Isolation And Identification Of Porcine Parvovirus(PPV) From Fetal Mummies In Henan Province And VP2 Gene Cloning And Sequence Analysis

Porcine Parvovirus disease, characterized by reproduction barrier of primiparous sows, is caused by porcine parvovirus leading to abortion, sterility, still births, fetal malformation, fetal mummies. This disease is widespread around the world and endemic in most pig farms, seriously affecting the development of the swine industry. It has been one of the hot pig diseases. A PCR method was established in this study for the detection of porcine parvovirus infection, and a strain of virus isolated from the diseased tissue by using the technology of cell culture. The major antigenic region of VP2 gene was amplified by PCR from the isolated porcine parvovirus, and then compared and analyzed with relative virus strains in order to lay the foundation for expression in prokaryotic of eukaryotic cell and the immune, prevention and application of genetic engineering vaccine.According to the reported porcine parvovirus genome sequence, a pair of oligonucleotide primers were designed and synthesized. A DNA fragment of 1124bp was successfully amplified by PCR from the tissues and cells infected with porcine parvovirus, and identified by Hindâ…¢digestion. When PCR was performed with the genomic DNA of PPV cytotoxicity, PCV2, PRRSV, PRV and PPV, only the PPV cytotoxicity even at 10-6 dilution of the template can produce an expected positive band, indicating good sensitivity of the method. By using this method, 6 out of 32 diseased materials of abortion were detected positive, while the use of HA, only 4 positive were detected. These results indicate that the established PCR assay has characteristics of high sensitivity and specificity. The PCR diagnostic method was used to detect samples of diseased pigs, the positive is expected to conduct a preliminary isolation and identification. the results revealed that the isolated virus in PK-15 cell for 4 blind passage can induce PK-15 cells stack together, pull nets, formation of plaque, and other typical lesions. A clear precipitation line can be seen in agar diffusion test; and positive reaction in indirect immunofluorescence; Application of the established PCR assay can amplify a DNA band from the extracted viral DNA of porcine parvovirus, and the amplified band is a specific fragment. The results prove that the virus isolated is porcine parvovirus, and named PPV ZK strains.The VP2 gene of porcine parvovirus containing the major epitope gene was amplified by PCR, and the DNA fragments were cloned and sequenced. The sequence was compared with other domestic and international isolates in GenBank by using DNA Blast net service and DNAstar software. The results showed that the isolated strain has nucleotide and amino acid homology of more than 97% with PPV-VP2 genome registered in GenBank, which proved PPV genome sequence is very conservative. But geographical differences can still be seen from phylogenetic tree of the sequence, it was in a branch with demestic isolates but has distant relationships with the sequence of Germany isolates. Sequence analysis of amino acid showed that the fragment encoded 352 amino acids with 14 potential antigenic plots uniformly distributed, and the hydrophobic and hydrophilic regions are also relatively uniform distribution. this result indicates that the VP2 gene of PPV-ZK strain has good prospects for vaccination.In this study, PPV PCR method was established for the detection of early phase and inapparent infection of PPV and its contamination to passage cell lines, which plays an important role in clinical diagnosis, control of porcine parvovirus disease and production of biological products. The cloning of VP2 structural protein gene, gene sequence analysis and amino acid sequence analysis and protein structure prediction lay the solid foundation for further study of the immunodiagnosis, molecular epidemiology, and vaccination and prevention, and also further molecular biology research of parvovirus.