Sequencing the complete genome of rabies virus CVS-11 strain and constructing its full-length infectious cDNA clone

Abstract:

Abstract:［Objective］To sequence the complete genome of CVS-11 strain and establish a reverse genetic system of CVS-11 to further study its pathogenic mechanism，virulence genes and antigenic sites．［Methods］We amplified12 fragments covering the complete genome of the CVS-11 strain by RT-PCR，and then cloned to pEASY-Blunt vector for sequencing the complete genome of CVS-11．We analyzed single restriction enzyme sites of the full length cDNA of the CVS-11 strain by DNAMAN and designed 4 pairs of specific primers．We amplified the full-length cDNA of CVS-11 by RT-PCR．We obtained four fragments and cloned into pcDNA3.1．We named the full-length cDNA plasmid pcDNA3. 1-CVS-11．We also cloned helper plasmids pcDNA3. 1-N，P，L and G expressing N，P，L and G protein of CVS-11 strain，respectively．We co-transfected NA cells with the full-length plasmid and four helper plasmids．We identified the supernatant of the transfected and then passaged NA cells by immunofluorescence staining and RT-PCR and found the recombinant virus rCVS-11 rescued successfully．［Results］Sequencing results showed that the complete genome of CVS-11 was composed of 11 927 nucleotides． The complete genome of CVS-11 encoded 5 structure proteins and gene array was the same as other reported rabies viruses． We successfully constructed a reverse genetic system of CVS-11，namely the full length plasmid pcDNA3. 1-CVS-11 and 4 help plasmids pcDNA3. 1-N，P，L，G and rescued the rCVS-11 from a full-length infectious cDNA clone．［Conclusion］ The reverse genetic system of the CVS-11 strain laid the foundation for future studies on rabies virus．