Establishment Of RT-PCR Methods To Defect Bovine Viral Diarrhea Virus&Construction Of Recombinant Swinepox Virusws Expressing Protective Antigen Of Bvdv

Bovine viral diarrheavirus(BVDV), as well as classical swine fever virus(hog cholera virus) and border disease virus, is the member of Pestivirus within the family Flaviviridae. BVDV can cause digestive disease, respiratory disease and reproductive losses. Infection, disease, or both have been described in cattle, sheep, goats, pigs, bison, alpacas, llamas, and white-tailed deer, and among other species. Animals with virus and vaccines contaminated with BVDV are responsible for the BVDV prevalence in pig populations. It is reported that BVDV is similar to the CSFV in the clinical symptom and the pathological change when they infected the pigs. Co-infection is more popular and complicated than old days in integrated pig industry. The identifical diagnosis between BVDV and CSFV is more and more important for the diagnosis of co-infection and secondary infection, the detection of exogenous gene in hog cholera vaccines, and the analysis of the failure of the immunity of CSFV. Swinepox virus(SPV) is the sole member of the genus Suipoxvirus, belonging to the family Poxviridae. SPV infects only swine. Natural SPV infections are typically mild and occasionally accompanied by localized skin lesions that heal naturally. SPV is well suited for the development of recombinant vaccines, because of its biological and clinical safety, large packaging capacity for recombinant DNA, and its ability to induce solid protective immunity.1Establishment and application of RT-PCR methods to detect BVDV from swineBased on the sequence of5’untranslated region (5’UTR) of the BVDV genome, primer was designed and synthesized. By optimization of the reaction condition, RT-PCR assay for detection of bovine viral diarrhea virus from swine was established. The specific PCR products was186bp in length. The detection limit of cDNA template in the RT-PCR was10TCID50; The high specificity of the method was demonstrated by detecting CSFV、 PRRSV、PEDV、SPV and PCV. Among169clinical samples, there were20samples with BVDV positive, with11positive of124feces samples,4positive of tissues samples, and5 positive of vaccine samples. It proved that RT-PCR assay can be applied value in practices.2Construction of recombinant Swinepox viruses expressing protective antigen of BVDVGlycoprotein E2is the protective antigen of BVDV,with the dominant antigen determinants,induces protective inmmuological reaction. In this study, we constructed transfer vector pUSG11/P28E2based on vector pUSGll/P28. The non-coding region between the SPV20gene and the SPV21gene in the SPV genome was used as a novel insertion locus for generating recombinant SPV, and a dominant selection procedure based onGFP gene expression was developed for recombinant virus isolation. We used this system to produce the recombinant viruses rSPV-E2. The recombinant viruses were identified by PCR, Western blotting and immunofluorescence assays, and their replication were compared with the wild-type virus. The results demonstrated that E2gene expression from the recombinant virus was stable over ten passages.It suggested that SPV recombinants have the potential as BVDV vaccines for the use in pigs.