Abstract
The complete nucleotide sequence of classical swine fever virus (CSFV) strain cF114 (F114 strain propagated on PK-15 cells) was cloned by RT-PCR. The analyses of nucleotide and amino acids identity between cF114 and F114, Brescia, Alfort or C strain were 99.41%, 96.80%, 86.03%, 95.70% and 99.28%, 98.54%, 93.33%, 97.41% respectively. The cDNA fragments with correct sequence were ligated into a full-length cDNA and inserted into pMC18 plasmid (pMC12297). A full-length infectious viral RNA was synthesized by runoff transcription and transfected to PK15 cells. Viruses were recovered from transfected cells which wese titrated on PK-15 cells by endpoint dilution and indirect immunofluorescence with a CSFV-specific monoclonal antibody. The antigenicity and replication kinetics of the plasmid-derived virus (vM12297) were similar to the parental virusin vitro. TheE01 orE2 gene was replaced with the genes from strain C and the pM/CE01 and pM/CE2 with chimeric full-length cDNA of cF114 were generated. The infectious viruses were obtained from pM/CE01 and pM/CE2. Both of the chimeric viruses can infect PK-15, SK-6 and primary testicle cell of swine. The chimeric viruses can grow to a titer of 8×105 F-PFU/mL. These results are very important for understanding the genes related to the CSFV propagation and pathogenesis.
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References
Regenmortel, M. H. V., Fauquet, C. M., Bishop, D. H. L. et al., The Seventh Report of the International Committee on Taxonomy of Viruses, 1st ed., San Diego: Academic Press, 2000, 859–878.
Thiel, H. J., Stark, R., Weiland, E. et al., Hog cholera virus: molecular composition of virions from a pestivirus, J. Virol., 1991, 65: 4705–4712.
Moormann, R. J. M., Gennip, H. G. P., Miedema, G. K. W. et al., Infectious RNA transcribed from an engineered full-length cDNA template of the genome of a pestivirus, J. Virol., 1996, 70: 763–770.
Ruggli, N., Tratschin, J., Mittelholzer, C. et al., Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA, J. Virol., 1996, 70: 3478–3487.
Mose, C., Tratschin, J. D., Hofmann, M. A., A recombinant classical swine fever virus stably expresses a marker gene, J. Virol., 1998, 72: 5318–5322.
Schnell, M. J., Mebatsion, T., Conzelmann, K. K., Infectious rabies viruses from cloned cDNA, EMBO J., 1994, 13: 4195–4203.
Whelan, S. P. J., Ball, L. A., Barr, J. N. et al., Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones, Proc. Natl. Acad. Sci. USA, 1995, 92: 8388–8392.
Garcin, D., Pelet, T., Calain, P. et al., A highly recombinogenic system for the recovery of infectious Sendai paramyxovirus from cDNA: generation of a novel copy-back non-defective interfering virus, EMBO J., 1995, 14: 6087–6094.
Hurrelbrink, R. J., Nestorowicz, A., McMinn, P. C., Characterization of infectious Murray Valley encephalitis virus derived from a stably cloned genome-length cDNA, J. Gen. Virol., 1999, 80: 3115–3125.
Major, M. E., Mihalik, K., Fernandez, J. et al., Long-term follow-up of Chimpanzees inoculated with the first infectious clone for hepatitis C virus, J. Virol., 1999, 73: 3317–3325.
Imada, T., Yamaguchi, S., Mase, M. et al., Avian nephritis virus (ANV) as a new member of the family Astroviridae and construction of infectious ANV cDNA, J. Virol., 2000, 74: 8487–8493.
Phillips, J. J., Chua, M. M., Lavi, E. et al., Pathogenesis of chimeric MHV4/MHV-A59 recombinant viruses: the murine Coronavirus spike protein is a major determinant of neurovirulence, J. Virol., 1999, 73: 7752–7760.
Peeters, B. P. H., Leeuw, O. S., Koch, G. et al., Rescue of Newcastle Disease Virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence, J. Virol., 1999, 73: 5001–5009.
Zhou, P. C., Chen, J. G., Zhai, Z. H. et al., Study on the cloning and expression of E0 of CSFV, Microbiology (in Chinese), 2000, 27(3): 165–170.
Sambrook, F., Fritsch, E. F., Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press, 1989, 401–417.
Wang, Z., Lu, Y., Ding, M. X., Study of characters of infectivity of CSFV C strain on primary bovine testicle cells, Virologica Sinica (in Chinese), 2000, 15(2): 170–179.
Huang, Q. H., Zhang, C. H., Wang, J. F. et al., Construction, sequencing and analysis of cDNA library of CSFV genome of Shi-men strain, Chinese Science Bulletin, 2000, (4): 367–369
Khromykh, A. A., Westaway, E. G., Completion of Kun** virus RNA sequence and recovery of an infectious RNA transcribed from stably cloned full-length cDNA, J. Virol., 1994, 68: 4580–4588.
Boyer, J., Haenni, A., Infectious transcripts and cDNA clones of RNA viruses, Virology, 1994, 198: 415–426.
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Nie, Y., Chen, J. & Ding, M. Sequencing and rescuing a highly virulent classical swine fever virus: Chinese strain cF114 from a full-length cDNA clone. Chin.Sci.Bull. 48, 1124–1128 (2003). https://doi.org/10.1007/BF03185766
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DOI: https://doi.org/10.1007/BF03185766