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
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ABSTRACT |
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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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MAIN TEXT |
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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 1081143 in the genome of BVDV strain NADL (Fig. 1
a). 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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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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ACKNOWLEDGEMENTS |
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REFERENCES |
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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, 29922998.[Abstract]
Becher, P., Orlich, M. & Thiel, H.-J. (1998). Ribosomal S27a-coding sequence upstream of ubiquitin-coding sequences in the genome of a pestivirus. J Virol 72, 86978704.
Becher, P., Orlich, M., Konig, M. & Thiel, H.-J. (1999). Nonhomologous RNA recombination in bovine viral diarrhea virus: molecular characterization of a variety of subgenomic RNAs isolated during an outbreak of fatal mucosal disease. J Virol 73, 56465653.
Becher, P., Orlich, M. & Thiel, H.-J. (2001). RNA recombination between persisting pestivirus and a vaccine strain: generation of cytopathogenic virus and induction of lethal disease. J Virol 75, 62566264.
Collett, M. S., Larson, R., Gold, C., Strick, D., Anderson, D. K. & Purchio, A. F. (1988). Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus. Virology 165, 191199.[Medline]
De Moerlooze, L., Lecomte, C., Brown-Shimmer, S. & 9 other authors (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, 14331438.[Abstract]
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, 867879.[Medline]
Fritzemeier, J., Haas, L., Liebler, E., Moennig, V. & Greiser-Wilke, I. (1997). The development of early vs. late onset mucosal disease is a consequence of two different pathogenic mechanisms. Arch Virol 142, 13351350.[CrossRef][Medline]
Heinz, F. X., Collett, M. S., Purcell, R. H., Gould, E. A., Howard, C. R., Houghton, M., Moormann, R. J. M., Rice, C. M. & Thiel, H.-J. (2000). Family Flaviviridae. In Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses, pp. 859878. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego: Academic Press.
Kodama, K., Sasaki, N., Fukuyama, S., Izumida, A. & Ishii, F. (1974). Studies on cytopathogenic bovine viral diarrhea virus recovery, identification, and properties of the isolates virus. Bull Nippon Vet Zootech Coll 23, 5160.
Kümmerer, B. M. & Meyers, G. (2000). Correlation between point mutations in NS2 and the viability and cytopathogenicity of bovine viral diarrhea virus strain Oregon analyzed with an infectious cDNA clone. J Virol 74, 390400.
Kümmerer, B. M., Stoll, D. & Meyers, G. (1998). Bovine viral diarrhea strain Oregon: a novel mechanism for processing of NS2-3 based on point mutations. J Virol 72, 41274138.
Kupfermann, H., Thiel, H.-J., Dubovi, E. J. & Meyers, G. (1996). Bovine viral diarrhea virus: characterization of a cytopathogenic defective interfering particle with two internal deletions. J Virol 70, 81758181.[Abstract]
Meyers, G. & Thiel, H.-J. (1996). Molecular characterization of pestiviruses. Adv Virus Res 47, 53118.[Medline]
Meyers, G., Tautz, N., Dubovi, E. J. & Thiel, H.-J. (1991). Viral cytopathogenicity correlated with integration of ubiquitin-coding sequences. Virology 180, 602616.[Medline]
Meyers, G., Tautz, N., Stark, R., Brownlie, J., Dubovi, E. J., Collett, M. S. & Thiel, H.-J. (1992). Rearrangement of viral sequences in cytopathogenic pestiviruses. Virology 191, 368386.[Medline]
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, 41394148.
Nagai, M., Ito, T., Sugita, S. & 7 other authors (2001). Genomic and serological diversity of bovine viral diarrhea virus in Japan. Arch Virol 146, 685696.[CrossRef][Medline]
Neill, J. D. & Ridpath, J. F. (2001). Recombination with a cellular mRNA encoding a novel DnaJ protein results in biotype conversion in genotype 2 bovine viral diarrhea viruses. Virus Res 79, 5969.[CrossRef][Medline]
Qi, F., Ridpath, J. F., Lewis, T., Bolin, S. R. & Berry, E. S. (1992). Analysis of the bovine viral diarrhea virus genome for possible cellular insertions. Virology 189, 285292.[Medline]
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, 19.[CrossRef][Medline]
Ridpath, J. F. & Bolin, S. R. (1995). Delayed onset postvaccinal mucosal disease as a result of genetic recombination between genotype 1 and genotype 2 BVDV. Virology 212, 259262.[CrossRef][Medline]
Ridpath, J. F. & Neill, J. D. (2000). Detection and characterization of genetic recombination in cytopathic type 2 bovine viral diarrhea viruses. J Virol 74, 87718774.
Rinck, G., Birghan, C., Harada, T., Meyers, G., Thiel, H.-J. & Tautz, N. (2001). A cellular J-domain protein modulates polyprotein processing and cytopathogenicity of a pestivirus. J Virol 75, 94709482.
Sakoda, Y., Yamaguchi, O. & Fukusho, A. (1998). A new assay for classical swinefever virus based on cytopathogenicity in porcine kidney cell line FS-L3. J Virol Methods 70, 93101.[CrossRef][Medline]
Sentsui, H., Nishimori, T., Kirisawa, R. & Morooka, A. (2001). Mucosal disease induced in cattle persistently infected with bovine viral diarrhea virus by antigenically different cytopathic virus. Arch Virol 146, 9931006.[CrossRef][Medline]
Shimizu, M. & Satou, K. (1987). Frequency of persistent infection of cattle with bovine viral diarrhea-mucosal disease virus in epidemic areas. Jpn J Vet Sci 49, 10451051.
Shimizu, M., Satou, K., Nishioka, N., Yoshino, T., Momotani, E. & Ishikawa, Y. (1989). Serological characterization of viruses isolated from experimental mucosal disease. Vet Microbiol 19, 1221.
Tautz, N., Thiel, H.-J., Dubovi, E. J. & Meyers, G. (1994). Pathogenesis of mucosal disease: a cytopathogenic pestivirus generated by internal deletion. J Virol 68, 32893297.[Abstract]
Tautz, N., Meyers, G., Stark, R., Dubovi, E. J. & Thiel, H.-J. (1996). Cytopathogenicity of a pestivirus correlated with a 27-nucleotide insertion. J Virol 70, 78517858.[Abstract]
Thiel, H.-J., Plagemann, P. G. W. & Moenning, V. (1996). Pestiviruses. In Fields Virology, 3rd edn, vol. 1, pp. 10591073. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: LippincottRaven.
Vilcek, S., Herring, A. J., Herring, J. A., Nettleton, P. F., Lowings, J. P. & Paton, D. J. (1994). Pestiviruses isolates from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Arch Virol 136, 309323.[Medline]
Vilcek, S., Greiser-Wilke, I., Nettleton, P. & Paton, D. J. (2000). Cellular insertions in the NS2-3 genome region of cytopathic bovine viral diarrhea virus (BVDV) isolates. Vet Microbiol 77, 129136.[CrossRef][Medline]
Received 14 August 2002;
accepted 8 October 2002.