Identification Of PCV2 Isolates And Development And Application Of Diagnosic Methods

Porcine circovirus disease is caused by porcine circovirus type 2 (PCV2), in which included postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome(PDNS), congenital tremors(CT), proliferative and necrotizing pneumonia(PNP). At present, pathology, epidemiology, diagnosis and immunology had been studied thoroughly worldwide, but available diagnostic method could not differentiate PCV2 from PCV1 during the earlier infection, there is no commercial vaccine available against PCV2 now, it is important to develop sensitive, specific diagnostic method for antigen and antibody detection.First, the native PCV2 strains from farms of typical PMWS-infected pig were isolated in China in the present study. Then their total genomes were cloned, sequenced and analyzed followed by building a PCV2 PCR-based diagnostic method. The genome and the Cap gene of the PCV2 isolates were amplified and cloned, and two recombinant plasmids containing partial Cap gene were obtained. The proteins were induced by IPTG and the recombinent protein was expressed highly in E. coli, respectively. Using the recombinant antigens, an indirect PCV2 ELISA method detecting PCV2 was built. Monoclonal antibodies specific to PCV2 were prepared by inoculating BALB/c mice with the recombinant antigens; double antibodies sandwhich ELISA detection method was built by using of PCV2 specific monoclonal antibody ascite fluid. All above methods were used in the field for detecting PCV2 in PMWS-suspected farms in China.Three strains of PCV2 were isolated from farms suffered from PMWS in Jiangxi and Heilongjiang provinces, and the genomic sequences of the three virus strains were submitted to GenBank with accession number AY288133 (S2), AY288134 (P11), AY288135 (L), respectively. The length of the genomes were 1767nt,1767nt,1768nt respectively. Comparison of nucleotide of PCV2 isolates was conducted with those of reference PCVs from GenBank, and phylogenic tree was constructed.A PCR diagnose method was developed by designing and using two sets of different primers specific to PCV1 and PCV2, respectively. The method was highly sensitive and specific by optimizing each condition in PCR assay. Since 2002, 513 field samples from pig farms of 12 provinces/citys including Shandong, Shanghai, Heilongjiang, Henan, Liaoning, Qinghai, Guangdong, Beijng, Jilin, Jiangxi, Shanxi and Tianjin suffered from PMWS-suspected were detected using PCV2 PCR. 115 out of 513 samples were positive, with the positive rate 22.0%. Among them, samples from Henan provinces had higher positive rate of 75.0%, respectively; Samples from Qinghai province with 0% and other provinces/citys were between 6.0% and 48.0%. 194 samples from 3 pig farms suffering PMWS at Daqing, Kaohezhai of Heilongjiang province and Gongzhuling of Jilin province were detected by PCR, resulting in the positive rate between 5.1% and 73.7%. In detail, 4 out of 78 samples from Daqing of Heflongjiang province were positive (5.1%) during March and April in 2002; 9 out of 78 samples from Kaohezhai of Heilongjiang province were positive (11.5%) during April and June in 2002; 28 out of 38 samples from Gongzhuling of Jilin province were positive (73.7%) between 2003 and 2005. These results suggest that PCV2 infection is different in aboved places, and indicated that there is relationship between PMWS and PCV2 infection, and PCV2 infection is increasing.The genome and Cap gene of strain L were amplified and cloned followed by obtaining recombinant plasmids of pMDT-PCV and pMDT-Cap. Using EcoRI site in the ORF2 gene, two recombinant expressed plasmids were obtained, designed as pGEX-PCV737-421 and pGEX-PCV421-37, respectively ,which harboring the NH₃-end and COO-end of ORF2 gene, and expressed highly in E. coli, respectively. The antiserum against recombinent proteins of PCV737-421 and PCV421-37 were prepared, and used for identifying PCV2 L strain by IFA assay. PCV2 indirect ELISA was developed by using the recombinant antigen of pGEX-PCV421-37. In 2005, almost 40 serum samples from 4 farms with PMWS located in Heilongjiang, Jilin and Henan provinces were detected. It was showed that 35 out of 40 samples were positive with positive rate 87.5% using the above recombinent protein as antigen in ELISA assayUsing the expressed protein of pGEX-PCV737-421 as immungen, 16 strains of monoclonal antibodies were screened by IFA methed specific to PCV2. Among them, 8B7-B1, 9E2-A10 and 9B6-A7 cannot detect PCV1 infected PK15, and were PCV2 specific, the other 13 strains can detect both PCV1 and PCV2 in IFA assay. Using the expressed protein of pGEX-PCV421-37 as immungen, 5 strains of monoclonal antibodies were prepared, and could detect both PCV1 and PCV2, were PCV specific. 3F4-D10, 9B4-D8 and 26H5-A3 belong to IgG1 while other 18 stains belong to IgM; all light strands of 21 strains belong to K.Using McAb 9B6-A7 as coating antibody, a double-antibody sandwhich ELISA method was built. Each component was optimized by board assay. Specific assay showed that the method didn't react with PCV1, SIV, PRV, PRRSV and PPV strains. When compared with PCR, 26 ELISA positive samples was also PCR positive, however, 14 ELISA negative samples were showed positive by PCR. Since 2003, there were 133 field samples have been detected from PCV2 suspected farms in Heilongjiang, Henan, Jilin and Jiangxi provinces by PCV2 double-antibody sandwhich ELSIA, 117 of them were positive with positive rate of 87.9%, indicating the high rate of infection by PCV2.