Experimental Infection Of Porcine Circovirus 2(PCV2) In Pigs And Mouse Immune Responses Against PCV2 Cap Protein

To establish quantitative real-time PCR (qPCR) to detect the Cap gene of PCV2, we designed specific primers, optimized reaction protocol, and performed specificity test, and determined that the 5×103 copies/ml as the critical value of the test. Then we detected the viral genomic levels in field serum samles using the established qPCR method, and found no direct correlation between the results of qPCR and PCV2 ELIS A or IFA antibody. Therfore, we defined<5×103 copies/ml as the standard of negative serum with no viremia. Further, we investingated the viral genomic dynamics and pathological changes in experimentally PCV2-infected 8-week pigs. qPCR revealed that the peak viremia level occurred at 1 week post challenge (p.c.) and then declined to level of around critical value at 4 weeks p.c.. The continuous viral load monitor up to 14 weeks p.c. revealed persistant infection in experimentally infected pigs. In addition, higher viral loads were detected in lung, spleen, lymph node, stomach of inoculated pigs than other tissues. The pathological studies revealed that the PCV2-inoculated pigs developed mild to moderate lesions, with hemorrhage and edema of lymph node, pulmonary edema and congestion as main gross lesions, and lymphocytic depletion, histiocytic and multinucleate giant cell (MGC) infiltrations in lymphoid tissues, bronchial pneumonia, hemorrhagic nephritis and pancreastitis, hepatocellular granular degeneration as microscopic lesions. These data suggest that the persistant infection is the important characteristic of PCV2 pathogenicity, and the viral genomic copy number in serum and tissues as well as the microscopic lesions in lymphoid tissues and lung may be use as the virulent standard of PCV2.ORF2 is major protective immunogenic gene of PCV2. We compared the immune responses induced by ORF2 plasmid (pORF2) and capsid protein (Cap) by using different vaccine combination. The results revealed that the ORF2 plasmid is superior to Cap protein in inducing CD8+ cells, when both the plasmid and protein showed comparable efficacy in eliciting lymphoproliferative responses and CD4+ cells. The pORF2 induced Thl biased responses, while the Cap protein induced Th2 responses. The Cap protein evoked stronger ELIS A antibody responses than the ORF2 plasmid, whereas induced weaker neutralizing responses than the plasmid after challenge. The combined use of ORF2 plasmid and Cap protein increased the requency of activated CD8+ cells and developed balanced Thl/Th2 responses, especially in pORF2 prime-Cap boost protocol. Finally, pORF2 plasmid provided more efficient protection than the Cap protein after PCV2 challeng. These results suggest that ORF2 plasmid induces CD8+ T cells and Thl responses, and Cap protein induces Th2 responses. CD8+ T cells and Thl responses may play crucial roles in the protective immunity against PCV2 infection.We constructed eukaryotic expression vector of PCV2 ORF1 (pORFl), ORF3 (pORF3) and ORF4 (pORF4) and mixed them with PCV2 ORF2 plasmids as gene vaccines, additionally, PCV2 ORF2 was fused to ORF1,3 and 4 to construct pORF2-ORF1, pORF2-ORF3 and pORF2-ORF4 expression vectors, and their potential to induce humoral and cellular immune responses and provide protection against PCV2 infection were compared. For cellular immune responses, the co-administration of ORF1,3 with ORF2 induced comparable Cap-specific lymphocyte proliferation responses and CD4+ cells but lower CD8+ cells frequencies as compared to the pORF2 plasmid alone, indicating the reduction effect of ORF1,3 on the efficiency of pORF2 in eliciting CD8+ cells. In contrast, the co-administration of pORF4 showed no obvious influences on the cellular immune responses of pORF2. For humoral immune response, the protocols of pORF2-ORF1, pORF2+pORF1, pORF2+pORF3 and pORF2+pORF4 induced specific antibodies against the correspondent PCV2 ORF1, ORF3 and ORF4 proteins, especially high levels of ORF3 and ORF4 antibodies. However, the ORF1 and pORF3 showed reduction effects on the Cap specific antibody responses induced by ORF2. The antibody isotype profiles revealed Th1 biased responses against PCV2 Cap protein, while non-significant biased Th1 or Th2 responses against PCV2 ORF1,3 or 4 protein. After challenging with the virulent PCV2, only protocols pORF2+pORF3 and pORF2+pORF4 showed protection efficacies, however, they were not stronger than the pORF2+pCI. These data suggest that ORF1,3 and 4 had no augmention effect on the immunogenicity of ORF2.To examine the adjuvant effects of porcine cytokines in humoral, cellular and protective immunity to PCV2, we constructed eukaryotic expression vector of porcine IL2 (pIL2), IL4 (pIL4), IFN-γ(pIFN-γ) and fusion vectors of pORF2-pIL2, pORF2-pIL4 and pORF2-pIFN-γ. While the combined uses of pIL2, pIL4 or pIFN-γwith pORF2 showed comparable efficacy in eliciting Cap protein specific lymphocyte proliferation responses and CD4+ CD8- cells as the pORF2 plasmid, the coinjection of pIL2 enhanced the activated CD4-CD8+ and CD4+CD8+ cells. The Cap specific humoral responses induced by the coinjection of pIL4 was stronger than the coinjection of pIL2 or pIFN-y, but all of them did not show enhance effect upon pORF2 plasmid. The antibody isotype profiles revealed Thl type responses in mice co-immunized with pORF2 and pIL2, pIL4 or pIFN-y plasmids, but the coadministration of pIL2 suppressed the IgG1 antobody production and altered the Th1 biased immune responses more stronger. After challenging with the virulent PCV2, mice immunized with pIL2 and pORF2 plasmid exhibited more effective protection than the coinjection of pIL4 or pIFN-γ. Unexpectedly, the fusion plasmids did not elicit any specific immune responses and protective immuneity in our study. Taken together, our results suggest that, in contrast to pIL4 and pIFN-γwhich did not exhibit adjuvant effects, pIL2 can act as adjuvant to enhance the Thl and the cellular responses, but all the three porcine cytokines failed to enhance the antibody responses and protective efficacy of pORF2 plasmid.