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Genetic grouping of classical swine fever virus by restriction fragment length polymorphism of the E2 gene
Journal of Virological Methods 87 (2000) 145 – 149 www.elsevier.com/locate/jviromet
Genetic grouping of classical swine fever virus by restriction fragment length polymorphism of the E2 gene Sujira Parchariyanon a,*, Ken Inui a, Wasana Pinyochon a, Sudarat Damrongwatanapokin a, Eiji Takahashi b b
a National Institute of Animal Health, Jatujak, Bangkok 10900, Thailand Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The Uni6ersity of Tokyo, Yayoi 1 -1 -1, Bunkyo-ku, Tokyo 113 -8657, Japan
Received 11 October 1999; received in revised form 8 March 2000; accepted 8 March 2000
Abstract A method for genetic grouping of classical swine fever viruses (CSFV) was developed based on the restriction fragment length polymorphism (RFLP) revealed by AvaII, BanII and PvuII digestion of RT-PCR amplified segments of the E2 gene. From inspection of the genetic sequences of Thai isolates and reference strains, the RFLP method was designed to be capable of differentiating all known genogroups and subgenogroups suggested by phylogenetic analysis of the CSFV E2 gene. The method was applied to 60 CSFV samples which included three genogroups and seven subgenogroups. Unlike previously described RFLP methods, the agarose gel patterns obtained from these samples were completely in agreement with the predicted RFLP patterns and enabled accurate genetic grouping of CSFV at the subgenogroup level. The simplicity of this method allows rapid CSFV genogrouping at diagnostic laboratories without sequencing facilities and provides a useful method for diagnosis as well as epidemiological investigation and control of classical swine fever outbreaks. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Classical swine fever; Genetic grouping; Restriction fragment length polymorphism
Classical swine fever (CSF) is an important disease economically for the pig industry in many parts of the world. The causative agent is classical swine fever virus (CSFV) which belongs to the Pesti6irus genus of the Fla6i6iridae family together with bovine viral diarrhea virus and border disease virus. Genetic variability of CSFV has been * Corresponding author. Tel.: + 66-2-579890814; fax: +662-579891819. E-mail address: p – [email protected] (S. Parchariyanon)
reported among isolates from different parts of the world (Lowings et al., 1996; Vilcek et al., 1996). The identification of virus strains and genotypes by genetic characterisation can be used conveniently to trace sources of infection and to improve our understanding of the epidemiology of CSF. Genetic characterisation based on genetic sequence data provides precise information on the relationships between field isolates, but it is not a practical method for all diagnostic laboratories, as it requires facilities for sequencing.
0166-0934/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 0 0 ) 0 0 1 6 2 - 2
S. Parchariyanon et al. / Journal of Virological Methods 87 (2000) 145–149
146
The polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis is an easy and quick system widely practiced for various viral diseases to identify the virus and to differentiate strains or genotypes. Vilcek et al. (1994) showed that RFLP of the 5% noncoding region (5%NCR) of pestiviruses can be used to differentiate CSFV from ruminant pestiviruses but not the genogroups within CSFV. Harding et al. (1994) demonstrated with a limited number of CSFV samples that RFLP of the NS23 nonstructural protein gene could differentiate isolates to some extent. Phylogenetic analysis of the E2 gene of Thai isolates when compared with those of world isolates (Lowings et al., 1996; Paton et al., 2000, in press) has demonstrated that these can be divided into three genogroups comprising five subgenogroups, termed 1.1, 1.2, 1.3, 2.2 and 3.3. No Thai isolates were found in subgenogroups 2.1 and 2.3 (Parchariyanon et al., 2000, in press). In the present study we describe a simple and rapid method combining RT-PCR and RFLP of the E2 gene to differentiate CSFV genogroups identified by phylogenetic analysis of the E2 gene. The method is compared to the RFLP approach based on NS23 described previously (Harding et al., 1994). Restriction enzymes and their cleavage sites were selected and determined by computer analysis of the E2 gene sequence information obtained from our previous study (Parchariyanon et al., Table 1 Predicted RFLP patterns of E2 gene PCR products digested by AvaII, BanII and PvuII Enzyme
RFLP patterns
Size of DNA fragments (bps)
AvaII
1 2 3 4 5 1 2 3 4 1 2
270 221 181 132, 116, 270 205 179 179, 270 194
BanII
PvuII
and 49 and 89 89 and 49 105 and 49 and 65 and 91 65 and 26 and 76
2000, in press) or from published sequences. The actual RFLP analysis was performed on 54 Thai field isolates, four vaccines and two reference strains for which sequences and genogroups had already been determined. The two reference strains representing subgenogroup 2.1 and 2.3 were kindly provided by Dr David Paton (Veterinary Laboratories Agency, UK). RNA extraction and RT-PCR were carried out as described previously (Parchariyanon et al., 2000, in press). Primers used to amplify 270 bases of the E2 gene (Alfort 187 nucleotides 2467–2716) were E2F (5%TCRWCAACC-AAYGAGATAGGG-3%) and E2R (5%-CACAGYCCRAAYCCRAAGTCATC-3%). Primers used to amplify 508 bases of the NS23 gene (Alfort 187 nucleotides 5067–5574) were HCV1 (5%-GCTCCTGGTTGGTAACCTCGG-3%) and HCV2 (5%TGATGCTGTCACACAGGTGAA-3%). RFLP analysis was carried out by digesting PCR products with AvaII, BanII and PvuII for the E2 gene and AvaII, MboI and NcoI for the NS23 gene under conditions specified by the manufacturer (Promega). Briefly, 5 ml of PCR product, 2 ml of 10 X buffer, 0.2 ml of BSA (1mg/ml), 0.5 ml of enzyme and 12.3 ml of distilled water were mixed and incubated at 37°C for 3 h. The digested DNA fragments were electrophoresed in Tris/Borate/EDTA on 4% Nusieve 3:1 agarose gels (FMC BioProduct, Rockland, ME). They were sized by comparison with the migration distances of standard size DNA markers (100 bp ladder) and visualized by GelStar (FMC BioProduct, Rockland, ME) staining. Analysis of the E2 gene sequences obtained from our previous studies and from published sequences predicted five AvaII RFLP patterns, four BanII patterns and two PvuII patterns (Table 1). It was anticipated that a combination of these RFLP patterns would identify each genogroup as in Table 3. The actual RFLP patterns obtained from the digestion of NS23 PCR products are shown in Table 2 based on examination of 60 CSFV samples using AvaII, MboI and NcoI. The RFLP patterns-AvaII-5, AvaII-6 and MboI-3 are novel and were not described by Harding et al. (1994).
S. Parchariyanon et al. / Journal of Virological Methods 87 (2000) 145–149 Table 2 Actual RFLP patterns of NS23 gene PCR products digested by AvaII, MboI and NcoI Enzyme
RFLP patterns
Size of DNA fragments (bps)
AvaII
1 2 3 4 5 6 1 2 3 1 2 3 4
268 268, 460 508 240, 240, 508 474 400, 456 306, 306 508
MboI
NcoI
and 240 200 and 40 and 48 200, 40 and 28 200 and 68 and 34 74 and 34 and 52 150 and 52 and 202
The combination of RFLP patterns observed after agarose gel electrophoresis of the digests from the 60 CSFV samples, are shown in Table 3. The RFLP patters for the E2 gene, can be divided into three major genogroups and seven subgenogroups, termed 1.1, 1.2, 1.3, 2.1, 2.2, 2.3 and 3.3. Subgenogroup 3.3 can be subdivided into 3.3.1, 3.3.2 and 3.3.3. This indicated that the actual combinations of RFLP patterns for the E2 gene completely agreed with the predicted ones
147
(Fig. 1). RFLP analysis of the E2 gene could identify the correct genogroup and subgenogroup of all 60 samples as suggested by phylogenetic analysis of the E2 gene. RFLP analysis of the NS23 gene showed a lesser ability to differentiate isolates. The same combinations of RFLP patterns were observed in subgenogroup 1.1 and 1.3, 1.2 and 3.3.2, 3.3.1 and 3.3.3. Therefore the genogroups suggested by phylogenetic analysis of the E2 gene could not be clearly identified by RFLP of the NS23 gene. This is perhaps not surprising, since the method described by Harding et al. (1994) was not designed for that purpose. It has been shown that phylogenetic analysis of various genes of CSFV, i.e. 5%NCR, E2, NS23 and 3%NCR gave similar results to one other (Hofmann et al., 1994; Lowings et al., 1994; Stadejek et al., 1996; Vilcek and Belak, 1997). However, the E2 gene is the most suitable for the differentiation of strains, as it is one of the most variable parts of the pestivirus genome (Paton, 1995) and the sequence of the E2 gene has been most extensively studied. The genogroups identified by phylogenetic analysis of the E2 gene may be considered as the standard ones (Van Rijn et al., 1997). The present report describes the development of a simple and quick RFLP method for genogrouping of CSFV. RFLP analysis is obviously not as
Table 3 Comparison of RFLP patterns obtained for E2 and NS23 genes for each CSFV subgenogroup Genogroup
No. of samples
RFLP patterns E2
1.1 1.2 1.3 2.1 2.2 2.3 3.3.1 3.3.2 3.3.3
8 4 7 1 14 1 13 7 5
NS23
AvaII
BanII
PvuII
AvaII
MboI
NcoI
4 2 1 5 4 5 3 3 1
2 2 2 2 1 or 3 1 4 4 4
– – – – – – 1 2 –
2 1 2 3 1 or 6 3 5 1 5
1 1 1 2 2 or 3 2 1 1 1
1 1 1 3 1 2 1 1 1
148
S. Parchariyanon et al. / Journal of Virological Methods 87 (2000) 145–149
Fig. 1. RFLP analysis of the E2 gene digested with the use of AvaII (A), BanII (B) or PvuII (P) enzyme. Gelstar stained agarose gel showing undigested and digested amplicons of a representative CSFV isolate. Lane 1, 100 bp ladder molecular weight markers; (1a) lane 2 – 3, 4 – 5 and 6–7: genogroup 1.1, 1.2, and 1.3; (1b) lane 8 – 9, 10 – 11, and 12 – 13: genogroup 2.1, 2.2, and 2.3; (1c) lane 14–15– 16, 17 – 18 – 19, and 20–21: genogroup 3.3.1, 3.3.2, and 3.3.3, respectively.
sensitive as sequence comparison for molecular epidemiological analysis but it is easy, quick and cheap to perform. Therefore the technique is suitable for mass screening or for use by basic diagnostic laboratories without sequencing facilities. It can be anticipated that due to the variable nature of the E2 gene, isolates may be found that give unexpected RFLP results or that cannot be successfully distinuguished. There is therefore a continued need to update the sequence database for CSFV. Accumulation of such data will increase the accuracy and the value of this RFLP method. Genogroups reflect the genetic relatedness among viral isolates, which should be useful for epidemiological studies and may facilitate tracing sources of infection. Such information derived from the routine application of this RFLP method will be of value for the control of CSF.
Acknowledgements The authors thank Dr David Paton for providing two reference CSFV strains and for critical reading of the manuscript.
References Harding, M., Lutze-Wallace, C., Homme, P.I., et al., 1994. Reverse transcriptase-PCR assay for detection of hog cholera virus. J. Clin. Microbiol. 32, 2600 – 2602. Hofmann, M.A., Brechtbuhl, K., Stauber, N., 1994. Rapid characterisation of new pestivirus strains by direct sequencing of PCR-amplified cDNA from the 5% noncoding region. Arch. Virol. 139, 217 – 229. Lowings, J.P., Ibata, G., Needham, J., et al., 1996. Classical swine fever virus diversity and evolution. J. Gen. Virol. 77, 1311 – 1321. Lowings, J.P., Paton, D.J., Sands, J.J., et al., 1994. Classical swine fever: genetic detection and analysis of differences between virus isolates. J. Gen. Virol. 75, 3460 – 3468. Parchariyanon, S., Inui, K., Damrongwatanapokin, S. et al., 2000. Sequence analysis of E2 glycoprotein genes of classical swine fever viruses: identification of a novel genogroup in Thailand. Vet. Med. (in press). Paton, D.J., 1995. Pestivirus diversity. J. Comp. Path. 112, 215 – 236. Paton, D.J., McGoldrick, A., Greiser-Wilke, I. et al., 2000. Genetic typing of classical swine fever virus. Vet. Micro. (in press). Stadejek, T., Warg, J., Ridpath, J.F., 1996. Comparative sequence analysis of the 5% noncoding region of classical swine fever virus strains from Europe, Asia and America. Arch. Virol. 141, 771 – 777. Van Rijn, P.A., Van Gennip, H.G.P., Leendertse, C.H., et al., 1997. Subdivision of the Pesti6irus genus based on envelope glycoprotein E2. Virology 237, 337 – 348.
S. Parchariyanon et al. / Journal of Virological Methods 87 (2000) 145–149
149
Vilcek, S., Stadejek, T., Ballagi-Pordany, A., et al., 1996. Genetic variability of classical swine fever virus. Virus Res. 43, 137 – 147. Vilcek, S., Belak, S., 1997. Organization and diversity of the 3%-noncoding region of classical swine fever virus genome. Virus Genes 15, 181 – 186.
Vilcek, S., Herring, A.J., Herring, J.A., et al., 1994. Pestiviruses isolated from pigs, cattle and can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Arch. Virol. 136, 309 – 323.
.
Genetic grouping of classical swine fever virus by restriction fragment length polymorphism of the E2 gene Sujira Parchariyanon a,*, Ken Inui a, Wasana Pinyochon a, Sudarat Damrongwatanapokin a, Eiji Takahashi b b
a National Institute of Animal Health, Jatujak, Bangkok 10900, Thailand Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The Uni6ersity of Tokyo, Yayoi 1 -1 -1, Bunkyo-ku, Tokyo 113 -8657, Japan
Received 11 October 1999; received in revised form 8 March 2000; accepted 8 March 2000
Abstract A method for genetic grouping of classical swine fever viruses (CSFV) was developed based on the restriction fragment length polymorphism (RFLP) revealed by AvaII, BanII and PvuII digestion of RT-PCR amplified segments of the E2 gene. From inspection of the genetic sequences of Thai isolates and reference strains, the RFLP method was designed to be capable of differentiating all known genogroups and subgenogroups suggested by phylogenetic analysis of the CSFV E2 gene. The method was applied to 60 CSFV samples which included three genogroups and seven subgenogroups. Unlike previously described RFLP methods, the agarose gel patterns obtained from these samples were completely in agreement with the predicted RFLP patterns and enabled accurate genetic grouping of CSFV at the subgenogroup level. The simplicity of this method allows rapid CSFV genogrouping at diagnostic laboratories without sequencing facilities and provides a useful method for diagnosis as well as epidemiological investigation and control of classical swine fever outbreaks. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Classical swine fever; Genetic grouping; Restriction fragment length polymorphism
Classical swine fever (CSF) is an important disease economically for the pig industry in many parts of the world. The causative agent is classical swine fever virus (CSFV) which belongs to the Pesti6irus genus of the Fla6i6iridae family together with bovine viral diarrhea virus and border disease virus. Genetic variability of CSFV has been * Corresponding author. Tel.: + 66-2-579890814; fax: +662-579891819. E-mail address: p – [email protected] (S. Parchariyanon)
reported among isolates from different parts of the world (Lowings et al., 1996; Vilcek et al., 1996). The identification of virus strains and genotypes by genetic characterisation can be used conveniently to trace sources of infection and to improve our understanding of the epidemiology of CSF. Genetic characterisation based on genetic sequence data provides precise information on the relationships between field isolates, but it is not a practical method for all diagnostic laboratories, as it requires facilities for sequencing.
0166-0934/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 0 0 ) 0 0 1 6 2 - 2
S. Parchariyanon et al. / Journal of Virological Methods 87 (2000) 145–149
146
The polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis is an easy and quick system widely practiced for various viral diseases to identify the virus and to differentiate strains or genotypes. Vilcek et al. (1994) showed that RFLP of the 5% noncoding region (5%NCR) of pestiviruses can be used to differentiate CSFV from ruminant pestiviruses but not the genogroups within CSFV. Harding et al. (1994) demonstrated with a limited number of CSFV samples that RFLP of the NS23 nonstructural protein gene could differentiate isolates to some extent. Phylogenetic analysis of the E2 gene of Thai isolates when compared with those of world isolates (Lowings et al., 1996; Paton et al., 2000, in press) has demonstrated that these can be divided into three genogroups comprising five subgenogroups, termed 1.1, 1.2, 1.3, 2.2 and 3.3. No Thai isolates were found in subgenogroups 2.1 and 2.3 (Parchariyanon et al., 2000, in press). In the present study we describe a simple and rapid method combining RT-PCR and RFLP of the E2 gene to differentiate CSFV genogroups identified by phylogenetic analysis of the E2 gene. The method is compared to the RFLP approach based on NS23 described previously (Harding et al., 1994). Restriction enzymes and their cleavage sites were selected and determined by computer analysis of the E2 gene sequence information obtained from our previous study (Parchariyanon et al., Table 1 Predicted RFLP patterns of E2 gene PCR products digested by AvaII, BanII and PvuII Enzyme
RFLP patterns
Size of DNA fragments (bps)
AvaII
1 2 3 4 5 1 2 3 4 1 2
270 221 181 132, 116, 270 205 179 179, 270 194
BanII
PvuII
and 49 and 89 89 and 49 105 and 49 and 65 and 91 65 and 26 and 76
2000, in press) or from published sequences. The actual RFLP analysis was performed on 54 Thai field isolates, four vaccines and two reference strains for which sequences and genogroups had already been determined. The two reference strains representing subgenogroup 2.1 and 2.3 were kindly provided by Dr David Paton (Veterinary Laboratories Agency, UK). RNA extraction and RT-PCR were carried out as described previously (Parchariyanon et al., 2000, in press). Primers used to amplify 270 bases of the E2 gene (Alfort 187 nucleotides 2467–2716) were E2F (5%TCRWCAACC-AAYGAGATAGGG-3%) and E2R (5%-CACAGYCCRAAYCCRAAGTCATC-3%). Primers used to amplify 508 bases of the NS23 gene (Alfort 187 nucleotides 5067–5574) were HCV1 (5%-GCTCCTGGTTGGTAACCTCGG-3%) and HCV2 (5%TGATGCTGTCACACAGGTGAA-3%). RFLP analysis was carried out by digesting PCR products with AvaII, BanII and PvuII for the E2 gene and AvaII, MboI and NcoI for the NS23 gene under conditions specified by the manufacturer (Promega). Briefly, 5 ml of PCR product, 2 ml of 10 X buffer, 0.2 ml of BSA (1mg/ml), 0.5 ml of enzyme and 12.3 ml of distilled water were mixed and incubated at 37°C for 3 h. The digested DNA fragments were electrophoresed in Tris/Borate/EDTA on 4% Nusieve 3:1 agarose gels (FMC BioProduct, Rockland, ME). They were sized by comparison with the migration distances of standard size DNA markers (100 bp ladder) and visualized by GelStar (FMC BioProduct, Rockland, ME) staining. Analysis of the E2 gene sequences obtained from our previous studies and from published sequences predicted five AvaII RFLP patterns, four BanII patterns and two PvuII patterns (Table 1). It was anticipated that a combination of these RFLP patterns would identify each genogroup as in Table 3. The actual RFLP patterns obtained from the digestion of NS23 PCR products are shown in Table 2 based on examination of 60 CSFV samples using AvaII, MboI and NcoI. The RFLP patterns-AvaII-5, AvaII-6 and MboI-3 are novel and were not described by Harding et al. (1994).
S. Parchariyanon et al. / Journal of Virological Methods 87 (2000) 145–149 Table 2 Actual RFLP patterns of NS23 gene PCR products digested by AvaII, MboI and NcoI Enzyme
RFLP patterns
Size of DNA fragments (bps)
AvaII
1 2 3 4 5 6 1 2 3 1 2 3 4
268 268, 460 508 240, 240, 508 474 400, 456 306, 306 508
MboI
NcoI
and 240 200 and 40 and 48 200, 40 and 28 200 and 68 and 34 74 and 34 and 52 150 and 52 and 202
The combination of RFLP patterns observed after agarose gel electrophoresis of the digests from the 60 CSFV samples, are shown in Table 3. The RFLP patters for the E2 gene, can be divided into three major genogroups and seven subgenogroups, termed 1.1, 1.2, 1.3, 2.1, 2.2, 2.3 and 3.3. Subgenogroup 3.3 can be subdivided into 3.3.1, 3.3.2 and 3.3.3. This indicated that the actual combinations of RFLP patterns for the E2 gene completely agreed with the predicted ones
147
(Fig. 1). RFLP analysis of the E2 gene could identify the correct genogroup and subgenogroup of all 60 samples as suggested by phylogenetic analysis of the E2 gene. RFLP analysis of the NS23 gene showed a lesser ability to differentiate isolates. The same combinations of RFLP patterns were observed in subgenogroup 1.1 and 1.3, 1.2 and 3.3.2, 3.3.1 and 3.3.3. Therefore the genogroups suggested by phylogenetic analysis of the E2 gene could not be clearly identified by RFLP of the NS23 gene. This is perhaps not surprising, since the method described by Harding et al. (1994) was not designed for that purpose. It has been shown that phylogenetic analysis of various genes of CSFV, i.e. 5%NCR, E2, NS23 and 3%NCR gave similar results to one other (Hofmann et al., 1994; Lowings et al., 1994; Stadejek et al., 1996; Vilcek and Belak, 1997). However, the E2 gene is the most suitable for the differentiation of strains, as it is one of the most variable parts of the pestivirus genome (Paton, 1995) and the sequence of the E2 gene has been most extensively studied. The genogroups identified by phylogenetic analysis of the E2 gene may be considered as the standard ones (Van Rijn et al., 1997). The present report describes the development of a simple and quick RFLP method for genogrouping of CSFV. RFLP analysis is obviously not as
Table 3 Comparison of RFLP patterns obtained for E2 and NS23 genes for each CSFV subgenogroup Genogroup
No. of samples
RFLP patterns E2
1.1 1.2 1.3 2.1 2.2 2.3 3.3.1 3.3.2 3.3.3
8 4 7 1 14 1 13 7 5
NS23
AvaII
BanII
PvuII
AvaII
MboI
NcoI
4 2 1 5 4 5 3 3 1
2 2 2 2 1 or 3 1 4 4 4
– – – – – – 1 2 –
2 1 2 3 1 or 6 3 5 1 5
1 1 1 2 2 or 3 2 1 1 1
1 1 1 3 1 2 1 1 1
148
S. Parchariyanon et al. / Journal of Virological Methods 87 (2000) 145–149
Fig. 1. RFLP analysis of the E2 gene digested with the use of AvaII (A), BanII (B) or PvuII (P) enzyme. Gelstar stained agarose gel showing undigested and digested amplicons of a representative CSFV isolate. Lane 1, 100 bp ladder molecular weight markers; (1a) lane 2 – 3, 4 – 5 and 6–7: genogroup 1.1, 1.2, and 1.3; (1b) lane 8 – 9, 10 – 11, and 12 – 13: genogroup 2.1, 2.2, and 2.3; (1c) lane 14–15– 16, 17 – 18 – 19, and 20–21: genogroup 3.3.1, 3.3.2, and 3.3.3, respectively.
sensitive as sequence comparison for molecular epidemiological analysis but it is easy, quick and cheap to perform. Therefore the technique is suitable for mass screening or for use by basic diagnostic laboratories without sequencing facilities. It can be anticipated that due to the variable nature of the E2 gene, isolates may be found that give unexpected RFLP results or that cannot be successfully distinuguished. There is therefore a continued need to update the sequence database for CSFV. Accumulation of such data will increase the accuracy and the value of this RFLP method. Genogroups reflect the genetic relatedness among viral isolates, which should be useful for epidemiological studies and may facilitate tracing sources of infection. Such information derived from the routine application of this RFLP method will be of value for the control of CSF.
Acknowledgements The authors thank Dr David Paton for providing two reference CSFV strains and for critical reading of the manuscript.
References Harding, M., Lutze-Wallace, C., Homme, P.I., et al., 1994. Reverse transcriptase-PCR assay for detection of hog cholera virus. J. Clin. Microbiol. 32, 2600 – 2602. Hofmann, M.A., Brechtbuhl, K., Stauber, N., 1994. Rapid characterisation of new pestivirus strains by direct sequencing of PCR-amplified cDNA from the 5% noncoding region. Arch. Virol. 139, 217 – 229. Lowings, J.P., Ibata, G., Needham, J., et al., 1996. Classical swine fever virus diversity and evolution. J. Gen. Virol. 77, 1311 – 1321. Lowings, J.P., Paton, D.J., Sands, J.J., et al., 1994. Classical swine fever: genetic detection and analysis of differences between virus isolates. J. Gen. Virol. 75, 3460 – 3468. Parchariyanon, S., Inui, K., Damrongwatanapokin, S. et al., 2000. Sequence analysis of E2 glycoprotein genes of classical swine fever viruses: identification of a novel genogroup in Thailand. Vet. Med. (in press). Paton, D.J., 1995. Pestivirus diversity. J. Comp. Path. 112, 215 – 236. Paton, D.J., McGoldrick, A., Greiser-Wilke, I. et al., 2000. Genetic typing of classical swine fever virus. Vet. Micro. (in press). Stadejek, T., Warg, J., Ridpath, J.F., 1996. Comparative sequence analysis of the 5% noncoding region of classical swine fever virus strains from Europe, Asia and America. Arch. Virol. 141, 771 – 777. Van Rijn, P.A., Van Gennip, H.G.P., Leendertse, C.H., et al., 1997. Subdivision of the Pesti6irus genus based on envelope glycoprotein E2. Virology 237, 337 – 348.
S. Parchariyanon et al. / Journal of Virological Methods 87 (2000) 145–149
149
Vilcek, S., Stadejek, T., Ballagi-Pordany, A., et al., 1996. Genetic variability of classical swine fever virus. Virus Res. 43, 137 – 147. Vilcek, S., Belak, S., 1997. Organization and diversity of the 3%-noncoding region of classical swine fever virus genome. Virus Genes 15, 181 – 186.
Vilcek, S., Herring, A.J., Herring, J.A., et al., 1994. Pestiviruses isolated from pigs, cattle and can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Arch. Virol. 136, 309 – 323.
.