Porcine Circovirus Type 2 And Development Of An Indirect ELISA For Antibody Detection

Porcine circovirus type 2 (PCV2), a member of the Circoviridae family, is the causative agent of porcine circovirus associated diseases (PCVAD) which include post-weaning multisystemic wasting syndrome (PMWS), porcine respiratory disease complex (PRDC), porcine dermatitis and nephropathy syndrome (PDNS), congenital tremors (CT), etc. In addition, PCV2 infection can cause immune suppression, leading to a variety of bacteria and virus multiple infections. PCV2 infection poses a threat to the development of the pig industry and causes significant economic losses in the world.The current commercialization vaccines can induce specific protective antibody, but PCV2 still circulates in the immunized pig population and the genomes of PCV2 is in a continuous variation state. So, the concerns are raised that the available commercial vaccines are not able to provide complete protection against variant PCV2 infection. As the PCV2 mutant strains and vaccine failure cases are constantly reported in China, the United States, South Korea and other countries, it is particularly important to establish the PCV2 specific detection method and develop the novel vaccine. To solve these problems, the following researches were performed:1. Construction of the porcine circovirus type 2 infectious cloneA Hind III restriction enzymes site (AAGCTT) was introduced into PCV2 ORF1 gene before the termination codon (TGA) through gene cloning technology, and the obtained genome was used to transfect PK-15 cells after circularized by ligation, then they were blindly passaged five generations. The results of IFA and whole genome sequencing showed that the H-PCV2 strain with a genetic marker was obtained through infectious clone technology, which could be differentiated from the parental strain by PCR restriction fragment length polymorphism analysis. The results of the biological characteristics of H-PCV2 revealed that the proliferation of the virus at the early stage of the replication was decreased by the insertion of Hind III sequence, but the rescued virus and the parental strain are similar in the proliferation characteristics, and the virus titer of 15th generation could reach 104.8TCID50/mL. Through sequencing the whole genome of H-PCV2 from different generations, the results revealed the embedded molecular genetic marker with high stability, which laid a solid foundation for the future to differentiate vaccine strains from wild strains.2. Development of an indirect ELISA for antibody detection against porcine circovirus type 2In order to explore new antigen epitopes of PCV2 genome which have the diagnosis function for PCV2 infection, the specific antigen fragments that may exist in PCV2 genome besides ORF2 were analyzed through bioinformatics analysis, and then 7 peptide sequences were designed and synthesized. The immunoreactivity of 7 synthesized peptides was confirmed by indirect ELISA and Dot-ELISA, respectively. The results indicated that only the synthesized ORF9p peptide possess relative antigenicity towards PCV2. Then an indirect ELISA with ORF9p as the envelope antigen to detect PCV2 antibody was established. The values of the ORF9p-based ELISA were compared with that determined by commercial ELISA Kit, and results showed that the sensitivity and specificity of the ORF9p-based ELISA were 84.5% and 92.9%, respectively. The coincident rate between the two methods was 86.7%. Then, the newly established indirect ELISA method was used to detect the serum from the PCV2 infection mice or the inactivated vaccine immunized mice or control group mice, and the results showed that both PCV2 infection and vaccine immunization could stimulate mice to produce specific antibodies against PCV2. Comparative analysis of the antibody levels of three groups, no notable differences between OD630nm values of serum in PCV2 infection or vaccine immunization mice groups were observed, and both groups had similar variation tendency in the values, while the values in the control group were negative. All these results suggested that the ORF9p possess a specific immunoreactivity towards PCV2, and the newly established ORF9p-based ELISA described in this study could be a sensitive, specific test for detection PCV2 infected serum.