Generation of a DNA-launched classical swine fever virus infectious clone packaged in bacterial artificial chromosome

Reverse genetics system offers powerful tool for the research of RNA viruses. The infectious clones of classical swine fever virus (CSFV) were commonly constructed either in high- or low-copy number plasmids and transcribed to infectious RNA using phage RNA-polymerases. Herein, the full-length genome of CSFV Shimen strain, flanked by cytomegalovirus immediate-early (CMV) promoter (a eukaryotic RNA polymerase II promoter) sequence at the 5′-end and the hepatitis delta virus ribozyme along with the bovine growth hormone termination and polyadenylation signal sequences at the 3′-end, was packaged in bacterial artificial chromosome vector to establish a CSFV infectious clone pBAC-smCSFV. This infectious cDNA clone maintained stability after passaged 20 times in bacteria. Transfection of PK15 cells with this cDNA clone facilitated recovery of infectious progeny virus which was identical to parent virus as characterized by RT-qPCR, western blotting, indirect immunofluorescence assay, one-step growth kinetics analysis and nucleotide sequencing. Based on this CSFV infectious cDNA clone, the mCherry was inserted between viral N(pro) and C protein to develop reporter virus CSFV-mCherry. The mCherry was stably expressed after CSFV-mCherry was passaged 10 times in PK15 cells. Taken together, this present study develops a concise and efficient CSFV infectious cDNA clone and a reporter virus CSFV-mCherry. To the best of our knowledge, this is the first combination of CMV promoter and BAC system in construction of CSFV reverse genetics system. The CSFV infectious cDNA clone and the reporter virus will be useful in the study of CSFV virus biology, virulence determinants, molecular pathogenesis, vaccine development and virus-host interaction.