Optimize Of Propagation And Study On Stability Of Immunogencity Of Porcine Circovirus Type 2 SH Strain

Porcine circovirus(PCV) is one kind of smallest viruses we have known by now, which has two gene types,PCV1 and PCV2.PCV1 can be detected in most of PK15 cells.Porcine circovirus 2(PCV2) is the main causative agent of postweaning multisystemic wasting syndrome(PMWS),porcine dermatitis nephropathy syndrome (PDNS),reproductive failure and porcine respiratory disease complex(PRDC) etc.We called these PCVD and PCVD caused huge loss in many nations and regions in the world.Currently,cultivation of PCV2 virus in vitro has been carried out in PK15 in laboratories world-wide.However,the virus titers,expressed as TCID50,obtained from PK15 cell cultures were low.The 300 mM D-Glucosamine could be used to enhance the replication of PCV2 in PK15 cells,but it could harm to the cells.In order to acquire the virus with high titres,in this study,the concentration of D-Glucosamine in the cultures was optimized,and the titers of virus were detected with IPMA which reaction condition was selected.The results showed that maximum virus titers were acquired when PCV2-SH strain was cultured in PK15 cell with RMPI-1640 containing 1-2 mM D(+)-Glucosamine at 37℃for 72 h.Moreover,PCV2-SH strain was passaged serially until 50 passage in PK15 cells.The virus titres of F20 and F50 were all 10^6.25 TCID₅₀/ml which detected by IPMA.To study on the stability of immunogenicity of PCV2 SH strain,F20 and F50 passage viruses were inactivated with 0.2%formaldehyde under 37℃.The inactivation effect was estimated by PCR after passaged on PK-15 cell by three times. Then the inactivate vaccines were individually made with the F20 and F50 passage virus and the immunogenicities of the viruses were evaluated in pigs.Twenty-three healthy piglets were assigned to four groups(group 1,2,3 had six pigs and group 4 five),F20 vaccined,F50 vaccined,PCV2 challenged control and empty control group. Group 1 and Group 2 were vaccinated individually with 2 ml killed vaccine and boosted 3 weeks later.Twenty-one days later,Group 1,2 and 3 were all challenged with 3×10^6.25TCID₅₀ of PCV2-SH.The pigs were weighed at the days of challenge and the end of the experiment.All groups were isolated and killed at 35 days after PCV2 challeng.PCV2-specific antibody were detected at every week after the first vaccination by indirect ELISA.The efficacy of inactivated vaccines were evaluated by seroconversion,clinical signs(fever),gross lesions,growth parameters and viremia.The results showed that the higher ELISA antibody titers of two vaccines could be detected in piglets after second immunization.After PCV2 challenged,the vaccinated and control groups had no clinical signs,challenged groups had increased rectal temperature and showed growth retardation..Viremia were detected in 4 piglets of challenged groups after PCV2 challenge,but no viremia in all piglets of vaccinated groups at 21 days.Gross leision and histopathological findings in all piglets of challenged groups were more severe than that of vaccinated groups.It demonstrated that PCV2 strain had stable immunogencity,and could provid protetive efficity.The PCV2 SH strain might be an candidate virus for PCV2 inactivated vaccine.The study laid the foundation of the reseach in killed vaccine against the disease associated with PCV2.