Construction And Immunogenicity To Piglets Of Chimeric Porcine Circovirus

Porcine circovirus (PCV) is divided into 2 genotypes, one is the nonpathogenic PCV type 1 (PCV1), another is the pathogenic PCV2, which is the primary causative agent of postweaning multisystemic wasting syndrome and other diseases. In this study, the complete genome sequence of one PCV1 was cloned, and its infectious DNA clone was constructed. Subsequently, the biologically pure and homogeneous PCV1-2 infectious virus stock was acquired by the construction of a chimeric PCV (PCV1-2) infectious DNA clones. Then, the immunogenicity to piglets was analyzed by animal experiment.The complete genome of one PCV1, named BJ-1 strain, was amplified and cloned by PCR assay. The complete genome sequence results showed that the BJ-1 strain shared 99.4% identity of nucleotide with other PCV1 strains from GenBank. The sequences of the main open reading frame (ORF) revealed that there were above 92.7% identity of nucleotide among the selected PCV1 isolates. Although the identity of nucleotide in the complete genomes and main ORFs of the selected PCV1 isolates were relatively high, there was a geographical correlation among them.The DNA clones of PCV1 was constructed after the insertion of the clone of complete PCV1 genome into the pBluescriptâ…¡sk(+)vector. After in vitro transfection of porcine kidney (PK)-15 cells with the PCV1 DNA clones, the rescue viruses of PCV1 were obtained. Its 50% tissue culture infectious doses (TCID50) of was 106.55/ml after 5 serial passages in PK-15 cells, which indicate its relatively high infectious titer.After the ORF2 gene of PCV1 was deleted by reverse PCR and was replaced of the ORF2 gene of PCV2, a chimeric PCV (PCV1-2) DNA clone was acquired. After in vitro transfection of PK-15 cells with the PCV1-2 DNA clones, the biologically pure PCV1-2 was produced. The TCID50 of the PCV1-2 was 106.05/ml after 5 serial passages in PK-15 cells, which suggested that the biologically pure and homogeneous PCV1-2 stock with relatively high infectious titer was successfully generated.After ten 35-day-old healthy piglets were inoculated with PCV1-2, the clinical effects of PCV1-2 on piglets were observed, the antibodies against PCV2 ORF2 in sera was determined by ELISA assay, the PCV2 DNA in sera and tissues were detected by conventional and real-time PCR. The results showed that there were not visible clinical signs and gross pathology in PCV1-2-inoculated piglets, and there were no obvious effects of PCV1-2 inoculation on the average gains and feed conversion efficiency of piglets. The antibodies against PCV2 ORF2 in sera was conversed on 14 day post inoculation (PI), and were 100% positive on 28 day PI, which demonstrated the good immunogenicity of PCV1-2 to piglets to the piglets. The DNA loads of PCV1-2 in sera increased gradually and peaked on 28 day PI, declined quickly thereafter. The DNA loads of PCV1-2 in lymph nodes is the higest but in heart is the lowest on 35 day PI, when the piglets were slaughtered. However, it was difficult to detect the PCV1-2 DNA in sera and tissues with conventional PCR, which indicated that the inoculation with PCV1-2 could only cause a slight infection.Taken together, a chimeric PCV1-2 with relatively high infectious titer and good immunogenicity to piglets was successfully generated in this study, which provided a strong base for the development of an attenuated PCV2 vaccine.