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1 College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong Heungduk-Ku, Cheongju 361-763, Republic of Korea
2 College of Veterinary Medicine, Chungnam National University, 220 Gung-Dong, Yuseoung-Gu, DaeJeon 305-764, Republic of Korea
3 Chungbuk Veterinary Service Laboratory, Cheongju 360-171, Republic of Korea
4 Division of Bioscience and Technology, College of Life and Environmental Science, Korea University, Seoul, Republic of Korea
Correspondence
Young-Ki Choi
choiki55{at}chungbuk.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this study are EU301177–EU301431.
A supplementary figure showing phylogenetic analysis of Korean H3 influenza viruses isolated from wild birds and live poultry markets is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Fouchier et al., 2005; Webster et al., 1992). Some of these subtypes have been transmitted to domestic poultry, causing severe or mild disease. From the early 1970s, live poultry markets (LPM) have been suspected to be a prolific source of AI viruses (Shortridge, 1992). Due to the high-density conditions in LPM, a variety of host species from backyard poultry are housed together, creating an ideal environment for reassortment and interspecies transfer of the viruses (Liu et al., 2003). Human infections with H5N1 (Chan, 2002; Peiris et al., 2004; Tam, 2002; Tran et al., 2004) and H9N2 viruses have been linked closely with LPM in Hong Kong (Butt et al., 2005; Peiris et al., 1999), and there have been reports of H7N7 viruses being transmitted to humans during an outbreak in commercial poultry farms in the Netherlands (Koopmans et al., 2004). Furthermore, LPM are hypothesized to be a missing link in the epidemiology of AI viruses (Senne et al., 1993). Although the role of LPM in the ecology of influenza viruses is being emphasized and the hypothesis that Asia is an influenza epicentre has been proposed (Shortridge & Stuart-Harris, 1982), little is known about the genetic information of influenza viruses circulating in LPM outside southern China.
We have reported previously that the H9N2 and H3N2 subtypes are the most common AI viruses in LPM, and that the novel H3N2 subtype is a reassortant between the H9N2 virus circulating in the Korean chicken population and H3N8 circulating in wild birds from south-eastern China and Japan (Choi et al., 2005). To investigate whether the H3N2 viruses were circulating continuously in LPM and whether further reassortment events had occurred with viruses from wild birds in Korea, we focused on the characterization of H3 influenza viruses isolated from these two bird populations. Furthermore, we conducted animal-challenge studies in chickens and mice to determine the replication potential and pathogenicity of representative H3 viruses. Here, we report the continuous evolution of novel H3 AI viruses by reassortment events through two-way transmission between domestic and wild hosts of AI viruses in LPM. These reassortment events may have altered the pathogenicity and host ranges of H3 viruses in experimentally infected animals.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The collected faecal samples were suspended in antibiotic solution and centrifuged at 3000 r.p.m. for 15 min. The supernatants were then inoculated into 10-day-old embryonated eggs and incubated at 37 °C for 48 h. Allantoic fluid from the incubated eggs was harvested and centrifuged for purification. The presence of the viruses was determined by haemagglutination assay and viruses were subtyped by partial or full sequencing. Of a total of 2880 faecal samples from LPM, 42 H3 viruses (39 H3N2 and three H3N6) were isolated from 35 ducks and seven chickens.
Genetic and phylogenetic analyses.
Viral RNA was extracted from allantoic fluid of the inoculated eggs by using an RNeasy mini kit (Qiagen) or TRIzol reagent (Invitrogen) as recommended by the manufacturers. The extracted RNA was reverse-transcribed at 42 °C for 60 min using SuperScript II reverse transcriptase (Invitrogen) and all PCRs were performed with Ex Taq polymerase (TaKaRa), according to the manufacturer's recommendations. The DNA fragments were extracted and purified with a QIAquick gel extraction kit (Qiagen) and sequencing of the template DNA was performed at Macrogen (Seoul, South Korea) with an ABI 373 XL DNA sequencer (Applied Biosystems). DNA sequences were compiled and edited by using the Lasergene sequence analysis software package (DNASTAR). Multiple sequence alignments were made by using CLUSTAL_X (Aiyar, 2000; Thompson et al., 1997). Rooted phylograms were prepared by using the neighbour-joining algorithm and then plotted by using the program NJPlot (Perriere & Gouy, 1996). Branch lengths are proportional to sequence divergence and can be measured relative to the scale bar shown (0.01 nucleotide changes per site). Branch labels record the stability of the branches over 100 bootstrap replicates. Complete sequences of all eight genes of the representative viruses (isolated from 2004 to 2006) and previously isolated wild (2003–2005 isolates) and domestic (2003 isolates) Korean H3 viruses were added to the phylogenetic analyses. Domestic and wild bird AI virus sequences from neighbouring China, Hong Kong and Japan were also added to investigate the introduction of novel influenza viruses. In addition, swine viruses were included in the analysis to investigate reassortment between mammalian and domestic avian viruses.
Antigenic analysis.
Polyclonal antibodies were obtained from 5-week-old BALB/c mice that had been inoculated with isolates Ck/Korea/LPM03/04, Dk/Korea/LPM38/05, Ab/Korea/KN-4/05, Ab/Korea/CN-1/04 and Ab/Korea/JN-2/06. To investigate the cross-reactivity of the isolated LPM viruses and Korean wild bird viruses, we performed a haemagglutination inhibition (HI) assay as described by Palmer et al. (1975), using the antibodies mentioned above. Sera that were generated from hosts other than chickens were pretreated with a receptor-destroying enzyme (Denka Seiken) to eliminate non-specific serum inhibitors.
In vivo growth characteristics.
Chickens (specific-pathogen-free white leghorn broilers) and mice (BALB/c) were inoculated intranasally with 103.0 or 102.5 EID50 of four H3N2 viruses (Ck/Korea/LPM03/04, Ck/Korea/LPM44/05, Dk/Korea/LPM39/05 and Dk/Korea/LPM91/06) and two H3N6 viruses (Dk/Korea/LPM36/05 and Dk/Korea/LPM38/05), respectively. At 1, 3, 5, 7 and 9 (up to 10 if necessary) days post-infection (p.i.), tracheal-swab samples of chickens and lung samples of mice (homogenized before inoculation) were taken and inoculated into 10-day-old embryonated eggs to investigate virus replication.
Virus transmission to contact birds.
Four H3N2 viruses (Ck/Korea/LPM03/04, Ck/Korea/LPM44/05, Dk/Korea/LPM39/05 and Dk/Korea/LPM91/06) and two H3N6 viruses (Dk/Korea/LPM36/05 and Dk/Korea/LPM38/05) were each inoculated into 10 chickens (60 chickens were used in total). Chickens infected with each virus were housed individually with five uninoculated chickens for 7 days (30 chickens were used in total). At 1, 3, 5 and 7 days p.i., tracheal samples of the contact chickens were collected and inoculated into 10-day-old embryonated eggs. After the inoculated eggs had been incubated at 37 °C for 48 h, the presence of the viruses was detected by haemagglutination assay.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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To understand the genetic diversity and molecular evolution of the H3 influenza viruses in LPM, we selected randomly (but including all subtype variation found) five H3 viruses isolated in 2004, nine isolated in 2005 and six isolated in 2006, characterized them genetically and tested their pathogenic potential in chickens and mice.
In addition, to investigate the ecological relationships between the H3 viruses from LPM and from wild birds, we selected randomly 11 H3 viruses isolated from wild birds during this study and compared them with LPM isolates (Table 1).
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). However, the LPM isolates could be separated into two groups: Ck/Korea/S6/03-like (n=10) and Dk/Korea/LPM09/04-like (n=10). On the other hand, the HA genes of the wild bird H3 isolates were separated into five groups with 82–85 % similarity to LPM isolates: Bantam/Nanchang/9-366/00-like (H3N3, n=5), Ab/Hong Kong/399/99-like (H3N8, n=2), Swan/Shimane/227/01-like (H3N9, n=2), Ck/Korea/S6/03-like (H3N2, n=1) and Dk/Korea/LPM09/04-like (H3N2, n=1). It is interesting to note that the HA genes of wild bird viruses Ab/Korea/JN-2/06 and Ab/Korea/CN-2/04 form a cluster with those of viruses isolated from LPM, because there have been no previous reports of the isolation of Ck/Korea/LPM-like viruses in wild birds in Korea.
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). The N6 genes of the H3N6 viruses were related closely to Ab/Korea/KN-5/06, a 2006 Korean wild bird isolate, and Ck/Nanchang/7-010/00-like wild bird isolates circulating in south-eastern China (data not shown). These results suggest that the HA and NA genes of H3 viruses from LPM tested in this study originated directly from those of wild bird isolates circulating in Korea.
Internal genes.
The matrix (M) genes of the H3 isolates separated into at least two different lineages: the Ck/Korea/25232-96006/96-like Korean lineage and the Ab/HK/399/99-like wild bird lineage circulating in south-eastern China. All LPM isolates and two wild bird isolates (Ab/Korea/CN-2/04 and Ab/Korea/JN-2/06) had the M genes of the Ck/Korea/25232-96006/96-like Korean lineage (see Supplementary Fig. S1a, available in JGV Online).
The non-structural (NS) genes were separated into three distinct lineages: the Ck/Korea/25232-96006/96-like Korean lineage, the Dk/HK/Y439/97-like wild bird lineage and the Dk/Hokkaido/8/80-like B-allele lineage. Only the wild bird isolates were clustered into the B-allele lineage. Like the other genes of H3 LPM isolates, most NS genes of LPM isolates and one wild bird isolate (Ab/Korea/KN-3/05) were related closely to the Ck/Korea/25232-96006/96-like Korean lineage, whilst two isolates (Ck/Korea/LPM88/06 and Ck/Korea/LPM03/04) clustered with the Dk/HK/Y439/97-like wild bird lineage (see Supplementary Fig. S1b, available in JGV Online).
In contrast, no nucleoprotein (NP) genes of H3 viruses were related closely to the Ck/Korea/25232-96006/96-like Korean lineage. Instead, four different lineages were found: Dk/HK/Y439/97-like (n=14), Dk/Nanchang/9-091/00-like (n=9), Ab/HK/399/99-like (n=4) and Dk/HK/7/75-like (n=4). The NP genes of five LPM isolates (Dk/Korea/LPM36/05, Dk/Korea/LPM38/05, Dk/Korea/LPM39/05, Dk/Korea/LPM91/06 and Dk/Korea/LPM92/06) were related closely to those of isolates Ab/Korea/KN-1/04 and Ab/Korea/KN-3/05 (Dk/Nanchang/9-091/00-like lineages). However, Ck/Korea/LPM88/06 was clustered in the Dk/HK/7/75-like lineage along with four wild bird isolates (see Supplementary Fig. S1c, available in JGV Online).
Most of the polymerase acidic protein (PA) genes of the LPM viruses (18 of 20 isolates) were clustered with the Ck/Korea/99029/99-like viruses, but two H3N2 viruses, Dk/Korea/LPM92/06 and Dk/Korea/LPM91/06, had PA gene sequences that were related closely to a wild bird isolate (Ab/Korea/KN-4/05) and an American turkey virus (Tu/MN/833/80) (see Supplementary Fig. S1d, available in JGV Online).
All polymerase basic protein 1 (PB1) genes of LPM viruses and of a wild bird isolate (Ab/Korea/CN-2/04) clustered with the Ck/Korea/99029/99-like Korean lineage of previous Korean isolates (see Supplementary Fig. S1e, available in JGV Online).
Most of the polymerase basic protein 2 (PB2) genes of LPM viruses and of two wild bird isolates (Ab/Korea/CN-1/04 and Ab/Korea/CN-2/04) were related closely to the Ck/Korea/25232-96006/96-like Korean lineage, except for one LPM isolate (Dk/Korea/LPM36/05), which was related closely to the Dk/Hong Kong/7/75-like lineage circulating in south-eastern China (see Supplementary Fig. S1f, available in JGV Online).
Antigenic analysis
To analyse antigenic diversity of the LPM-isolated H3 viruses and their relationship with wild bird viruses, antisera against six selected viruses, based on the phylogenetic analysis of the HA gene, were used to perform HI assays (Palmer et al., 1975). As expected from the phylogenetic analysis, the H3 AI viruses in Korea could be separated into at least four antigenic groups. Interestingly, the group I viruses (Ck/Korea/LPM03/04 and Ab/Korea/JN-2/06) showed relatively high cross-reactivity (HI titre 
640) with all H3 isolates, including wild bird isolates (Table 2). Dk/Korea/LPM38/05 (H3N6, group II) cross-reacted relatively well with all LPM viruses of groups I and II (HI titre 640), whereas it cross-reacted weakly with group III and group IV viruses. Antisera to Ab/Korea/CN-1/04 and Ab/Korea/CN-3/05 (group III) and Ab/Korea/KN-1/04 (group IV) reacted more strongly with group I viruses than with group II viruses (Table 2).
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160) and Sw/Korea/JNS06/04 (H3N2) (HI titre 160); the titres were at least fourfold lower than with the homologous viruses. These results suggested that the avian H3 viruses circulating in LPM cross-reacted serologically relatively well with some wild bird isolates, but not with those of swine.
Genotypes
To examine the genetic diversity of these viruses, we compared full or partial sequences of all eight segments of each virus, including several Korean wild bird isolates. Based on phylogenetic analysis, we identified at least four different genotypes in the H3N2 subtypes, designated A, B, C and D, and two different genotypes in the H3N6 isolates from LPM in Korea (Fig. 2). Compared with our previous study, at least three more genotypes of the H3N2 subtype and a novel H3N6 subtype had been created in LPM viruses by reassortment with wild bird viruses. These genotypes possessed a combination of genes from wild bird viruses and previously circulating domestic Korean chicken and duck viruses. It is interesting to note that the Ck/Korea/S6/03-like H3N2 genotype was not detected during this study, whereas at least one of the gene segments was derived from wild bird viruses. Although only two isolates of the H3N6 subtype were detected in ducks, their PB2 gene segments originated from two different lineages (Fig. 2). Taken together, these results suggest that active reassortment events among AI viruses occur in LPM.
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Growth and virus transmission in chickens
Three H3N2 viruses [Ck/Korea/LPM44/05 (genotype A), Dk/Korea/LPM39/05 (genotype B) and Ck/Korea/LPM03/04 (genotype C)] replicated relatively well in the tracheas of infected chickens. Viral titres peaked at 3 days p.i. [2.0–2.7 log10(EID50) per 0.1 ml] and viruses were recovered from tracheal swabs until 5 days p.i., but none were detected from the swabs beyond 7 days p.i. In contrast, the genotype D (Dk/Korea/LPM91/06) isolate of the H3N2 subtype was detected only at 1 day p.i. at a titre of 1.3 log10(EID50) per 0.1 ml. The H3N6 (Dk/Korea/LPM38/05 and Dk/Korea/LPM36/05) isolates were recovered from tracheal swabs of infected chickens at titres 1.5 log10(EID50) per 0.1 ml until 3 days p.i. and became undetectable at 5 and 7 days p.i., respectively (Fig. 3). These data suggest that genotypes A, B and C of the H3N2 subtype replicate relatively well in chickens, but genotype D of H3N2 and all H3N6 subtypes replicate poorly in this host. Thus, we investigated the chicken-to-chicken transmissibility of these H3 viruses. Ten chickens were inoculated with 3.0 log10(EID50) per 0.1 ml of each virus. Chickens infected with each virus were housed individually with five uninfected chickens for 7 days. Two sentinel chickens in contact with Ck/Korea/LPM44/05 (H3N2, genotype A)-infected chickens started to shed viruses 3 days post-contact (p.c.) and, by 7 days p.c., three contact chickens were positive for viruses (Table 3). One sentinel chicken in contact with each of Dk/Korea/LPM39/05 (H3N2, genotype B) and Ck/Korea/LPM03/04 (H3N2, genotype C) started to shed viruses at 5 days p.c., whereas none of the chickens in contact with Dk/Korea/LPM91/06 (H3N2, genotype D), Dk/Korea/LPM36/05 (H3N6) or Dk/Korea/LPM38/05 (H3N6) was positive for viral shedding up to the end of the experiment. In summary, LPM H3N2 viruses of genotypes A, B and C replicated readily in chickens, but genotype D of the H3N2 LPM virus and H3N6 isolates replicated poorly or did not replicate at all in this host.
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All H3N2 viruses replicated well at titres 2 log10(EID50) per 0.1 ml at 3 days p.i. In contrast to the results observed in the chicken-inoculation experiments, Dk/Korea/LPM91/06 (H3N2, genotype D) showed the highest replication in murine lungs: >5 log10(EID50) per 0.1 ml virus was recovered at 5 days p.i. Among the four H3N2 viruses tested, Ck/Korea/LPM03/04 (genotype C) replicated to the lowest titre [2.5 log10(EID50) per 0.1 ml] at 3 days p.i., and no detectable viruses were found beyond 7 days p.i. (Fig. 4). Ck/Korea/LPM44/05 (H3N2, genotype A), Dk/Korea/LPM39/05 (H3N2, genotype B) and Dk/Korea/LPM91/06 (H3N2, genotype D) viruses were recovered from lungs until 9 days p.i. For the H3N6 subtype, viral titres of murine lungs infected with Dk/Korea/LPM36/05 peaked at 3 days p.i. at 2.6 log10(EID50) per 0.1 ml, and viruses were recovered until 5 days p.i. Viral detection from Dk/Korea/LPM38/05-infected lungs was possible until 5 days p.i., with titres gradually decreasing from 2.3 log10(EID50) per 0.1 ml (1 day p.i.) to no detectable virus at 7 days p.i. (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Shortridge & Stuart-Harris, 1982), and similar studies have demonstrated that LPM in the USA have also played a pivotal role in the distribution and genetic interaction of influenza viruses (Senne et al., 1993). Here, we investigated the evolution of novel H3 subtype AI viruses present in LPM in Korea. We isolated two different subtypes (H3N2 and H3N6) of H3 AI virus; H3N2 predominated (comprising >92 % of viruses isolated).
Serological analysis of representative viruses (Table 2) showed that at least four distinguishable serotypes of H3 AI virus were co-circulating in Korea during the study period. Interestingly, group I viruses showed relatively high cross-reactivity with all avian H3 isolates, including wild bird isolates, but not with swine isolates (Table 2). Furthermore, all segments of two swine isolates (Sw/Korea/JNS06/04 and Sw/Korea/PZ72-1/06), including the HA genes, clustered differently from those of other avian H3 viruses, but shared a root with USA swine H3N2 subtypes in phylogenetic analysis (Fig. 1; Supplementary Fig. S1, available in JGV Online) (Shin et al., 2006). The results of serological and phylogenetic analysis suggest that there were no relationships between avian and swine H3 viruses, despite the fact that they were isolated in a similar time period and from similar regions.
Phylogenetic analysis demonstrated that there is active evolution of H3 AI viruses in LPM in Korea and that reassortments occur frequently between viruses circulating in aquatic birds of south-eastern China and viruses found previously to be circulating in chickens in Korea, as described in earlier studies (Choi et al., 2004, 2005). We identified four genotypes of H3N2 virus and two genotypes of H3N6 virus. Most of the H3N2 LPM viruses isolated in 2006 were categorized into genotype A, a different genotype from the 2003 Korean LPM viruses, revealing the occurrence of continuous evolution. It is interesting that several gene segments (HA, NS, M, PB1 and PB2) of Korean wild bird isolates were included in the same cluster as Ck/Korea/25232-96006/96-like viruses, commonly isolated from poultry farms in Korea. It is not clear, however, whether these gene segments of wild bird isolates originated from H3 or H9 viruses of domestic poultry or from another lineage of wild bird Ck/Korea/S6/03-like viruses, if they exist.
In Italy, there have been multiple H3N2 viruses isolated from chickens with mild respiratory disease. Campitelli et al. (2002) first reported that these avian-like H3N2 viruses could replicate and cause mild disease in experimentally inoculated chickens. Although most Korean LPM viruses isolated in 2006 were from ducks (83 %), chickens that were inoculated experimentally with two representative H3N6 viruses (Dk/Korea/LPM36/05 and Dk/Korea/LPM38/05) and three genotypes (A, B, C) of H3N2 viruses showed viral replication in the upper respiratory tract without pre-adaptation of the viruses. This result is consistent with previous studies of chickens inoculated experimentally with Korean LPM H3 viruses from 2003 (Choi et al., 2005). Interestingly, genotype D virus (Dk/Korea/LPM91/06), which did not replicate in chickens, replicated to the highest titre in experimentally inoculated mice [5 log10(EID50) per 0.1 ml]. Understanding the mechanism behind these different replication characteristics of the same virus in two different species is a matter of interest, and more study is needed.
The H3N2 viruses isolated in Italy did not establish a stable lineage, due to the disappearance of the virus; the origin of the viruses that infected the Italian chickens also remains unknown (Campitelli et al., 2002). Previous studies (Choi et al., 2005) have speculated on whether the H3N2 viruses of Korea would spread within the regional or national poultry populations and establish a stable lineage. In this study, we revealed that Korean LPM H3 viruses, unlike Italian chicken H3N2 viruses, seem to have established a steady lineage since the year that they were first isolated. Continuous virus isolation covering the whole Korean peninsula is needed to understand the gene pool of national LPM viruses.
In summary, the four genotypes of H3N2 and two genotypes of H3N6 AI viruses co-circulating in LPM in Korea seem to have the potential to replicate in mammalian hosts. The geographical proximity of the Korean peninsula to southern China and Japan may allow the introduction of new influenza virus genes into this region by migrating birds, as occurred in Japan (Liu et al., 2003). Continuous monitoring of viruses in domestic and wild bird populations is essential to better understand interspecies transmission, such as recent human infections with H5N1 (Peiris et al., 2004; Tran et al., 2004), H9N2 (Peiris et al., 1999), H7N3 and H7N7 (Koopmans et al., 2004) AI viruses. Further related studies and continued monitoring will also give a better understanding of the importance of avian hosts in the ecology of influenza viruses.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 20 September 2007;
accepted 28 November 2007.
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, CK/Korea/LPM03/04; 
, DK/Korea/LPM39/05; 
, CK/Korea/LPM44/05; x, DK/Korea/LPM91/06. (b) 