Construction Of Infectious DNA Clone Of Chimeric Porcine Circovirus Type 2 And Its Immunogenicity

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS) and other associated diseases, whereas the porcine circovirus type 1 (PCVl) is ubiquitous and nonpathogenic for pigs.The ORF2 gene of PCV encodes the major immunogenic capsid protein. Sequence analyses revealed that ORF2 gene of PCV2 shares about 67% nucleotide sequence identity with that of PCV 1. There are common antigenic determinants in the Cap protein between the two types of PCVs, however, no cross-reaction of antigenicity is observed between them. Therefore, the ORF2 gene can be used to differentiate PCVl and PCV2.The full-length PCVl genome was amplified by polymerase chain reacion (PCR) from the supernatant of PCVl infected cells, while the ORF2 gene of PCV2 was amplified by PCR from the supernatant of PCV2 infected cells. The amplified fragments were then cloned into pCR2.1 vector respectively. PCR, restriction endonuclease analysis and DNA sequencing were used to identify the recombinant plasmids. The results indicated that the fragments were successfully inserted. The full-length genomic DNA of PCVl was cloned into pBluescript SK vector (pSK) . Then recombinant plasmid pSK-PCVl â–³ ORF2-PCV2-ORF2 (pSK-sPCVl-2) was finally constructed by replacing the PCVl capsid gene with that of PCV2 in the backbone recombinant containing the complete genomic DNA of PCVl. The full-length chimeric fragment PCV 1-2 was excised from pSK-sPCVl-2 and cloned into the same recombinant to form the dimerized tandem DNA clone pSK-dPCVl-2. The PCVl non-infected Dulac cells and PCVl infected PK-15 cells were transfected with pSK-dPCVl-2 using Lipofectamine transfection reagent. The expression of viralcapsid protein was confirmed by indirect immunofluorescence assay (IFA). The IFA results showed that the PCV1-2 DNA clone was infectious and PCV2 capsid was expressed in the nucleus and cytoplasma of the transfected cells.BALB/c mice were immunized with recombinant virus PCV1-2 and sera samples collected from all control and vaccinated animals at -2, 7, 14, 21, 28, 35, 42 day post-inoculation (dpi) were assayed for anti-PCV2 capsid antibodies by ELISA. These data indicated that the chimeric PCV1-2 virus with the immunogenic ORF2 capsid gene of pathogenic PCV2 cloned into the nonpathogenic PCV1 genomic backbone induced specific antibodies against the pathogenic PCV2 capsid antigen in almost all mice at 42dpi. The results indicated that the recombinant virus strain of PCV1-2 were of some immunogencity.