An Improved Methodology For Rapid Recovery Of Classical Swine Fever Virus From Cloned CDNA

Classical swine fever (CSF), a highly contagious and often lethal disease of pigs, is caused by classical swine fever virus (CSFV) and one of OIE-listed diseases. Reverse genetics systems, with the ability to manipulate viral genomes at the DNA molecular level, are an important platform for study of the assembly and function of viruses. An efficient, convenient and economical virus rescue method is undoubtedly required for rapid rescuing recombinant viruses.To develop an efficient, helper virus-free viral recovery system, the T7 RNA polymerase (T7 RNAP) gene was amplified using PCR from E. coli BL21(DE3) cells and inserted into the retroviral vector pLXSN creating the recombinant plasmid pLXSN-T7. The pLXSN-T7 was transfected into PT67 cells to prepare the recombinant retrovirus (rMLV-T7) in the presence of G418. The PK-15 cells stably expressing the T7 RNAP designated as PK/T7 was established by rMLV-T7 transduction and G418 selection followed by idenfication by immunofluorescence assay (IFA). T7 RNAP transcriptional activity was detected in the PK/T7 cells transfected with pET-RFP harboring red fluorescent protein gene under the control of T7 promoter. The PK/T7 cell line was stable during multiple passages in the presence of G418.Six cDNA fragments covering the complete genome of CSFV Shimen strain were amplified by RT-PCR and cloned into pOK12 vector, generating pOKShimen. The genomic RNA was in vitro transcribed from linearized pOKShimen and transfected into PK-15 cells, rescuing the progeny virus vShimen with an introduced genetic tag. The pOKShimen-RzTΦ, flanked at the 3′end by ribozyme (Rz) of hepatitis D virus and T7 terminator (TΦ) sequences, was transfected directly into PK/T7 cells, resulting in the recovered virus designated as vShimen-RzTΦ. vShimen-RzTΦshowed indistinguishable characteristics compared to vShimen and wild type Shimen strain, as demonstrated by RT-PCR, real-time PCR, IFA, antigen-capture ELISA, electron microscope and one-step replication kinetics. Multiple gene sequencing showed that vShimen-RzTΦwas genetically stable.In conclusion, the present study developed an efficient methodology for rapid recovery of CSFV from cloned cDNA in the cell line stably expressing T7 RNAP and the improved reverse genetics system will be useful in the pathogenesis study and new vaccine development of CSF.