| Porcine parvovirus infection(PPI), called the pig breeding disorder disease,is caused a swine breeding disorder disease by porcine parvovirus(PPV). The only animal susceptibility is the pig with the characteristics of abortion, stillbirth, mummied foetus of pregnancy sow. The main characteristics is death of the embryo and fetus, especially the first-timer sows stillbirth, deformities and mummy foetus, but sows have no obvious symptoms by itself. This caused a great economic loss to the pig industry. Therefore, it is of great significance to control the PPI for pig industry.A PPV strain, named PPVGD was gained in this study through sample of positive PPV by the polymerase chain reaction( PCR) amplification being isolated in Swine testicular cells(ST cells). Cell cultures of PPVGD were evaluated by physical and chemical properties of solvent resistance, acid resistance, heat resistance and not sensitive to the pancreatic enzyme.These characteristics are consistent with PPV.This study have got 4251 bp genes include all the gene sequence encoding genes,according to PPV genome sequence published by the Gen Bank, primers designed and synthesized for PCR amplification of PPVGD genome and sequencing.We analyzed nucleotide, amino acid sequences of PPVGD and 44 NS1 and 55 VP2 gene sequences of PPV isolated, reference strains from Gen Bank, and draw the evolutionary tree. NS1 nucleotide sequence of PPVGD isolate has the highest similarity(99.9%) with Chinese isolates included HN-G, HN-H and HN-K, and has the lowest similarity(98.2%) with33760005-3a strain. VP2 nucleotide sequence of PPVGD isolate has the highest similarity(99.7%) with American isolates included NADL-2 and POVNADL-2, and has the lowest similarity(98.1%) with 15425 strain. NS1 amino acids sequence of PPVGD isolate has the highest similarity(99.7%)with Chinese isolates(HN-G, J-PPV, NJ and HN-K) and American isolates(NADL-2, POVNADL-2), and has the lowest similarity(98.0%) with33760005-3a strain. However, VP2 amino acids sequence of PPVGD isolate has the highest similarity(99.0%) with Chinese isolate HN-I and American isolates included NADL-2 and POVNADL-2, and has the lowest similarity(96.4%) with WB-773 ? 21620005-1h strains.PPVGD isolate was proliferated in swine testicular cells in this study. We gained a lot of high-titer virus after conditions of viral proliferation were optimized. This laid the foundation for the preparation of PPV vaccine. The results of Hemagglutination Assay(HA)showed that the PPVGD virus titers can reach up to1: 29. In strict accordance with China Biological Products, we prepared promising oil emulsion inactivated PPVGD vaccine by high-titer virus.Inactivated PPVGD vaccine from this study was inculated in about 350 g guinea pigs and about 4 months replacement gilts, respectively. Serum antibody titers was determined through HI test that blood came from guinea pigs of 28 d after immunization; Serum antibody titers was determined through HI test that blood was from replacement gilts of 14,28 and 42 d after immunization(it was time to reimmune replacement gilts on 28 d after immunization). Results showed that serum antibody levels of guinea pigs and replacement gilts 28 d after immunization of inactivated PPVGD vaccine were greater than 1:26, which proved inactivated PPVGD vaccine has good immunogenicity. And no adverse reaction occurred on any subject after the inactivated PPVGD vaccine immuned to the animals,which proved inactivated PPVGD vaccine has good security. The study provided with appropriate safety, laying solid foundation for further study of PPVGD vaccine. |
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