Abstract
We characterized low pathogenic avian influenza (LPAI) viruses of the H7 subtype that were isolated from domestic ducks and wild birds in South Korea from 2008 to 2011. A total of 20 H7 viruses were collected from live-bird markets (LBMs), duck farms and wild-bird habitats using avian influenza (AI) surveillance and epidemiological approaches. A phylogenetic analysis of the H7 viruses that were isolated from domestic ducks and wild birds demonstrated that they were separated into 12 genotypes (A–D and Wb-1–8, respectively), indicating genetic diversity. These H7 viruses were related to the recently isolated Eurasian LPAI H7 viruses and various influenza viruses that are circulating in Asia, including southern China and South Korea. The same genotype was not found between domestic poultry and wild-bird isolates; however, most of the H7 viruses in poultry (genotypes B and C) were closely related to the H7 virus isolated from a wild bird (genotype Wb-3). Animal-challenge studies revealed that certain H7 AI viruses replicated well only in chickens or ducks depending on the genotype, indicating that the pathogenicity of H7 viruses has the potential to be altered due to multiple reassortments, and these viruses can potentially expand their host range. Our results are evidence of abundant and frequent reassortment between H7 viruses in poultry and wild birds and emphasize the continuing need to monitor the evolutionary genetics of the influenza virus in poultry and wild birds.
Introduction
Avian influenza viruses (AIV) are classified in the family Orthomyxoviridae, genus Influenzavirus A. To date, 16 haemagglutinin (HA) and nine neuraminidase (NA) subtypes have been identified, and all known subtypes of AIV have been implicated in various clinical indications that range from asymptomatic infections to respiratory diseases with low mortality to severe pathogenicity with high mortality (Swayne & Halvorson, 2003).
Low pathogenic avian influenza (LPAI) viruses of the H5 and H7 subtypes have been reported to evolve into highly pathogenic avian influenza (HPAI) viruses once they are transmitted to domestic poultry (Banks et al., 2001; Horimoto & Kawaoka, 1995; Lee et al., 2005). In Chile and Canada, the circulation of an H7 LPAI virus was detected shortly before the HPAI virus outbreak of the same subtype and was concluded to be evolved from an LPAI virus through a recombination event (Hirst et al., 2004; Suarez et al., 2004). H5 and H7 LPAI viruses in migratory ducks have been introduced to poultry and caused HPAI H5N2 and H7N7 outbreaks in northern Europe (Munster et al., 2005). The Italian H7N3 poultry viruses have been shown to be the result of a direct introduction of a wild-bird virus (Campitelli et al., 2004).
However, there were no reports of outbreaks of H7 subtype HPAI in Asian countries unlike the continued circulation of HPAI H5N1 except for the outbreak of H7 HPAI in North Korea in 2005 (Swayne, 2008). There have been limited reports of LPAI H7 viruses detected in poultry in several Asian countries. Campitelli et al. (2008) analysed LPAI H7 viruses in Italy and southern China and determined that they were most closely related to each other according to phylogenetic evidence. In 2009, seven quail farms in Japan were found to be infected with an H7N6 virus of unknown origin (Sugiura et al., 2009). In Korea, two LPAI H7 viruses were isolated from domestic ducks in 2007 (Kim et al., 2010), but there have been no reports on the characteristics of the virus.
In this study, we are the first to characterize H7 viruses (nine isolates) in domestic ducks in Korea, together with H7 AIV isolated from wild birds, as the result of a systematic surveillance programme conducted between 2008 and 2011. We performed a phylogenetic analysis to investigate their genetic relationships and conducted animal experiments to determine their pathogenicity.
Results
Epidemiological background
Twenty H7 viruses and one H3 virus analysed in this study were collected from South Korea from domestic ducks and wild birds through active surveillance between July 2008 and May 2011 (Table 1).
In December 2009, an H7N2 strain (A/duck/Korea/A349/09) was isolated from domestic ducks at the live-bird market (LBM) in the Jeonnam province located in the southern part of Korea (Fig. 1, no. 1). All of the poultry that were kept in this infected poultry shop were destroyed according to the control policy although no clinical signs of disease were observed in any of the birds. Five farms with an epidemiological relationship to this poultry shop were inspected as a follow-up survey, but we could not detect any additional viruses. In May 2010, two H7N7 viruses (A/duck/Korea/A75/10 and A/duck/Korea/A76/10) were simultaneously isolated in two duck farms located in the Jeonnam province (Fig. 1, nos 2 and 3). These two farms are located 75 and 47 km, respectively, from the site of the previous poultry shop and the distance between the two farms is 46 km. The A/duck/Korea/A79/10 (H7N7) virus was confirmed in one farm (Fig. 1, no. 4) that supplied or received ducklings or embryonated duck eggs from farm no. 3. In June 2010, the A/duck/Korea/LSY/10 (H7N7) and A/duck/Korea/JSM/10 (H7N7) viruses were isolated in duck farms (Fig. 1, nos 5 and 6) and had an epidemiological correlation with farm no. 2. In November 2010, another domestic duck farm (Fig. 1, no. 8) in the Jeonnam province was found to be infected by the A/duck/Korea/A117/10 (H7N6) virus. No H7 viruses were isolated from the 11 farms with an epidemiological relationship to farm no. 8, but the A/duck/Korea/A122/10 (H3N6) virus was identified in an agistment farm (Fig. 1, no. 10) 8 km away. The A/duck/Korea/A112/10 (H7N7) and A/duck/Korea/109/11 (H7N7) viruses were isolated from a domestic duck farm (Fig. 1, nos 7 and 9) in the Chungnam province located in the middle part of Korea by ordinary active surveillance, but no additional AIV were detected through epidemiological surveillance.
Location of H7 subtype AIV that were isolated from 2009 to 2011 in South Korea. The blue circles represent the locations of H7 subtype viral isolation from domestic ducks, and the green circles represent the locations of H7 subtype viral isolation from wild birds. The numbering is indicated by ‘no. virus’ in Table 1. The lines illustrate a direct epidemiological relationship between different locations as indicated by the same colour, and the dotted lines illustrate an indirect epidemiological relationship that was assumed from the genetic analysis.
In addition, 11 H7 viruses were isolated from the faeces of wild birds in wild-bird habitats and of migratory birds that were captured in several regions in Korea. These viruses were genetically analysed and were compared with H7 viruses in domestic ducks to elucidate the genetic relationship with those viruses.
Antigenic and genetic analyses
A haemagglutinin-inhibition (HI) assay of the H7 viruses that were isolated from poultry and wild birds in South Korea using antisera revealed no significant differences in their antigenic patterns, but the A/duck/Korea/A75/10 virus showed a high titre only against its homologous antiserum virus and A/duck/Korea/109/11 virus weakly reacted with H7 viruses excluding A349 antiserum (Table S1 available in JGV Online).
A phylogenetic analysis of the HA1 gene showed that the Korean H7 viruses were clustered into the Eurasian lineage, which correlated with the serological reactivity that was observed in the HI assay; however, these viruses could be divided into two subgroups (Fig. 2). The A/turkey/Italy/4169/99 and A/duck/JX/1742/03 viruses were selected to represent the European H7 viruses and Asian H7 viruses, respectively, through subclade division. The Korean H7 strains of domestic ducks and four viruses of wild birds that were isolated between 2009 and 2010 belonged to the A/duck/JX/1742/03-like lineage, together with the viruses that were isolated in South Korea in 2007 (95.0–99.8 % homology). Seven viruses of wild birds that were isolated between 2010 and 2011 formed the same group with the A/turkey/Italy/4169/99 virus, and the highest degree of divergence was observed with 92.9–94.6 % homology. These data suggest that most of the H7 viruses in domestic ducks originated from H7 viruses that were continuously circulating in poultry and wild birds from Asian regions, but some of the H7 viruses in wild birds have been affected by newly introduced viruses from Europe. A variety of NA subtypes were detected among 20 H7 viruses: N2 (n = 1), N3 (n = 2), N6 (n = 1), N7 (n = 12) and N9 (n = 4). In the NA gene tree, all of the N7 and N9 genes of the H7 viruses were related to the H7 viruses of Eurasian lineage, and the N3 and N6 genes were closely related to the influenza viruses of several HA subtypes that were isolated from wild migratory birds in Asia. Notably, the N6 gene of the A/duck/Korea/A117/10 (H7N6) virus was similar (99.9 % homology) to that of the A/duck/Korea/A122/10 (H3N6) strain that was isolated through epidemiological surveillance. The N2 gene of the A/duck/Korea/A349/09 (H7N2) strain was related to that of the LPAI H5N2 and H9N2 strains that were recently isolated from poultry and wild birds in South Korea and China (data not shown). These data suggest that the various NA genes of the H7 viruses have originated from the gene pool of AIV in poultry and wild birds in Eurasia.
Phylogenetic diagram of the H7 HA1 genes analysed using maximum-likelihood. The numbers above and below the branches indicate neighbour-joining distances with 1000 bootstrap replicates. Of the H7 viruses that were isolated in South Korea from 2009 to 2011, the H7 isolates from domestic ducks are indicated in blue and the H7 viruses from wild birds are represented in green. In addition, the H7 influenza viruses that were isolated in areas of Eurasia, including China, Hong Kong, Japan, Mongolia, Italy and England were added to the analysis. Ck, Chicken; Dk, duck; SBD, spot-billed duck; Tk, turkey; Wb, wild bird; JX, Jiangxi; Kr, Korea; NJ, New Jersey; HK, Hong Kong; NY, New York.
The phylogeny of the PB2 genes demonstrated that the PB2 genes of the AIV that were in this study were separated into two distinct lineages (Fig. S1a). Most of the H7 viruses in the domestic ducks and three H7 strains in wild birds were clustered into an aquatic-bird lineage of AIV that have been circulating recently in the Eurasian region, including South Korea. The other viruses formed a sister group with the A/turkey/Italy/4169/99-like lineage and the A/duck/Jiangxi/1742/03-like lineage. Seven H7 viruses that were isolated from wild birds were grouped with the A/turkey/Italy/4169/99 virus. The A/duck/Korea/A117/10, A/duck/Korea/A122/10 and A/mallard/Korea/822/10 viruses were similar to the H7 viruses that were isolated from wild birds in South Korea in 2007 and belonged to the A/duck/Jiangxi/1742/03-like lineage. The PB1 genes of all the poultry and wild-bird strains were related to two different lineages: an A/turkey/Italy/4169/99-like lineage and an A/duck/JX/1742/03-like lineage (Fig. S1b). The PB1 genes of all the duck strains and two wild-bird H7 viruses were the same lineage, but most of the H7 viruses of the wild birds were of the other lineage. All of the PA genes shared a very recent common ancestor: the A/duck/Jiangxi/1742/03 virus, together with various AIV that have been circulating in the Eurasian region). Regarding the NP genes, the Korean H7 viruses that were isolated from domestic ducks formed two distinct subgroups: an aquatic-bird lineage and an A/duck/Jiangxi/1742/03-like lineage. Most of the H7 viruses that were isolated from wild birds were clustered into the A/duck/Jiangxi/1742/03-like lineage, but two H7 viruses formed a group with the H5 and H7 subtype AIV of Italy and Denmark (Fig. S1d). A phylogenetic analysis of the M genes showed that the Korean H7 viruses that were isolated from domestic ducks were divided into two different lineages analogous to the NP genes. Most of the H7 viruses in wild birds formed a group with the A/duck/Jiangxi/1742/03 and A/turkey/Italy/4169/99 viruses, except for the A/mandarinduck/Korea/468/11 virus. The NS genes of most of the viruses that were tested in this study were clustered with the allele A of various subtype AIV that have been isolated in Eurasia, but only the A/wild bird/Korea/A9/11 virus belonged to the allele B.
Molecular characterization
In the Korean H7 viruses, the absence of a multi-basic amino acid motif at the HA cleavage site, which is known to confer a highly pathogenic phenotype of H5 and H7 subtype viruses, was identified. All of the H7 viruses from poultry and four viruses from wild birds had a PEIPKGR motif, but other H7 viruses from wild birds had different motifs by changing one or two amino acids (Table 1). The HA peptide motif of the selected H3 isolate (A/duck/Korea/A122/10) was PEKQTR, which was similar to that of the H3 viruses that were isolated from poultry at LBMs in Korea between 2004 and 2006 (Song et al., 2008).
Genotypes
The genetic diversity of the H7 viruses was determined based on a phylogenetic analysis of eight genes of each strain, including the viruses of both domestic poultry and wild-bird isolates. Four (A–D) and eight (Wb-1–8) genotypes from domestic poultry and wild-bird isolates, respectively, were identified (Fig. 3). The H7N7 viruses that were isolated in wild birds from 2009 to 2010 were divided into four genotypes. Compared with the H7N7 viruses that were isolated in South Korea in 2007, the A/pintail/Korea/1173/09 (genotype Wb-2) virus consisted of the same gene constellation, whereas the genotypes Wb-1 and Wb-4 viruses had different NP and PB1 genes from the 2007 H7N7 viruses, respectively. The genotype Wb-3 virus had different PB2 and PB1 genes compared with the 2007 H7N7 viruses. The H7 viruses from wild birds in 2011 were observed to have many genotypes; four genotypes were detected in the same year. The H7N9 genotype Wb-6 viruses and the A/wild bird/Korea/A4/11(H7N3; genotype Wb-7) virus had a different NA gene only from each other although both viruses were isolated from faeces in the same place at the same time. In addition, both viruses consisted of a similar gene constellation to the H7N7 viruses that were isolated in South Korea in 2007, excluding the NA gene. The genotypes Wb-5 and Wb-8 viruses were different from the 2007 H7N7 viruses according to three genes: PB2, NA and M or NS.
Genetic diversity of LPAI H7 viruses tested in this study. The full sequences of the eight segments were compared to determine the genetic diversity among these viruses. From the first bar downward in each genotype circle: PB2, PB1, PA, HA, NP, NA, M and NS are indicated. The abbreviations are listed in the legend for Fig. 2.
The same genotype was not found between domestic poultry and wild-bird isolates; however, the gene constellation of the Wb-3 viral genotype was similar to that of the genotype B isolates except for the NP gene, and it was similar to that of the genotype C isolates except for the M gene. The NP and M genes of the genotypes B and C viruses, which were the predominant genotypes of the H7 viruses from domestic ducks, were different from each other despite being isolated from samples that had been collected over similar durations and from similar locations. Hence, two genotypes showed a clear distinction among the H7N7 viruses according to the epidemiological background. This finding suggests that two genotype H7 viruses have been circulated to the nearby duck farms from May to June 2010, which had affected an additional H7 viral detection in the Chungnam province in October 2010 and January 2011. Seven gene segments of genotype A, the H7N2 strain that was isolated at the LBM, were closely related to those of genotype C H7N7 viruses except for the NA gene. The A/duck/Korea/A117/10 (H7N6) virus was identified as genotype D because it consisted of six genes of the genotype B H7N7 viruses and the other PB2 and NA genes of viruses from aquatic birds. In addition, this virus and the A/duck/Korea/A122/10 (H3N6) virus were closely related to each other, excluding the HA gene.
Replication and transmission in chickens and ducks
We compared the replication capacity of the H7 subtype AIV in chickens and ducks with four viruses that were selected from each of the four genotypes of viruses that were isolated from poultry.
In chickens that were inoculated with viruses through nostril, the viral titres of the A/duck/Korea/A349/09 (genotype A) virus in the oropharyngeal and cloacal swabs peaked after 3 days post-infection (p.i.), and virus was recovered until 7 days p.i. This virus was shed to higher titres by the cloacal versus oropharyngeal routes. In contrast, genotypes B, C and D viruses were only detected in swabs collected at 1 or 3 days p.i. at low titres that could be interpreted as a lack of replication (Table 2). In the contact group, only the A/duck/Korea/A349/09 virus-infected chickens became positive for the virus from 3 to 10 days p.i. (Table 3).
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, 7 and 10 after inoculation. The dose of inoculation for chickens was 6.3 logEID50 per 0.1 ml. OP, Oropharyngeal; CL, cloacal.
Values shown are the number of infected animals per number contacted (virus titre: logEID50 per 0.1 ml). Virus titre is the mean of positive samples taken on days 1, 3, 5, 7 and 10 after inoculation. The dose of inoculation for chickens was 6.3 logEID50 per 0.1 ml. OP, Oropharyngeal; CL, cloacal.
In the duck experiment, the viral titres of the A/duck/Korea/A76/10 (genotype C) and A/duck/Korea/A117/10 (genotype D) viruses in the oropharyngeal swabs peaked at 3 days p.i., and these two viruses were recovered until 14 and 7 days p.i., respectively. However, the A/duck/Korea/A349/09 (genotype A) virus replicated to low titres, which was different from the results of the chicken experiment, and the A/duck/Korea/A75/10 (genotype B) virus was not detected through 14 days p.i. (Table 4). Using c-ELISA, we observed seropositivity rates of 50, 25, 62.5 and 62.5 % in inoculated ducks for genotypes A, B, C and D viruses, respectively.
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, 7, 10 and 14 after inoculation. The dose of inoculation for ducks was 6.3 logEID50 per 0.1 ml. OP, Oropharyngeal; CL, cloacal.
In the study of inoculation by the intravenous route, four genotype viruses were recovered from caecal tonsils although the titre peaks differed. The livers and spleens were negative for viruses or were infected at low titres of genotypes A, B and C viruses, but these viruses replicated well in the kidneys. In contrast, the A/duck/Korea/A117/10 (genotype D) virus was not recovered from the kidneys. The finding of the A/duck/Korea/A349/09 virus in the brain of one chicken is of interest, but the level was lower than what would be expected for a typical HPAI virus (Table 5) (Wood et al., 1995). None of the H7 subtype viruses induced signs of disease or killed the inoculated chickens and ducks.
Values shown are the number of infected animals per number inoculated (virus titre: logEID50per 0.1 ml). Virus titre is the mean of positive samples taken on days 3 and 5 after inoculation. The dose of inoculation for chickens was 1/10 diluent of allantoic fluid (6.3–6.9 logEID50 per 0.1 ml). CT, Caecal tonsil.
Discussion
In South Korea, the results of the previous surveillance that was performed only on wild birds and breeder ducks during the winter season indicated that LPAI H7 viruses were rarely detected in poultry (two H7 viruses during 4 years) despite frequent viral isolation from wild birds (Kim et al., 2010). The findings of the present study reveal that LPAI H7 viruses were isolated predominantly from domestic ducks and wild birds year-round by fortified active surveillance to expanded targets, including poultry at the LBMs and domestic duck farms since July 2008. A genetic analysis was performed to elucidate this high rate of H7 viral isolation in domestic poultry, which either resulted from interspecies transmission from wild birds to poultry or from the endemicity of H7 viruses in South Korea.
All of the H7 AIV that were isolated in Eurasian countries between 1999 and 2005 have HA genes that share a common ancestor because of the potential overlap of migratory flyways according to Campitelli et al. (2008). However, the H7 strains of South Korea were subdivided into two restricted sublineages based on the HA sequence data that were generated in our study (Fig. 2), although they did not reveal any significant antigenic differences. This result indicates that the H7 viruses of South Korea were continuously circulating in poultry and wild birds and that the antigenic characteristics of these viruses have the potential to be altered by a continuous drift of the HA gene.
Phylogenetic analyses demonstrated that 12 genotypes were identified from the Korean H7 viruses as a result of multiple reassortment events between H7 viruses that were isolated in Eurasian regions and various influenza viruses of waterfowl (Fig. 2, Fig. S1 and Fig. 3). Of the H7 viruses that were isolated from wild birds between 2009 and 2011, some of the viruses were the same as or similar to H7N7 viruses that were isolated in South Korea in 2007, excluding one or two genes, and other viruses had appeared as a result of at least triple reassortment. Although the same genotype was not found between domestic poultry and wild-bird isolates in South Korea, the genotype Wb-3 virus was closely related to the genotypes B and C viruses in domestic ducks. The H7 viruses that were detected in poultry were divided into four genotypes with only one or two gene differences, and they had affected several duck farms for only 3 years through a direct or indirect epidemiological relationship. These results suggested that prolonged co-circulation of an H7 virus and other AIV in the poultry and wild-bird population must be considered which has enabled reassortment and, hence, led to a new genotype. In addition, the H7 subtype viruses that were isolated from wild birds showed more genotypes as indicated by a more dynamic influenza viral gene pool than those of domestic ducks, and this gene pool affected the generation of new viral reassortments.
The Korean H7 LPAI viruses that were isolated from domestic ducks demonstrated no clinically significant signs of disease in ducks. Additionally, no pathological or gross lesions were observed in the experimentally inoculated chickens and ducks. The genotype A virus (A/duck/Korea/A349/09) showed high titres of viral replication in the inoculated chickens and was transmitted to the contact chickens, whereas this virus inoculated into duck were detected at low titres over 5 days (Table 2, Table 3 and Table 4). The genotype B virus (A/duck/Korea/A75/10), one of the H7N7 viruses that were isolated predominantly in 2010, was found not to replicate in both inoculated chickens and ducks. The H7 viruses of genotypes C (A/duck/Korea/A76/10) and D (A/duck/Korea/A117/10) were poorly replicated in chickens, but were reisolated at high titres in ducks. Genotypes A and B viruses had a relatively lower seropositivity rate than the other viruses, which meant both viruses did not induce the infection and replication well in domestic ducks.
Four genotype H7 viruses of poultry demonstrated various replication distinctions and differences of tissue tropism depending on the species. In particular, the genotype A virus that was isolated from domestic ducks at the LBM, in which the genetic interaction of influenza viruses frequently occurred, carried the potential risk of changing its pathogenicity, which could have resulted in the implementation of a rapid eradication policy to the infected herd. In several countries, HPAI viruses of the H7 subtype have been demonstrated to be derived from LPAI viruses of the same subtype that was circulating in chickens by genetic reassortment and repetitive interspecies transmission (Hirst et al., 2004; Suarez et al., 2004; Wood et al., 1995). Understanding the mechanisms behind these different replication characteristics is a matter of interest, and further study is needed to determine whether this finding suggests a genetic distinction.
We identified many LPAI H7-positive duck farms through active surveillance and confirmed that at least four genotypes of H7 subtype AIV had been isolated from poultry in South Korea. Because these viruses carry the risk of increasing in virulence or reassorting to create novel viruses among poultry in South Korea, continuous systemic surveillance and genetic analyses are essential to understand the additional evolution of these H7 subtypes in the Korean peninsula.
Methods
Surveillance and viral isolation.
A total of 4327 captive migratory birds, 10 820 duck farms, 24 309 oropharyngeal swabs and faeces from LBM) and 57 856 faeces of aquatic birds were tested according to the active surveillance programme of HPAI conducted in South Korea from July 2008 to May 2011. The capturing and sampling of migratory birds were conducted at migratory bird habitats, and the sampling of domestic ducks and LBMs was conducted at farms that were adjacent to these migratory bird sites for the rapid detection of HPAI viruses. The swabs and faecal samples were suspended in an antibiotic solution and centrifuged at 1942 g for 10 min. The supernatants were filtered with 0.45 µm syringe filters and then inoculated into 9- to 11-day-old specific-pathogen-free (SPF) embryonated chicken eggs (ECEs) (Hy-Vac). The presence of AIV was determined using a haemagglutination assay after incubating the eggs for 4 days at 37 °C. AIV were subtyped using RT-PCR methods as described previously (Fereidouni et al., 2009; Fouchier et al., 2000; Lee et al., 2001; Munch et al., 2001). The 50 % egg infectious dose (EID50) titres were determined in 11-day-old SPF ECEs, and the viral titres were calculated by the Reed and Muench method (Reed & Muench, 1938).
Antigenic analysis.
The hyperimmune sera were prepared in chickens by intramuscular inoculation with 0.5 ml doses of inactivated vaccines that were constructed with the allantoic fluid of the viruses and an oil adjuvant. The hyperimmune sera were collected 2 weeks after injection with the vaccines. To investigate the cross-reactivity of the isolated H7 viruses, we performed a haemagglutinin-inhibition (HI) assay (Palmer et al., 1975) using chicken polyclonal antibodies to the following four H7 Korean AIV: A/duck/Korea/A349/09 (Dk/Kr/A349/09), A/duck/Korea/A75/10 (Dk/Kr/A75/10), A/wild bird/Korea/A330/09 (Wb/Kr/A330/09) and A/mallard/Korea/822/10 (mallard/Kr/822/10).
Sequencing and phylogenetic analysis.
The viral RNA was extracted directly from the HA-positive allantoic fluid using the Viral Gene-spin viral DNA/RNA extraction kit (iNtRON Biotechnology) according to the manufacturer’s instructions. A one-step RT-PCR was conducted using the PrimeScript one-step RT-PCR kit (TaKaRa) as recommended by the manufacturer. The primers that were used 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 using a QIAquick gel extraction kit (Qiagen), and the PCR product was sequenced at Cosmogenetech using an ABI 3730 XL DNA sequencer (Applied Biosystems). The sequences of the isolated viruses were aligned and edited using the Vector NTI and BioEdit software programs. The phylogenetic trees were generated by the neighbour-joining method using the mega 4.0 software (Tamura et al., 2007) with 1000 bootstrap replications.
Ethics statement.
All experiments were approved by the Animal Ethics Committee of the Animal, Plant and Fisheries Quarantine and Inspection Agency (QIA), in Anyang, Korea under approval number 2010-4.
Animal experiments.
For the replication capacity studies, 4-week-old SPF white leghorn broiler chickens and 4-week-old domestic ducks were inoculated intranasally with allantoic fluid that contained 5.5 logEID50 per 0.1 ml of each viral genotype as described previously (Li et al., 2003). Four H7 viruses that were isolated from domestic ducks [Dk/Kr/A349/09 (H7N2), Dk/Kr/A75/10 (H7N7), Dk/Kr/A76/10 (H7N7) and Dk/Kr/A117/10 (H7N6)] were inoculated into four chickens and eight ducks. Oropharyngeal and cloacal swabs were taken from chickens at 1, 3, 5, 7 and 10 days p.i. and from ducks at 1, 3, 5, 7, 10 and 14 days p.i. and inoculated into 9- to 11-day-old SPF ECE. For viral transmission to contact birds, four uninfected chickens were housed with inoculated chickens for 10 days. At 1, 3, 5, 7 and 10 days p.i., oropharyngeal and cloacal swabs were taken from contact chickens and inoculated into 9- to 11-day-old SPF ECE. In addition, the blood samples of ducks were collected to determine the viral replication at 14 days p.i. Group-specific antibodies were detected using a commercial c-ELISA test (AIV Ab ELISA kit; BioNote, Inc.) based on the detection of antibodies to the avian influenza virus type A (NP gene), following the manufacturer’s instructions.
To investigate the viral distribution in infected chickens, eight 6-week-old SPF white leghorn broiler chickens were inoculated via the intravenous route with 0.2 ml of 1/10 diluent of allantoic fluids from the four viruses described previously. We collected the trachea, caecal tonsil, kidney, spleen, liver and brain samples at 3 and 5 days p.i. All of the tissues were collected using separate scissors to prevent cross-contamination.
Acknowledgements
The authors thank Hyuk-Man Kwon for his excellent technical assistance. We also thank the colleges of Veterinary Medicine (Gyeongsang National University, Kangwon National University, Konkuk University, Kyungpook National University and Seoul National University), regional veterinary institutes (Jeonnam province, Jeonbuk province and Chungnam province) and the Veterinary Epidemiology Division of QIA for providing the samples. This work was supported by a grant from the National Animal Disease Control Project of the Ministry of Food, Agriculture, Forest and Fisheries of Korea.