Characterization Of Porcine Parvovirus A PK-15Cell-adapted Strain In Different Cells And The Preparation Of Inactivated Vaccine

Porcine parvovirus (PPV) is one major etiological agent of reproductive failure in pigs and inoculating the inactivated vaccine is the important countermeasure for the prevention and control of PPV infection. To study the proliferation characteristics of PPV, acquire porcine parvovirus PK-15cell culture-adapted strain and screen inactivated vaccine according to the standard Chinese Veterinary Pharmacopoeia2010edition and The Veterinary Biological Products Quality Standards of the People’s Republic of China2001edition. A virulent strain of porcine parvovirus(PPV) was isolated from the liver, kidney and mesenteric lymph node of the abortion piglet using PK-15cell, the virus was blindly passaged to cytopathic effect(CPE) and acquired porcine parvovirus PK-15cell culture-adapted strain PPV-SC-L. According to the NS1gene of PPV, a pair of specific primers was designed, and Real-time fluorescent quantitative PCR (FQ-PCR) was introduced to quantify the PPV. The FQ-PCR was used to detect the viral content infected by synchronization asynchronous, drawing One-step growth curves; detected the viral content in different cells at the same time. According to the standard established local virus species and inactivation by formalin through24hours, preparation inactivated vaccine with the adjuvant of white oil, alumina gel and propolis and Screened the best inactivated vaccine through the tests of immune efficiency.The result showed that PPV strain produced CPE after12generations, and the CPE appeared after infected40h, the CPE rised to80%at56h, successfully acquired a PK-15cell culture-adapted strain of PPV, Successfully established a Real-Time FQ-PCR method after various optimization. The one-step growth curve showed that the virus content of synchronous inoculation was higher than the asynchronous inoculation, and the cell cycle of synchronous inoculation was shorter. The PPV cultured in different cells showed that the ranking of time of CPE appeared was PK-15, ST, Hela and MDBK, Ranking of virus content from high to low was ST, PK-15, MDBK and Hela. But the strain couldn’t proliferate in F81, BHK-21and Marc-145cell. The tests of virus species showed that bacteria, fungus, mycoplasma and exogenous virus were negative, the content of viral come up to the standard. Examination of inactivated vaccine showed that it was purity and had a good security to guinea pigs and piglets. The tests of immune efficiency showed that the white oil had the best efficacy of immunity, hemagglutination inhibition (HI) reached1:1024, then was the adjuvant of alumina gel, and without any clinical response after injected virulent strain. The adjuvant of propolis induced low levels of antibodies, piglets could not protected against virulent strain. Research has revealed that the pattern of proliferation was different when PPV infected in one cell by different ways. The time of appeared CPE and the content of virus are different when PPV proliferate in different cells, but the length of CPE time is irrelevant with the level of viral titer. PPV-SC-L strain inactivation by formalin and preparation inactivated vaccine with white oil could defense against PPV virulent strain infection, it could be develop a novel vaccine candidate of Porcine Parvovirus (PPV).