Isolation And Identification Of Tembusu Virus SD/02Strain,Construction And Vaccination Test Of Tembusu Virus DNA Vaccine

From June to November2010, a disease characterized by a sudden onset was observed inmany egg-laying and breeder duck farms in China. The affected ducks manifest a clinicalsymptom with heavy egg drop. Hyperemia, hemorrhage, degeneration, distortion andlymphocyte infiltration in the ovaries and portal area interstitial inflammation in the liverswere the main pathological changes observed consistently in almost all diseased ducks. Basedon the isolation and identification, the new disease was found to be caused by Tembusu virus(TMUV). In the study, a strain virus (SD/02) was isolated and identified as TMUV. The DNAvaccines were successfully constructed via inserting the prM/E gene and CpG-ODN. Theexpression efficacy was examined by eukaryotic cell transfection and animal experiments.Our study demonstrated that the DNA vaccines could induce humoral and cell-mediateimmune response in ducks. Compared with TMUV oil-emulsion inactivated vaccines, DNAvaccines play a better protective effect on the disease. In conclusion, our study providesimportant experimental and useful data for further development of vaccines against TMUV.Part1: Isolation and identification of Tembusu virus SD/02strainA strain virus (SD/02) isolated from a suspected case of TMUV was identified as TMUVby RT-PCR identification, the sequencing analysis of prM/E gene and pathogenicity testanimal experimental infection. It was identified by RT-PCR, and the amplification productswere sequenced. It revealed that the amplification result is right, compared with the standardstrain, the gene of TMUV SD/02strain has the homology99%in nucleic acids level. The12-day Cherry Valley Pekin ducks are attacked by the SD/02strain, the incidence were100％,and the mortality were40%. Cherry Valley Pekin ducks in the pathogenicity test showed thesame or similar clinical symptoms and pathological changes as the natural infections. Toxicitytests showed that ELD50of the isolated virus was10-2.67/0.2mL, ICPI was1.238.Part2: Construction and vaccination test of Tembusu virus DNA vaccineSynthetic oligodeoxynucleotide carrying immunostimulatory CpG motifs were insertedinto the eukaryotic expression plasmid pVAX1.One pair of special primer were designed toamplify the prM/E gene of TMUV SD/02strain isolated from SHANDONG. And the prM/Egene were inserted into the pVAX1-CpG and controlled by the PCMV promoter to construct the recombined plasmid pVAX1-prM/E-CpG. pVAX1-prM/E-CpG were transfected into293Tcells by Lipofectamine2000Transfection Kit. Expressions of prM/E genes were detected byindirect immuno-fluorescence experiment, and the mRNA transmitted from prM/E gene wasdetected by RT-PCR. These experiments indicated that DNA vaccines work well in vitro,serving as a prerequisite for further animal experiments.DNA vaccines pVAX1-prM/E-CpG and TMUV oil-emulsion inactivated vaccine wereinjected into three-week Cherry Valley Peking ducks individually. The dose of DNA vaccinesis200μg per duck. Blood serums were taken once a week after immunization and theantibody were monitored by indirect ELISA method. The results showed that the antibodylevels of groups which immunized with pVAX1-prM/E-CpG and TMUV oil-emulsioninactivated vaccine were higher very significantly than that of the negative control(P<0.01).There is not denotes significant difference in antibody levels between the groupimmunized with pVAX1-prM/E-CpG and the group immunized with TMUV oil-emulsioninactivated vaccine (P>0.05).DNA vaccines pVAX1-prM/E-CpG and TMUV oil-emulsion inactivated vaccine wereinjected into four-day Cherry Valley Peking ducks individually. The dose of DNA vaccines is200μg per duck. The ducks were challenged by TMUV SD/02after8days fromimmunization. The mortality and relative protection rate, value changes of CD4﹢, CD8﹢,CD4﹢/CD8﹢T-lymphocyte subsets in the peripheral blood, blood biochemical indexes, thecontent of IFN-γ and IL-6in the serums among groups in different days post challenge weredetected respectively. Result showed that, compared with TMUV oil-emulsion inactivatedvaccine group, the relative protection rate of DNA vaccine group can increase42.8%. Theratio of CD4﹢in the peripheral blood increased significantly (P<0.01), the ratio of CD4﹢/CD8﹢subsets increased very significantly (P<0.01) compared with the group challenged byTMUV SD/02. Compared with oil-emulsion inactivated vaccine groups, levels of serum ALT,AST, UA of DNA vaccine groups were lower very significantly (P<0.01), levels of serum ALP,TP were higher very significantly (P<0.01), the content of IFN-γ increased very significantly(P<0.01) and the content of IL-6decreased very significantly (P<0.01).