- Email: [email protected]
Genetic characterization of porcine circovirus type 2 in Republic of Korea
Available online at www.sciencedirect.com
Research in Veterinary Science 84 (2008) 497–501 www.elsevier.com/locate/rvsc
Genetic characterization of porcine circovirus type 2 in Republic of Korea Kyoung-Seong Choi
a,*
, Joon-Seok Chae
b
a
b
Department of Animal Science, College of Life Science and Natural Resources, Sangju National University, 386 Gajang-Dong, Sangju 742-711, Republic of Korea Department of Veterinary Internal Medicine, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea Accepted 22 May 2007
Abstract Porcine circovirus type 2 (PCV2) is a major causative agent of postweaning multisystemic wasting syndrome (PMWS). Sequence and phylogenetic analyses based on the ORF2 capsid protein gene fragment showed that field isolate in Republic of Korea (ROK), PCV2 YJK 0703, was closely related with the PCV2 Fh18 isolate. PCV2 YJK 0703 was genetically distinct and not related to previously reported ROK isolates. Therefore, genotypic variation exists among prevailing PCV2 in ROK. This result suggests that several PCV2 genotypes exist in Korean pig farms. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Porcine circovirus type 2; Postweaning multisystemic wasting syndrome; Phylogenetic analysis; ORF2
Postweaning multisystemic wasting syndrome (PMWS) is an economically important disease characterized by progressive weight loss, jaundice, respiratory signs and skin paleness that affects late nursery and early fattening pigs (Allan and Ellis, 2000; Calsamiglia et al., 2002; Fenaux et al., 2000). Porcine circovirus type 2 (PCV2) is recognized as a major causal agent of PMWS, one of the significant health problems of the swine industry (Kim and Lyoo, 2002; Krakowka et al., 2001; Morozov et al., 1998; Nayar et al., 1998; Olvera et al., 2007; Staebler et al., 2005). Diagnosis of PMWS is based on the presence of compatible clinical signs, characteristic microscopic lesions and detection of PCV2 within characteristic lesions (Calsamiglia et al., 2002; Chae, 2004; Rosell et al., 1999). These three criteria separately are non-diagnostic of PMWS since PCV2 infection does not necessarily imply disease, and clinical signs are nonspecific and variable. Typical histopathological findings of PMWS are lymphocyte depletion, histiocytic infiltration, cytoplasmic inclusion body, and *
Corresponding author. Tel.: +82 54 530 5222; fax: +82 54 530 5229. E-mail address: [email protected] (K.-S. Choi).
0034-5288/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2007.05.017
granulomatous inflammation in lymphoid organs (Mankertz et al., 2000; Rosell et al., 1999). To date, a large number of PCV2 isolates from different countries and from pigs of various health states have been published (Choi et al., 2002; De boisseson et al., 2004; Grierson et al., 2004; Knell et al., 2005; Larochelle et al., 2002; Wen et al., 2005), but no genotyping study from Republic of Korea (ROK) isolates has been undertaken. Therefore, this study was done to report PMWS cases in ROK, to conduct sequencing analysis of ROK field isolates, to determine phylogenetic comparisons with other PCV2 isolates, and to estimate a source of domestic pig circovirus infection. Various tissue samples (lung, spleen, liver and mesenteric lymph node) were collected from three different commercial pig farms with 10 PMWS affected pigs (growth retardation, dyspnea, paleness of the skin, and enlarged lymph node at necropsy) and aged 6–8 weeks that originated from Gyeonggi province, ROK. The tissues were immediately frozen at 70 °C until use. The remaining tissue samples were fixed in 10% buffered formalin, embedded in paraffin, and routinely processed for histopathology.
498
K.-S. Choi, J.-S. Chae / Research in Veterinary Science 84 (2008) 497–501
DNA was extracted from the tissues using DNeasy tissue kit (Qiagen Inc.,Valencia, CA) according to the manufacturer’s instructions. For each DNA extraction, 20 mg of tissue sample was used. PCR was performed using PCV2 specific primers as previously described (Caprioli et al., 2006): The forward and reverse primers used were 5 0 -CACGGATATTGTAGTCCTGGT-30 and 50 -CCGCACCTTCGGATATACTGTC-3 0 . The predicted size of the amplified PCR product was 494 bp. PCR amplification was carried out for 39 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. The amplicons were subjected to electrophoresis in 1% agarose gels and visualized by ethidium bromide. Amplified DNAs were purified for direct sequencing using the QIAquick Gel Extraction kit (Qiagen) and sequenced both 3 0 and 5 0 by dideoxy termination with an automated sequencer (ABI PRISMÒ 3700 DNA analyzer, applied biosystems, Foster, CA). The sequence data were collected using ABI prism data collection software (v2.1), and analyzed by ABI prism sequence analysis software (ver. 2.1.1) and chromas software (v1.51) (Technelysium Pty Ltd., Mt. Gravatt Plaza, Queensland, Australia). Sequence homology searches
were made via the National Center for Biotechnology Information (National Institute of Health, Bethesda, MD) BLAST network service. The sequences were translated to amino acid sequences and were aligned initially using ClustalX (1.60) (Thompson et al., 1997). Aligned sequences were examined with a similarity matrix. Relationships between individuals were assessed by neighbor-joining (NJ) method with nucleotides distances (p-distance) with 100 replications in the bootstrap test. Phylogenetic analysis based on the obtained sequences was conducted using maximum-likelihood (PAUP* 4.0b; Swofford, 2002). PCV2 YJK 0703 capsid gene sequence of this study in ROK has been deposited in GenBank under accession number EF055264. The GenBank accession numbers of PCV2 capsid gene sequences used to analyze percent identity and to construct the phylogenetic tree were: Fh18, France (AY321987); ROM, Romania (DQ233257); Hun B, Hungary (DQ648031); AUT5, Austria (AY424405); am9, Brazil (DQ861899); NL_PMWS_3, Netherlands (AY484415); ZC, China (AY682997); Shanghai, China (DQ195679); Fh21, France (AY322001); 304, Hungary,
1
90
YJK 0703/Korea
RTFGYTVKRT TVKTPSWAVD MMRFNINDFL PPGGGSNPRS VPFEYYRIRK VKVEFWPCSP ITQGDRGVGS SAVILDDNFV TKATALTYDP
Fh18/France
RTFGYTVKRT TVKTPSWAVD MMRFNINDFL PPGGGSNPRS VPFEYYRIRK VKVEFWPCSP ITQGDRGVGS SAVILDDNFV TKATALTYDP
NL_PMWS_1/Netherlands
RTFGYTVKRT TVRTPSWAVD MMRFNINDFL PPGGGSNPRS VPFEYYRIRK VKVEFWPCSP ITQGDRGVGS SAVILDDNFV TKATALTYDP
375/Hungary
RTFGYTVKRT TVRTPSWAVD MMRFNINDFL PPGGGSNPRS VPFEYYRIRK VKVEFWPCSP ITQGDRGVGS SAVILDDNFV TKATALTYDP
KSY-2/Korea
RTFGYTVKRT TVTTPSWAVD MMRFTIDDFV PPGGGTNKIS IPFEYYRIRK VKVEFWPCSP ITQGDRGVGS TAVILDDNFF PKTTALTYDP
JHP/Korea
RTFGYTVKRT TVTTPSWAVD MMRFTIGDFV PPGGGTNKIS IPFEYYRIRK VKVEFWPCSP ITQGDRGVGS TAVILDDNFV PKATALTYDP
KSY-1/Korea
RTFGYTVRRT TVTTPSWAVD MMRFKLDDFV PPGGGTNKIS IPFEYYRIRK VKVEFWPCSP ITQGDRGVGS TAVILDDNFV PKANALTYDP
CNU2/Korea
RTFGYTVKRT TVTTPSWAVD MMRFKLDDFV PPGGGTNKIS IPFEYYRIRK VKVEFWPCSP ITQGDRGVGS TAVILDDNFV PKANALTYDP
CNU/Korea
RTFGYTVKRT TVTTPSWAVD MMRFKLDDFV PPGGGTNKIS IPFEYYRIRK VKVEFWPCSP ITQGDRGVGS TAVILDDNFV PKANALTYDP *
*
***
*
* **
*
91
* * ** 164
YJK 0703/Korea
YVNYSSRHTI TQPFSYHSRY FTPKPVLDST IDYFQPNNKR NQLWLRLQTA GNVDHVGLGT AFENSIYDQD YNIR
Fh18/France
YVNYSSRHTI TQPFSYHSRY FTPKPVLDST IDYFQPNNKR NQLWLRLQTA GNVDHVGLGT AFENSIYDQE YNIR
NL_PMWS_1/Netherlands
YVNYSSRHTI TQPFSYHSRY FTPKPVLDST IDYFQPNNKR NQLWLRLQTA GNVDHVGLGT AFENSIYDQE YNIR
375/Hungary
YVNYSSRHTI TQPFSYHSRY FTPKPVLDST IDYFQPNNKR NQLWLRLQTA GNVDHVGLGT AFENSIYDQE YNIR
KSY-2/Korea
YVNYSSRHTI PQPFSYHSRY FTPKPVLDST IDYFQPNNKR NQLWLRLQTS RNVDHVGLGT AFENSKYDQD YNIR
JHP/Korea
YVNYSSRHTI PQPFSYHSRY FTPKPVLDST IDYFQPNNKR NQLWLRLQTS RNVDHVGLGT AFENSKYDQD YNIR
KSY-1/Korea
YVNYSSRHTI PQPFSYHSRY FTPKPVLDST IDYFQPNNKR NQLWLRLQTS RNVDHVGLGT AFENSKYDQD YNIR
CNU2/Korea
YVNYSSRHTI PQPFSYHSRY FTPKPVLDST IDYFQPNNKR NQLWLRLQTS RNVDHVGLGT AFENSKYDQD YNIR
CNU/Korea
YVNYSSRHTI PQPFSYHSRY FTPKPVLDST IDYFQPNNKR NQLWLRLQTS RNVDHVGLGT AFENNKYDQD YNIR *
* *
*
*
Fig. 1. Comparison of the putative capsid protein coded by ORF2 gene fragment of YJK 0703 with France, Netherlands, Hungary and five ROK isolates. Differences in amino acid sequence (bold letters) are indicated with asterisks below the alignment.
K.-S. Choi, J.-S. Chae / Research in Veterinary Science 84 (2008) 497–501
(AY256457); BRA1, Brazil (DQ364650); HZ0202, China (AY217743); am21, Brazil (DQ861902); NL_PMWS_1, Netherlands (AY484413); 375, Hungary (AY256460); CNU2, South Korea (AY672601); CNU, South Korea (AY672600); KSY-2, South Korea (AF544024); KSY-1, South Korea (AF454546); JHP, South Korea (AF520783). To determine the extent of genetic heterogeneity among PCV2 ROK field isolates, 10 capsid gene fragments of PCV2 were amplified and sequenced. The pigs included in this study possessed clinical signs with PMWS, and PCV2 DNA was detected by PCR in several tissues including lymph node, lung, liver, and spleen, and as well as confirmed by histopathology (data not shown). No porcine reproductive and respiratory syndrome (PRRS) was detected in pigs with PMWS. Total RNA was extracted from the same tissues by using the QIAamp viral RNA mini kit (Qiagen). The entire ORF5 of PRRS was amplified by RT-RCR (Cha et al., 2004) All of the amplified PCV2 capsid gene fragment sequences from spleen, liver, lung and mesenteric lymph
499
node collected from 10 PMWS affected pigs were exactly identical among amplicons and deposited to GenBank as YJK 0703 (GenBank accession number, EF055264). The YJK 0703 (494 bp) sequence was closely related to isolate Fh18, displaying 99.8% nucleotide sequence identity and one nucleotide mutation in position 14. However, the translated amino acid sequence (164 amino acid residues) from YJK 0703 had similar identity (99.4%) to the PCV2 ORF2 amino acid fragment from isolate Fh18 with a discrepancy at position 160 (Fig. 1). To understand the genetic relationship, phylogenetic analysis was performed on the basis of the genomic sequences of 21 PCV2 isolates worldwide, including YJK 0703 sequenced in this study. In the neighbor-joining phylogram of PCV2 isolates, the tree shows two ROK branch isolates outside of all others. Five ROK isolates were within one cluster, and still showed micro-heterogeneity. YJK 0703 was more closely related to French, Netherlands, Hungary and China isolates. (Fig. 2). There were clear divergences among other ROK isolates.
375, Hungary KSY—1, Korea 97 100
52
58
CNU, Korea CNU2, Korea KSY—2, Korea JHP, Korea
YJK 0703, Korea HZ0202,China Fh21, France 68 52
Hun B, Hungary AUT5, Austria ROM, Romania 304, Hungary China NL_PMWS_3, Netherlands ZC, China BRA1, Brazil AM21, Brazil AM9, Brazil Fh18, France
NL_PMWS_1, Netherlands 0.005 substitutions/site Fig. 2. The phylogram of neighbor-joining tree for 21 PCV2 isolates. The tree was made using PAUP* 4.0b software after alignment of ORF2 DNA sequences (494 bp) obtained from GenBank and sequenced during this study by the ClustalX program. The numbers present at the nodes of the tree represent the number of bootstrap replicates (out of 100) that display the indicated sequence groupings.
500
K.-S. Choi, J.-S. Chae / Research in Veterinary Science 84 (2008) 497–501
Few PCV2 isolates from ROK have been genetically characterized (Kim and Lyoo, 2002). Most phylogenetic analysis of PCV2 isolates reported to date have been performed on PCV2 isolates from PMWS worldwide (Grierson et al., 2004; Knell et al., 2005; Larochelle et al., 2002; Mankertz et al., 2000). We report here the phylogenetic analysis of 21 PCV2 sequences, including YJK 0703, from GenBank representing mostly isolates from PMWS cases in different countries. This phylogenetic analysis revealed one large cluster composed of isolates from Europe, Brazil and China. However, ROK PCV2 isolates sequenced were differentially and genetic variation was observed. Five of ROK PCV2 isolates form a distinguishable branch that is separate from other isolates. The clinical significance of this divergence among ROK isolates is not known. ORF2 of PCV2 is believed to encode for the putative capsid protein and found to be highly variable compared with ORF1 (Fenaux et al., 2000). Furthermore, it is available for ORF2 gene of PCV2 as target fragment, since ORF2 is shorter and its sequencing is less labor extensive than the whole genome, which is used for epidemiological and phylogenetic studies (De boisseson et al., 2004; Opriessnig et al., 2006; Olvera et al., 2007). Our amino acid sequence alignment of the capsid protein revealed that the highest sequence-convergence existed between YJK 0703 and Fh18 however; there was greater heterogeneity in sequence analysis among ROK isolates. Therefore, our data strongly suggest that genetic variations do exist among ROK PCV2 isolates. These PCV2 isolates from PMWS affected pigs have the greatest homology with isolate Fh18. Although this homology with Fh18 is interesting, it remains unclear whether these ROK isolates evolved within other domestic sources such as semen, if they were introduced from outside of ROK, and whether they indicate the route of transmission. It is possible that the Fh18-like isolates are the most prevalent type in ROK. Further research should focus on the characterization of PCV2 sequences from different regions of ROK to better understand the reason for adaptive advantages of these genetic variants. Acknowledgements We thank Jin-Sun Kim at DaeSang Farmsco for field samples, Ki-Jung Yun at Department of Pathology, College of Medicine, WonKwang University for histopathological expertise, and Dong-Ho Nho for assistance. This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) KRF-2006-331-E00358. References Allan, G.M., Ellis, J.A., 2000. Porcine circoviruses: a review. Journal of Veterinary Diagnostic Investigation 12, 3–14. Calsamiglia, M., Segales, J., Quintana, J., Rosell, C., Domingo, M., 2002. Detection of porcine circovirus types 1 and 2 in serum and tissue
samples of pigs with and without postweaning multisystemic wasting syndrome. Journal of Clinical Microbiology 40, 1848–1850. Caprioli, A., Mcneilly, F., Mcnair, I., Lagan-Tregaskis, P., Ellis, J., Krakowka, S., Mckillen, J., Ostanello, F., Allen, G., 2006. PCR detection of porcine circovirus type 2 (PCV2) DNA in blood, tonsillar and faecal swabs from experimentally infected pigs. Research in Veterinary Science 81, 287–292. Cha, S.H., Chang, C.C., Yoon, K.J., 2004. Instability of the restriction fragment length polymorphism pattern of open reading frame 5 of porcine reproductive and respiratory syndrome virus during sequential pig-to-pig passages. Journal of Clinical Microbiology 42, 4462– 4467. Chae, C., 2004. Postweaning multisystemic wasting syndrome: a review of aetiology, diagnosis and pathology. Veterinary Journal 168, 41–49. Choi, J., Stevenson, G.W., Kiupel, M., Harrach, B., Anothayanontha, L., Kanitz, C.L., Mittal, S.K., 2002. Sequence analysis of old and new strains of porcine circovirus associated with congenital tremors in pigs and their comparison with strains involved with postweaning multisystemic wasting syndrome. Canadian Journal of Veterinary Research 66, 217–224. De boisseson, C., Beven, V., Bigarre, L., Thiery, R., Rose, N., Eveno, E., Madec, F., Jestin, A., 2004. Molecular characterization of Porcine circovirus type 2 isolates from post-weaning multisystemic wasting syndrome-affected and non-affected pigs. Journal of General Virology 85, 293–304. Fenaux, M., Halbur, P.G., Gill, M., Toth, T.E., Meng, X.J., 2000. Genetic characterization of type 2 porcine circovirus (PCV-2) from pigs with postweaning multisystemic wasting syndrome in different geographic regions of North America and development of a differential PCRrestriction fragment length polymorphism assay to detect and differentiate between infections with PCV-1 and PCV-2. Journal of Clinical Microbiology 38, 2494–2503. Grierson, S.S., King, D.P., Wellenberg, G.J., Banks, M., 2004. Genome sequence analysis of 10 Dutch porcine circovirus type 2 (PCV-2) isolates from a PMWS case-control study. Research in Veterinary Science 77, 265–268. Kim, J.H., Lyoo, Y.S., 2002. Genetic characterization of porcine circovirus-2 field isolates from PMWS pigs. Journal of Veterinary Science 3, 31–39. Knell, S., Willems, H., Hertrampf, B., Reiner, G., 2005. Comparative genetic characterization of Porcine Circovirus type 2 samples from German wild boar populations. Veterinary Microbiology 109, 169–177. Krakowka, S., Ellis, J.A., Mcneilly, F., Ringler, S., Rings, D.M., Allan, G., 2001. Activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus-2 (PCV-2). Veterinary Pathology 38, 31–42. Larochelle, R., Magar, R., D’allaire, S., 2002. Genetic characterization and phylogenetic analysis of porcine circovirus type 2 (PCV2) strains from cases presenting various clinical conditions. Virus Research 90, 101–112. Mankertz, A., Domingo, M., Folch, J.M., Lecann, P., Jestin, A., Segales, J., Chmielewicz, B., Plana-Duran, J., Soike, D., 2000. Characterisation of PCV-2 isolates from Spain, Germany and France. Virus Research 66, 65–77. Morozov, I., Sirinarumitr, T., Sorden, S.D., Halbur, P.G., Morgan, M.K., Yoon, K.J., Paul, P.S., 1998. Detection of a novel strain of porcine circovirus in pigs with postweaning multisystemic wasting syndrome. Journal of Clinical Microbiology 36, 2535–2541. Nayar, G.P., Hamel, A.L., Lin, L., 1998. Detection and characterization of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs. Canadian Veterinary Journal 38, 385–386. Olvera, A., Cortey, M., Segales, J., 2007. Molecular evolution of porcine circovirus type 2 genomes: phylogeny and clonality. Virology 357, 175–185. Opriessnig, T., McKeown, N.E., Zhou, E.M., Meng, X.J., Halbur, P.G., 2006. Genetic and experimental comparison of porcine circovirus type 2 (PCV2) isolates from cases with and without PCV2-associated lesions
K.-S. Choi, J.-S. Chae / Research in Veterinary Science 84 (2008) 497–501 provides evidence for differences in virulence. Journal of General Virology 87, 2923–2932. Rosell, C., Segales, J., Plana-Duran, J., Balasch, M., Rodriguez-Arrioja, G.M., Kennedy, S., Allan, G.M., Mcneilly, F., Latimer, K.S., Domingo, M., 1999. Pathological, immunohistochemical, and in-situ hybridization studies of natural cases of postweaning multisystemic wasting syndrome (PMWS) in pigs. Journal of Comparative Pathology 120, 59–78. Staebler, S., Sydler, T., Buergi, E., Mccullough, K., Mcneilly, F., Allan, G., Pospischil, A., 2005. PMWS: an emerging disease identified in archived porcine tissues. Veterinary Journal 170, 132–134.
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Swofford, D.L., 2002. PAUP*: Phylogenetic analysis using parasimony (* and other methods), Version 4. Sinauer Associates, Sunderland, Massachusetts. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Research 24, 4876–4882. Wen, L., Guo, X., Yang, H., 2005. Genotyping of porcine circovirus type 2 from a variety of clinical conditions in China. Veterinary Microbiology 110, 141–146.
Research in Veterinary Science 84 (2008) 497–501 www.elsevier.com/locate/rvsc
Genetic characterization of porcine circovirus type 2 in Republic of Korea Kyoung-Seong Choi
a,*
, Joon-Seok Chae
b
a
b
Department of Animal Science, College of Life Science and Natural Resources, Sangju National University, 386 Gajang-Dong, Sangju 742-711, Republic of Korea Department of Veterinary Internal Medicine, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea Accepted 22 May 2007
Abstract Porcine circovirus type 2 (PCV2) is a major causative agent of postweaning multisystemic wasting syndrome (PMWS). Sequence and phylogenetic analyses based on the ORF2 capsid protein gene fragment showed that field isolate in Republic of Korea (ROK), PCV2 YJK 0703, was closely related with the PCV2 Fh18 isolate. PCV2 YJK 0703 was genetically distinct and not related to previously reported ROK isolates. Therefore, genotypic variation exists among prevailing PCV2 in ROK. This result suggests that several PCV2 genotypes exist in Korean pig farms. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Porcine circovirus type 2; Postweaning multisystemic wasting syndrome; Phylogenetic analysis; ORF2
Postweaning multisystemic wasting syndrome (PMWS) is an economically important disease characterized by progressive weight loss, jaundice, respiratory signs and skin paleness that affects late nursery and early fattening pigs (Allan and Ellis, 2000; Calsamiglia et al., 2002; Fenaux et al., 2000). Porcine circovirus type 2 (PCV2) is recognized as a major causal agent of PMWS, one of the significant health problems of the swine industry (Kim and Lyoo, 2002; Krakowka et al., 2001; Morozov et al., 1998; Nayar et al., 1998; Olvera et al., 2007; Staebler et al., 2005). Diagnosis of PMWS is based on the presence of compatible clinical signs, characteristic microscopic lesions and detection of PCV2 within characteristic lesions (Calsamiglia et al., 2002; Chae, 2004; Rosell et al., 1999). These three criteria separately are non-diagnostic of PMWS since PCV2 infection does not necessarily imply disease, and clinical signs are nonspecific and variable. Typical histopathological findings of PMWS are lymphocyte depletion, histiocytic infiltration, cytoplasmic inclusion body, and *
Corresponding author. Tel.: +82 54 530 5222; fax: +82 54 530 5229. E-mail address: [email protected] (K.-S. Choi).
0034-5288/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2007.05.017
granulomatous inflammation in lymphoid organs (Mankertz et al., 2000; Rosell et al., 1999). To date, a large number of PCV2 isolates from different countries and from pigs of various health states have been published (Choi et al., 2002; De boisseson et al., 2004; Grierson et al., 2004; Knell et al., 2005; Larochelle et al., 2002; Wen et al., 2005), but no genotyping study from Republic of Korea (ROK) isolates has been undertaken. Therefore, this study was done to report PMWS cases in ROK, to conduct sequencing analysis of ROK field isolates, to determine phylogenetic comparisons with other PCV2 isolates, and to estimate a source of domestic pig circovirus infection. Various tissue samples (lung, spleen, liver and mesenteric lymph node) were collected from three different commercial pig farms with 10 PMWS affected pigs (growth retardation, dyspnea, paleness of the skin, and enlarged lymph node at necropsy) and aged 6–8 weeks that originated from Gyeonggi province, ROK. The tissues were immediately frozen at 70 °C until use. The remaining tissue samples were fixed in 10% buffered formalin, embedded in paraffin, and routinely processed for histopathology.
498
K.-S. Choi, J.-S. Chae / Research in Veterinary Science 84 (2008) 497–501
DNA was extracted from the tissues using DNeasy tissue kit (Qiagen Inc.,Valencia, CA) according to the manufacturer’s instructions. For each DNA extraction, 20 mg of tissue sample was used. PCR was performed using PCV2 specific primers as previously described (Caprioli et al., 2006): The forward and reverse primers used were 5 0 -CACGGATATTGTAGTCCTGGT-30 and 50 -CCGCACCTTCGGATATACTGTC-3 0 . The predicted size of the amplified PCR product was 494 bp. PCR amplification was carried out for 39 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. The amplicons were subjected to electrophoresis in 1% agarose gels and visualized by ethidium bromide. Amplified DNAs were purified for direct sequencing using the QIAquick Gel Extraction kit (Qiagen) and sequenced both 3 0 and 5 0 by dideoxy termination with an automated sequencer (ABI PRISMÒ 3700 DNA analyzer, applied biosystems, Foster, CA). The sequence data were collected using ABI prism data collection software (v2.1), and analyzed by ABI prism sequence analysis software (ver. 2.1.1) and chromas software (v1.51) (Technelysium Pty Ltd., Mt. Gravatt Plaza, Queensland, Australia). Sequence homology searches
were made via the National Center for Biotechnology Information (National Institute of Health, Bethesda, MD) BLAST network service. The sequences were translated to amino acid sequences and were aligned initially using ClustalX (1.60) (Thompson et al., 1997). Aligned sequences were examined with a similarity matrix. Relationships between individuals were assessed by neighbor-joining (NJ) method with nucleotides distances (p-distance) with 100 replications in the bootstrap test. Phylogenetic analysis based on the obtained sequences was conducted using maximum-likelihood (PAUP* 4.0b; Swofford, 2002). PCV2 YJK 0703 capsid gene sequence of this study in ROK has been deposited in GenBank under accession number EF055264. The GenBank accession numbers of PCV2 capsid gene sequences used to analyze percent identity and to construct the phylogenetic tree were: Fh18, France (AY321987); ROM, Romania (DQ233257); Hun B, Hungary (DQ648031); AUT5, Austria (AY424405); am9, Brazil (DQ861899); NL_PMWS_3, Netherlands (AY484415); ZC, China (AY682997); Shanghai, China (DQ195679); Fh21, France (AY322001); 304, Hungary,
1
90
YJK 0703/Korea
RTFGYTVKRT TVKTPSWAVD MMRFNINDFL PPGGGSNPRS VPFEYYRIRK VKVEFWPCSP ITQGDRGVGS SAVILDDNFV TKATALTYDP
Fh18/France
RTFGYTVKRT TVKTPSWAVD MMRFNINDFL PPGGGSNPRS VPFEYYRIRK VKVEFWPCSP ITQGDRGVGS SAVILDDNFV TKATALTYDP
NL_PMWS_1/Netherlands
RTFGYTVKRT TVRTPSWAVD MMRFNINDFL PPGGGSNPRS VPFEYYRIRK VKVEFWPCSP ITQGDRGVGS SAVILDDNFV TKATALTYDP
375/Hungary
RTFGYTVKRT TVRTPSWAVD MMRFNINDFL PPGGGSNPRS VPFEYYRIRK VKVEFWPCSP ITQGDRGVGS SAVILDDNFV TKATALTYDP
KSY-2/Korea
RTFGYTVKRT TVTTPSWAVD MMRFTIDDFV PPGGGTNKIS IPFEYYRIRK VKVEFWPCSP ITQGDRGVGS TAVILDDNFF PKTTALTYDP
JHP/Korea
RTFGYTVKRT TVTTPSWAVD MMRFTIGDFV PPGGGTNKIS IPFEYYRIRK VKVEFWPCSP ITQGDRGVGS TAVILDDNFV PKATALTYDP
KSY-1/Korea
RTFGYTVRRT TVTTPSWAVD MMRFKLDDFV PPGGGTNKIS IPFEYYRIRK VKVEFWPCSP ITQGDRGVGS TAVILDDNFV PKANALTYDP
CNU2/Korea
RTFGYTVKRT TVTTPSWAVD MMRFKLDDFV PPGGGTNKIS IPFEYYRIRK VKVEFWPCSP ITQGDRGVGS TAVILDDNFV PKANALTYDP
CNU/Korea
RTFGYTVKRT TVTTPSWAVD MMRFKLDDFV PPGGGTNKIS IPFEYYRIRK VKVEFWPCSP ITQGDRGVGS TAVILDDNFV PKANALTYDP *
*
***
*
* **
*
91
* * ** 164
YJK 0703/Korea
YVNYSSRHTI TQPFSYHSRY FTPKPVLDST IDYFQPNNKR NQLWLRLQTA GNVDHVGLGT AFENSIYDQD YNIR
Fh18/France
YVNYSSRHTI TQPFSYHSRY FTPKPVLDST IDYFQPNNKR NQLWLRLQTA GNVDHVGLGT AFENSIYDQE YNIR
NL_PMWS_1/Netherlands
YVNYSSRHTI TQPFSYHSRY FTPKPVLDST IDYFQPNNKR NQLWLRLQTA GNVDHVGLGT AFENSIYDQE YNIR
375/Hungary
YVNYSSRHTI TQPFSYHSRY FTPKPVLDST IDYFQPNNKR NQLWLRLQTA GNVDHVGLGT AFENSIYDQE YNIR
KSY-2/Korea
YVNYSSRHTI PQPFSYHSRY FTPKPVLDST IDYFQPNNKR NQLWLRLQTS RNVDHVGLGT AFENSKYDQD YNIR
JHP/Korea
YVNYSSRHTI PQPFSYHSRY FTPKPVLDST IDYFQPNNKR NQLWLRLQTS RNVDHVGLGT AFENSKYDQD YNIR
KSY-1/Korea
YVNYSSRHTI PQPFSYHSRY FTPKPVLDST IDYFQPNNKR NQLWLRLQTS RNVDHVGLGT AFENSKYDQD YNIR
CNU2/Korea
YVNYSSRHTI PQPFSYHSRY FTPKPVLDST IDYFQPNNKR NQLWLRLQTS RNVDHVGLGT AFENSKYDQD YNIR
CNU/Korea
YVNYSSRHTI PQPFSYHSRY FTPKPVLDST IDYFQPNNKR NQLWLRLQTS RNVDHVGLGT AFENNKYDQD YNIR *
* *
*
*
Fig. 1. Comparison of the putative capsid protein coded by ORF2 gene fragment of YJK 0703 with France, Netherlands, Hungary and five ROK isolates. Differences in amino acid sequence (bold letters) are indicated with asterisks below the alignment.
K.-S. Choi, J.-S. Chae / Research in Veterinary Science 84 (2008) 497–501
(AY256457); BRA1, Brazil (DQ364650); HZ0202, China (AY217743); am21, Brazil (DQ861902); NL_PMWS_1, Netherlands (AY484413); 375, Hungary (AY256460); CNU2, South Korea (AY672601); CNU, South Korea (AY672600); KSY-2, South Korea (AF544024); KSY-1, South Korea (AF454546); JHP, South Korea (AF520783). To determine the extent of genetic heterogeneity among PCV2 ROK field isolates, 10 capsid gene fragments of PCV2 were amplified and sequenced. The pigs included in this study possessed clinical signs with PMWS, and PCV2 DNA was detected by PCR in several tissues including lymph node, lung, liver, and spleen, and as well as confirmed by histopathology (data not shown). No porcine reproductive and respiratory syndrome (PRRS) was detected in pigs with PMWS. Total RNA was extracted from the same tissues by using the QIAamp viral RNA mini kit (Qiagen). The entire ORF5 of PRRS was amplified by RT-RCR (Cha et al., 2004) All of the amplified PCV2 capsid gene fragment sequences from spleen, liver, lung and mesenteric lymph
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node collected from 10 PMWS affected pigs were exactly identical among amplicons and deposited to GenBank as YJK 0703 (GenBank accession number, EF055264). The YJK 0703 (494 bp) sequence was closely related to isolate Fh18, displaying 99.8% nucleotide sequence identity and one nucleotide mutation in position 14. However, the translated amino acid sequence (164 amino acid residues) from YJK 0703 had similar identity (99.4%) to the PCV2 ORF2 amino acid fragment from isolate Fh18 with a discrepancy at position 160 (Fig. 1). To understand the genetic relationship, phylogenetic analysis was performed on the basis of the genomic sequences of 21 PCV2 isolates worldwide, including YJK 0703 sequenced in this study. In the neighbor-joining phylogram of PCV2 isolates, the tree shows two ROK branch isolates outside of all others. Five ROK isolates were within one cluster, and still showed micro-heterogeneity. YJK 0703 was more closely related to French, Netherlands, Hungary and China isolates. (Fig. 2). There were clear divergences among other ROK isolates.
375, Hungary KSY—1, Korea 97 100
52
58
CNU, Korea CNU2, Korea KSY—2, Korea JHP, Korea
YJK 0703, Korea HZ0202,China Fh21, France 68 52
Hun B, Hungary AUT5, Austria ROM, Romania 304, Hungary China NL_PMWS_3, Netherlands ZC, China BRA1, Brazil AM21, Brazil AM9, Brazil Fh18, France
NL_PMWS_1, Netherlands 0.005 substitutions/site Fig. 2. The phylogram of neighbor-joining tree for 21 PCV2 isolates. The tree was made using PAUP* 4.0b software after alignment of ORF2 DNA sequences (494 bp) obtained from GenBank and sequenced during this study by the ClustalX program. The numbers present at the nodes of the tree represent the number of bootstrap replicates (out of 100) that display the indicated sequence groupings.
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Few PCV2 isolates from ROK have been genetically characterized (Kim and Lyoo, 2002). Most phylogenetic analysis of PCV2 isolates reported to date have been performed on PCV2 isolates from PMWS worldwide (Grierson et al., 2004; Knell et al., 2005; Larochelle et al., 2002; Mankertz et al., 2000). We report here the phylogenetic analysis of 21 PCV2 sequences, including YJK 0703, from GenBank representing mostly isolates from PMWS cases in different countries. This phylogenetic analysis revealed one large cluster composed of isolates from Europe, Brazil and China. However, ROK PCV2 isolates sequenced were differentially and genetic variation was observed. Five of ROK PCV2 isolates form a distinguishable branch that is separate from other isolates. The clinical significance of this divergence among ROK isolates is not known. ORF2 of PCV2 is believed to encode for the putative capsid protein and found to be highly variable compared with ORF1 (Fenaux et al., 2000). Furthermore, it is available for ORF2 gene of PCV2 as target fragment, since ORF2 is shorter and its sequencing is less labor extensive than the whole genome, which is used for epidemiological and phylogenetic studies (De boisseson et al., 2004; Opriessnig et al., 2006; Olvera et al., 2007). Our amino acid sequence alignment of the capsid protein revealed that the highest sequence-convergence existed between YJK 0703 and Fh18 however; there was greater heterogeneity in sequence analysis among ROK isolates. Therefore, our data strongly suggest that genetic variations do exist among ROK PCV2 isolates. These PCV2 isolates from PMWS affected pigs have the greatest homology with isolate Fh18. Although this homology with Fh18 is interesting, it remains unclear whether these ROK isolates evolved within other domestic sources such as semen, if they were introduced from outside of ROK, and whether they indicate the route of transmission. It is possible that the Fh18-like isolates are the most prevalent type in ROK. Further research should focus on the characterization of PCV2 sequences from different regions of ROK to better understand the reason for adaptive advantages of these genetic variants. Acknowledgements We thank Jin-Sun Kim at DaeSang Farmsco for field samples, Ki-Jung Yun at Department of Pathology, College of Medicine, WonKwang University for histopathological expertise, and Dong-Ho Nho for assistance. This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) KRF-2006-331-E00358. References Allan, G.M., Ellis, J.A., 2000. Porcine circoviruses: a review. Journal of Veterinary Diagnostic Investigation 12, 3–14. Calsamiglia, M., Segales, J., Quintana, J., Rosell, C., Domingo, M., 2002. Detection of porcine circovirus types 1 and 2 in serum and tissue
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