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A novel method for pestivirus genotyping based on palindromic nucleotide substitutions in the 5′-untranslated region
Journal of Virological Methods 70 (1998) 225 – 230
Short communication
A novel method for pestivirus genotyping based on palindromic nucleotide substitutions in the 5%-untranslated region Ryoˆ Harasawa a,*, Massimo Giangaspero b a
Animal Center for Biomedical Research, Faculty of Medicine, The Uni6ersity of Tokyo, Bunkyo-ku, Tokyo, 113, Japan Special Pathology and Veterinary Medical Clinic Institute, Faculty of Veterinary Medicine, The Uni6ersity of Milan, Via Celoria 10, I-20133, Milan, Italy
b
Received 9 July 1997; received in revised form 13 October 1997; accepted 14 October 1997
Abstract A simple and practical method was developed for pestivirus genotyping based on analysis of the secondary structures in the 5%-untranslated region (UTR). Three stable stem-loop structures, V1, V2 and V3, predicted by computer in the 5%-UTR, included strictly conserved consensus base-pairings which are shared by all the genotypes of pestivirus or are characteristic to each genotype of pestivirus. On the basis of the palindromic nucleotide substitution at the secondary structural level, six genotypes have been identified among pestivirus strains, irrespective of the cytopathic and non-cytopathic biotypes. They are genotypes Ia, Ib, Ic and II in bovine viral diarrhea virus, genotype III in border disease virus, and genotype IV in classical swine fever virus. The stable stem-loop structures, which were maintained by palindromic nucleotide substitutions in the stem region, may represent references for the classification and identification of pestivirus species and/or genotypes. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Genotyping; Palindromic sequence; Pestivirus; Secondary structure; Untranslated region
Pestiviruses are small enveloped viruses containing a single-stranded, positive-sense RNA genome of about 12.5 kb in length, and have been classified into three species, border disease virus (BDV) in sheep and goats, bovine [viral] diarrhea * Corresponding author. Tel.: +81 3 38165680; fax: + 81 3 38165680; e-mail: [email protected]
virus (BVDV) in cattle, and hog cholera virus (HCV) in swine, in the genus Pesti6irus of the family Fla6i6iridae (Wengler et al., 1995). Although these viral species are identified primarily according to the species of animal host from which they are isolated, there is extensive antigenic cross-reactivity among them. Furthermore, they can cross the host-species barrier, infecting
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R. Harasawa, M. Giangaspero / Journal of Virological Methods 70 (1998) 225–230
different species from among the cloven-hoofed ungulates (Paton et al., 1992; Edwards et al., 1995; Wensvoort and Terpstra, 1988). While pestivirus infections in humans have often been suspected (Yolken et al., 1989; Wilks et al., 1989; Yolken et al., 1993), a pestivirus strain was isolated recently from a human buffy coat sample (Giangaspero et al., 1993). Such cross-infections may weaken the definition of pestivirus species by the host. BVDV has been classified into two genetically distinct clusters, BVDV-1 and BVDV-2 (Ridpath et al., 1994). It is, therefore, essential to establish satisfactory criteria for the definition of pestivirus species and/or genotypes (Horzinek, 1995). The Fla6i6iridae Study Group of the International Committee on Taxonomy of Viruses proposed recently four species names: pestivirus 1 for BVDV-1, pestivirus 2 for BVDV-2, pestivirus 3 for BDV, and pestivirus 4 for classical swine fever virus (CSFV) or HCV (Brownlie et al., 1997). The pestivirus genome has a relatively long 5%-untranslated region (UTR) upstream of the polyprotein open reading frame. Although the nucleotide sequence of the 5%-UTR is well conserved among the members of the Pesti6irus genus (Meyers et al., 1989; Qi et al., 1993), the 5%-UTR has been shown to contain at least three variable loci (Harasawa, 1996). The nucleotide substitutions in these variable loci are particularly important because the 5%-UTR of positive-sense RNA viruses generally includes regulatory motifs which are indispensable to viral survival. Therefore, random mutations at the 5%-UTR have a high probability of incompatibility with viral survival. Point mutations occur continuously and at random throughout the virus genome at every replication phase. Mutation rates in RNA viruses are higher than those in DNA viruses, 10 − 3 10 − 4 and 10 − 8 10 − 11 per incorporated nucleotide, respectively (Raming, 1990). Although point mutations must occur in both the translated and untranslated regions at the same rate, incidence of some nucleotide substitutions, observed in the controlling regions of virus genomes, are biased by the selection of lethal mutations. These lethal mutations are not apparent in critical regions of the 5%-UTR, which are responsible for translational, transcriptional and replicational events in
pestiviruses (Brown et al., 1992; Deng and Brock, 1993; Le et al., 1995). Nucleotide sequences at the three variable loci in the 5%-UTR of pestiviruses have been shown to be palindromic and capable of forming a stable stem-loop structure peculiar to each genotype and/or species of pestivirus (Harasawa, 1996). On the basis of these characteristics, a genotyping procedure based on palindromic nucleotide substitutions (PNS) in the 5%UTR was developed. This method may have further applications for genotyping other positivestranded RNA viruses as well as pestiviruses. Nucleotide sequences in the 5%-UTR of the pestivirus strains representing BDV, BVDV and CSFV were obtained from our previous publications and from DNA databases. They were aligned using the method of Higgins et al. (1992). The sequence alignment indicated that the pestivirus genome includes three variable loci, V1, V2 and V3, in the 5%-UTR (data not shown). These variable loci were palindromic, and formed a stem-loop structure to minimize free energy (Fig. 1). Nucleotide substitutions occurred in the stem regions, thereby maintaining a palindromic structure. These PNS were easily identified from the predicted secondary structures by using a DNASIS software package (Hitachi, Japan). According to the PNS, the pestivirus strains were segregated into six genotypes Ia, Ib, Ic, II, III and IV. The proposed keys of identification consist of three aspects. (1) Palindromic structures. Three strictly conserved regions representing palindromic sequences can be identified in the 5%-UTR of the same genotype. (2) Variable loci. Three variable loci, V1, V2 and V3, can be identified in the 5%-UTR, which correspond to the three palindromic structures. These variations are characteristic and well conserved, either among all the different genotypes or specifically within a single genotype. (3) Nucleotide sequences. In the nucleotide sequence of each variable locus, three kinds of base-pairings can be classified. They are (i) inter-genotype specific base-pairings in the stem regions, (ii) genotype specific base-pairs (bp) in the stem regions and (iii) highly variable and uncharacteristic bp. Nucleotide substitutions in the stem regions always occur to conserve the palindromic structure and thereby form a stable stem-loop structure.
R. Harasawa, M. Giangaspero / Journal of Virological Methods 70 (1998) 225–230
Observation of the variable loci reveals several important characteristics. The quantity of nucleotides in the three variable loci are peculiar to each species or genotype. Genotypes Ia, Ib and Ic included in BVDV-1 [pestivirus 1] share a U:A pairing which is common to the V1 locus, a G:C pairing common to the V2 region and U:A and G:C pairings common to the V3 region. A stem at the V1 locus, divided into top and bottom helices by a consensus C:C bulge, is composed of 9 bp in genotypes Ib and Ic, 9 – 11 bp in genotypes Ia, II [pestivirus 2] and III [pestivirus 3] (Sullivan et al.,
Fig. 1. A representative secondary structure at the 5%-UTR of the pestivirus 3 or genotype III (BDV) strain Ch1Es. Palindromic sequences are depicted in stem-loop structures V1, V2 and V3. Minimum free energy (DG) was calculated to be −84.51 kcal/mol.
227
1997; Vilcek et al., 1997) and 11 bp in genotype IV [pestivirus 4]. The loop region of V1 is relatively longer in genotypes Ib, Ic and II [pestivirus 2], composed of seven–nine nucleotides. In genotype Ia, the loop region at V1 has six–seven nucleotides. In genotypes III [pestivirus 3] and IV [pestivirus 4], there are five–six and four nucleotides, respectively. The V2 locus has a strictly conserved number of 23 nucleotides with a stem of 9 bp and a loop of five defined nucleotides, 5%-GGGGU-3%. The V3 locus is shortest among the three variable loci. It is shorter in genotypes III [pestivirus 3] and IV [pestivirus 4], with 16 and 15 nucleotides, respectively, than in genotypes I [pestivirus 1] and II [pestivirus 2], with 19–20 and 18 nucleotides, respectively. A stem structure at the V3 locus consisting of 7 bp in genotypes I and II, 6 bp in genotype III [pestivirus 3] and 5 bp in genotype IV [pestivirus 4]. A loop of the V3 locus consists of five–six nucleotides in pestivirus 1 [genotype I], four nucleotides in pestivirus 2 [genotype II] and pestivirus 3 [genotype III] and three nucleotides in pestivirus 4 [genotype IV]. The folding energy of the three variable loci V1, V2 and V3 in the 5%-UTR does not particularly differ among the pestivirus strains and shows substantial negative free energy (Freier et al., 1986). Regarding consensus motifs shared by all the pestivirus genotypes, it was possible to identify six typical base-pairings and a 5-nucleotide sequence forming a loop (Fig. 2). In the V1 locus, there was a common C:G pairing in the second position from the bottom of the stem, an A:U pairing in position 4 and a C:C bulge in position 5. In the V2 locus, two common C:G pairings were located at positions 2 and 8, and the loop was formed by a 5-nucleotide sequence, 5%-GGGGU-3%. In the V3 locus, a common C:G pairing was present in the third position from the bottom of the stem. The nucleotide sequences forming a loop at V1 and V3 were highly variable and not suitable for genotyping parameters, while the remaining nucleotide base-pairings in the stems were deemed viable (Fig. 2). While palindromic structures can be located visually within primary structures, it is much easier to find them within the secondary structures
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Fig. 2. Key patterns for pestivirus genotyping in the typical predicted secondary structures at the V1, V2 and V3 variable loci in the 5%-UTR of RNA. Characteristic PNS to each genotype are shown by light shadow. Some nucleotide changes by transitional substitution were observed among different strains within the same genotype. Genus-specific base-pairings are shown by heavy shadow. The Watson – Crick base-pairing is indicated by a dash and the G:U-tolerated pairing by asterisk. Possible nucleotide changes by transition within the same genotype are parenthesized. Minimum free energy of the proto-genotype is expressed in kcal/mol (DG), shown in brackets.
using a computer to predict their locations according to the algorithm of Zuker and Stiegler (1981). Palindromic nucleotide substitutions in base-pairings correspond to radical evolutionary
changes, which can generate new genotypes or species. At the level of the G:U pairing tolerated in the secondary structure, transitional substitutions were possible within a single genotype. Sim-
R. Harasawa, M. Giangaspero / Journal of Virological Methods 70 (1998) 225–230
ilarly, limited single nucleotide transitions which maintain a stable palindromic structure were allowed within a single genotype. Although the observation of sequence variations in the palindromic regions can show similarities among certain groups of genotypes, it is clear that the present method is not suitable for phylogenetic studies on isolates. Different approaches have been proposed (De Moerlooze et al., 1993; Pellerin et al., 1994; Ridpath et al., 1994; Becher et al., 1995; Stadejek et al., 1996), though they were based on nucleotide sequence alignment of the primary structures (Higgins et al., 1988; Devereux et al., 1994) and on phylogenetic trees generated by different software packages such as PileUp (Genetics Computer Group, University of Wisconsin), Clustal (Hitachi) and Gene Work (IntelliGenetics, University of Geneva). For phylogenetic analysis, translated regions such as the gp125 or Npro region have also be considered (Becher et al., 1995). According to phylogenetic procedures, the degree of homology among the different genotypes was calculated by computer, and gave a clear quantitative assessment of these methods. The method described in this paper may prove as complex as phylogenetics, since both approaches require nucleotide sequencing and computer analysis. It also requires a subjective judgment on the part of the investigator since genotype allocation is carried out visually rather than by computer, although a subjective bias judgment may also be present in computer software used for phylogenetic analysis. The results of our method are essentially qualitative and provide the exact genotype classification of an isolate, although they offer no percentage values for homology among genotypes. Thus, the method can provide a clear picture of genotype boundaries, due to the exclusive consideration of strategic and highly conserved regions, and consequently helps to avoid unclear classification. In the method based on PNS the ovine isolates D1120/1 and D1432/P, which have been reported as a third cluster in BVDV by Vilcek et al. (1997), are assigned to the genotype Ia. Therefore, the degree of homology might have an application for the evaluation of strains belonging to the same geno-
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type, or for defining phylogenetic relationships between genotypes, but might be less meaningful for genotyping since its concept (classification based on genetic markers) is fundamentally different than that of phylogeny (quantitative estimation of evolutionary history). In conclusion, the proposed method of genotyping provides results basically compatible with phylogenetic classifications based on primary structures in the 5%-UTR, but it differs from these in that only the palindromic structures are strictly conserved in the 5%-UTR. Therefore, the crucial nucleotide sequences, compared visually in terms of PNS, become a novel genetic marker for genotyping.
References Becher, P., Ko¨nig, M., Paton, D.J., Thiel, H.J., 1995. Further characterization of border disease virus isolates: Evidence for the presence of more than three species within the genus pestivirus. Virology 209, 200 – 206. Brown, E.A., Zhang, H., Ping, L.H., Lemon, S.M., 1992. Secondary structure of the 5% nontranslated regions of hepatitis C virus and pestivirus genomic RNAs. Nucleic Acids Res. 20, 5041 – 5045. Brownlie, J., Donis, R., Nettleton, P., Paton, D.J., Wensvoort, G., 1997. Third ESVV symposium on pestiviruses, Conclusions and recommendations for future research. In: Edwards, S., Paton, D.J., Wensvoort G. (Eds.), Proceedings on the third ESVV symposium on pestivirus infections, Lelystad, 19 – 20 September 1996. Central Veterinary Laboratory, Weybridge, UK, pp 188 – 189. De Moerlooze, L., Lecomte, C., Brown-Shimmer, S., Schmetz, D., Guiot, C., Vandenbergh, D., Allaer, D., Rossius, M., Chappuis, G., Dina, D., Renard, A., Martial, J.A., 1993. Nucleotide sequence of the bovine viral diarrhoea virus Osloss strain: comparison with related viruses and identification of specific DNA probes in the 5% untranslated region. J. Gen. Virol. 74, 1433 – 1438. Deng, R., Brock, K.V., 1993. 5% and 3% untranslated regions of pestivirus genome: primary and secondary structure analyses. Nucleic Acids Res. 21, 1949 – 1957. Devereux, J., Haeberli, P., Smithies, O., 1994. A comprehensive set of sequence analysis programs for the Vax. Nucleic Acid Res. 12, 387 – 395. Edwards, S., Roehe, P.M., Ibata, G., 1995. Comparative studies of border disease and closely related virus infections in experimental pigs and sheep. Brit. Vet. J. 151, 181 – 188. Freier, S.M., Kierzek, R., Jaeger, J.A., Sugimoto, M., Caruthers, M.H., Nielson, T., Turner, D.H., 1986. Improved free-energy parameters for predictions of RNA duplex stability. Proc. Natl. Acad. Sci. U.S.A. 83, 9373 – 9377.
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Giangaspero, M., Vacirca, G., Buttner, M., Wolf, G., Vanopdenbosch, E., Muyldermans, G., 1993. Serological and antigenical findings indicating pestivirus in man. Arch. Virol. 7 (Suppl.), 53 –62. Harasawa, R., 1996. Phylogenetic analysis of pestivirus based on the 5%-untranslated region. Acta Virol. 40, 49–54. Higgins, D.G., Sharp, P.M., 1988. Clustal: a package for performing multiple alignment on a microcomputer. Gene 73, 237 – 244. Higgins, D.G., Bleasby, A.J., Fouchs, R., 1992. Clustal V: Improved software for multiple sequence alignment. Comput. Appl. Biosci. 8, 189–191. Horzinek, M.C., 1995. Pestivirus diversity. Arch. Virol. 134, 216 – 217. Le, S.Y., Sonenberg, N., Maizel, J.V. Jr., 1995. Unusual folding regions and ribosomial landing pad within hepatitis C virus and pestivirus RNAs. Gene 154, 137–143. Meyers, G., Rumenapf, T., Thiel, H.J., 1989. Molecular cloning and nucleotide sequence of the genome of hog cholera virus. Virology 171, 555–567. Paton, D.J., Simpson, V., Done, S.H., 1992. Infection of pigs and cattle with bovine viral diarrhea virus on a farm in England. Vet. Rec. 131, 185–188. Pellerin, C., Van Den Hurk, J., Lecomte, J., Tijssen, P., 1994. Identification of a new group of bovine viral diarrhea virus strains associated with severe outbreaks and high mortalities. Virology 203, 260–268. Qi, F., Gustad, T., Lewis, T.L., Berry, E.S., 1993. The nucleotide sequence of the 5%-untranslated region of bovine viral diarrhoea virus: its use as a probe in rapid detection of bovine viral diarrhoea viruses and border disease viruses. Mol. Cell. Probes 7, 349–356. Raming, R.F., 1990. Principles of animal virus genetics, mutations. In: Fields, B.N., Knipe, D.M. (Eds.), Fields Virol-
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ogy, Second edition, vol. 1. Raven Press, New York, pp. 96 – 99. Ridpath, J.F., Bolin, S.R., Dubovi, E.J., 1994. Segregation of bovine viral diarrhea virus into genotypes. Virology 205, 66 – 74. Stadejek, T., Warg, J., Ridpath, J.F., 1996. Comparative sequence analysis of the 5% noncoding region of the classical swine fever virus strains from Europe, Asia, and America. Arch. Virol. 141, 771 – 777. Sullivan, D.G., Chang, G., Akkina, R.K., 1997. Genetic characterization of ruminant pestiviruses: Sequence analysis of viral genotypes isolated from sheep. Virus Res. 47, 19 – 29. Vilcek, S., Nettleton, P.F., Paton, D.J., Belak, S., 1997. Molecular characterization of ovine pestiviruses. J. Gen. Virol. 78, 725 – 735. Wengler, G., Bradley, D.W., Collett, M.S., Heinz, F.X., Schlesinger, R.W., Strauss, J.H., 1995. Family Fla6i6iridae Arch. Virol. 10 (Suppl.), 415 – 427. Wensvoort, G., Terpstra, C., 1988. Bovine viral diarrhea virus infections in piglets born to sows vaccinated against swine fever with contaminated vaccine. Res. Vet. Sci. 45, 143 – 148. Wilks, C.R., Abraham, G., Blackmore, D.K., 1989. bovine pestivirus and human infection. Lancet i, 107. Yolken, R.H., Leister F., Almeido-Hill, J., Dubovi, E., Reid, R., Santosham, M., 1989. Infantile gastroenteritis associated with excretion of pestivirus antigens. Lancet i, 517 – 519. Yolken, R.H., Petric, M., Collett, M., Fuller Torrey, E., 1993. Pestivirus infection in identical twins discordant for schizophrenia. In: Proceedings of the IXth International Congress of Virology, Glasgow, UK, p. 148. Zuker, M., Stiegler, P., 1981. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary. Nucleic Acids Res. 9, 133 – 148.
Short communication
A novel method for pestivirus genotyping based on palindromic nucleotide substitutions in the 5%-untranslated region Ryoˆ Harasawa a,*, Massimo Giangaspero b a
Animal Center for Biomedical Research, Faculty of Medicine, The Uni6ersity of Tokyo, Bunkyo-ku, Tokyo, 113, Japan Special Pathology and Veterinary Medical Clinic Institute, Faculty of Veterinary Medicine, The Uni6ersity of Milan, Via Celoria 10, I-20133, Milan, Italy
b
Received 9 July 1997; received in revised form 13 October 1997; accepted 14 October 1997
Abstract A simple and practical method was developed for pestivirus genotyping based on analysis of the secondary structures in the 5%-untranslated region (UTR). Three stable stem-loop structures, V1, V2 and V3, predicted by computer in the 5%-UTR, included strictly conserved consensus base-pairings which are shared by all the genotypes of pestivirus or are characteristic to each genotype of pestivirus. On the basis of the palindromic nucleotide substitution at the secondary structural level, six genotypes have been identified among pestivirus strains, irrespective of the cytopathic and non-cytopathic biotypes. They are genotypes Ia, Ib, Ic and II in bovine viral diarrhea virus, genotype III in border disease virus, and genotype IV in classical swine fever virus. The stable stem-loop structures, which were maintained by palindromic nucleotide substitutions in the stem region, may represent references for the classification and identification of pestivirus species and/or genotypes. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Genotyping; Palindromic sequence; Pestivirus; Secondary structure; Untranslated region
Pestiviruses are small enveloped viruses containing a single-stranded, positive-sense RNA genome of about 12.5 kb in length, and have been classified into three species, border disease virus (BDV) in sheep and goats, bovine [viral] diarrhea * Corresponding author. Tel.: +81 3 38165680; fax: + 81 3 38165680; e-mail: [email protected]
virus (BVDV) in cattle, and hog cholera virus (HCV) in swine, in the genus Pesti6irus of the family Fla6i6iridae (Wengler et al., 1995). Although these viral species are identified primarily according to the species of animal host from which they are isolated, there is extensive antigenic cross-reactivity among them. Furthermore, they can cross the host-species barrier, infecting
0166-0934/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 6 - 0 9 3 4 ( 9 7 ) 0 0 1 8 0 - 8
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different species from among the cloven-hoofed ungulates (Paton et al., 1992; Edwards et al., 1995; Wensvoort and Terpstra, 1988). While pestivirus infections in humans have often been suspected (Yolken et al., 1989; Wilks et al., 1989; Yolken et al., 1993), a pestivirus strain was isolated recently from a human buffy coat sample (Giangaspero et al., 1993). Such cross-infections may weaken the definition of pestivirus species by the host. BVDV has been classified into two genetically distinct clusters, BVDV-1 and BVDV-2 (Ridpath et al., 1994). It is, therefore, essential to establish satisfactory criteria for the definition of pestivirus species and/or genotypes (Horzinek, 1995). The Fla6i6iridae Study Group of the International Committee on Taxonomy of Viruses proposed recently four species names: pestivirus 1 for BVDV-1, pestivirus 2 for BVDV-2, pestivirus 3 for BDV, and pestivirus 4 for classical swine fever virus (CSFV) or HCV (Brownlie et al., 1997). The pestivirus genome has a relatively long 5%-untranslated region (UTR) upstream of the polyprotein open reading frame. Although the nucleotide sequence of the 5%-UTR is well conserved among the members of the Pesti6irus genus (Meyers et al., 1989; Qi et al., 1993), the 5%-UTR has been shown to contain at least three variable loci (Harasawa, 1996). The nucleotide substitutions in these variable loci are particularly important because the 5%-UTR of positive-sense RNA viruses generally includes regulatory motifs which are indispensable to viral survival. Therefore, random mutations at the 5%-UTR have a high probability of incompatibility with viral survival. Point mutations occur continuously and at random throughout the virus genome at every replication phase. Mutation rates in RNA viruses are higher than those in DNA viruses, 10 − 3 10 − 4 and 10 − 8 10 − 11 per incorporated nucleotide, respectively (Raming, 1990). Although point mutations must occur in both the translated and untranslated regions at the same rate, incidence of some nucleotide substitutions, observed in the controlling regions of virus genomes, are biased by the selection of lethal mutations. These lethal mutations are not apparent in critical regions of the 5%-UTR, which are responsible for translational, transcriptional and replicational events in
pestiviruses (Brown et al., 1992; Deng and Brock, 1993; Le et al., 1995). Nucleotide sequences at the three variable loci in the 5%-UTR of pestiviruses have been shown to be palindromic and capable of forming a stable stem-loop structure peculiar to each genotype and/or species of pestivirus (Harasawa, 1996). On the basis of these characteristics, a genotyping procedure based on palindromic nucleotide substitutions (PNS) in the 5%UTR was developed. This method may have further applications for genotyping other positivestranded RNA viruses as well as pestiviruses. Nucleotide sequences in the 5%-UTR of the pestivirus strains representing BDV, BVDV and CSFV were obtained from our previous publications and from DNA databases. They were aligned using the method of Higgins et al. (1992). The sequence alignment indicated that the pestivirus genome includes three variable loci, V1, V2 and V3, in the 5%-UTR (data not shown). These variable loci were palindromic, and formed a stem-loop structure to minimize free energy (Fig. 1). Nucleotide substitutions occurred in the stem regions, thereby maintaining a palindromic structure. These PNS were easily identified from the predicted secondary structures by using a DNASIS software package (Hitachi, Japan). According to the PNS, the pestivirus strains were segregated into six genotypes Ia, Ib, Ic, II, III and IV. The proposed keys of identification consist of three aspects. (1) Palindromic structures. Three strictly conserved regions representing palindromic sequences can be identified in the 5%-UTR of the same genotype. (2) Variable loci. Three variable loci, V1, V2 and V3, can be identified in the 5%-UTR, which correspond to the three palindromic structures. These variations are characteristic and well conserved, either among all the different genotypes or specifically within a single genotype. (3) Nucleotide sequences. In the nucleotide sequence of each variable locus, three kinds of base-pairings can be classified. They are (i) inter-genotype specific base-pairings in the stem regions, (ii) genotype specific base-pairs (bp) in the stem regions and (iii) highly variable and uncharacteristic bp. Nucleotide substitutions in the stem regions always occur to conserve the palindromic structure and thereby form a stable stem-loop structure.
R. Harasawa, M. Giangaspero / Journal of Virological Methods 70 (1998) 225–230
Observation of the variable loci reveals several important characteristics. The quantity of nucleotides in the three variable loci are peculiar to each species or genotype. Genotypes Ia, Ib and Ic included in BVDV-1 [pestivirus 1] share a U:A pairing which is common to the V1 locus, a G:C pairing common to the V2 region and U:A and G:C pairings common to the V3 region. A stem at the V1 locus, divided into top and bottom helices by a consensus C:C bulge, is composed of 9 bp in genotypes Ib and Ic, 9 – 11 bp in genotypes Ia, II [pestivirus 2] and III [pestivirus 3] (Sullivan et al.,
Fig. 1. A representative secondary structure at the 5%-UTR of the pestivirus 3 or genotype III (BDV) strain Ch1Es. Palindromic sequences are depicted in stem-loop structures V1, V2 and V3. Minimum free energy (DG) was calculated to be −84.51 kcal/mol.
227
1997; Vilcek et al., 1997) and 11 bp in genotype IV [pestivirus 4]. The loop region of V1 is relatively longer in genotypes Ib, Ic and II [pestivirus 2], composed of seven–nine nucleotides. In genotype Ia, the loop region at V1 has six–seven nucleotides. In genotypes III [pestivirus 3] and IV [pestivirus 4], there are five–six and four nucleotides, respectively. The V2 locus has a strictly conserved number of 23 nucleotides with a stem of 9 bp and a loop of five defined nucleotides, 5%-GGGGU-3%. The V3 locus is shortest among the three variable loci. It is shorter in genotypes III [pestivirus 3] and IV [pestivirus 4], with 16 and 15 nucleotides, respectively, than in genotypes I [pestivirus 1] and II [pestivirus 2], with 19–20 and 18 nucleotides, respectively. A stem structure at the V3 locus consisting of 7 bp in genotypes I and II, 6 bp in genotype III [pestivirus 3] and 5 bp in genotype IV [pestivirus 4]. A loop of the V3 locus consists of five–six nucleotides in pestivirus 1 [genotype I], four nucleotides in pestivirus 2 [genotype II] and pestivirus 3 [genotype III] and three nucleotides in pestivirus 4 [genotype IV]. The folding energy of the three variable loci V1, V2 and V3 in the 5%-UTR does not particularly differ among the pestivirus strains and shows substantial negative free energy (Freier et al., 1986). Regarding consensus motifs shared by all the pestivirus genotypes, it was possible to identify six typical base-pairings and a 5-nucleotide sequence forming a loop (Fig. 2). In the V1 locus, there was a common C:G pairing in the second position from the bottom of the stem, an A:U pairing in position 4 and a C:C bulge in position 5. In the V2 locus, two common C:G pairings were located at positions 2 and 8, and the loop was formed by a 5-nucleotide sequence, 5%-GGGGU-3%. In the V3 locus, a common C:G pairing was present in the third position from the bottom of the stem. The nucleotide sequences forming a loop at V1 and V3 were highly variable and not suitable for genotyping parameters, while the remaining nucleotide base-pairings in the stems were deemed viable (Fig. 2). While palindromic structures can be located visually within primary structures, it is much easier to find them within the secondary structures
228
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Fig. 2. Key patterns for pestivirus genotyping in the typical predicted secondary structures at the V1, V2 and V3 variable loci in the 5%-UTR of RNA. Characteristic PNS to each genotype are shown by light shadow. Some nucleotide changes by transitional substitution were observed among different strains within the same genotype. Genus-specific base-pairings are shown by heavy shadow. The Watson – Crick base-pairing is indicated by a dash and the G:U-tolerated pairing by asterisk. Possible nucleotide changes by transition within the same genotype are parenthesized. Minimum free energy of the proto-genotype is expressed in kcal/mol (DG), shown in brackets.
using a computer to predict their locations according to the algorithm of Zuker and Stiegler (1981). Palindromic nucleotide substitutions in base-pairings correspond to radical evolutionary
changes, which can generate new genotypes or species. At the level of the G:U pairing tolerated in the secondary structure, transitional substitutions were possible within a single genotype. Sim-
R. Harasawa, M. Giangaspero / Journal of Virological Methods 70 (1998) 225–230
ilarly, limited single nucleotide transitions which maintain a stable palindromic structure were allowed within a single genotype. Although the observation of sequence variations in the palindromic regions can show similarities among certain groups of genotypes, it is clear that the present method is not suitable for phylogenetic studies on isolates. Different approaches have been proposed (De Moerlooze et al., 1993; Pellerin et al., 1994; Ridpath et al., 1994; Becher et al., 1995; Stadejek et al., 1996), though they were based on nucleotide sequence alignment of the primary structures (Higgins et al., 1988; Devereux et al., 1994) and on phylogenetic trees generated by different software packages such as PileUp (Genetics Computer Group, University of Wisconsin), Clustal (Hitachi) and Gene Work (IntelliGenetics, University of Geneva). For phylogenetic analysis, translated regions such as the gp125 or Npro region have also be considered (Becher et al., 1995). According to phylogenetic procedures, the degree of homology among the different genotypes was calculated by computer, and gave a clear quantitative assessment of these methods. The method described in this paper may prove as complex as phylogenetics, since both approaches require nucleotide sequencing and computer analysis. It also requires a subjective judgment on the part of the investigator since genotype allocation is carried out visually rather than by computer, although a subjective bias judgment may also be present in computer software used for phylogenetic analysis. The results of our method are essentially qualitative and provide the exact genotype classification of an isolate, although they offer no percentage values for homology among genotypes. Thus, the method can provide a clear picture of genotype boundaries, due to the exclusive consideration of strategic and highly conserved regions, and consequently helps to avoid unclear classification. In the method based on PNS the ovine isolates D1120/1 and D1432/P, which have been reported as a third cluster in BVDV by Vilcek et al. (1997), are assigned to the genotype Ia. Therefore, the degree of homology might have an application for the evaluation of strains belonging to the same geno-
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type, or for defining phylogenetic relationships between genotypes, but might be less meaningful for genotyping since its concept (classification based on genetic markers) is fundamentally different than that of phylogeny (quantitative estimation of evolutionary history). In conclusion, the proposed method of genotyping provides results basically compatible with phylogenetic classifications based on primary structures in the 5%-UTR, but it differs from these in that only the palindromic structures are strictly conserved in the 5%-UTR. Therefore, the crucial nucleotide sequences, compared visually in terms of PNS, become a novel genetic marker for genotyping.
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