Abstract:

Seven cDNA fragments covered the whole genome of classical swine fever virus (CSFV) Thiverval strain were amplified by reverse transcription PCR with PfuUltraTM HighFidelity DNA Polymerase. Products were ligated into pMD18T vector and sequenced. Using the appropriate ligation strategy, the whole genome cDNA were constructed into a lowcopy pAC/F101 vector. The recombinant plasmid was designated as pAC/F101/T17, which contained the complete genome of CSFV Thiverval strain with T7 promoter, hammerhead ribozyme and HDV ribozyme, T7 termination sequence at both sides. The viral RNA was synthesized in vitro with T7 RNA polymerase, and transfected into PK15 cells with lipofectamine 2000. After the cell passage, RTPCR detection and immunoperoxidase monolayer assay with E2 monoclonal antibody, the results indicated that the virus had been rescued from the Thiverval strain fulllength cDNA clone. Successful construction of infectious clone of CSFV Thiverval strain provided a useful tool for further study the attenuated mechanism of CSFV vaccine strains.