Construction of a Chimeric Virus Expressing Mutation Sequences of Classical Swine Fever Virus Yunnan Strain

摘要

Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. To explore the role of mutations in Classical Swine Fever Virus (CSFV) Yunnan strain which caused the atypical clinical signs in pigs, a new pSM derived from CSFV Shiman strain has been constructed and subsequently replaced by the mutation sequences of CSFV YN strain isolated by the laboratory at the positions 1510-1532, 2471-2658, 3152-3176 and 11785-11816 using the targeted recombination strategy to enable rescue of chimeric CSFV. The results showed that chimeric CSFV (vSM-YN) was successfully rescued from PK-15 cells by transfection of the chimeric CSFV RNA transcripts and identified by whole genome sequence analysis, immunofluorescence antibody assay and ELISA detection. Sequencing of the pAC-SM-YN revealed a high genetic stability and the complete genome sequences of rescued viruses vSM-YN after extensive passages in PK-15 cells showed that modifications in pSM were stably maintained. The results indicate that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should facilitate further study of the pathogenic mechanism of CSFV leading to atypical CSF.