In a previous study, it has been shown that a Semliki Forest virus (SFV) replicon vectored DNA vaccine (pSFV1CS-E2) expressing the E2 glycoprotein of classical swine fever virus (CSFV) conferred full protection for pigs immunized three times with 600 microg of the vaccine. This study was designed to evaluate further the efficacy of the vaccine with lower dosage and fewer inoculations. Pigs were immunized twice with 100 microg of pSFV1CS-E2 (n=5) or control plasmid pSFV1CS (n=3), respectively, and challenged with virulent Shimen strain 6 weeks following the booster immunization. Pigs immunized with pSFV1CS-E2 developed high titers of specific neutralizing antibodies against CSFV after the booster, and the antibody titers increased rapidly upon challenge. The immunized animals showed no clinical symptoms except short-term fever and low-level viremia, whereas, the control pigs immunized with the control plasmid produced no detectable antibody prior to challenge, and showed obvious clinical signs following challenge, and two pigs died of illness. All control animals developed extended viremia as detected by nested RT-PCR and real-time RT-PCR. Severe pathologic lesions typical of CSFV infection were observed at necropsy. It is concluded that the alphavirus replicon-vectored DNA-based vaccine can be a potential marker vaccine against CSFV.