Abstract
Conventional reverse genetics for classical swine fever virus (CSFV) is based on the transfection of permissive cells with either in vitro or intracellularly synthesized RNA transcripts from a viral genomic cDNA clone. These strategies are complicated, inefficient and time-consuming. This study is aimed to develop an improved reverse genetics method for the direct, rapid and efficient recovery of CSFV from cloned cDNA. The cDNA clone pBRCISM was constructed, which harbors the full-length genomic sequence from the CSFV Shimen strain flanked by the cytomegalovirus promoter (an RNA polymerase II promoter), a chimeric intron, and hammerhead ribozyme sequences at the 5′-end and the hepatitis delta virus ribozyme and SV40 polyadenylation signal sequences at the 3′-end. Infectious progeny virus was rescued from PK-15 cells directly transfected with pBRCISM, and its morphology, one-step growth characteristics and pathogenicity were indistinguishable from the parent virus and virus rescued from classical reverse genetics. The reverse genetics based on RNA polymerase II yielded a 120-fold increase in the titer of nascent virus in 12-h less time than a reverse genetics method based on in vitro transcription. The full-length cDNA clone remained stable and infectious after 20 passages in bacterial cells, in contrast to the instability of the full-length clone without the intron after 9 passages. The improved reverse genetics method developed in the present study is efficient, stable, convenient and cost-effective and will be valuable for the rapid recovery of CSFV mutants.
References
Ben Abdeljelil N, Khabouchi N, Mardassi H (2008) Efficient rescue of infectious bursal disease virus using a simplified RNA polymerase II-based reverse genetics strategy. Arch Virol 153:1131–1137
Everett H, Salguero FJ, Graham SP, Haines F, Johns H, Clifford D, Nunez A, La Rocca SA, Parchariyanon S, Steinbach F, Drew T, Crooke H (2010) Characterisation of experimental infections of domestic pigs with genotype 2.1 and 3.3 isolates of classical swine fever virus. Vet Microbiol 21:26–33
Huang JH, Li Y, He F, Li D, Sun Y, Han W, Qiu HJ (2013) An improved methodology for rapid recovery of classical swine fever virus from cloned cDNA. J Int Agri (in press)
Lee C, Calvert JG, Welch SK, Yoo D (2005) A DNA-launched reverse genetics system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity. Virology 331:47–62
Li BY, Li XR, Lan X, Yin XP, Li ZY, Yang B, Liu JX (2011) Rescue of Newcastle disease virus from cloned cDNA using an RNA polymerase II promoter. Arch Virol 156:979–986
Martin A, Staeheli P, Schneider U (2006) RNA polymerase II-controlled expression of antigenomic RNA enhances the rescue efficacies of two different members of the Mononegavirales independently of the site of viral genome replication. J Virol 80:5708–5715
Meyers G, Thiel HJ, Rumenapf T (1996) Classical swine fever virus: recovery of infectious viruses from cDNA constructs and generation of recombinant cytopathogenic defective interfering particles. J Virol 70:1588–1595
Mishin VP, Cominelli F, Yamshchikov VF (2001) A ‘minimal’ approach in design of flavivirus infectious DNA. Virus Res 81:113–123
Moormann RJ, van Gennip HG, Miedema GK, Hulst MM, van Rijn PA (1996) Infectious RNA transcribed from an engineered full-length cDNA template of the genome of a pestivirus. J Virol 70:763–770
Peng WP, Hou Q, Xia ZH, Chen D, Li N, Sun Y, Qiu HJ (2008) Identification of a conserved linear B-cell epitope at the N-terminus of the E2 glycoprotein of classical swine fever virus by phage-displayed random peptide library. Virus Res 135:267–272
Pu SY, Wu RH, Yang CC, Jao TM, Tsai MH, Wang JC, Lin HM, Chao YS, Yueh A (2011) Successful propagation of flavivirus infectious cDNAs by a novel method to reduce the cryptic bacterial promoter activity of virus genomes. J Virol 85:2927–2941
Qi X, Gao Y, Gao H, Deng X, Bu Z, Wang X, Fu C (2007) An improved method for infectious bursal disease virus rescue using RNA polymerase II system. J Virol Methods 142:81–88
Reed LJ, Münch MH (1938) A simple method of estimating fifty percent end points. Am J Hyg 27:709–716
van Gennip HG, van Rijn PA, Widjojoatmodjo MN, Moormann RJ (1999) Recovery of infectious classical swine fever virus (CSFV) from full-length genomic cDNA clones by a swine kidney cell line expressing bacteriophage T7 RNA polymerase. J Virol Methods 78:117–128
Vilcek S, Stadejek T, Ballagi-Pordány A, Lowings JP, Paton DJ, Belák S (1996) Genetic variability of classical swine fever virus. Virus Res 43:137–147
Yamshchikov V, Mishin V, Cominelli F (2001) A new strategy in design of +RNA virus infectious clones enabling their stable propagation in E. coli. Virology 281:272–280
Yanai H, Hayashi Y, Watanabe Y, Ohtaki N, Kobayashi T, Nozaki Y, Ikuta K, Tomonaga K (2006) Development of a novel Borna disease virus reverse genetics system using RNA polymerase II promoter and SV40 nuclear import signal. Microbes Infect 8:1522–1529
Zhao JJ, Cheng D, Li N, Sun Y, Shi Z, Zhu QH, Tu C, Tong GZ, Qiu HJ (2008) Evaluation of a multiplex real-time RT-PCR for quantitative and differential detection of wild-type viruses and C-strain vaccine of classical swine fever virus. Vet Microbiol 126:1–10
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This study was supported by the National 973 Project of China (No. 2005CB523202).
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C. Li and J. Huang contributed equally to this work.
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Li, C., Huang, J., Li, Y. et al. Efficient and stable rescue of classical swine fever virus from cloned cDNA using an RNA polymerase II system. Arch Virol 158, 901–907 (2013). https://doi.org/10.1007/s00705-012-1548-8
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DOI: https://doi.org/10.1007/s00705-012-1548-8