17, 18). They found that duck H6 viruses with a group II HA gene have replaced the previously predominant lineage, i.e., group I (18). In this study, the HA gene of the H6 virus DK/Hunan/S1661/12 (H6N6) clustered in the phylogenetic tree with those of the group II H6 viruses from Huang et al.'s study.

With the exception of CK/Heilongjiang/35/00 virus, which derived its HA gene from the North American lineage (27), the HA genes of H9N2 viruses isolated in mainland China belong to the Eurasian lineage, in which CK/Beijing/1/94-like viruses are the dominant sublineage (27, 49). The H9N2 virus in this study, DK/Hunan/S4111/11, belonged to the CK/Beijing/1/94 sublineage and was located at the extremity of the phylogenetic tree (Fig. 2; see Fig. S1E), indicating that the H9N2 viruses in China are evolving over time.

The HA gene of the H10N3 virus DK/Hunan/S11205/12 belonged to the Eurasian lineage (Fig. 2; see Fig. S1F) (50). Its closest relatives were the H10 viruses isolated from Korea, represented by WB/Korea/A12/10 (H10N1), with approximately 96% identity. However, it is unknown whether these HA genes originated from a common ancestor.

The HA genes of the H11 viruses were divided into the North American and Eurasian lineages (51, 52). In this study, the HA genes of the six H11 viruses clustered in the Eurasian lineage and were separated into groups 1 and 2 (Fig. 2; see Fig. S1G). The HA gene of DK/Hunan/S4443/11 (H11N9) virus belonged to group 1. It clustered with the H11 viruses isolated from Vietnam and Thailand and shared 96.7% identity with its closest relative in GenBank, DK/Vietnam/OIE-2329/09 (H11N3). The other 5 H11 viruses shared nucleotide sequence similarities ranging from 96.6% to 99.9% and belonged to group 2.

The HA gene of the H12N7 virus EN/Hunan/S4484/11 belonged to the Eurasian lineage and was most closely related to WG/Dongting/C1037/11 (H12N8) virus, with 98.3% identity (Fig. 2; see Fig. S1H). Notably, EN/Hunan/S4484/11 is the only H12N7 virus isolated in nature to date, highlighting the role of the Dongting Lake region in the genesis of new influenza viruses.

Genetic and phylogenetic analyses of NA genes.

The NA genes of the four Dongting Lake H5N1 viruses possessed a 20-amino-acid deletion (residues 49 to 68) (Fig. 3) which is correlated with the increased pathogenicity of H5N1 influenza viruses (53, 54). The number of H5N1 viruses with this 20-amino-acid deletion increased dramatically in 2002, and by 2007, all avian isolates, including those from wild birds, had short NA stalks (53).

Of the 28 Dongting Lake viruses sequenced in this study, 7 possessed NA genes of the N2 subtype, and they were phylogenetically diversified (Fig. 3; see Fig. S1I in the supplemental material). Similar to the HA genes, the NA genes of the three H5N2 viruses, i.e., DK/Hunan/S4101/11, DK/Hunan/S4120/11, and DK/Hunan/S4124/11, shared the highest nucleotide sequence identities (98.3% to 98.8%) with those of WB/Korea/L60-2/08 and EWC/Vietnam/8/07 in GenBank, which distinguished them from other N2 NA genes. The other 4 N2 NA genes belonged to groups 1, 2, and 3 of the Eurasian lineage. EN/Hunan/S4304/11 (H3N2), in group 1, shared the highest identity (96.1%) with MDK/Vietnam/LBM189/12 (H3N2) in GenBank. Group 2 included viruses with N2 NA genes in combination with different subtypes of the HA gene, for example, EN/Hunan/S1798/12 (H11N2) and DK/Hunan/S11313/12 (H4N2). The NA gene of DK/Hunan/S4111/11 (H9N2) in group 3 formed a cluster with those of other H9N2 viruses.

The N3 NA gene of the DK/Hunan/S11205/12 (H10N3) virus belonged to the Eurasian lineage and was most closely related to the NA genes of the H11N3 viruses isolated from Vietnam, i.e., DK/Vietnam/OIE-2329/09 and DK/Vietnam/OIE-0068/12 (Fig. 3; see Fig. S1J), with identities of 97.2% and 99.0%, respectively.

The phylogenetic tree of the N6 NA gene was separated into the North American and Eurasian lineages (Fig. 3; see Fig. S1K). The four N6 NA genes of the Dongting Lake viruses were separated into three groups. The NA gene of DK/Hunan/S11893/12 (H4N6) belonged to group 1 and shared 99.8% identity with its closest relative in GenBank, DK/Mongolia/OIE-7438/11 (H4N6). The NA genes of DK/Hunan/S11090/12 (H4N6) and DK/Hunan/S11200/12 (H4N6) clustered in group 2, whereas the NA gene of the third virus, DK/Hunan/S1661/12 (H6N6), formed group 3 with the NA genes of other closely related H6N6 viruses.

The NA gene of the H12N7 virus EN/Hunan/S4484/11 belonged to the Eurasian lineage and shared the highest identity (98.9%) with the WG/Dongting/PC0360/12 (H7N7) virus (Fig. 3; see Fig. S1L). As described above, its HA gene was a close relative of those of the H12 isolates from Thailand and Vietnam. However, with this limited information, we are unable to understand how this H12N7 virus emerged in nature.

The phylogenetic tree of the avian N8 NA genes was separated into the North American and Eurasian lineages (55). The N8 NA gene of DK/Hunan/S1256/12 belonged to the Eurasian lineage (Fig. 3; see Fig. S1M). In contrast, the NA genes of DK/Hunan/S1824/12 and EN/Hunan/S4350/11 belonged to two different groups in the North American lineage. Note that the NA gene of DK/Hunan/S1824/12 shared only 92.9% identity with its closest relative in GenBank, DK/Nanchang/1681/92 (H3N8).

The topology of the phylogenetic tree of the N9 NA gene was formed by three lineages, the North American lineage, Eurasian lineage I, and Eurasian lineage II (Fig. 3; see Fig. S1N). The NA genes of the 3 H4N9 viruses and the 5 H11N9 viruses were all grouped in Eurasian lineage II. With the exception of DK/Hunan/S4443/11 (H11N9) virus, the NA genes of the other 4 H11N9 viruses and 3 H4N9 viruses shared identities between 99.1% and 100%, suggesting that they originated from a common ancestor.

Phylogenetic analysis of internal genes.

To better understand the evolution of AIV in the Dongting Lake region, each of the 6 internal genes of the 28 Dongting Lake viruses was phylogenetically analyzed as an entity. The PB2 genes of the Dongting Lake AIV were considerably diverse, forming 8 distinct groups in the phylogenetic tree (see Fig. S1O in the supplemental material). The PB2 genes shared nucleotide sequence identities of <94.5% among the different groups.

Phylogenetic analysis of the 28 Dongting Lake PB1 genes revealed that they formed 11 groups in the tree (see Fig. S1P). The 28 PB1 genes among the different groups shared identities of <96.1%. Notably, the closest relatives of the DK/Hunan/S11205/12 PB1 gene (group 4) were the PB1 gene of WG/Dongting/C1037/11 (H12N8) in GenBank and that of DK/Hunan/S1256/12 (H3N8) in this study, both with an identity of 95.3%. The two PB1 genes in group 5, DK/Hunan/S1798/12 (H11N2) and DK/Hunan/S4443/11 (H11N9), shared 97.0% identity but had only 93.4% and 93.3% identity, respectively, with the closest PB1 gene in GenBank, that of DK/Nanchang/1941/93 (H4N4). These three PB1 genes from groups 4 and 5 were therefore previously unidentified, with unknown origins.

The PA genes of the 28 Dongting Lake viruses clustered into 9 groups in the phylogenetic tree and shared identities of <96.3% among the different groups (see Fig. S1Q). As with the PB1 gene, we did not find PA genes in GenBank similar to the two PA genes in group 4, i.e., those of DK/Hunan/S4443/11 (H11N9) and DK/Hunan/S11205/12 (H10N3). These genes shared 97.3% identity but had only 94.4% and 94.6% identity, respectively, with the closest PA gene in GenBank, SN/Hokkaido/51/96 (H5N3).

The NP genes of the 28 Dongting Lake viruses were divided into 10 groups in the phylogenetic tree and shared less than 94.9% identity among the different groups (see Fig. S1R). Unlike the three polymerase genes, the NP genes of six of the Dongting Lake viruses, i.e., DK/Hunan/S4013/11 (H11N9), DK/Hunan/4137/11 (H11N9), EN/Hunan/S4474/11 (H11N9), DK/Hunan/S4101/11 (H5N2), DK/Hunan/S4120/11 (H5N2), and DK/Hunan/S4124/11 (H5N2), formed a distinct group far away from the main body of the tree. These six NP genes clustered with the NP gene of WB/Korea/L60-2/08 virus, indicating that the WB/Korea/L60-2/08-like H5N2 viruses had reassorted with local isolates of other subtypes (H11N9 in this case) in the Dongting Lake region. The single NP gene in group 2, DK/Hunan/S11893/12, was previously unidentified. It shared only 93.5% identity with its closest relative in GenBank, DK/Vietnam/G17-1/11 (H11N9).

The M genes of the 28 viruses clustered into 7 groups, with nucleotide sequence similarities of <95.8% among the different groups (see Fig. S1S). Similar to the other gene segments, the M genes of the three Dongting Lake H5N2 viruses also distinguished themselves by clustering with those of WB/Korea/L60-2/08 (H5N2) and EWC/Vietnam/8/07 (H5N2). Among these 7 groups, group 7 was dominant, containing half of the 28 M genes. One of the four H5N1 viruses, DK/Hunan/S4234/11, and the only H9N2 virus, DK/Hunan/S4111/11, had M genes that were descendants of the CK/Beijing/1/94 (H9N2)-like M genes. The other three H5N1 M genes, from DK/Hunan/S4030/11, DK/Hunan/S4150/11, and DK/Hunan/S4220/11, evolved from the GS/Guangdong/1/96 (H5N1)-like viruses.

The NS genes of influenza A viruses were separated into two alleles: A and B (22). Two viruses in this study, DK/Hunan/S1256/12 (H3N8) and EN/Hunan/S4484/11 (H12N7), possessed the NS gene of allele A (see Fig. S1T). The other 26 viruses possessed NS genes from allele B and clustered into 6 groups. Group 1 contained only the NS genes of the three H5N2 viruses derived from the WB/Korea/L60-2/08-like or EWC/Vietnam/8/07 H5N2-like virus. The NS gene of DK/Hunan/S4111/11 (H9N2) virus belonged to group 2, which was a descendant of the CK/Beijing/1/94 (H9N2)-like virus. Group 5 contained exclusively the NS gene of 4 H5N1 viruses, which was characterized by a 5-amino-acid deletion at residues 80 to 84. The 16 NS genes in group 6 contained multiple virus subtypes and shared nucleotide sequence identities ranging from 95.1% to 100%.

To better understand the evolutionary relationships among the Dongting Lake viruses, we analyzed the six internal genes of the 28 viruses as a single entity. On the basis of the phylogenetic diversity of these six internal genes, the 28 Dongting Lake viruses were divided into a complex of 21 genotypes (Fig. 4). Among the four H5N1 viruses, 3 genotypes coexisted. Genotype J contained two H5N1 viruses, namely, DK/Hunan/S4030/11 and DK/Hunan/S4220/11. The two H5N1 viruses in genotypes K and L differed from those in genotype J by the introduction of PA or M genes from different origins. These data show that AIV of subtypes other than H5N1 in the Dongting Lake region could serve as donors of internal genes for H5N1 viruses. Of the 13 subtypes of Dongting Lake virus in this study, 6 subtypes contained more than one isolate. Only 2 subtypes, H4N9 and H5N2, formed a single genotype, whereas the other 4 subtypes presented with 2 or 3 genotypes. These results demonstrate that complex reassortment between different subtypes of AIV has actively occurred in the Dongting Lake region.

Fig 4 — Genotypes of the six internal genes of the avian influenza viruses isolated from the Dongting Lake region. (A) Genotypes were defined on the basis of the gene phylogenies of the six internal genes, shown in Fig. S1O to T in the supplemental material. (B) Color designations for groups in the phylogenetic trees. Genes of the same group are shown in the same color. Note that the NS genes of groups 1 to 6 in allele B are colored normally, whereas the two NS genes in allele A are colored purple (the same as group 7).

DISCUSSION

Southern China has long been considered an epicenter for the emergence of pandemic influenza viruses (56, 57). Of the four human pandemic influenza viruses, at least half, the 1957 H2N2 “Asian flu” and the 1968 “Hong Kong flu,” emerged in southern China (58). It is generally accepted that live poultry markets play an important role in the ecology of AIV (17, 18, 59–65). In 1996, the first avian H5N1 influenza virus was isolated in Guangdong Province, in southern China (22, 66). This H5N1 virus contaminated the live bird markets in Hong Kong and caused the first known outbreak of human infections with H5N1 influenza viruses; 18 people were infected, among whom 6 died (67, 68). Live poultry markets bring together a number of bird species from different locations in a high-density setting, thus providing an ideal environment for gene exchange among AIV of different subtypes.

Several studies have shown that domestic ducks in live poultry markets have played an important role in the ecosystem of AIV in southern China (17, 18, 62). However, the role of domestic ducks on duck farms in the ecology of AIV remains unclear. In this study, we conducted active avian influenza surveillance of duck farms in the Dongting Lake region. This region is located on the flyway of migratory birds, and a large population of domestic ducks breeds there annually. The combination of these features makes the Dongting Lake region an ideal location for the cocirculation of AIV of different origins and the generation of new influenza viruses through reassortment. Here we demonstrated that 8 HA subtypes of AIV were circulating in the duck farms of the Dongting Lake region. Phylogenetic analyses of 28 representative viruses revealed that extensive reassortment had occurred for the AIV in the duck farms of this region. Surprisingly, when the evolutionary relationships of the six internal genes of the 28 AIV in this study were analyzed as a single entity, we observed a complex of 21 genotypes. Several studies have shown that reassortment is a very frequent event in wild birds, with very short survival times of lineages or genotypes of AIV (69–71). It is not clear from the present study whether the evolution of AIV in domestic ducks in the Dongting Lake region is occurring at a faster or slower pace than that in wild bird reservoirs. The answer lies in continued active surveillance of AIV in this region, which will enable us to observe the transition of different lineages or genotypes.

In this study, we found that AIV of subtypes other than H5N1 could serve as gene donors for H5N1 viruses in the duck farms of the Dongting Lake region, which would increase the genetic diversity of H5N1 viruses (72–74). We identified three H5N2 viruses, all eight gene segments of which were derived from the WB/Korea/L60-2/08-like (H5N2) virus. Notably, the WB/Korea/L60-2/08-like virus had reassorted with local strains in the Dongting Lake region, as demonstrated by the introduction of its NP gene into the three H11N9 viruses. In addition, our findings showed that the HA gene of DK/Hunan/S4443/11 (H11N9) virus was most closely related to those of the H11 viruses isolated from Vietnam and Thailand; DK/Hunan/S11205/12 (H10N3) virus possessed an NA gene closely related to those of the H11N3 viruses from Vietnam, and the NA genes of two H3N8 viruses clustered into the North American lineage. These findings demonstrate that migratory birds may have spread AIV to the Dongting Lake region from other countries and contributed to the complexity of the ecology of AIV in this region. We believe that the circulation of influenza viruses in a given region involves the two-way traffic of both virus import and virus export. Although we have no direct evidence, we speculate that AIV that emerge from domestic ducks in the Dongting Lake region could be transmitted to wild aquatic birds and spread to other countries via the flyway of bird migrations. Furthermore, our study identified gene segments of unknown origin, such as H3 HA, PB1, PA, and NP genes and a new HA/NA combination (H12N7), demonstrating that the Dongting Lake region is actively involved in the genesis of novel influenza viruses. Our discovery of novel viruses and viral gene segments sheds light on the limitations of currently available surveillance data, which restricted our ability to trace the origins of these viruses and viral genes. There is a clear need for long-term surveillance of AIV in the Dongting Lake region, as well as other lake regions of China.

Huang et al. isolated a large number of AIV from live poultry markets in several provinces of southern China (17, 18). They genetically and phylogenetically characterized the H6 AIV and showed that these H6 viruses were dominant in Guangdong and Fujian Provinces but that the isolation rates in other provinces, such as Hunan Province, were low. We isolated only two H6 viruses in this study. The eight gene segments of the H6N6 virus, DK/Hunan/S1661/12, grouped exclusively with those of the other H6 viruses in the phylogenetic trees, with the exception of the NP gene. Our findings therefore agree with the observations of Huang et al. that H6 viruses are not the dominant subtype in Hunan Province and that they seldom reassort with AIV of other subtypes. In contrast to the H6 viruses, the AIV of the other subtypes in this study, such as H3, H4, and H11, have undergone extensive reassortment.

The virus isolation rate in domestic ducks was 3.5% in this study. This is considerably lower than the rates reported for domestic ducks housed in live poultry markets (17, 18). Domestic ducks and other aquatic birds, such as geese and wild waterfowl, are housed together with chickens and other terrestrial poultry in the live poultry markets. In addition, most of the live poultry markets in China and Southeast Asia are characterized by poor sanitation. Therefore, the higher rate of virus isolation from domestic ducks in live poultry markets might be caused at least in part by virus infections occurring in the live poultry markets. In fact, AIV reassortment in the live poultry markets of Nanchang, China, has previously been reported (62).

In summary, the central message from our study is that domestic duck farms in the Dongting Lake region play an important role in the ecology of AIV by supporting complex reassortment of multiple subtypes of AIV during their circulation. Our study emphasizes the need for continued active surveillance to monitor the evolution of AIV in this region. Duck farming may have significantly contributed to the complexity of AIV in this region, thus jeopardizing food security and public health. Therefore, efforts above and beyond surveillance, such as fortifying the biosafety measures of duck farms, are needed to eradicate AIV from domestic ducks in this region.

Supplementary Material

Supplemental material

supp_87_17_9452__index.html^{(1.1KB, html)}

ACKNOWLEDGMENTS

We thank Susan Watson for editing the manuscript.

This work was supported by the disease control program of the Ministry of Agriculture, China, by the National Science and Technology Major Project (2012ZX10004214), and by the Founding Research Centers for Emerging and Reemerging Infectious Diseases.

Footnotes

Published ahead of print 26 June 2013

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JVI.00776-13.

REFERENCES

1.Hinshaw VS, Webster RG, Turner B. 1980. The perpetuation of orthomyxoviruses and paramyxoviruses in Canadian waterfowl. Can. J. Microbiol. 26:622–629 [DOI] [PubMed] [Google Scholar]
2.Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y. 1992. Evolution and ecology of influenza A viruses. Microbiol. Rev. 56:152–179 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Tong S, Li Y, Rivailler P, Conrardy C, Castillo DA, Chen LM, Recuenco S, Ellison JA, Davis CT, York IA, Turmelle AS, Moran D, Rogers S, Shi M, Tao Y, Weil MR, Tang K, Rowe LA, Sammons S, Xu X, Frace M, Lindblade KA, Cox NJ, Anderson LJ, Rupprecht CE, Donis RO. 2012. A distinct lineage of influenza A virus from bats. Proc. Natl. Acad. Sci. U. S. A. 109:4269–4274 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Piaggio AJ, Shriner SA, VanDalen KK, Franklin AB, Anderson TD, Kolokotronis SO. 2012. Molecular surveillance of low pathogenic avian influenza viruses in wild birds across the United States: inferences from the hemagglutinin gene. PLoS One 7:e50834. 10.1371/journal.pone.0050834 [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Chen H, Li Y, Li Z, Shi J, Shinya K, Deng G, Qi Q, Tian G, Fan S, Zhao H, Sun Y, Kawaoka Y. 2006. Properties and dissemination of H5N1 viruses isolated during an influenza outbreak in migratory waterfowl in western China. J. Virol. 80:5976–5983 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Liu J, Xiao H, Lei F, Zhu Q, Qin K, Zhang XW, Zhang XL, Zhao D, Wang G, Feng Y, Ma J, Liu W, Wang J, Gao GF. 2005. Highly pathogenic H5N1 influenza virus infection in migratory birds. Science 309:1206. [DOI] [PubMed] [Google Scholar]
7.Fauci AS. 2006. Pandemic influenza threat and preparedness. Emerg. Infect. Dis. 12:73–77 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Fusaro A, Nelson MI, Joannis T, Bertolotti L, Monne I, Salviato A, Olaleye O, Shittu I, Sulaiman L, Lombin LH, Capua I, Holmes EC, Cattoli G. 2010. Evolutionary dynamics of multiple sublineages of H5N1 influenza viruses in Nigeria from 2006 to 2008. J. Virol. 84:3239–3247 [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Kilpatrick AM, Chmura AA, Gibbons DW, Fleischer RC, Marra PP, Daszak P. 2006. Predicting the global spread of H5N1 avian influenza. Proc. Natl. Acad. Sci. U. S. A. 103:19368–19373 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Lee YJ, Choi YK, Kim YJ, Song MS, Jeong OM, Lee EK, Jeon WJ, Jeong W, Joh SJ, Choi KS, Her M, Kim MC, Kim A, Kim MJ, Ho Lee E, Oh TG, Moon HJ, Yoo DW, Kim JH, Sung MH, Poo H, Kwon JH, Kim CJ. 2008. Highly pathogenic avian influenza virus (H5N1) in domestic poultry and relationship with migratory birds, South Korea. Emerg. Infect. Dis. 14:487–490 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Lipatov AS, Evseenko VA, Yen HL, Zaykovskaya AV, Durimanov AG, Zolotykh SI, Netesov SV, Drozdov IG, Onishchenko GG, Webster RG, Shestopalov AM. 2007. Influenza (H5N1) viruses in poultry, Russian Federation, 2005–2006. Emerg. Infect. Dis. 13:539–546 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Salzberg SL, Kingsford C, Cattoli G, Spiro DJ, Janies DA, Aly MM, Brown IH, Couacy-Hymann E, De Mia GM, Dung Do H, Guercio A, Joannis T, Maken Ali AS, Osmani A, Padalino I, Saad MD, Savic V, Sengamalay NA, Yingst S, Zaborsky J, Zorman-Rojs O, Ghedin E, Capua I. 2007. Genome analysis linking recent European and African influenza (H5N1) viruses. Emerg. Infect. Dis. 13:713–718 [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Shestopalov AM, Durimanov AG, Evseenko VA, Ternovoi VA, Rassadkin YN, Razumova YV, Zaykovskaya AV, Zolotykh SI, Netesov SV. 2006. H5N1 influenza virus, domestic birds, western Siberia, Russia. Emerg. Infect. Dis. 12:1167–1169 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Watanabe Y, Ibrahim MS, Ellakany HF, Kawashita N, Mizuike R, Hiramatsu H, Sriwilaijaroen N, Takagi T, Suzuki Y, Ikuta K. 2011. Acquisition of human-type receptor binding specificity by new H5N1 influenza virus sublineages during their emergence in birds in Egypt. PLoS Pathog. 7:e1002068. 10.1371/journal.ppat.1002068 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Kandeel A, Manoncourt S, Abd el Kareem E, Mohamed Ahmed AN, El-Refaie S, Essmat H, Tjaden J, de Mattos CC, Earhart KC, Marfin AA, El-Sayed N. 2010. Zoonotic transmission of avian influenza virus (H5N1), Egypt, 2006–2009. Emerg. Infect. Dis. 16:1101–1107 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Oner AF, Bay A, Arslan S, Akdeniz H, Sahin HA, Cesur Y, Epcacan S, Yilmaz N, Deger I, Kizilyildiz B, Karsen H, Ceyhan M. 2006. Avian influenza A (H5N1) infection in eastern Turkey in 2006. N. Engl. J. Med. 355:2179–2185 [DOI] [PubMed] [Google Scholar]
17.Huang K, Bahl J, Fan XH, Vijaykrishna D, Cheung CL, Webby RJ, Webster RG, Chen H, Smith GJ, Peiris JS, Guan Y. 2010. Establishment of an H6N2 influenza virus lineage in domestic ducks in southern China. J. Virol. 84:6978–6986 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Huang K, Zhu H, Fan X, Wang J, Cheung CL, Duan L, Hong W, Liu Y, Li L, Smith DK, Chen H, Webster RG, Webby RJ, Peiris M, Guan Y. 2012. Establishment and lineage replacement of H6 influenza viruses in domestic ducks in southern China. J. Virol. 86:6075–6083 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Kim HR, Park CK, Lee YJ, Oem JK, Kang HM, Choi JG, Lee OS, Bae YC. 2012. Low pathogenic H7 subtype avian influenza viruses isolated from domestic ducks in South Korea and the close association with isolates of wild birds. J. Gen. Virol. 93:1278–1287 [DOI] [PubMed] [Google Scholar]
20.Li KS, Guan Y, Wang J, Smith GJ, Xu KM, Duan L, Rahardjo AP, Puthavathana P, Buranathai C, Nguyen TD, Estoepangestie AT, Chaisingh A, Auewarakul P, Long HT, Hanh NT, Webby RJ, Poon LL, Chen H, Shortridge KF, Yuen KY, Webster RG, Peiris JS. 2004. Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia. Nature 430:209–213 [DOI] [PubMed] [Google Scholar]
21.Van Boeckel TP, Prosser D, Franceschini G, Biradar C, Wint W, Robinson T, Gilbert M. 2011. Modelling the distribution of domestic ducks in monsoon Asia. Agric. Ecosyst. Environ. 141:373–380 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Chen H, Deng G, Li Z, Tian G, Li Y, Jiao P, Zhang L, Liu Z, Webster RG, Yu K. 2004. The evolution of H5N1 influenza viruses in ducks in southern China. Proc. Natl. Acad. Sci. U. S. A. 101:10452–10457 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Gu M, Liu W, Cao Y, Peng D, Wang X, Wan H, Zhao G, Xu Q, Zhang W, Song Q, Li Y, Liu X. 2011. Novel reassortant highly pathogenic avian influenza (H5N5) viruses in domestic ducks, China. Emerg. Infect. Dis. 17:1060–1063 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Hotta K, Takakuwa H, Le QM, Phuong SL, Murase T, Ono E, Ito T, Otsuki K, Yamashiro T. 2012. Isolation and characterization of H6N1 and H9N2 avian influenza viruses from ducks in Hanoi, Vietnam. Virus Res. 163:448–453 [DOI] [PubMed] [Google Scholar]
25.Kang HM, Choi JG, Kim KI, Park HY, Park CK, Lee YJ. 2013. Genetic and antigenic characteristics of H4 subtype avian influenza viruses in Korea and their pathogenicity in quails, domestic ducks and mice. J. Gen. Virol. 94:30–39 [DOI] [PubMed] [Google Scholar]
26.Kim HR, Lee YJ, Lee KK, Oem JK, Kim SH, Lee MH, Lee OS, Park CK. 2010. Genetic relatedness of H6 subtype avian influenza viruses isolated from wild birds and domestic ducks in Korea and their pathogenicity in animals. J. Gen. Virol. 91:208–219 [DOI] [PubMed] [Google Scholar]
27.Li C, Yu K, Tian G, Yu D, Liu L, Jing B, Ping J, Chen H. 2005. Evolution of H9N2 influenza viruses from domestic poultry in mainland China. Virology 340:70–83 [DOI] [PubMed] [Google Scholar]
28.Song J, Feng H, Xu J, Zhao D, Shi J, Li Y, Deng G, Jiang Y, Li X, Zhu P, Guan Y, Bu Z, Kawaoka Y, Chen H. 2011. The PA protein directly contributes to the virulence of H5N1 avian influenza viruses in domestic ducks. J. Virol. 85:2180–2188 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Sturm-Ramirez KM, Hulse-Post DJ, Govorkova EA, Humberd J, Seiler P, Puthavathana P, Buranathai C, Nguyen TD, Chaisingh A, Long HT, Naipospos TS, Chen H, Ellis TM, Guan Y, Peiris JS, Webster RG. 2005. Are ducks contributing to the endemicity of highly pathogenic H5N1 influenza virus in Asia? J. Virol. 79:11269–11279 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Kim JK, Seiler P, Forrest HL, Khalenkov AM, Franks J, Kumar M, Karesh WB, Gilbert M, Sodnomdarjaa R, Douangngeun B, Govorkova EA, Webster RG. 2008. Pathogenicity and vaccine efficacy of different clades of Asian H5N1 avian influenza A viruses in domestic ducks. J. Virol. 82:11374–11382 [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Li Y, Shi J, Zhong G, Deng G, Tian G, Ge J, Zeng X, Song J, Zhao D, Liu L, Jiang Y, Guan Y, Bu Z, Chen H. 2010. Continued evolution of H5N1 influenza viruses in wild birds, domestic poultry, and humans in China from 2004 to 2009. J. Virol. 84:8389–8397 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Songserm T, Jam-On R, Sae-Heng N, Meemak N, Hulse-Post DJ, Sturm-Ramirez KM, Webster RG. 2006. Domestic ducks and H5N1 influenza epidemic, Thailand. Emerg. Infect. Dis. 12:575–581 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Shortridge KF. 1992. Pandemic influenza: a zoonosis? Semin. Respir. Infect. 7:11–25 [PubMed] [Google Scholar]
34.Choi YK, Ozaki H, Webby RJ, Webster RG, Peiris JS, Poon L, Butt C, Leung YH, Guan Y. 2004. Continuing evolution of H9N2 influenza viruses in southeastern China. J. Virol. 78:8609–8614 [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Perez DR, Lim W, Seiler JP, Yi G, Peiris M, Shortridge KF, Webster RG. 2003. Role of quail in the interspecies transmission of H9 influenza A viruses: molecular changes on HA that correspond to adaptation from ducks to chickens. J. Virol. 77:3148–3156 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Zhong F, Wang Y, Deng X, Li L, Huang L, Jiang Y. 2007. Species diversity of rare, endangered and national key protected waterfowls in Dongting Lake wetlands. Ecol. Environ. 16:1485–1491 (In Chinese.) [Google Scholar]
37.Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR. 2001. Universal primer set for the full-length amplification of all influenza A viruses. Arch. Virol. 146:2275–2289 [DOI] [PubMed] [Google Scholar]
38.Page RD. 1996. TreeView: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12:357–358 [DOI] [PubMed] [Google Scholar]
39.Senne DA, Panigrahy B, Kawaoka Y, Pearson JE, Suss J, Lipkind M, Kida H, Webster RG. 1996. Survey of the hemagglutinin (HA) cleavage site sequence of H5 and H7 avian influenza viruses: amino acid sequence at the HA cleavage site as a marker of pathogenicity potential. Avian Dis. 40:425–437 [PubMed] [Google Scholar]
40.Subbarao K, Chen H, Swayne D, Mingay L, Fodor E, Brownlee G, Xu X, Lu X, Katz J, Cox N, Matsuoka Y. 2003. Evaluation of a genetically modified reassortant H5N1 influenza A virus vaccine candidate generated by plasmid-based reverse genetics. Virology 305:192–200 [DOI] [PubMed] [Google Scholar]
41.Tian G, Zhang S, Li Y, Bu Z, Liu P, Zhou J, Li C, Shi J, Yu K, Chen H. 2005. Protective efficacy in chickens, geese and ducks of an H5N1-inactivated vaccine developed by reverse genetics. Virology 341:153–162 [DOI] [PubMed] [Google Scholar]
42.Li Y, Liu L, Zhang Y, Duan Z, Tian G, Zeng X, Shi J, Zhang L, Chen H. 2011. New avian influenza virus (H5N1) in wild birds, Qinghai, China. Emerg. Infect. Dis. 17:265–267 [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Uchida Y, Mase M, Yoneda K, Kimura A, Obara T, Kumagai S, Saito T, Yamamoto Y, Nakamura K, Tsukamoto K, Yamaguchi S. 2008. Highly pathogenic avian influenza virus (H5N1) isolated from whooper swans, Japan. Emerg. Infect. Dis. 14:1427–1429 [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Jiang WM, Liu S, Chen J, Hou GY, Li JP, Cao YF, Zhuang QY, Li Y, Huang BX, Chen JM. 2010. Molecular epidemiological surveys of H5 subtype highly pathogenic avian influenza viruses in poultry in China during 2007–2009. J. Gen. Virol. 91:2491–2496 [DOI] [PubMed] [Google Scholar]
45.Kim HR, Lee YJ, Park CK, Oem JK, Lee OS, Kang HM, Choi JG, Bae YC. 2012. Highly pathogenic avian influenza (H5N1) outbreaks in wild birds and poultry, South Korea. Emerg. Infect. Dis. 18:480–483 [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Nagarajan S, Tosh C, Smith DK, Peiris JS, Murugkar HV, Sridevi R, Kumar M, Katare M, Jain R, Syed Z, Behera P, Cheung CL, Khandia R, Tripathi S, Guan Y, Dubey SC. 2012. Avian influenza (H5N1) virus of clade 2.3.2 in domestic poultry in India. PLoS One 7:e31844. 10.1371/journal.pone.0031844 [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Choi JG, Kang HM, Kim MC, Paek MR, Kim HR, Kim BS, Kwon JH, Kim JH, Lee YJ. 2012. Genetic relationship of H3 subtype avian influenza viruses isolated from domestic ducks and wild birds in Korea and their pathogenic potential in chickens and ducks. Vet. Microbiol. 155:147–157 [DOI] [PubMed] [Google Scholar]
48.Karasin AI, Brown IH, Carman S, Olsen CW. 2000. Isolation and characterization of H4N6 avian influenza viruses from pigs with pneumonia in Canada. J. Virol. 74:9322–9327 [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Sun Y, Pu J, Jiang Z, Guan T, Xia Y, Xu Q, Liu L, Ma B, Tian F, Brown EG, Liu J. 2010. Genotypic evolution and antigenic drift of H9N2 influenza viruses in China from 1994 to 2008. Vet. Microbiol. 146:215–225 [DOI] [PubMed] [Google Scholar]
50.Kim HR, Lee YJ, Oem JK, Bae YC, Kang MS, Kang HM, Choi JG, Park CK, Kwon YK. 2012. Characterization of H10 subtype avian influenza viruses isolated from wild birds in South Korea. Vet. Microbiol. 161:222–228 [DOI] [PubMed] [Google Scholar]
51.Karamendin K, Kydyrmanov A, Zhumatov K, Asanova S, Ishmukhametova N, Sayatov M. 2011. Phylogenetic analysis of avian influenza viruses of H11 subtype isolated in Kazakhstan. Virus Genes 43:46–54 [DOI] [PubMed] [Google Scholar]
52.Pawar S, Chakrabarti A, Cherian S, Pande S, Nanaware M, Raut S, Pal B, Jadhav S, Kode S, Koratkar S, Thite V, Mishra A. 2010. An avian influenza A(H11N1) virus from a wild aquatic bird revealing a unique Eurasian-American genetic reassortment. Virus Genes 41:14–22 [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Matsuoka Y, Swayne DE, Thomas C, Rameix-Welti MA, Naffakh N, Warnes C, Altholtz M, Donis R, Subbarao K. 2009. Neuraminidase stalk length and additional glycosylation of the hemagglutinin influence the virulence of influenza H5N1 viruses for mice. J. Virol. 83:4704–4708 [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Zhou H, Yu Z, Hu Y, Tu J, Zou W, Peng Y, Zhu J, Li Y, Zhang A, Ye Z, Chen H, Jin M. 2009. The special neuraminidase stalk-motif responsible for increased virulence and pathogenesis of H5N1 influenza A virus. PLoS One 4:e6277. 10.1371/journal.pone.0006277 [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Rimondi A, Xu K, Craig MI, Shao H, Ferreyra H, Rago MV, Romano M, Uhart M, Sutton T, Ferrero A, Perez DR, Pereda A. 2011. Phylogenetic analysis of H6 influenza viruses isolated from rosy-billed pochards (Netta peposaca) in Argentina reveals the presence of different HA gene clusters. J. Virol. 85:13354–13362 [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Shortridge KF. 1997. Is China an influenza epicentre? Chin. Med. J. (Engl.) 110:637–641 [PubMed] [Google Scholar]
57.Shortridge KF, Stuart-Harris CH. 1982. An influenza epicentre? Lancet ii:812–813 [DOI] [PubMed] [Google Scholar]
58.Cox NJ, Subbarao K. 2000. Global epidemiology of influenza: past and present. Annu. Rev. Med. 51:407–421 [DOI] [PubMed] [Google Scholar]
59.Choi YK, Seo SH, Kim JA, Webby RJ, Webster RG. 2005. Avian influenza viruses in Korean live poultry markets and their pathogenic potential. Virology 332:529–537 [DOI] [PubMed] [Google Scholar]
60.Jadhao SJ, Nguyen DC, Uyeki TM, Shaw M, Maines T, Rowe T, Smith C, Huynh LP, Nghiem HK, Nguyen DH, Nguyen HK, Nguyen HH, Hoang LT, Nguyen T, Phuong LS, Klimov A, Tumpey TM, Cox NJ, Donis RO, Matsuoka Y, Katz JM. 2009. Genetic analysis of avian influenza A viruses isolated from domestic waterfowl in live-bird markets of Hanoi, Vietnam, preceding fatal H5N1 human infections in 2004. Arch. Virol. 154:1249–1261 [DOI] [PubMed] [Google Scholar]
61.Kim JA, Cho SH, Kim HS, Seo SH. 2006. H9N2 influenza viruses isolated from poultry in Korean live bird markets continuously evolve and cause the severe clinical signs in layers. Vet. Microbiol. 118:169–176 [DOI] [PubMed] [Google Scholar]
62.Liu M, He S, Walker D, Zhou N, Perez DR, Mo B, Li F, Huang X, Webster RG, Webby RJ. 2003. The influenza virus gene pool in a poultry market in South Central China. Virology 305:267–275 [DOI] [PubMed] [Google Scholar]
63.Moon HJ, Song MS, Cruz DJ, Park KJ, Pascua PN, Lee JH, Baek YH, Choi DH, Choi YK, Kim CJ. 2010. Active reassortment of H9 influenza viruses between wild birds and live-poultry markets in Korea. Arch. Virol. 155:229–241 [DOI] [PubMed] [Google Scholar]
64.Nguyen DC, Uyeki TM, Jadhao S, Maines T, Shaw M, Matsuoka Y, Smith C, Rowe T, Lu X, Hall H, Xu X, Balish A, Klimov A, Tumpey TM, Swayne DE, Huynh LP, Nghiem HK, Nguyen HH, Hoang LT, Cox NJ, Katz JM. 2005. Isolation and characterization of avian influenza viruses, including highly pathogenic H5N1, from poultry in live bird markets in Hanoi, Vietnam, in 2001. J. Virol. 79:4201–4212 [DOI] [PMC free article] [PubMed] [Google Scholar]
65.Wan XF, Dong L, Lan Y, Long LP, Xu C, Zou S, Li Z, Wen L, Cai Z, Wang W, Li X, Yuan F, Sui H, Zhang Y, Dong J, Sun S, Gao Y, Wang M, Bai T, Yang L, Li D, Yang W, Yu H, Wang S, Feng Z, Wang Y, Guo Y, Webby RJ, Shu Y. 2011. Indications that live poultry markets are a major source of human H5N1 influenza virus infection in China. J. Virol. 85:13432–13438 [DOI] [PMC free article] [PubMed] [Google Scholar]
66.Xu X, Subbarao Cox NJ, Guo Y. 1999. Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96 (H5N1) virus: similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong. Virology 261:15–19 [DOI] [PubMed] [Google Scholar]
67.Claas EC, Osterhaus AD, van Beek R, De Jong JC, Rimmelzwaan GF, Senne DA, Krauss S, Shortridge KF, Webster RG. 1998. Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 351:472–477 [DOI] [PubMed] [Google Scholar]
68.Subbarao K, Klimov A, Katz J, Regnery H, Lim W, Hall H, Perdue M, Swayne D, Bender C, Huang J, Hemphill M, Rowe T, Shaw M, Xu X, Fukuda K, Cox N. 1998. Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. Science 279:393–396 [DOI] [PubMed] [Google Scholar]
69.Dugan VG, Chen R, Spiro DJ, Sengamalay N, Zaborsky J, Ghedin E, Nolting J, Swayne DE, Runstadler JA, Happ GM, Senne DA, Wang R, Slemons RD, Holmes EC, Taubenberger JK. 2008. The evolutionary genetics and emergence of avian influenza viruses in wild birds. PLoS Pathog. 4:e1000076. 10.1371/journal.ppat.1000076 [DOI] [PMC free article] [PubMed] [Google Scholar]
70.Hatchette TF, Walker D, Johnson C, Baker A, Pryor SP, Webster RG. 2004. Influenza A viruses in feral Canadian ducks: extensive reassortment in nature. J. Gen. Virol. 85:2327–2337 [DOI] [PubMed] [Google Scholar]
71.Macken CA, Webby RJ, Bruno WJ. 2006. Genotype turnover by reassortment of replication complex genes from avian influenza A virus. J. Gen. Virol. 87:2803–2815 [DOI] [PubMed] [Google Scholar]
72.Guan Y, Peiris M, Kong KF, Dyrting KC, Ellis TM, Sit T, Zhang LJ, Shortridge KF. 2002. H5N1 influenza viruses isolated from geese in southeastern China: evidence for genetic reassortment and interspecies transmission to ducks. Virology 292:16–23 [DOI] [PubMed] [Google Scholar]
73.Guan Y, Shortridge KF, Krauss S, Webster RG. 1999. Molecular characterization of H9N2 influenza viruses: were they the donors of the “internal” genes of H5N1 viruses in Hong Kong? Proc. Natl. Acad. Sci. U. S. A. 96:9363–9367 [DOI] [PMC free article] [PubMed] [Google Scholar]
74.Hoffmann E, Stech J, Leneva I, Krauss S, Scholtissek C, Chin PS, Peiris M, Shortridge KF, Webster RG. 2000. Characterization of the influenza A virus gene pool in avian species in southern China: was H6N1 a derivative or a precursor of H5N1? J. Virol. 74:6309–6315 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental material

supp_87_17_9452__index.html^{(1.1KB, html)}

JVI.00776-13_zjv999098007so1.pdf^{(101.2KB, pdf)}

JVI.00776-13_zjv999098007so2.pdf^{(441.7KB, pdf)}

[B1] 1.Hinshaw VS, Webster RG, Turner B. 1980. The perpetuation of orthomyxoviruses and paramyxoviruses in Canadian waterfowl. Can. J. Microbiol. 26:622–629 [DOI] [PubMed] [Google Scholar]

[B2] 2.Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y. 1992. Evolution and ecology of influenza A viruses. Microbiol. Rev. 56:152–179 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3.Tong S, Li Y, Rivailler P, Conrardy C, Castillo DA, Chen LM, Recuenco S, Ellison JA, Davis CT, York IA, Turmelle AS, Moran D, Rogers S, Shi M, Tao Y, Weil MR, Tang K, Rowe LA, Sammons S, Xu X, Frace M, Lindblade KA, Cox NJ, Anderson LJ, Rupprecht CE, Donis RO. 2012. A distinct lineage of influenza A virus from bats. Proc. Natl. Acad. Sci. U. S. A. 109:4269–4274 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4.Piaggio AJ, Shriner SA, VanDalen KK, Franklin AB, Anderson TD, Kolokotronis SO. 2012. Molecular surveillance of low pathogenic avian influenza viruses in wild birds across the United States: inferences from the hemagglutinin gene. PLoS One 7:e50834. 10.1371/journal.pone.0050834 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5.Chen H, Li Y, Li Z, Shi J, Shinya K, Deng G, Qi Q, Tian G, Fan S, Zhao H, Sun Y, Kawaoka Y. 2006. Properties and dissemination of H5N1 viruses isolated during an influenza outbreak in migratory waterfowl in western China. J. Virol. 80:5976–5983 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6.Liu J, Xiao H, Lei F, Zhu Q, Qin K, Zhang XW, Zhang XL, Zhao D, Wang G, Feng Y, Ma J, Liu W, Wang J, Gao GF. 2005. Highly pathogenic H5N1 influenza virus infection in migratory birds. Science 309:1206. [DOI] [PubMed] [Google Scholar]

[B7] 7.Fauci AS. 2006. Pandemic influenza threat and preparedness. Emerg. Infect. Dis. 12:73–77 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8.Fusaro A, Nelson MI, Joannis T, Bertolotti L, Monne I, Salviato A, Olaleye O, Shittu I, Sulaiman L, Lombin LH, Capua I, Holmes EC, Cattoli G. 2010. Evolutionary dynamics of multiple sublineages of H5N1 influenza viruses in Nigeria from 2006 to 2008. J. Virol. 84:3239–3247 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9.Kilpatrick AM, Chmura AA, Gibbons DW, Fleischer RC, Marra PP, Daszak P. 2006. Predicting the global spread of H5N1 avian influenza. Proc. Natl. Acad. Sci. U. S. A. 103:19368–19373 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10.Lee YJ, Choi YK, Kim YJ, Song MS, Jeong OM, Lee EK, Jeon WJ, Jeong W, Joh SJ, Choi KS, Her M, Kim MC, Kim A, Kim MJ, Ho Lee E, Oh TG, Moon HJ, Yoo DW, Kim JH, Sung MH, Poo H, Kwon JH, Kim CJ. 2008. Highly pathogenic avian influenza virus (H5N1) in domestic poultry and relationship with migratory birds, South Korea. Emerg. Infect. Dis. 14:487–490 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11.Lipatov AS, Evseenko VA, Yen HL, Zaykovskaya AV, Durimanov AG, Zolotykh SI, Netesov SV, Drozdov IG, Onishchenko GG, Webster RG, Shestopalov AM. 2007. Influenza (H5N1) viruses in poultry, Russian Federation, 2005–2006. Emerg. Infect. Dis. 13:539–546 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12.Salzberg SL, Kingsford C, Cattoli G, Spiro DJ, Janies DA, Aly MM, Brown IH, Couacy-Hymann E, De Mia GM, Dung Do H, Guercio A, Joannis T, Maken Ali AS, Osmani A, Padalino I, Saad MD, Savic V, Sengamalay NA, Yingst S, Zaborsky J, Zorman-Rojs O, Ghedin E, Capua I. 2007. Genome analysis linking recent European and African influenza (H5N1) viruses. Emerg. Infect. Dis. 13:713–718 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13.Shestopalov AM, Durimanov AG, Evseenko VA, Ternovoi VA, Rassadkin YN, Razumova YV, Zaykovskaya AV, Zolotykh SI, Netesov SV. 2006. H5N1 influenza virus, domestic birds, western Siberia, Russia. Emerg. Infect. Dis. 12:1167–1169 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14.Watanabe Y, Ibrahim MS, Ellakany HF, Kawashita N, Mizuike R, Hiramatsu H, Sriwilaijaroen N, Takagi T, Suzuki Y, Ikuta K. 2011. Acquisition of human-type receptor binding specificity by new H5N1 influenza virus sublineages during their emergence in birds in Egypt. PLoS Pathog. 7:e1002068. 10.1371/journal.ppat.1002068 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15.Kandeel A, Manoncourt S, Abd el Kareem E, Mohamed Ahmed AN, El-Refaie S, Essmat H, Tjaden J, de Mattos CC, Earhart KC, Marfin AA, El-Sayed N. 2010. Zoonotic transmission of avian influenza virus (H5N1), Egypt, 2006–2009. Emerg. Infect. Dis. 16:1101–1107 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16.Oner AF, Bay A, Arslan S, Akdeniz H, Sahin HA, Cesur Y, Epcacan S, Yilmaz N, Deger I, Kizilyildiz B, Karsen H, Ceyhan M. 2006. Avian influenza A (H5N1) infection in eastern Turkey in 2006. N. Engl. J. Med. 355:2179–2185 [DOI] [PubMed] [Google Scholar]

[B17] 17.Huang K, Bahl J, Fan XH, Vijaykrishna D, Cheung CL, Webby RJ, Webster RG, Chen H, Smith GJ, Peiris JS, Guan Y. 2010. Establishment of an H6N2 influenza virus lineage in domestic ducks in southern China. J. Virol. 84:6978–6986 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B18] 18.Huang K, Zhu H, Fan X, Wang J, Cheung CL, Duan L, Hong W, Liu Y, Li L, Smith DK, Chen H, Webster RG, Webby RJ, Peiris M, Guan Y. 2012. Establishment and lineage replacement of H6 influenza viruses in domestic ducks in southern China. J. Virol. 86:6075–6083 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B19] 19.Kim HR, Park CK, Lee YJ, Oem JK, Kang HM, Choi JG, Lee OS, Bae YC. 2012. Low pathogenic H7 subtype avian influenza viruses isolated from domestic ducks in South Korea and the close association with isolates of wild birds. J. Gen. Virol. 93:1278–1287 [DOI] [PubMed] [Google Scholar]

[B20] 20.Li KS, Guan Y, Wang J, Smith GJ, Xu KM, Duan L, Rahardjo AP, Puthavathana P, Buranathai C, Nguyen TD, Estoepangestie AT, Chaisingh A, Auewarakul P, Long HT, Hanh NT, Webby RJ, Poon LL, Chen H, Shortridge KF, Yuen KY, Webster RG, Peiris JS. 2004. Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia. Nature 430:209–213 [DOI] [PubMed] [Google Scholar]

[B21] 21.Van Boeckel TP, Prosser D, Franceschini G, Biradar C, Wint W, Robinson T, Gilbert M. 2011. Modelling the distribution of domestic ducks in monsoon Asia. Agric. Ecosyst. Environ. 141:373–380 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22.Chen H, Deng G, Li Z, Tian G, Li Y, Jiao P, Zhang L, Liu Z, Webster RG, Yu K. 2004. The evolution of H5N1 influenza viruses in ducks in southern China. Proc. Natl. Acad. Sci. U. S. A. 101:10452–10457 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B23] 23.Gu M, Liu W, Cao Y, Peng D, Wang X, Wan H, Zhao G, Xu Q, Zhang W, Song Q, Li Y, Liu X. 2011. Novel reassortant highly pathogenic avian influenza (H5N5) viruses in domestic ducks, China. Emerg. Infect. Dis. 17:1060–1063 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24.Hotta K, Takakuwa H, Le QM, Phuong SL, Murase T, Ono E, Ito T, Otsuki K, Yamashiro T. 2012. Isolation and characterization of H6N1 and H9N2 avian influenza viruses from ducks in Hanoi, Vietnam. Virus Res. 163:448–453 [DOI] [PubMed] [Google Scholar]

[B25] 25.Kang HM, Choi JG, Kim KI, Park HY, Park CK, Lee YJ. 2013. Genetic and antigenic characteristics of H4 subtype avian influenza viruses in Korea and their pathogenicity in quails, domestic ducks and mice. J. Gen. Virol. 94:30–39 [DOI] [PubMed] [Google Scholar]

[B26] 26.Kim HR, Lee YJ, Lee KK, Oem JK, Kim SH, Lee MH, Lee OS, Park CK. 2010. Genetic relatedness of H6 subtype avian influenza viruses isolated from wild birds and domestic ducks in Korea and their pathogenicity in animals. J. Gen. Virol. 91:208–219 [DOI] [PubMed] [Google Scholar]

[B27] 27.Li C, Yu K, Tian G, Yu D, Liu L, Jing B, Ping J, Chen H. 2005. Evolution of H9N2 influenza viruses from domestic poultry in mainland China. Virology 340:70–83 [DOI] [PubMed] [Google Scholar]

[B28] 28.Song J, Feng H, Xu J, Zhao D, Shi J, Li Y, Deng G, Jiang Y, Li X, Zhu P, Guan Y, Bu Z, Kawaoka Y, Chen H. 2011. The PA protein directly contributes to the virulence of H5N1 avian influenza viruses in domestic ducks. J. Virol. 85:2180–2188 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29.Sturm-Ramirez KM, Hulse-Post DJ, Govorkova EA, Humberd J, Seiler P, Puthavathana P, Buranathai C, Nguyen TD, Chaisingh A, Long HT, Naipospos TS, Chen H, Ellis TM, Guan Y, Peiris JS, Webster RG. 2005. Are ducks contributing to the endemicity of highly pathogenic H5N1 influenza virus in Asia? J. Virol. 79:11269–11279 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30.Kim JK, Seiler P, Forrest HL, Khalenkov AM, Franks J, Kumar M, Karesh WB, Gilbert M, Sodnomdarjaa R, Douangngeun B, Govorkova EA, Webster RG. 2008. Pathogenicity and vaccine efficacy of different clades of Asian H5N1 avian influenza A viruses in domestic ducks. J. Virol. 82:11374–11382 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B31] 31.Li Y, Shi J, Zhong G, Deng G, Tian G, Ge J, Zeng X, Song J, Zhao D, Liu L, Jiang Y, Guan Y, Bu Z, Chen H. 2010. Continued evolution of H5N1 influenza viruses in wild birds, domestic poultry, and humans in China from 2004 to 2009. J. Virol. 84:8389–8397 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B32] 32.Songserm T, Jam-On R, Sae-Heng N, Meemak N, Hulse-Post DJ, Sturm-Ramirez KM, Webster RG. 2006. Domestic ducks and H5N1 influenza epidemic, Thailand. Emerg. Infect. Dis. 12:575–581 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33.Shortridge KF. 1992. Pandemic influenza: a zoonosis? Semin. Respir. Infect. 7:11–25 [PubMed] [Google Scholar]

[B34] 34.Choi YK, Ozaki H, Webby RJ, Webster RG, Peiris JS, Poon L, Butt C, Leung YH, Guan Y. 2004. Continuing evolution of H9N2 influenza viruses in southeastern China. J. Virol. 78:8609–8614 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B35] 35.Perez DR, Lim W, Seiler JP, Yi G, Peiris M, Shortridge KF, Webster RG. 2003. Role of quail in the interspecies transmission of H9 influenza A viruses: molecular changes on HA that correspond to adaptation from ducks to chickens. J. Virol. 77:3148–3156 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B36] 36.Zhong F, Wang Y, Deng X, Li L, Huang L, Jiang Y. 2007. Species diversity of rare, endangered and national key protected waterfowls in Dongting Lake wetlands. Ecol. Environ. 16:1485–1491 (In Chinese.) [Google Scholar]

[B37] 37.Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR. 2001. Universal primer set for the full-length amplification of all influenza A viruses. Arch. Virol. 146:2275–2289 [DOI] [PubMed] [Google Scholar]

[B38] 38.Page RD. 1996. TreeView: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12:357–358 [DOI] [PubMed] [Google Scholar]

[B39] 39.Senne DA, Panigrahy B, Kawaoka Y, Pearson JE, Suss J, Lipkind M, Kida H, Webster RG. 1996. Survey of the hemagglutinin (HA) cleavage site sequence of H5 and H7 avian influenza viruses: amino acid sequence at the HA cleavage site as a marker of pathogenicity potential. Avian Dis. 40:425–437 [PubMed] [Google Scholar]

[B40] 40.Subbarao K, Chen H, Swayne D, Mingay L, Fodor E, Brownlee G, Xu X, Lu X, Katz J, Cox N, Matsuoka Y. 2003. Evaluation of a genetically modified reassortant H5N1 influenza A virus vaccine candidate generated by plasmid-based reverse genetics. Virology 305:192–200 [DOI] [PubMed] [Google Scholar]

[B41] 41.Tian G, Zhang S, Li Y, Bu Z, Liu P, Zhou J, Li C, Shi J, Yu K, Chen H. 2005. Protective efficacy in chickens, geese and ducks of an H5N1-inactivated vaccine developed by reverse genetics. Virology 341:153–162 [DOI] [PubMed] [Google Scholar]

[B42] 42.Li Y, Liu L, Zhang Y, Duan Z, Tian G, Zeng X, Shi J, Zhang L, Chen H. 2011. New avian influenza virus (H5N1) in wild birds, Qinghai, China. Emerg. Infect. Dis. 17:265–267 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B43] 43.Uchida Y, Mase M, Yoneda K, Kimura A, Obara T, Kumagai S, Saito T, Yamamoto Y, Nakamura K, Tsukamoto K, Yamaguchi S. 2008. Highly pathogenic avian influenza virus (H5N1) isolated from whooper swans, Japan. Emerg. Infect. Dis. 14:1427–1429 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B44] 44.Jiang WM, Liu S, Chen J, Hou GY, Li JP, Cao YF, Zhuang QY, Li Y, Huang BX, Chen JM. 2010. Molecular epidemiological surveys of H5 subtype highly pathogenic avian influenza viruses in poultry in China during 2007–2009. J. Gen. Virol. 91:2491–2496 [DOI] [PubMed] [Google Scholar]

[B45] 45.Kim HR, Lee YJ, Park CK, Oem JK, Lee OS, Kang HM, Choi JG, Bae YC. 2012. Highly pathogenic avian influenza (H5N1) outbreaks in wild birds and poultry, South Korea. Emerg. Infect. Dis. 18:480–483 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B46] 46.Nagarajan S, Tosh C, Smith DK, Peiris JS, Murugkar HV, Sridevi R, Kumar M, Katare M, Jain R, Syed Z, Behera P, Cheung CL, Khandia R, Tripathi S, Guan Y, Dubey SC. 2012. Avian influenza (H5N1) virus of clade 2.3.2 in domestic poultry in India. PLoS One 7:e31844. 10.1371/journal.pone.0031844 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B47] 47.Choi JG, Kang HM, Kim MC, Paek MR, Kim HR, Kim BS, Kwon JH, Kim JH, Lee YJ. 2012. Genetic relationship of H3 subtype avian influenza viruses isolated from domestic ducks and wild birds in Korea and their pathogenic potential in chickens and ducks. Vet. Microbiol. 155:147–157 [DOI] [PubMed] [Google Scholar]

[B48] 48.Karasin AI, Brown IH, Carman S, Olsen CW. 2000. Isolation and characterization of H4N6 avian influenza viruses from pigs with pneumonia in Canada. J. Virol. 74:9322–9327 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B49] 49.Sun Y, Pu J, Jiang Z, Guan T, Xia Y, Xu Q, Liu L, Ma B, Tian F, Brown EG, Liu J. 2010. Genotypic evolution and antigenic drift of H9N2 influenza viruses in China from 1994 to 2008. Vet. Microbiol. 146:215–225 [DOI] [PubMed] [Google Scholar]

[B50] 50.Kim HR, Lee YJ, Oem JK, Bae YC, Kang MS, Kang HM, Choi JG, Park CK, Kwon YK. 2012. Characterization of H10 subtype avian influenza viruses isolated from wild birds in South Korea. Vet. Microbiol. 161:222–228 [DOI] [PubMed] [Google Scholar]

[B51] 51.Karamendin K, Kydyrmanov A, Zhumatov K, Asanova S, Ishmukhametova N, Sayatov M. 2011. Phylogenetic analysis of avian influenza viruses of H11 subtype isolated in Kazakhstan. Virus Genes 43:46–54 [DOI] [PubMed] [Google Scholar]

[B52] 52.Pawar S, Chakrabarti A, Cherian S, Pande S, Nanaware M, Raut S, Pal B, Jadhav S, Kode S, Koratkar S, Thite V, Mishra A. 2010. An avian influenza A(H11N1) virus from a wild aquatic bird revealing a unique Eurasian-American genetic reassortment. Virus Genes 41:14–22 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B53] 53.Matsuoka Y, Swayne DE, Thomas C, Rameix-Welti MA, Naffakh N, Warnes C, Altholtz M, Donis R, Subbarao K. 2009. Neuraminidase stalk length and additional glycosylation of the hemagglutinin influence the virulence of influenza H5N1 viruses for mice. J. Virol. 83:4704–4708 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B54] 54.Zhou H, Yu Z, Hu Y, Tu J, Zou W, Peng Y, Zhu J, Li Y, Zhang A, Ye Z, Chen H, Jin M. 2009. The special neuraminidase stalk-motif responsible for increased virulence and pathogenesis of H5N1 influenza A virus. PLoS One 4:e6277. 10.1371/journal.pone.0006277 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B55] 55.Rimondi A, Xu K, Craig MI, Shao H, Ferreyra H, Rago MV, Romano M, Uhart M, Sutton T, Ferrero A, Perez DR, Pereda A. 2011. Phylogenetic analysis of H6 influenza viruses isolated from rosy-billed pochards (Netta peposaca) in Argentina reveals the presence of different HA gene clusters. J. Virol. 85:13354–13362 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B56] 56.Shortridge KF. 1997. Is China an influenza epicentre? Chin. Med. J. (Engl.) 110:637–641 [PubMed] [Google Scholar]

[B57] 57.Shortridge KF, Stuart-Harris CH. 1982. An influenza epicentre? Lancet ii:812–813 [DOI] [PubMed] [Google Scholar]

[B58] 58.Cox NJ, Subbarao K. 2000. Global epidemiology of influenza: past and present. Annu. Rev. Med. 51:407–421 [DOI] [PubMed] [Google Scholar]

[B59] 59.Choi YK, Seo SH, Kim JA, Webby RJ, Webster RG. 2005. Avian influenza viruses in Korean live poultry markets and their pathogenic potential. Virology 332:529–537 [DOI] [PubMed] [Google Scholar]

[B60] 60.Jadhao SJ, Nguyen DC, Uyeki TM, Shaw M, Maines T, Rowe T, Smith C, Huynh LP, Nghiem HK, Nguyen DH, Nguyen HK, Nguyen HH, Hoang LT, Nguyen T, Phuong LS, Klimov A, Tumpey TM, Cox NJ, Donis RO, Matsuoka Y, Katz JM. 2009. Genetic analysis of avian influenza A viruses isolated from domestic waterfowl in live-bird markets of Hanoi, Vietnam, preceding fatal H5N1 human infections in 2004. Arch. Virol. 154:1249–1261 [DOI] [PubMed] [Google Scholar]

[B61] 61.Kim JA, Cho SH, Kim HS, Seo SH. 2006. H9N2 influenza viruses isolated from poultry in Korean live bird markets continuously evolve and cause the severe clinical signs in layers. Vet. Microbiol. 118:169–176 [DOI] [PubMed] [Google Scholar]

[B62] 62.Liu M, He S, Walker D, Zhou N, Perez DR, Mo B, Li F, Huang X, Webster RG, Webby RJ. 2003. The influenza virus gene pool in a poultry market in South Central China. Virology 305:267–275 [DOI] [PubMed] [Google Scholar]

[B63] 63.Moon HJ, Song MS, Cruz DJ, Park KJ, Pascua PN, Lee JH, Baek YH, Choi DH, Choi YK, Kim CJ. 2010. Active reassortment of H9 influenza viruses between wild birds and live-poultry markets in Korea. Arch. Virol. 155:229–241 [DOI] [PubMed] [Google Scholar]

[B64] 64.Nguyen DC, Uyeki TM, Jadhao S, Maines T, Shaw M, Matsuoka Y, Smith C, Rowe T, Lu X, Hall H, Xu X, Balish A, Klimov A, Tumpey TM, Swayne DE, Huynh LP, Nghiem HK, Nguyen HH, Hoang LT, Cox NJ, Katz JM. 2005. Isolation and characterization of avian influenza viruses, including highly pathogenic H5N1, from poultry in live bird markets in Hanoi, Vietnam, in 2001. J. Virol. 79:4201–4212 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B65] 65.Wan XF, Dong L, Lan Y, Long LP, Xu C, Zou S, Li Z, Wen L, Cai Z, Wang W, Li X, Yuan F, Sui H, Zhang Y, Dong J, Sun S, Gao Y, Wang M, Bai T, Yang L, Li D, Yang W, Yu H, Wang S, Feng Z, Wang Y, Guo Y, Webby RJ, Shu Y. 2011. Indications that live poultry markets are a major source of human H5N1 influenza virus infection in China. J. Virol. 85:13432–13438 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B66] 66.Xu X, Subbarao Cox NJ, Guo Y. 1999. Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96 (H5N1) virus: similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong. Virology 261:15–19 [DOI] [PubMed] [Google Scholar]

[B67] 67.Claas EC, Osterhaus AD, van Beek R, De Jong JC, Rimmelzwaan GF, Senne DA, Krauss S, Shortridge KF, Webster RG. 1998. Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 351:472–477 [DOI] [PubMed] [Google Scholar]

[B68] 68.Subbarao K, Klimov A, Katz J, Regnery H, Lim W, Hall H, Perdue M, Swayne D, Bender C, Huang J, Hemphill M, Rowe T, Shaw M, Xu X, Fukuda K, Cox N. 1998. Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. Science 279:393–396 [DOI] [PubMed] [Google Scholar]

[B69] 69.Dugan VG, Chen R, Spiro DJ, Sengamalay N, Zaborsky J, Ghedin E, Nolting J, Swayne DE, Runstadler JA, Happ GM, Senne DA, Wang R, Slemons RD, Holmes EC, Taubenberger JK. 2008. The evolutionary genetics and emergence of avian influenza viruses in wild birds. PLoS Pathog. 4:e1000076. 10.1371/journal.ppat.1000076 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B70] 70.Hatchette TF, Walker D, Johnson C, Baker A, Pryor SP, Webster RG. 2004. Influenza A viruses in feral Canadian ducks: extensive reassortment in nature. J. Gen. Virol. 85:2327–2337 [DOI] [PubMed] [Google Scholar]

[B71] 71.Macken CA, Webby RJ, Bruno WJ. 2006. Genotype turnover by reassortment of replication complex genes from avian influenza A virus. J. Gen. Virol. 87:2803–2815 [DOI] [PubMed] [Google Scholar]

[B72] 72.Guan Y, Peiris M, Kong KF, Dyrting KC, Ellis TM, Sit T, Zhang LJ, Shortridge KF. 2002. H5N1 influenza viruses isolated from geese in southeastern China: evidence for genetic reassortment and interspecies transmission to ducks. Virology 292:16–23 [DOI] [PubMed] [Google Scholar]

[B73] 73.Guan Y, Shortridge KF, Krauss S, Webster RG. 1999. Molecular characterization of H9N2 influenza viruses: were they the donors of the “internal” genes of H5N1 viruses in Hong Kong? Proc. Natl. Acad. Sci. U. S. A. 96:9363–9367 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B74] 74.Hoffmann E, Stech J, Leneva I, Krauss S, Scholtissek C, Chin PS, Peiris M, Shortridge KF, Webster RG. 2000. Characterization of the influenza A virus gene pool in avian species in southern China: was H6N1 a derivative or a precursor of H5N1? J. Virol. 74:6309–6315 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Complex Reassortment of Multiple Subtypes of Avian Influenza Viruses in Domestic Ducks at the Dongting Lake Region of China

Guohua Deng

Dan Tan

Jianzhong Shi

Pengfei Cui

Yongping Jiang

Liling Liu

Guobin Tian

Yoshihiro Kawaoka

Chengjun Li

Hualan Chen

Abstract

INTRODUCTION

MATERIALS AND METHODS

Sample collection and virus isolation.

HA subtyping of isolates.

Genetic and phylogenetic analyses.

Nucleotide sequence accession numbers.

RESULTS

Geographic location of sampling sites.

Fig 1.

Table 1.

Genetic and phylogenetic analyses of HA genes.

Table 2.

Fig 2.

Genetic and phylogenetic analyses of NA genes.

Fig 3.

Phylogenetic analysis of internal genes.

Fig 4.

DISCUSSION

Supplementary Material

ACKNOWLEDGMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases