Abstract
The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are GQ414775[GenBank] –GQ414974.
Supplementary figures showing phylogenetic analysis of Korean H6 influenza viruses isolated from wild birds and poultry are available with the online version of this paper.
H6 subtypes of influenza viruses are the most abundantly detected influenza virus subtype, and they have a broader host range than any other subtype (Munster et al., 2007; Spackman et al., 2005). A/teal/Hong Kong/W312/97(W312/97) (H6N1), a virus isolated from a duck at a live poultry market (LPM) in Hong Kong, was identified as a potential precursor to the A/Hong Kong/156/97 (HK/97) H5N1 virus, as seven of eight gene segments of the two viruses have a common source (Hoffmann et al., 2000). H6 viruses continue to circulate worldwide in areas including North America and South Africa (Abolnik et al., 2007; Hatchette et al., 2004; Spackman et al., 2005; Winker et al., 2007; Woolcock et al., 2003), as well as in Asian countries including southern China and Taiwan (Cheung et al., 2007; Lee et al., 2006). In addition, an invasion of Eurasian H6 viruses into North America that were found to replace American H6 clades has recently been reported (zu Dohna et al., 2009).
Moreover, some H6 viruses have the ability to replicate to high titres in mammals without prior adaptation (Gillim-Ross et al., 2008), and H6 viruses isolated from waterfowl have been shown to be able to infect humans by experimental inoculation (Beare & Webster, 1991). These facts raise the possibility that the H6 viruses have the potential to expand their host range into mammals, including humans. Also, recent studies reporting the detection of positive antibody to H6 avian influenza (AI) virus in humans could provide evidence of zoonotic aspects of avian H6 viruses (Kayali et al., 2009; Myers et al., 2007).
In Korea, H6 subtypes were largely not reported, with the exception of one virus [duck/Korea/S17/2003 (H6N1); Dk/Kr/S17/03] isolated from an LPM in Korea in 2003 (Choi et al., 2005). In this study, we report the abundant isolation of H6 AI viruses (42 isolates) from domestic poultry, LPMs and wild birds in Korea between April 2008 and April 2009, as the result of a systematic surveillance programme. We performed a phylogenetic analysis of the H6 viruses to investigate their relationship with those from domestic poultry farms, LPMs and wild birds. In addition, animal experiments were performed to elucidate their pathogenicity, which was dependent upon their genetic characterization based on the phylogenetic results.
Virus isolation and selectionAll viruses were isolated by the NVRQS and local veterinary services through active surveillance programmes from April 2008 to April 2009. In total, 42 H6 influenza viruses were isolated: 10 from oropharyngeal and cloacal swabs of migratory birds, 24 from faeces of domestic ducks, five from oropharyngeal swabs and faeces of poultry at LPMs and three from faeces of wild birds. Three NA subtypes of H6 AI virus, H6N1 (28.6 %), H6N2 (69.0 %) and H6N8 (2.4 %), were found. We selected 25 H6 viruses for genetic characterization and tested eight of those viruses to investigate their pathogenic potential in chickens and mice, based on the genotyping results (Table 1).
Table 1. H6 AI viruses
Phylogenetic analyses of surface genes
A phylogenetic analysis of the HA gene showed that the 25 H6 viruses used in this study were clustered in the Eurasian gene pool and could be separated into three groups: A (n=11), B (n=13) and C (n=1) (Fig. 1). Group A viruses had high similarity (>99 %) within the same lineage, H6 AI viruses classified in group B were generally related to previous Dk/Kr/S17/03-like viruses (94–99 % similarity), and the HA gene of group C viruses belonged to the same lineage as that of Dk/Kingmen/E322/04 (93 % similarity). These data suggest that most of the isolates from domestic ducks were clustered with group A, and most of the viruses of wild birds were close to group B. Although some viruses from domestic ducks are related to group B, most H6N2 viruses in domestic ducks originated from H6 AI viruses of unknown aquatic birds.
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Three different NA subtypes were detected among the 25 viruses: N1 (n=11), N2 (n=13) and N8 (n=1). In the NA gene tree, all of the N1 genes from the tested H6N1 viruses were related to previous Dk/Kr/S17/03 (H6N1) viruses isolated from LPMs in 2003, but different from the N1 genes of H5N1 viruses such as Gs/GD/1/96 (H5N1) (Fig. 2). The N2 genes of the tested H6N2 viruses were related closely to the Korean H3N2 virus isolated from LPMs, with the exception of SBD/Kr/619/08 (Fig. 3). Therefore, most of the H6N2 viruses from domestic ducks were generated by reassortment between H3N2 from domestic ducks and H6 AI viruses of unknown aquatic birds. The N8 gene of SBD/Kr/528/08 (H6N8) was similar to that of swan/Shimane/42/99 (H7N8) and Dk/Nanchang/1681/92(H3N8) (data not shown).
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Phylogenetic analyses of internal genes
A phylogenetic analysis of polymerase basic protein 2 (PB2) demonstrated that most of the H6N1 viruses and one H6N2 virus from domestic ducks were similar to the previous Korean H5N1 viruses (Ck/Kr/ES/03 and Ck/Kr/IS/06), and most of the H6N2 viruses from domestic ducks clustered with the Ck/Kr/96006/96-like and Dk/Kr/S8/03-like domestic poultry lineage [see Supplementary Fig. S1(a), available in JGV Online]. The polymerase basic protein 1 (PB1) gene of the H6 viruses of wild birds and two H6N1 viruses of domestic ducks clustered with the Dk/Kr/S17/03-like wild bird virus group, but the PB1 gene of the H6N2 viruses of domestic ducks, except for Dk/Kr/412-4/08, was related to that of the Ck/Kr/96006/96-like Korean lineage [see Supplementary Fig. S1(b), available in JGV Online]. In the polymerase acidic protein (PA) phylogeny, most of the H6 viruses from wild birds and two viruses from domestic ducks belonged to the Dk/Kr/S17/03 (H6N1)-like group, whereas most domestic viruses were related to mDk/HK/MP2437/03 (H6N1). The PA gene of Dk/Korea/460-5/08 was only similar to the Dk/Kr/LPM91/06 (H3N2) virus isolated from an LPM [see Supplementary Fig. S1(c), available in JGV Online]. The nucleoprotein (NP) genes were separated into four distinct lineages: the Ck/Kr/96006/96-like Korean lineage, the H3N2 Korean LPM lineage and two different wild bird lineages [see Supplementary Fig. S2(a), available in JGV Online]. A phylogenetic analysis of the matrix (M) and non-structural (NS) genes showed that most of the H6N1 viruses of wild birds clustered with the Dk/Kr/S17/03-like lineage and most of the H6N2 viruses of domestic ducks belonged to the Ck/Kr/96006/96-like Korean lineage. However, the M genes of isolates from wild birds [SBD/Kr/625/08 (H6N1) and SBD/Kr/619/08 (H6N2)] were similar to those of the H6 viruses circulating in Hong Kong [see Supplementary Fig. S2(b, c), available in JGV Online].
The NS gene of SBD/Kr/625/08 belonged to a different NS allele (allele B) that was distinct from the others. Two distinct alleles of the NS gene are present in avian influenza viruses (Scholtissek & von Hoyningen-Huene, 1980; Treanor et al., 1989). Although influenza viruses containing the B allele of the NS gene do not differ in their geographical location, host or year of isolation from viruses that have the other allele of the NS gene, they do not replicate well in mammalian hosts (Kawaoka et al., 1998; Webby et al., 2002).
Molecular characterization
All viruses tested had the identified H6 amino acid motif (PQIETRG) at the HA cleavage site, and glutamine (Q) and glycine (G) at positions 224 and 226 of the HA1, respectively; these bind to the α-2,3NeuAcGal receptors (Ha et al., 2001). No viruses in this study had a glutamic acid (E) 92 substitution in the NS1 protein, which is related to the ability of HK/97 viruses to escape the host antiviral cytokine response (Seo et al., 2002). The Korean H6N1/N2 viruses had no deletions in their NA stalk regions, a trait that is typical of an adaptation to poultry (Matrosovich et al., 1999). All of the H6 viruses studied retained a glutamic acid (E) at aa 627 in the PB2 protein; they did not contain lysine (K), which has been associated with increased virulence in mice (Hatta et al., 2001). Only the Dk/Kr/A127/09 (H6N2) and SBD/Kr/619/08 (H6N2) isolates of H6 viruses isolated in Korea had an isoleucine (I) 27 substitution in the M2 gene, which can lead to amantadine resistance (Belshe et al., 1988).
Genotypes
The genetic diversity of the H6 viruses was recognized based on a phylogenetic analysis of eight genes of each virus, including virus isolates of both domestic poultry and wild birds. Genotypes K-1 and K-2a were identified as the predominant genotypes in the H6 AI viruses of wild birds and domestic ducks, respectively. Most of the H6N1 viruses of wild birds and two H6N1 viruses of domestic ducks were of the same genotype (K-1). The gene segments of genotype K-1 were similar to those of Dk/Kr/S17/03 (H6N1), with the exception of the PB2 and NP genes. Seven gene segments of the SBD/Kr/528/08 (H6N8) (genotype WB-1) virus were related closely to those of the H6N1 viruses tested, except for the NA gene. Dk/Kr/625/08 was identified as genotype WB-2, as it consisted of PB1, HA and NA genes that were the same as those of genotype K-1 and the other genes of viruses from aquatic birds. SBD/Kr/619/08 (H6N2) was designated genotype WB-3, which was generated by combination of the NP and NA genes from the Korean H3N2 and H9N2 viruses and the PB2 and PB1 segments from genotype K-1 viruses. On the other hand, we identified four distinct genotypes in the H6N2 subtypes of domestic ducks, designated genotypes K-2a, 2b, 2c and 2d. These genotypes had been created by reassortments with wild bird viruses and circulating Korean poultry H3N2 or H9N2 viruses (Fig. 4). These results showed that H6 AI viruses in the wild birds were derived from a variety of gene pools, although genotype K-1 was identified as the major isolate. The H6 viruses from wild birds were transmitted directly to domestic poultry or generated novel genotypes, as the result of multiple reassortments with H9N2 and H3N2 viruses that circulate substantially in domestic poultry farms and LPMs. In particular, H6N2 viruses in domestic ducks seem to have been created by at least a triple reassortment between the other unknown AI viruses of aquatic birds and the Korean H3N2 viruses, which have previously been reported as the reassortments between H9N2 viruses of Korean chickens and unknown AI viruses of wild birds (Song et al., 2008).
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Antigenic analysis
A haemagglutination inhibition (HI) assay was performed to investigate the antigenic diversity of H6 viruses isolated from poultry and wild birds in Korea, using antisera against eight selected viruses based on the phylogenetic analysis and genotypes. As expected from the phylogenetic analysis, the H6 AI viruses studied separated into three antigenically distinct groups: domestic bird viruses (group A), domestic/wild bird viruses (group B) and wild bird viruses (group C) (Table 2). The SBD/Kr/619/08 (H6N2) virus isolated from a wild bird (group C) cross-reacted weakly with both groups A and B.
Table 2. Antigenic analysis of H6 AI viruses isolated in Korea Values shown are HI titres. The titre of the homologous antigen group is shown in bold.
Replication of selected viruses in chickens and mice
We compared the replication capacity of the H6 subtype AI viruses in chickens and mice, with eight viruses selected from each of the eight genotypes (Table 3).
Table 3. Replication of selected H6 isolates in 4-week-old chickens and 5-week-old mice Values shown are the number of infected animals per number inoculated (virus titre: logEID50 per 0.1 ml). Virus titre is the mean of positive samples taken on days 1, 3, 5 and 7 after inoculation. The dose of inoculation for chickens and mice was 5.5 logEID50 per 0.1 ml. Abbreviations: CL, cloacal; OP, oropharyngeal; WL, weight loss.
In chickens, the virus titres of Dk/Kr/334-15/08 (genotype K-1) in oropharyngeal and cloacal swabs peaked at 3 and 5 days post-infection (p.i.), respectively, and virus was recovered until 7 days p.i. Of the four H6N2 viruses, the genotype K-2a (Dk/Kr/A39/08) virus was not detected in swabs up to 7 days p.i., and a genotype K-2c (Dk/Kr/460-5/08) virus replicated to low titres for just 3 days and only in the upper respiratory tract. On the other hand, viruses of genotypes K-2b (Dk/Kr/412-4/08) and K-2d (Dk/Kr/A127/09) replicated better in the intestine than the trachea and to relatively high titres. Virus titres of Dk/Kr/412-4/08 and Dk/Kr/A127/09 in cloacal swabs peaked at 3 days p.i., and these two viruses were recovered until 7 days p.i. SBD/Kr/625/08 (H6N1) was not detected up to 7 days p.i., and SBD/Kr/619/08 (H6N2) and SBD/Kr/528/08 (H6N8) replicated poorly and to low titres. None of the H6 subtype viruses tested induced signs of disease in inoculated chickens.
In mice, the H6 viruses, with exceptions of SBD/Kr/528/08 (H6N8) and SBD/Kr/625/08 (H6N1), induced transient weight loss; no deaths occurred during the experiment. Dk/Kr/334-15/08 (H6N1) (genotype K-1) virus was replicated to the highest titre at 5 days p.i. Dk/Kr/A39/08 (genotype K-2a) virus peaked to its highest titre at 5 days p.i. in murine lungs, and the Dk/Kr/A127/09 (genotype K-2d) virus was recovered at a constant titre until 5 days p.i. In addition, the Dk/Kr/412-4/08 (genotype K-2b) virus was recovered from lungs until 7 days p.i. However, Dk/Kr/460-5/08 (genotype K-2c) and two wild bird isolates (SBD/Kr/619/08 and SBD/Kr/528/08) showed poor replication, which was analogous to the results observed in the chicken-inoculation experiments (Table 3).
Transmission in chickens
Only chickens in the contact group with SBD/Kr/619/08(H6N2)-infected chickens became positive for the virus (two of four) in their oropharyngeal swabs at 3 days p.i. (data not shown). None of the chickens in the contact groups with chickens infected with the other seven H6 viruses became positive for the virus up to 7 days p.i. No chickens died during the course of this experiment.
Thirteen H6N2 viruses were isolated during the same duration, and at least five genotypes (K-2a, 2b, 2c, 2d and WB-3) were identified. Genotype K-2a has predominantly been isolated at domestic duck farms and LPMs. H6N2 viruses underwent broad reassortment with other influenza viruses of Korean poultry (H9N2 and H3N2) and various influenza viruses of waterfowl, including the H6N1 viruses. These results are similar to those of H3 AI viruses from backyard poultry, which are generated by genetic reassortment between H9N2 AI viruses from chickens and unknown influenza viruses from migratory birds in Korea (Song et al., 2008). Although Choi et al. (2005) reported only one H6N1 AI virus in 2003, there were no reports of H6 viruses in Korean poultry until now. Therefore, our results indicate that the H6N1 (genotype K-1) virus might have been established and co-circulating in wild birds around Korea since 2003, whereas the H6N2 viruses were newly generated through multiple and continuous reassortments between the H9N2 and H3N2 viruses from domestic poultry and AI viruses from aquatic birds.
In California, H6N2 viruses have commonly been isolated from chickens with clinical signs of infection (Webby et al., 2002). A drop in egg production and mild respiratory distress were observed in chickens infected with Taiwanese H6N1 viruses (Lee et al., 2006). The Korean H6 AI viruses were mostly isolated from ducks that showed no clinically significant signs of disease, but chickens that were inoculated experimentally with a representative H6N1 virus (Dk/Kr/334-15/08) (genotype K-1) and two genotypes (K-2b and 2d) of H6N2 virus showed virus replication in the respiratory tract and intestine without pre-adaptation of the viruses. Influenza viruses in aquatic birds have been found to replicate preferentially in the gastrointestinal tract, usually without producing clinical signs of infection and mainly transmitted via the faecal–oral route (Hinshaw et al., 1980; Webster et al., 1978). We also observed similar results in this study, as virus titres in cloacal swabs of the inoculated birds were higher than those in oropharyngeal swabs, which is different from most highly pathogenic H5N1 viruses isolated since late 2002 (Brown et al., 2006; Hulse-Post et al., 2005; Sturm-Ramirez et al., 2005). In addition, Korean H6 AI viruses replicated well in murine lung without pre-adaptation, much like the H6N1 viruses isolated in Hong Kong did in ferrets (Gillim-Ross et al., 2008). Interestingly, genotype K-2a virus (Dk/Kr/A39/08) and genotype WB-2 virus (SBD/Kr/625/08), which did not replicate in chickens, replicated to the highest titres in experimentally inoculated mice (>3.7 logEID50 per 0.1 ml virus). More in-depth studies on the difference in virus replication between chickens and mice are needed. In particular, genotype K-2a viruses, such as Dk/Kr/A39/08, have been isolated predominantly from domestic ducks in Korea, but they have not been found to replicate in experimentally infected chickens. In order to understand the mechanisms of intra- and interspecies transmission of these viruses, additional experiments in different animals, such as ducks and small poultry, are necessary.
South Korea experienced outbreaks of HPAI in 2003/2004, 2006/2007 and 2008 (C.-W. Lee et al., 2005; Y.-J. Lee et al., 2008). As these cases were controlled by effective policy combined with early detection and large-scale culling of infected poultry, the H5N1 HPAI virus is not endemic in Korea and endemics are unlikely in China and South-East Asia. Although the PA and PB2 genes of H5N1 HPAI viruses from Korea clustered with genes of the tested H6 AI viruses, various genotypes of H6 have been constructed, including those of the Korean H3N2 and H9N2 low-pathogenicity AI (LPAI) viruses (Lee et al., 2007; Song et al., 2008).
In summary, Korean H6 AI viruses were not related to W312/97 virus or the H5N1 HPAI viruses that caused outbreaks in Korea in 2003/2004, 2006/2007 and 2008. Phylogenetic and genetic analyses of our study showed that the H6 AI viruses isolated from poultry in Korea were from at least five genotypes, as a result of direct introduction by unknown wild birds, and reassortments between endemic Korean LPAI (H3N2, H9N2) viruses and AI viruses from wild birds. As H6 AI viruses isolated from wild birds have contained gene segments from various gene pools, the genes of these viruses have the possibility to cause additional exchanges with AI viruses from domestic poultry and aquatic birds. The pathogenicity and replication of certain H6 influenza viruses in chickens and mice were altered due to reassortment. When one considers the various genotypes of H6 subtype AI viruses that are endemic and have the possibility to cause infections in mammals and chickens, it is easy to see that continuous systemic surveillance is essential to understand the additional evolution of these subtypes in the Korean peninsula.
Surveillance and virus isolation.In total, 974 migratory birds, 2935 duck farms, 8060 oropharyngeal swabs and faeces from LPMs and 11 505 faeces of aquatic birds were tested according to an active systematic HPAI-surveillance programme that was conducted in South Korea for 13 months (from April 2008 to April 2009). The capturing and sampling of migratory birds were conducted at migratory bird aggregation sites, and the sampling of domestic ducks and LPMs was conducted at farms adjacent to these migratory bird sites for rapidly detecting HPAI virus. The swabs and faecal samples were suspended in antibiotic solution (0.4 mg gentamicin ml–1) and centrifuged at 3000 r.p.m. for 10 min. The supernatants were filtered with 0.45 µm syringe filters and then inoculated into 9–11-day-old specific-pathogen-free (SPF) embryonated chicken eggs (ECEs; Charles River Laboratories). The presence of AI viruses was determined by using a haemagglutination assay after incubating the eggs for 4 days at 37 °C. AI viruses were subtyped by using RT-PCR methods, as described previously (Fereidouni et al., 2009; Fouchier et al., 2000; Lee et al., 2001; Munch et al., 2001). Fifty per cent egg infectious dose (EID50) titres were determined in 11-day-old SPF ECEs, and virus titres were calculated by the method of Reed & Muench (1938).
Sequencing and phylogenetic analysis.
Virus RNA was extracted directly from HA-positive allantoic fluid by using a Viral Gene-spin viral DNA/RNA extraction kit (iNtRON Biotechnology) according to the manufacturer's instructions. A one-step RT-PCR was conducted using a PrimeScript one-step RT-PCR kit (TaKaRa) as recommended by the manufacturer. Primers for sequencing were identical to those described previously (Fereidouni et al., 2009; Fouchier et al., 2000; Lee et al., 2001; Munch et al., 2001). The DNA fragments were extracted and purified by using a QIAquick gel extraction kit (Qiagen) and the PCR product was sequenced at Macrogen (Seoul, South Korea) using an ABI 3730 XL DNA sequencer (Applied Biosystems). The sequences of the isolated viruses were aligned and edited by using Vector NTI and BioEdit software. Phylogenetic trees were generated by the neighbour-joining method using the MEGA 4.0 software (Tamura et al., 2007) with 1000 bootstrap replications.
Antigenic analysis.
Hyperimmune sera were prepared in chickens by intramuscular inoculation of 0.5 ml doses of inactivated vaccines, which were made with allantoic fluid of virus and oil adjuvant. The hyperimmune sera were collected 2 weeks after injection of the vaccine. To investigate the cross-reactivity of the isolated H6 viruses, we performed an HI assay (Palmer et al., 1975) using chicken polyclonal antibodies to eight H6 Korean AI viruses: A/spot-billed duck/Korea/625/08 (SBD/Kr/625/08), A/duck/Korea/334-15/08 (Dk/Kr/334-15/08), A/spot-billed duck/Korea/619/08 (SBD/Kr/619/08), A/duck/Korea/A39/08 (Dk/Kr/A39/08), A/duck/Korea/460-5/08 (Dk/Kr/460-5/08), A/duck/Korea/412-4/08 (Dk/Kr/412-4/08), A/duck/Korea/A127/09 (Dk/Kr/A127/09) and A/spot-billed duck/528/08 (SBD/Kr/528/08).
Virus replication in animals.
Chickens [SPF white leghorn broiler chickens (Gallus gallus domesticus; YoungSung Inc.)] and mice (BALB/c, Mus musculus; Orient Bio Inc.) were inoculated intranasally with allantoic fluid containing 5.5 logEID50 per 0.1 ml of each virus genotype, as described previously (Li et al., 2003). Two H6N1 (SBD/Kr/625/08 and Dk/Kr/334-15/08), five H6N2 (SBD/Kr/619/08, Dk/Kr/A39/08, Dk/Kr/460-5/08, Dk/Kr/412-4/08 and Dk/Kr/A127/09) and one H6N8 (SBD/Kr/528/08) viruses were inoculated into eight chickens and 15 mice. At 1, 3, 5, 7 and 9 days p.i., oropharyngeal and cloacal swabs of chickens and lung tissues of mice were taken and inoculated into 9–11-day-old SPF ECEs. To investigate the virus distribution in infected chickens, we collected samples of trachea, lung, caecal tonsil, kidney and spleen at 5 days p.i. All tissues were collected by using separate scissors to prevent cross-contamination. For virus transmission to contact birds, four uninfected chickens were housed with inoculated chickens for 7 days. At 1, 3, 5 and 7 days p.i., oropharyngeal and cloacal swabs were taken from contact chickens and inoculated into 9–11-day-old SPF ECEs.
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Received 20 August 2009; accepted 3 October 2009.