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Cellular insertions in the NS2-3 genome region of cytopathic bovine viral diarrhoea virus (BVDV) isolates
Veterinary Microbiology 77 (2000) 129±136
Cellular insertions in the NS2-3 genome region of cytopathic bovine viral diarrhoea virus (BVDV) isolates SÏtefan VilcÏeka,*, Irene Greiser-Wilkeb, Peter Nettletonc, David J. Patond b
a University of Veterinary Medicine, SK-041 81 Kosice, Slovak Republic School of Veterinary Medicine, Institute of Virology, BuÈnteweg 17, D-30559 Hannover, Germany c Moredun Research Institute, Edinburgh EH17 7JH, UK d Veterinary Laboratories Agency-Weybridge, Addlestone, Surrey KT15 3NB, UK
Abstract When compared to noncytopathic (ncp) bovine viral diarrhoea virus (BVDV), some cytopathic (cp) BVDV contain additional sequences in the NS2-3 genomic region. One of these insertions, which is 270 nucleotides long and of host origin (cINS), was ®rst described for strain NADL. To ®nd out how frequently this type of insertion occurs in other cp BVDV, 32 cp BVDV ®eld isolates and the BVDV reference cp strain Indiana were screened using RT-PCR which detected cINS in NADL. For most cp viruses an RT-PCR product of 402 bp indicated the presence of NS2-3 genes without insertions. In addition, one or two DNA fragments, around 600±850 bp in size, were ampli®ed from the genomes of 13 cp viruses indicating the presence of insertions. Sequencing of the PCR products, i.e. 402 bp DNA fragment (with no insertion) and longer fragments (with insertion) revealed the location of the insertions in the NS2-3 coding region of eight cp BVDV genomes. All of the insertions were con®rmed to be of the cINS type and were located in a very similar position to that found previously in the NADL genome. They were in the same reading frame as the viral polypeptide and they encoded 90±140 amino acids. The 50 and 30 ends of the insertions were different in most of the cp isolates studied. Interestingly, a 14-amino-acid stretch at the 50 -end of the insertion in the cp 5569 isolate as well as 15 amino acids at the 30 -end of the insertion in the cp 5.19516 isolate were not homologous to the cINS sequence. No signi®cant matches for these stretches were found in the EMBL and Swissprot databases. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Bovine viral diarrhoea virus; Cytopathic strain; Cellular insertion; NS2-3 region
*
Corresponding author. Tel.: 42-1-95-6339227; fax: 42-1-95-6323666 E-mail address: [email protected] (SÏ. VilcÏek). 0378-1135/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 0 ) 0 0 2 6 9 - 8
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SÏ. VilcÏek et al. / Veterinary Microbiology 77 (2000) 129±136
1. Introduction The Pestivirus genus comprises bovine viral diarrhoea virus (BVDV), classical swine fever virus (CSFV) and border disease virus (BDV), which together with Flaviruses and hepatitis C make up the family Flaviviridae (Wengler et al., 1995). The genome of these viruses is composed of a single stranded RNA with positive orientation and a size of approximately 12.3 kb. The ORF encodes a polyprotein around 4000 amino acids which is ¯anked at the 50 and 30 ends with untranslated regions (Collett et al., 1988). The polyprotein is co- and post-translationally processed by both viral and cellular proteases to form 11±12 structural and nonstructural (NS) proteins (Meyers and Thiel, 1996). Pestiviruses can be divided into two biotypes, which differ by their action in permissive cell cultures. Noncytopathic (ncp) BVDV predominate in vivo, and are invariably present in persistently infected cattle. Both ncp and cytopathic (cp) BVDV are present in cattle with the fatal form of infection known as mucosal disease (Brownlie et al., 1984; Donis and Dubovi, 1987). The ncp BVDV are characterised by the synthesis of the NS2-3 (formerly p125) protein. On the other hand, the cp BVDV express the NS3 (p80) protein, which is colinear with the C-terminal fragment of the NS2-3 protein (Collett et al., 1988; Donis and Dubovi, 1987; Greiser-Wilke et al., 1992; Meyers et al., 1991). Some cp strains seem to be generated within persistently infected animals by recombination between the genome of ncp BVDV and viral or cellular nucleotide sequences (Meyers and Thiel, 1996). The most commonly described insertions were found in two reference BVDV strains (Meyers et al., 1989, 1991). The ®rst type of insertion was identi®ed as the ubiquitin coding sequence, within the genome of BVDV strain Osloss. A second type of insertion (cINS) was discovered in the genome of the NADL strain. It encodes 270 bp (90 amino acids) of a cellular sequence of unknown function. Recently, cINS insertions were also found in some cp BDV strains (Becher et al., 1996). We have shown that the cINS type of insertions within the NS2-3 region can be detected in other cp BVDV isolates using RT-PCR (Greiser-Wilke et al., 1993), but the insertions found were not characterised in detail. The aim of this study was to search for additional insertions of this kind in cp BVDV ®eld isolates and to analyse them at the genetic level. 2. Materials and methods 2.1. Virus isolates Thirty-two biologically de®ned cp BVDV isolates (in cell culture) were analysed in this study. They were obtained from cases of suspected mucosal disease in cattle: 10 isolates originated from Scotland, ®ve isolates from England, 15 isolates from Germany and two isolates from New Zealand. The viruses were not biologically cloned. The cp strain Indiana originated from the USA.
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2.2. Ampli®cation of insertions using RT-PCR method To search for the cellular insertions within the BVDV genome, an RT-PCR method was used. Total RNA was isolated from infected cells (5106±10106) using Trizol (Sigma) according to the manufacturer's instructions. RNA was dissolved in 10 ml of ddH2O. Synthesis of cDNA was carried out using random hexamers starting with 3 ml of RNA in a ®nal reaction volume of 20 ml. The PCR reaction employed the A (position in NADL: 4937±4960, sense) and C (position in NADL: 5609±5591, antisense) primers ¯anking cINS in the NS2-3 of NADL strain (Greiser-Wilke et al., 1993). Three microlitres of cDNA were used for ampli®cation in a thermal pro®le involving 36 cycles of 948C for 1 min, 528C for 1 min and 728C for 1 min. PCR products were analysed on 2% agarose gels. The procedures for synthesis of cDNA and PCR are given elsewhere (Vilcek et al., 1994). 2.3. Sequencing of PCR products and computer analysis PCR products resolved as individual electrophoretic bands were desalted using the Wizard PCR preps kit (Promega). Where two or more electrophoretic bands were observed, the 402 bp and one larger fragment (of the strongest intensity) were excised from the gel and puri®ed using the Sephaglass kit (Pharmacia) according to the manufacturer's instructions. Pure DNA products were sequenced in both directions using the A and C primers on an ABI PRISM sequencing device using a Big Dye sequencing kit (Perkin Elmer). Nucleotide sequences were proof-read and aligned using the SeqMan and Clustal programs of the DNASTAR computer package. Searches for sequence similarities were performed using the BLAST and FASTA programs in the EMBL and Swissprot databases. 3. Results In all cp BVDV, except three isolates and Indiana strain, a 402 bp DNA fragment was detected. In addition, usually one or two DNA fragments, around 600±850 bp in size, were detected in 13 of 33 cp (Table 1) BVDV analysed. The presence of a 402 bp DNA fragment in cp isolates in which was also observed additional longer fragment(s) indicated that those viral samples also contain an ncp isolate and represent a mixture of ncp and cp pairs. This phenomenon helped us to determine precisely the position of cINS in cp isolates. Due to the variable quality of the PCR products the 600±850 bp size amplicons were sequenced from only eight of the 13 viruses with insertions (see Table 1). In the sequences of the remaining ®ve cp isolates, we observed unreadable stretches of mixed nucleotides, which indicated co-ampli®cation of different products. Sequencing of PCR products, e.g. 402 bp fragment (no insertion) longer fragments (with insertion) revealed the position of the insertions in virus genome (Fig. 1). With reference to the sequence of the ncp SD-1 strain of BVDV, the start of the insertion in different isolates equates to
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Table 1 In vitro ampli®cation of DNA fragments obtained by RT-PCR with primers A and C from the genomes of some cp BVDV strainsa BVDV isolate
DNA fragments (bp)b
Indiana 5.19516 7923 869 5569 88055 2324/94 A2146 3422/92 8078/92 A4411/4 X0092/1 A493/1
750 (S) 402 (S), 800 (W), 850 (S) 402 (S), 700 (W), 750 (S) 750 (S) 800 (S) 750 (S) 402 (S), 650 (W), 850 (S) 402 (S), 650 (W), 750 (S) 402, 600 (W), 700 (W) 402, 600 (W), 700 (W) 402, 700, 800 402, 650, 750 (W) 402, 750 (W)
a b
In other 20 cp BVDV field isolates only strong 402 bp DNA fragment was detected. W: weak DNA electrophoretic band; S: DNA fragment sequenced in this work.
Fig. 1. Position of cINS in the NS2-3 coding region in the genome of cp BVDV isolates. Sequences for SD-1 were taken from Deng and Brock (1992), for NADL from Collett et al. (1988). The cp 2324_94 and cp A2146 viruses originate from Scotland, cp 5569, cp 869 and cp 88055 from Germany, cp 5.19516 and cp 7923 from New Zealand and the Indiana strain from USA. The number above the amino acid sequences indicates the equivalent position in strain SD-1.
SÏ. VilcÏek et al. / Veterinary Microbiology 77 (2000) 129±136 Fig. 2. Alignment of deduced amino acid sequences of the insertions within the genome of cp BVDV strains. Sequence for cINS was taken from Becher et al. (1996).
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nucleotide position 4994±5012 (or 1537±1543 amino acid) of the NS2-3 region. For comparison, the start of cINS in NADL is located at position 4994 (or 1537 at the amino acid level). The viral sequences for all isolates were colinear to those of ncp SD-1 strain, but mutations of single amino acids were observed near to the inserted cellular sequences (see Fig. 1). The size of cellular insertions varied between 90 (cp 88055 and NADL) to 140 (cp 2324_94) amino acids, all being in the same reading frame as the viral polypeptide. All cINS had a common core of 89 amino acids which is very similar to the cellular insertion within NADL (Fig. 2). However, the 50 and 30 ends of insertions were unique, except for cp 88055, which was found to be the same as in NADL. Interestingly, neither a 14-aminoacid stretch (APRRQRHRVPAKED) at the 50 -end of the insertion found in cp 5569 nor 15 amino acids (RVFLKICRPRPTSTT) at the 30 -end of the insertion in cp 5.19516 (nor their corresponding nucleotide sequences), were homologous to the cINS sequence (Fig. 2). No signi®cant matches for these stretches were found in EMBL and Swissprot databases. 4. Discussion The results presented in our work show that the speci®c RT-PCR method revealed an insertion within the NS2-3 coding region in 12 of 32 cp BVDV ®eld isolates and in the cp Indiana strain. The cINS type insertions was con®rmed in eight cp BVDV genomes. This work documents for the ®rst time that the cINS insertion appears with a similarly high frequency as has been previously described for the insertion of ubiquitin-coding sequences. According to our data all cINS contain a very conserved core of 89 amino acids, as was observed in strain NADL, with only a small number of amino acid differences between the isolates. The conservation of this core sequence suggests that it has biological signi®cance. Recently it has been demonstrated that the cellular insertion in the genome of NADL is essential for the generation of NS3 by NS2-3 cleavage and for cytopathogenicity of this virus strain (Mendez et al., 1998). The exact mechanism by which the insertion induces cytopathogenicity is not known yet. From ®ve of the ®eld isolates analysed, the RT-PCR products could not be sequenced. We assume that this was due to the coampli®cation of more than one product, which suggests that the isolate consisted of more than one cp virus subpopulation. This is in agreement with the ®nding by radioimmunoprecipitation that some uncloned ®eld isolates show heterogeneous expression of the NS2-3 protein (Greiser-Wilke et al., 1992). Alternatively, the other mechanisms of cytopathogenicity, such as genomic rearrangement can also occur in those cp strains. The insertions studied differ signi®cantly at the 50 and 30 ends. Interestingly, additional ``in reading frame'' sequences were detected in two strains at the 50 or 30 ends of cINS. However, their origins could not be identi®ed. It seems that the combination of two peptide coding sequences is not so rare in BVDV insertions, because the same phenomenon has been observed in other BVDV strains (Becher et al., 1998; Qi et al., 1998). It is not surprising that cINS are not found in the NS2-3 region of all cp isolates, since other mechanisms are known to lead to the conversion of BVDV strains from ncp to cp. Different genomic rearrangements have been detected, as well as the insertions of a
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sequence encoding light chain 3 of microtubule-associated proteins 1A and 1B (Meyers et al., 1998), ribosomal S27a coding sequence (Becher et al., 1998) or SMT3B bovine gene (Qi et al., 1998). Furthermore, other cp BVDV seem to be the result of rearrangement in the virus genome, involving duplications, mutations and deletions (see review Meyers and Thiel, 1996). In conclusion, the variable length cINS type cellular insertions occur in some cp BVDV isolates. Although we do not know the exact mechanism of their incorporation into the virus genome, their presence is correlated to the expression of the cp biotype. A more systematic search is required to ®nd other insertions and rearrangements in pestivirus genomes and to explain the phenomenon of pestivirus cytopathogenicity. Acknowledgements SÏtefan VilcÏek's work was supported by an International Research Development Fellowship from The Wellcome Trust (Grant No. 049929/Z/96/Z) and VEGA of SR (Grant No. 1/6215/99). Irene Greiser-Wilke was supported by the SFB 280 (``Sonderforschungsbereich Gastroitestinale Barriere'').
References Becher, P., Meyers, G., Shannon, A.D., Thiel, H.-J., 1996. Cytopathogenicity of border disease virus is correlated with integration of cellular sequences into the viral genome. J. Virol. 70, 2992±2998. Becher, P., Orlich, M., Thiel, H.-J., 1998. Ribosomal S27a coding sequences upstream of ubiquitin coding sequences in the genome of a pestivirus. J. Virol. 72, 8697±8704. Brownlie, J., Clarke, M.C., Howard, C.J., 1984. Experimental production of fatal mucosal disease in cattle. Vet. Rec. 114, 535±536. Collett, M.S., Larson, R., Gold, C., Strinck, D., Anderson, D.K., Purchio, A.F., 1988. Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus. Virology 165, 191±199. Deng, R., Brock, K.V., 1992. Molecular cloning and nucleotide sequence of a pestivirus genome, noncytopathic bovine viral diarrhea virus strain SD-1. Virology 191, 867±879. Donis, R.O., Dubovi, E.J., 1987. Characterization of bovine viral diarrhoea-mucosal disease virus-speci®c proteins in bovine cells. J. Gen. Virol. 68, 1597±1605. Greiser-Wilke, I., Dittmar, K.E., Liess, B., Moennig, V., 1992. Heterogenous expression of the non-structural protein p80/p125 in cells infected with different pestiviruses. J. Gen. Virol. 73, 47±52. Greiser-Wilke, I., Haas, L., Dittmar, K., Liess, B., Moennig, V., 1993. RNA insertions and gene duplications in the nonstructural protein p125 region of pestivirus strains and isolates in vitro and in vivo. Virology 193, 977±980. Mendez, E., Ruggli, N., Collett, M.S., Rice, C.M., 1998. Infectious bovine viral diarrhea virus (strain NADL) RNA from stable cDNA clones: a cellular insert determines NS3 production and viral cytopathogenicity. J. Virol. 72, 4737±4745. Meyers, G., Thiel, H.-J., 1996. Molecular characterization of pestiviruses. Adv. Virus Res. 47, 53±118. Meyers, G., Rumenapf, T., Thiel, H.-J., 1989. Ubiquitin in a togavirus. Nature 341, 491. Meyers, G., Tautz, N., Dubovi, E.J., Thiel, H.-J., 1991. Viral cytopathogenicity correlated with integration of ubiquitin-coding sequences. Virology 180, 602±616. Meyers, G., Stoll, D., Gunn, M., 1998. Insertion of a sequence encoding light chain 3 of microtubule-associated proteins 1A and 1B in a pestivirus genome: connection with virus cytopathogenicity and induction of lethal disease in cattle. J. Virol. 72, 4139±4148.
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Qi, F., Ridpath, J.F., Berry, E.S., 1998. Insertion of a bovine SMT3B gene in NS4B and duplication of NS3 in a bovine viral diarrhea virus genome correlate with the cytopathogenicity of the virus. Virus Res. 57, 1±9. Vilcek, S., Herring, A.J., Herring, J.A., Nettleton, P.F., Lowings, J.P., Paton, D.J., 1994. Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Arch. Virol. 136, 309±323. Wengler, G., Bradley, D.W., Collett, M.S., Heinz, F.X., Schlesinger, R.W., Strauss, J.H., 1995. Family Flaviviridae. Arch. Virol. 10 (Suppl.), 415±427.
Cellular insertions in the NS2-3 genome region of cytopathic bovine viral diarrhoea virus (BVDV) isolates SÏtefan VilcÏeka,*, Irene Greiser-Wilkeb, Peter Nettletonc, David J. Patond b
a University of Veterinary Medicine, SK-041 81 Kosice, Slovak Republic School of Veterinary Medicine, Institute of Virology, BuÈnteweg 17, D-30559 Hannover, Germany c Moredun Research Institute, Edinburgh EH17 7JH, UK d Veterinary Laboratories Agency-Weybridge, Addlestone, Surrey KT15 3NB, UK
Abstract When compared to noncytopathic (ncp) bovine viral diarrhoea virus (BVDV), some cytopathic (cp) BVDV contain additional sequences in the NS2-3 genomic region. One of these insertions, which is 270 nucleotides long and of host origin (cINS), was ®rst described for strain NADL. To ®nd out how frequently this type of insertion occurs in other cp BVDV, 32 cp BVDV ®eld isolates and the BVDV reference cp strain Indiana were screened using RT-PCR which detected cINS in NADL. For most cp viruses an RT-PCR product of 402 bp indicated the presence of NS2-3 genes without insertions. In addition, one or two DNA fragments, around 600±850 bp in size, were ampli®ed from the genomes of 13 cp viruses indicating the presence of insertions. Sequencing of the PCR products, i.e. 402 bp DNA fragment (with no insertion) and longer fragments (with insertion) revealed the location of the insertions in the NS2-3 coding region of eight cp BVDV genomes. All of the insertions were con®rmed to be of the cINS type and were located in a very similar position to that found previously in the NADL genome. They were in the same reading frame as the viral polypeptide and they encoded 90±140 amino acids. The 50 and 30 ends of the insertions were different in most of the cp isolates studied. Interestingly, a 14-amino-acid stretch at the 50 -end of the insertion in the cp 5569 isolate as well as 15 amino acids at the 30 -end of the insertion in the cp 5.19516 isolate were not homologous to the cINS sequence. No signi®cant matches for these stretches were found in the EMBL and Swissprot databases. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Bovine viral diarrhoea virus; Cytopathic strain; Cellular insertion; NS2-3 region
*
Corresponding author. Tel.: 42-1-95-6339227; fax: 42-1-95-6323666 E-mail address: [email protected] (SÏ. VilcÏek). 0378-1135/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 0 ) 0 0 2 6 9 - 8
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1. Introduction The Pestivirus genus comprises bovine viral diarrhoea virus (BVDV), classical swine fever virus (CSFV) and border disease virus (BDV), which together with Flaviruses and hepatitis C make up the family Flaviviridae (Wengler et al., 1995). The genome of these viruses is composed of a single stranded RNA with positive orientation and a size of approximately 12.3 kb. The ORF encodes a polyprotein around 4000 amino acids which is ¯anked at the 50 and 30 ends with untranslated regions (Collett et al., 1988). The polyprotein is co- and post-translationally processed by both viral and cellular proteases to form 11±12 structural and nonstructural (NS) proteins (Meyers and Thiel, 1996). Pestiviruses can be divided into two biotypes, which differ by their action in permissive cell cultures. Noncytopathic (ncp) BVDV predominate in vivo, and are invariably present in persistently infected cattle. Both ncp and cytopathic (cp) BVDV are present in cattle with the fatal form of infection known as mucosal disease (Brownlie et al., 1984; Donis and Dubovi, 1987). The ncp BVDV are characterised by the synthesis of the NS2-3 (formerly p125) protein. On the other hand, the cp BVDV express the NS3 (p80) protein, which is colinear with the C-terminal fragment of the NS2-3 protein (Collett et al., 1988; Donis and Dubovi, 1987; Greiser-Wilke et al., 1992; Meyers et al., 1991). Some cp strains seem to be generated within persistently infected animals by recombination between the genome of ncp BVDV and viral or cellular nucleotide sequences (Meyers and Thiel, 1996). The most commonly described insertions were found in two reference BVDV strains (Meyers et al., 1989, 1991). The ®rst type of insertion was identi®ed as the ubiquitin coding sequence, within the genome of BVDV strain Osloss. A second type of insertion (cINS) was discovered in the genome of the NADL strain. It encodes 270 bp (90 amino acids) of a cellular sequence of unknown function. Recently, cINS insertions were also found in some cp BDV strains (Becher et al., 1996). We have shown that the cINS type of insertions within the NS2-3 region can be detected in other cp BVDV isolates using RT-PCR (Greiser-Wilke et al., 1993), but the insertions found were not characterised in detail. The aim of this study was to search for additional insertions of this kind in cp BVDV ®eld isolates and to analyse them at the genetic level. 2. Materials and methods 2.1. Virus isolates Thirty-two biologically de®ned cp BVDV isolates (in cell culture) were analysed in this study. They were obtained from cases of suspected mucosal disease in cattle: 10 isolates originated from Scotland, ®ve isolates from England, 15 isolates from Germany and two isolates from New Zealand. The viruses were not biologically cloned. The cp strain Indiana originated from the USA.
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2.2. Ampli®cation of insertions using RT-PCR method To search for the cellular insertions within the BVDV genome, an RT-PCR method was used. Total RNA was isolated from infected cells (5106±10106) using Trizol (Sigma) according to the manufacturer's instructions. RNA was dissolved in 10 ml of ddH2O. Synthesis of cDNA was carried out using random hexamers starting with 3 ml of RNA in a ®nal reaction volume of 20 ml. The PCR reaction employed the A (position in NADL: 4937±4960, sense) and C (position in NADL: 5609±5591, antisense) primers ¯anking cINS in the NS2-3 of NADL strain (Greiser-Wilke et al., 1993). Three microlitres of cDNA were used for ampli®cation in a thermal pro®le involving 36 cycles of 948C for 1 min, 528C for 1 min and 728C for 1 min. PCR products were analysed on 2% agarose gels. The procedures for synthesis of cDNA and PCR are given elsewhere (Vilcek et al., 1994). 2.3. Sequencing of PCR products and computer analysis PCR products resolved as individual electrophoretic bands were desalted using the Wizard PCR preps kit (Promega). Where two or more electrophoretic bands were observed, the 402 bp and one larger fragment (of the strongest intensity) were excised from the gel and puri®ed using the Sephaglass kit (Pharmacia) according to the manufacturer's instructions. Pure DNA products were sequenced in both directions using the A and C primers on an ABI PRISM sequencing device using a Big Dye sequencing kit (Perkin Elmer). Nucleotide sequences were proof-read and aligned using the SeqMan and Clustal programs of the DNASTAR computer package. Searches for sequence similarities were performed using the BLAST and FASTA programs in the EMBL and Swissprot databases. 3. Results In all cp BVDV, except three isolates and Indiana strain, a 402 bp DNA fragment was detected. In addition, usually one or two DNA fragments, around 600±850 bp in size, were detected in 13 of 33 cp (Table 1) BVDV analysed. The presence of a 402 bp DNA fragment in cp isolates in which was also observed additional longer fragment(s) indicated that those viral samples also contain an ncp isolate and represent a mixture of ncp and cp pairs. This phenomenon helped us to determine precisely the position of cINS in cp isolates. Due to the variable quality of the PCR products the 600±850 bp size amplicons were sequenced from only eight of the 13 viruses with insertions (see Table 1). In the sequences of the remaining ®ve cp isolates, we observed unreadable stretches of mixed nucleotides, which indicated co-ampli®cation of different products. Sequencing of PCR products, e.g. 402 bp fragment (no insertion) longer fragments (with insertion) revealed the position of the insertions in virus genome (Fig. 1). With reference to the sequence of the ncp SD-1 strain of BVDV, the start of the insertion in different isolates equates to
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Table 1 In vitro ampli®cation of DNA fragments obtained by RT-PCR with primers A and C from the genomes of some cp BVDV strainsa BVDV isolate
DNA fragments (bp)b
Indiana 5.19516 7923 869 5569 88055 2324/94 A2146 3422/92 8078/92 A4411/4 X0092/1 A493/1
750 (S) 402 (S), 800 (W), 850 (S) 402 (S), 700 (W), 750 (S) 750 (S) 800 (S) 750 (S) 402 (S), 650 (W), 850 (S) 402 (S), 650 (W), 750 (S) 402, 600 (W), 700 (W) 402, 600 (W), 700 (W) 402, 700, 800 402, 650, 750 (W) 402, 750 (W)
a b
In other 20 cp BVDV field isolates only strong 402 bp DNA fragment was detected. W: weak DNA electrophoretic band; S: DNA fragment sequenced in this work.
Fig. 1. Position of cINS in the NS2-3 coding region in the genome of cp BVDV isolates. Sequences for SD-1 were taken from Deng and Brock (1992), for NADL from Collett et al. (1988). The cp 2324_94 and cp A2146 viruses originate from Scotland, cp 5569, cp 869 and cp 88055 from Germany, cp 5.19516 and cp 7923 from New Zealand and the Indiana strain from USA. The number above the amino acid sequences indicates the equivalent position in strain SD-1.
SÏ. VilcÏek et al. / Veterinary Microbiology 77 (2000) 129±136 Fig. 2. Alignment of deduced amino acid sequences of the insertions within the genome of cp BVDV strains. Sequence for cINS was taken from Becher et al. (1996).
133
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nucleotide position 4994±5012 (or 1537±1543 amino acid) of the NS2-3 region. For comparison, the start of cINS in NADL is located at position 4994 (or 1537 at the amino acid level). The viral sequences for all isolates were colinear to those of ncp SD-1 strain, but mutations of single amino acids were observed near to the inserted cellular sequences (see Fig. 1). The size of cellular insertions varied between 90 (cp 88055 and NADL) to 140 (cp 2324_94) amino acids, all being in the same reading frame as the viral polypeptide. All cINS had a common core of 89 amino acids which is very similar to the cellular insertion within NADL (Fig. 2). However, the 50 and 30 ends of insertions were unique, except for cp 88055, which was found to be the same as in NADL. Interestingly, neither a 14-aminoacid stretch (APRRQRHRVPAKED) at the 50 -end of the insertion found in cp 5569 nor 15 amino acids (RVFLKICRPRPTSTT) at the 30 -end of the insertion in cp 5.19516 (nor their corresponding nucleotide sequences), were homologous to the cINS sequence (Fig. 2). No signi®cant matches for these stretches were found in EMBL and Swissprot databases. 4. Discussion The results presented in our work show that the speci®c RT-PCR method revealed an insertion within the NS2-3 coding region in 12 of 32 cp BVDV ®eld isolates and in the cp Indiana strain. The cINS type insertions was con®rmed in eight cp BVDV genomes. This work documents for the ®rst time that the cINS insertion appears with a similarly high frequency as has been previously described for the insertion of ubiquitin-coding sequences. According to our data all cINS contain a very conserved core of 89 amino acids, as was observed in strain NADL, with only a small number of amino acid differences between the isolates. The conservation of this core sequence suggests that it has biological signi®cance. Recently it has been demonstrated that the cellular insertion in the genome of NADL is essential for the generation of NS3 by NS2-3 cleavage and for cytopathogenicity of this virus strain (Mendez et al., 1998). The exact mechanism by which the insertion induces cytopathogenicity is not known yet. From ®ve of the ®eld isolates analysed, the RT-PCR products could not be sequenced. We assume that this was due to the coampli®cation of more than one product, which suggests that the isolate consisted of more than one cp virus subpopulation. This is in agreement with the ®nding by radioimmunoprecipitation that some uncloned ®eld isolates show heterogeneous expression of the NS2-3 protein (Greiser-Wilke et al., 1992). Alternatively, the other mechanisms of cytopathogenicity, such as genomic rearrangement can also occur in those cp strains. The insertions studied differ signi®cantly at the 50 and 30 ends. Interestingly, additional ``in reading frame'' sequences were detected in two strains at the 50 or 30 ends of cINS. However, their origins could not be identi®ed. It seems that the combination of two peptide coding sequences is not so rare in BVDV insertions, because the same phenomenon has been observed in other BVDV strains (Becher et al., 1998; Qi et al., 1998). It is not surprising that cINS are not found in the NS2-3 region of all cp isolates, since other mechanisms are known to lead to the conversion of BVDV strains from ncp to cp. Different genomic rearrangements have been detected, as well as the insertions of a
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sequence encoding light chain 3 of microtubule-associated proteins 1A and 1B (Meyers et al., 1998), ribosomal S27a coding sequence (Becher et al., 1998) or SMT3B bovine gene (Qi et al., 1998). Furthermore, other cp BVDV seem to be the result of rearrangement in the virus genome, involving duplications, mutations and deletions (see review Meyers and Thiel, 1996). In conclusion, the variable length cINS type cellular insertions occur in some cp BVDV isolates. Although we do not know the exact mechanism of their incorporation into the virus genome, their presence is correlated to the expression of the cp biotype. A more systematic search is required to ®nd other insertions and rearrangements in pestivirus genomes and to explain the phenomenon of pestivirus cytopathogenicity. Acknowledgements SÏtefan VilcÏek's work was supported by an International Research Development Fellowship from The Wellcome Trust (Grant No. 049929/Z/96/Z) and VEGA of SR (Grant No. 1/6215/99). Irene Greiser-Wilke was supported by the SFB 280 (``Sonderforschungsbereich Gastroitestinale Barriere'').
References Becher, P., Meyers, G., Shannon, A.D., Thiel, H.-J., 1996. Cytopathogenicity of border disease virus is correlated with integration of cellular sequences into the viral genome. J. Virol. 70, 2992±2998. Becher, P., Orlich, M., Thiel, H.-J., 1998. Ribosomal S27a coding sequences upstream of ubiquitin coding sequences in the genome of a pestivirus. J. Virol. 72, 8697±8704. Brownlie, J., Clarke, M.C., Howard, C.J., 1984. Experimental production of fatal mucosal disease in cattle. Vet. Rec. 114, 535±536. Collett, M.S., Larson, R., Gold, C., Strinck, D., Anderson, D.K., Purchio, A.F., 1988. Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus. Virology 165, 191±199. Deng, R., Brock, K.V., 1992. Molecular cloning and nucleotide sequence of a pestivirus genome, noncytopathic bovine viral diarrhea virus strain SD-1. Virology 191, 867±879. Donis, R.O., Dubovi, E.J., 1987. Characterization of bovine viral diarrhoea-mucosal disease virus-speci®c proteins in bovine cells. J. Gen. Virol. 68, 1597±1605. Greiser-Wilke, I., Dittmar, K.E., Liess, B., Moennig, V., 1992. Heterogenous expression of the non-structural protein p80/p125 in cells infected with different pestiviruses. J. Gen. Virol. 73, 47±52. Greiser-Wilke, I., Haas, L., Dittmar, K., Liess, B., Moennig, V., 1993. RNA insertions and gene duplications in the nonstructural protein p125 region of pestivirus strains and isolates in vitro and in vivo. Virology 193, 977±980. Mendez, E., Ruggli, N., Collett, M.S., Rice, C.M., 1998. Infectious bovine viral diarrhea virus (strain NADL) RNA from stable cDNA clones: a cellular insert determines NS3 production and viral cytopathogenicity. J. Virol. 72, 4737±4745. Meyers, G., Thiel, H.-J., 1996. Molecular characterization of pestiviruses. Adv. Virus Res. 47, 53±118. Meyers, G., Rumenapf, T., Thiel, H.-J., 1989. Ubiquitin in a togavirus. Nature 341, 491. Meyers, G., Tautz, N., Dubovi, E.J., Thiel, H.-J., 1991. Viral cytopathogenicity correlated with integration of ubiquitin-coding sequences. Virology 180, 602±616. Meyers, G., Stoll, D., Gunn, M., 1998. Insertion of a sequence encoding light chain 3 of microtubule-associated proteins 1A and 1B in a pestivirus genome: connection with virus cytopathogenicity and induction of lethal disease in cattle. J. Virol. 72, 4139±4148.
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Qi, F., Ridpath, J.F., Berry, E.S., 1998. Insertion of a bovine SMT3B gene in NS4B and duplication of NS3 in a bovine viral diarrhea virus genome correlate with the cytopathogenicity of the virus. Virus Res. 57, 1±9. Vilcek, S., Herring, A.J., Herring, J.A., Nettleton, P.F., Lowings, J.P., Paton, D.J., 1994. Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Arch. Virol. 136, 309±323. Wengler, G., Bradley, D.W., Collett, M.S., Heinz, F.X., Schlesinger, R.W., Strauss, J.H., 1995. Family Flaviviridae. Arch. Virol. 10 (Suppl.), 415±427.