Construction and characterization of porcine circovirus type 2 carrying a genetic marker strain

Porcine circovirus type 2 (PCV2) is the primary causative agent of post-weaning multisystemic wasting syndrome in pigs. To generate a genetic marker strain of PCV2, the full-length genome of the virus was amplified using PCR, and two copies of the genome were ligated in tandem to construct an infectious molecular clone. A Sal I restriction enzyme site was inserted into the clone as a genetic marker, and the recombinant plasmid was transfected into porcine kidney cells to generate mutant virus. The antigenicity of the recovered virus was confirmed by immunoperoxidase monolayer assay. The viral antigen was visualized in the nucleus and cytoplasm of the virus-infected cells. The viral genome could be differentiated from the wild-type parent by PCR and restriction fragment length polymorphism (PCR-RFLP). The mutant virus was stable on multiplication through 60 passages in cell culture; the highest titer reached was 106.6 TCID50/ml. Four 35-day-old unvaccinated piglets were inoculated with the virus by the intranasal and intravenous routes. Two of the virus-infected pigs developed high temperatures, progressive weight loss, and swollen lymph nodes. The viral antigen and nucleic acid were detected in numerous tissues of the pigs. The results indicate that the genetic marker strain should be a useful tool in studies on pathogenesis, vaccination, and molecular diagnosis of PCV2.