Insertion of cellular sequence and RNA recombination in the structural protein coding region of cytopathogenic bovine viral diarrhoea virus

Makoto Nagai1, Yoshihiro Sakoda2, Masashi Mori3, Michiko Hayashi1, Hiroshi Kida2 and Hiroomi Akashi4,{dagger}

1 Ishikawa Nanbu Livestock Hygiene Service Center, Kanazawa, Ishikawa 920-3101, Japan
2 Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
3 Laboratory of Plant Molecular Genetics Research Institute of Agricultural Resources, Ishikawa Agricultural College, Ishikawa 921-8836, Japan
4 National Institute of Animal Health, Kannondai, Tsukuba, Ibaraki 305-0856, Japan

Correspondence
Hiroomi Akashi
akashih{at}mail.ecc.u-tokyo.ac.jp


   ABSTRACT
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The cytopathogenic bovine viral diarrhoea virus (cp BVDV) strain KS86-1cp was isolated from a calf persistently infected with the noncytopathogenic (ncp) strain KS86-1ncp after it was exposed to cp BVDV strain Nose and developed mucosal disease (MD). Molecular analysis revealed that an insertion of a cellular gene and a duplication of the viral RNA encoding the nucleocapsid protein C and part of Npro had occurred in the C coding region of the Nose and KS86-1cp genomes. The inserted cellular gene was closely related to the cINS sequence. Remarkably, the 5' upstream region from the insertion of KS86-1cp had high sequence identity to that of Nose, but differed from that of KS86-1ncp. In contrast, the region downstream from the insertion of KS86-1cp showed high identity to KS86-1ncp, but not to Nose. These data reveal that KS86-1cp is a chimeric virus generated by homologous RNA recombination in a calf with MD.

{dagger}Present address: Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan.


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Bovine viral diarrhoea virus (BVDV) belongs to the genus Pestivirus of the family Flaviviridae (Heinz et al., 2000). The pestivirus genome consists of a single-stranded positive-sense RNA approximately 12·3 kb long. The single large open reading frame (ORF) flanked by the 5' and 3' noncoding regions (NCR) encodes a polyprotein of approximately 3900 amino acids (Meyers & Thiel, 1996; Thiel et al., 1996). Infection of foetuses with ncp BVDV during the first trimester of gestation may produce a persistently infected (PI) calf that is apparently immunotolerant. PI cattle may succumb later to fatal mucosal disease (MD). Cytopathogenic (cp) and noncytopathogenic (ncp) biotypes of BVDV can be isolated from an animal exhibiting MD, whereas persistent infection can be established only by ncp BVDV. Extensive data suggest that cp BVDV is derived from ncp BVDV by various RNA recombination events (Meyers & Thiel, 1996).

It has been shown that experimental induction of MD can be achieved by superinfection with a cp BVDV of cattle persistently infected with an antigenically different ncp BVDV, and several types of genetic recombination between ncp and cp BVDVs have been reported (Fritzemeier et al., 1997). However, the precise location of the homologous or nonhomologous recombination events has never been elucidated. BVDV strain Nose, a cp isolate from a case of chronic diarrhoea (Kodama et al., 1974), was used to superinfect a calf persistently infected with the ncp BVDV strain KS86-1ncp (Shimizu & Satou, 1987), which is antigenically distinct from Nose. The calf developed MD and became moribund 32 days post-inoculation. A cp BVDV (KS86-1cp) was isolated from the carcass. The antigenicity of KS86-1cp differed from that of the challenge virus but was similar to that of the ncp persistent virus (Shimizu et al., 1989). Since preliminary studies suggested that the 5' NCR sequences of Nose and KS86-1cp were similar, but differed from that of KS86-1ncp, we sequenced the full genomes of these three BVDVs. Here we report the first identification of a cellular insertion and a duplication of viral sequences within the structural protein coding-region of a cp BVDV resulting from RNA recombination between the persistent virus and the antigenically distinct cp BVDV.

Viruses were propagated for experiments using primary bovine foetal muscular (BFM) and bovine testicle (BT) cell cultures. The cp BVDVs, KS86-1cp and Nose, were cloned four times by plaque purification, and the ncp BVDV, KS86-1ncp, was obtained by three rounds of limiting dilution. The antigenic properties were compared by serum neutralization (SN) test using antisera prepared from sheep against KS86-1ncp, Nose and KS86-1cp. The SN test was performed by the microtitration method using BFM cell cultures (Shimizu & Satou, 1987). Production of NS2-3 was determined by immunodetection with an anti-NS3 monoclonal antibody cf10 (TropBio) and a polyclonal rabbit anti-NS3 specific serum. For molecular characterization, Northern blot analysis of RNA from BT cells infected with viruses was performed as described by Sakoda et al. (1998). Total RNA from BFM cells infected with viruses were used for RT-PCR, which was carried out as described previously (Nagai et al., 2001). The sense and antisense primers used for amplification of the viral genomes are based upon published sequences of three BVDV strains, NADL (Collett et al., 1988), Osloss (De Moerlooze et al., 1993) and SD-1 (Deng & Brock, 1992). The purified cDNA fragments were sequenced directly, at least twice. The 5' and 3' end sequences were determined by the rapid amplification cDNA end method using the 5' RACE system version 2.0 and the 3' RACE system for rapid amplification of cDNA ends (Invitrogen). The products were analysed on a model 373S automated DNA sequencer (Applied Biosystems). Computer analysis of sequence data were performed using the GENETYX-MAC sequence analysis program (Software Development Co. Ltd).

After plaque or limited dilution purification of the three viruses, the antigenic properties were compared by cross-SN tests. KS86-1ncp and KS86-1cp were antigenically similar to each other but had a 16-fold lower antibody titre in comparison with Nose, as reported previously (Shimizu et al., 1989). The Nose and KS86-1cp viruses caused a clear cytopathic effect (CPE) in BFM and BT cells, whereas no CPE was observed in the KS86-1ncp-infected cells.

Immunodetection with an anti-NS3 monoclonal antibody revealed that proteins of approximately 80 kDa (NS3) and 120 kDa (NS2-3) were detected in the KS86-1cp-infected BT cells and an 80 kDa protein (NS3) was detected in the Nose-infected BT cells, whereas only the 120 kDa NS2-3 precursor was detected in BT cells infected with KS86-1ncp (data not shown). Several cp BVDVs contain RNA genomes significantly longer or shorter than ncp BVDVs; the genome alterations are due to either large duplications or deletions (Baroth et al., 2000; Becher et al., 1998, 1999, 2001; Kupfermann et al., 1996; Meyers et al., 1991, 1992, 1998; Qi et al., 1992, 1998; Ridpath & Neill, 2000; Ridpath & Bolin, 1995; Tautz et al., 1994; Vilcek et al., 2000). RNA hybridization with the NS3-derived cDNA probe showed that the genomic RNA of KS86-1ncp, approximately 12·3 kb, was about 0·9 kb shorter than those of Nose and KS86-1cp (data not shown). Thus, it seemed likely that the Nose and KS86-1cp genomes would contain an insertion and/or duplication. No subgenomic smaller RNA was found in cells infected by any of the three viruses. On the basis of our previous 5' NCR sequence analysis and serological comparison data, it seemed probable that an homologous RNA recombination event had occurred between KS86-1ncp and Nose RNAs between nucleotide position 108 (the sense primer position used for 5' NCR amplification) and the E2-coding sequence.

We thus performed RT-PCR using sense primer 324 (Vilcek et al., 1994) and antisense primer 1124R [5'-GCTTT(C/T)TC(A/C)AGT(A/T)TCTTGCG-3'], flanking positions 108–1143 in the genome of BVDV strain NADL (Fig. 1a). The size of products when the Nose and KS86-1cp RNAs were used as template was about 1·9 kb, some 0·9 kb longer than the product obtained with the KS86-1ncp RNA (Fig. 1b). Further, we performed RT-PCR using sense primer 864F (5'-GGGTCCACAACAGGCTCAA-3') and antisense primer 767R (5'-CACATAAATGTGGTACAG-3'). With these primers no amplification of cDNA will be obtain from a BVDV genome without duplication (Fig. 1a). cDNA fragments of approximately 0·8 kb were generated from Nose and KS86-1cp, but not from KS86-1ncp (Fig. 1c). For further characterization, the amplified cDNA fragments of these BVDVs were directly sequenced (Fig. 2). An insertion of non-viral sequences was found 78 bases downstream of the start site of the C-protein coding sequence in the Nose and KS86-1cp RNAs. The 330 nucleotides of inserted sequences were identified as a cellular sequence, cINS, encoding a DnaJ protein.



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Fig. 1. RT-PCR analysis of KS86-1ncp, Nose and KS86-1cp. (a) Strategy for identification of the insertion and duplication in the C coding region. (b) RT-PCR products derived from KS86-1 ncp, Nose and KS86-1 cp with a primer pair, 324 (->) and 1124R (<-), for amplification of the 5' NCR, and the Npro and C coding regions. (c) RT-PCR products amplified with a primer pair, 864F (->) and 767R (<-), for amplification of the duplicated region. The PCR products were separated on a 1·5 % agarose gel and stained with ethidium bromide.

 


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Fig. 2. Comparison of the nucleotide sequences corresponding to the 5' NCR, and the Npro, C, cINS, Npro-2 and C-2 coding regions of KS86-1ncp, Nose and KS86-1cp. The positions of the 5' NCR, Npro, C, cINS, Npro-2 and C-2 are indicated. A dot indicates identity with the KS86-1ncp sequence, and a dash indicates a gap.

 
To our knowledge, this is the first report describing an insertion of a cellular sequence in the structural protein coding region of BVDV. A duplication of Npro (Npro-2) and C (C-2) sequences was also found downstream of the cINS sequence. Although the 3'-terminal 228 nucleotides of the C protein coding sequence and the 5'-terminal 15 nucleotides of the Npro-2 sequence of Nose and KS86-1cp were lacking, the complete coding sequences for the Npro and C-2 proteins were present. Thus, it is considered that the cp BVDVs could replicate using C protein produced from the C-2 coding sequence. cINS was identified previously in the NS2 sequence of BVDV NADL (Qi et al., 1992) and several other BVDV-1 isolates (Vilcek et al., 2000), BVDV-2 isolates (Ridpath & Neill, 2000) and a cp border disease virus (BDV) (Becher et al., 1996). The size of these insertions varied from 270 to 441 nucleotides. The insertion of cINS identified in the Nose and KS86-1cp RNAs was 330 nucleotides in length. The predicted amino acid sequence showed almost 100 % identity to that previously reported for the cINS insertion found in BVDV-1, BVDV-2 and BDV isolates as well as that of the cellular mRNA derived from bovine cells (Neill & Ridpath, 2001; Rinck et al., 2001) (Fig. 3). The cellular J-domain protein, Jiv, encoded from within the cINS insertion, can induce a conformational change in the NS2-3 protein depending upon the intracellular protein level of Jiv. The Jiv protein, and a fragment of Jiv (Jiv 90) identified as an insertion in the BVDV NADL RNA, interact stably with the NS2 protein and stimulate cleavage of the ncp BVDV derived NS2-3 in trans (Rinck et al., 2001). An alignment of the amino acid sequences of the cINS insertions found in several BVDVs showed that the cINS insertions of Nose and KS86-1cp completely included the Jiv 90 domain reported in BVDV NADL (Fig. 3). Therefore, it seems likely that a fragment of Jiv, which is expressed from cINS in the structural protein coding region of Nose and KS86-1cp, is responsible for promoting NS2-3 cleavage and hence causing the cytopathogenicity of these viruses.



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Fig. 3. Alignment of the amino acid sequences of the cINS insertions found in Nose, KS86-1cp and other cp pestiviruses. Sequences of jiv-mRNA, BVDV 1 NADL, BVDV 2 296C and BDV Cummock are quoted from Rinck et al. (2001), Collett et al. (1988), Ridpath & Neill (2000) and Becher et al. (1996) respectively. Underlined amino acids are the Jiv 90 domain.

 
Since no defective interfering particles, nor insertion of cellular RNA sequences and/or duplication of viral sequence in the NS2-3 coding region were identified in the Nose and KS86-1cp genomes (from the results of the full genome sequences), another possible mechanism for stimulating NS2-3 cleavage is the generation of point mutations within the NS2 coding sequence as reported for the BVDV Oregon genome (Kümmerer & Meyers, 2000; Kümmerer et al., 1998). Although three amino acid differences (at residues corresponding to 1298, 1376 and 1506 in the KS86-1ncp sequence) were observed between the NS2 sequences of KS86-1ncp and KS86-1cp, neither the small insertions (Tautz et al., 1996) nor the point mutations found in BVDV Oregon that affect the cleavage of NS2-3 were found in the NS2 coding region of the Nose and KS86-1cp sequences. Further studies to clarify the significance for NS2-3 cleavage of the mutations found in KS86-1cp will be needed.

To complete the genetic characterization of these viruses, we determined the full genome sequences of KS86-1ncp, Nose and KS86-1cp. The full genome sizes of KS86-1ncp, Nose and KS86-1cp were 12 306, 13 196 and 13 203 bp, and the accession numbers in the DDBJ, EMBL and GenBank nucleotide sequence databases are AB078950, AB078951 and AB078952, respectively. The RNA genome size of these three viruses was consistent with the results of the Northern blot analysis. The average percentage nucleotide sequence identity in the 5' upstream region from the insertion of KS86-1cp was 85·7 % compared to that of KS86-1ncp and 99·7 % compared to that of Nose. On the other hand, the average percentage identity in the region downstream from the insertion of KS86-1cp was 99·7 % to KS86-1ncp, and 82·1 % to Nose. These data clearly show that KS86-1cp represents a chimeric virus generated by homologous recombination between the KS86-1ncp and Nose RNAs, and that the recombination point is placed in the structural protein coding gene region. The genome organization of the KS86-1ncp, Nose and KS86-1cp RNAs and a putative RNA recombination point are shown in Fig. 4. It is well known that animals persistently infected with ncp BVDV succumb to fatal MD following superinfection with cp BVDV. Although a close antigenic relationship between the ncp and cp viruses is of crucial importance for development of acute MD, it has been suggested that chronic MD may occur as a result of superinfection of PI animals with cp viruses that have partial antigenic homology to the persisting ncp virus (Fritzemeier et al., 1997; Sentsui et al., 2001). However, the calf from which KS86-1cp was isolated produced SN antibody to Nose, but not to KS86-1ncp and KS86-1cp (Shimizu et al., 1989). These findings suggest that the superinfecting cp virus was eliminated or inactivated by host immunity, and it was a recombinant virus with the same antigenicity as the persistent ncp virus that induced MD. Although recombination of viral RNA from persisting virus with either vaccine or exogenous BVDV to produce recombinant cp viruses has been reported previously (Becher et al., 2001; Fritzemeier et al., 1997; Ridpath & Bolin, 1995), the precise mechanism(s) has not yet been identified. Our data showed that KS86-1cp was produced by a homologous recombination event between KS86-1ncp and Nose in the structural protein coding region. The new recombinant virus possessed the cINS sequence derived from the cp virus and had the same E2 amino acid sequence as the persistent ncp virus. Therefore the virus was both cytopathogenic and tolerated by host immune system. These findings suggest a new mechanism for the acquisition of cytopathogenicity by BVDV and the development of MD in cattle.



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Fig. 4. (a) Genome organization of KS86-1ncp, Nose and KS86-1cp virus RNAs. The grey and white boxes represent the KS86-1ncp and Nose genomic regions, respectively. The inserted cINS genes are indicated as black boxes. The putative homologous RNA recombination position between Nose and KS86-1ncp is indicated by an open arrow. (b) Nucleotide sequences of the recombination region between KS86-1ncp and Nose. The region of sequence identity is indicated by the vertical lines between the sequences. The putative RNA recombination position is indicated by an open arrow.

 


   ACKNOWLEDGEMENTS
 
We thank Dr M. Shimizu (National Institute of Animal Health) for providing KS86-1cp and KS86-1ncp. We also gratefully thank Dr Graham Belsham for critical review of the manuscript.


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Received 14 August 2002; accepted 8 October 2002.