The Isolation And Identification Of Porcine Circovirus 2 And Sequence Analysis Of ORF2 Genes Of Porcine Circovirus 2 Shaanxi Trains

The infection of porcine circovirus type2 (PCV2) is an epidemic disease which was recognized in recent years, including postweaning multisystemic wasting syndrome (PMWS), reproductive failure/sow abortion and mortality syndrome (SAMS), porcine respiratory disease syndrome (PRDS), porcine dermatitis and nephropathy syndrome (PDNS), congenital tremor type A2(CT), intersticial pneumonia(IP), proliferative and necrotizing pneumonia (PNP), etc. The infection of PCV2 may reduce the immunological ability of pigs, leading to the secondary infection of other pathogens, and the wide prevalence and spreading of this disease posed a great threat to pig industry and caused great loss in many nations and regions all over the world, and handicaped the progress of pig industry. PCV was first reported in 2000 by Hongwu Lang in China, then PCV2 infection was reported in Bei jing, Zhe Jiang, Si Chuan, Hei Long Jiang and so on. PCV2 prevail strains were isolated from these regions. Many suspectable cases like infection of PCV2 were found in Shaanxi, and only one paper was published on serosurvey, but no etiological and molecular biological report about PCV2 till now. In order to discover the characteristic and epidemic rule and control the epidemic disease, Shaanxi strains of PCV2 were isolated and identified, and ORF2 genes were cloned and analyzed. The results were as follows:1. According to the published sequences of PCV2 ORF2 genes, two pairs of PCR primers were designed and synthesized. About 124 samples from 8 pig farms in some regions of Shaanxi province were detected with the nested-PCR. The results showed that there were 78 positive samples, the average of positive rate was 62.9%. Together with clinical incidence, PCR detection and patho-observation, that the PCV2 infection is prevalent in Shaanxi province.2. The PCV2 positive samples detected by PCR were inoculated to the contaminated PK-15 cells without PCV contamination. PCV2 can be detected in the cells of the sixth generation by IFA and PCR. The results indicated the virus obtained in this experiment was PCV2, And 5 strains of PCV2 were isolated in Shaanxi province.3. One pair of primers was designed according to PCV2 ORF2 sequences published in GenBank. The ORF2 gene was amplified from PCV2 Shaanxi isolates. Then the PCR products were cloned into pMD18-T vector, and the recombinant plasmid pMD-T-ORF2 was obtained. The sequence of the cloned PCR products was analyzed and compared with other PCV2 in GenBank. The results indicated that ORF2 gene was composed of 702 nucleotides encoding a polypeptide of 233 amino acids. Nucleotide homogenecity of ORF2 gene ranged from 90.5%~99.7%, while 81.2%~97.4% identities was found on amino acid level. Phylogenetic analysis revealed that the isolated strains is close to each other, nucleotide homogenecity of ORF2 gene ranged from 98.3%~99.7%; far related to the other strains of PCV2 in China, nucleotide homogenecity of ORF2 gene ranged from 95.6%~99.3%; while the strains are the farthest to the strains from Thailand and Canada, nucleotide homogenecity of ORF2 gene ranged from 90.5%~94.2%.In this study, 5 strains of PCV2 were isolated, and analysised through molecular epidemiology. The research revealed the epidemic, variation of PCV2 in Shaanxi, as well as the specificity for the main structural gene on molecular level. The results provided the molecular epidemiological datas of PCV2 for better control and prevention of PCV2 related disease.