Study On ShRNA Interference, Wild/Vaccine Viruses Differentiation And Engineered Vaccine Of Classical Swine Fever

Classical swine fever (CSF) is a serious contagious disease of pig caused by classical swine fever virus (CSFV). CSFV is a single-strain positive RNAvirus, the 3′-nontranslation region(3′-NTR)plays an important role in virus replication. Three sites in 3′-NTR of CSFV shimen strain were selected by sequence analysis to study the function of 3′-NTR on virus replication. Three recombinant plasmids expressing the shRNAs target to 3′-NTR of CSFV shimen strain were constructed and tranfected into PK-15 cells. Three recombinant PK-15 cell strains targeting the 3′-NTR of CSFV were obtained. The interferences of CSFV replication were researched in the recombinant PK-15 cells infected with CSFV.Vaccination-based strategies are the main ways for prevention and control CSF at present. The riskes of virus variation and virulence increasing with the CSFV attenuated vaccine are widely used at present. Some people suspected largely to use attenuated vaccine are the one of reasons of more cases of atypical CSF are found in swine population. It needs to develop more new typic vaccines to control CSF. A suicide DNA vaccine of CSFV E2 gene and a recombinant fowlpox virus harboring E0 and E2 genes of CSFV were constructed (FPV-pSY-E0-E2) by analysis of the advances of CSFV enginerred vaccine researches. The preliminary immune effect of the constrcted vaccines were assessed in this study.Attenuated vaccines of CSF are widely used in the swine population in some countries. It is difficult to effectively differentiate the pigs or their products infected with CSFV wild virus or vaccine virus just by determining the CSF antibody in swine sera. This would has an influence on the trade of pigs and their products. A novel RT-PCR was developed for detecting and differentiating the wild and vaccine types of CSFV. The results that the specificity of developed RT-PCR was high and could be used to differentiate the CSFV infected pigs and vaccinated pigs.1. Three short hairpin RNAs (shRNAs) of single-stranded nucleotides target to the sequences at 115-132,137-154 and 210-228 of CSFV shimen strain 3′-NTR mRNA were designed according the sequences analysis respectively. The double-stranded shRNAs were formed by annealing and inserted into plasmid expressing vectors. Three recombinant plasmids expressing the shRNAs target to 3′-NTR of CSFV were obtained. There positive PK-15 cell lines target to 3′-NTR shRNA were developed by transfecting PK-15cells with recombinant plasmid respectively. The positive cells (P1, P2, P3) were infected with CSFV shimen virus respectively. The detection results showed that all of the three cell lines could interfere with proliferation of CSFV at transcriptional and protein level.2. RT-PCRs were developed for detecting and differentiating the wild CSFV from vaccine virus. A 187 bp PCR fragment response to CSFV Shimen strain and a 492 bp fragment response to vaccine virus were obntained using this PCR. No PCR products could be amplified in PCV2, TGEV and PRRSV, showed the designed primers were speicific to CSFV. The RT-PCRs can be used to detect and differentiate the wild from vaccine CSFV in the field.3. The suicidal vectored DNA vaccine (pSCA1-E2) was constructed by inserting E2 gene of CSFV into pSCA1 plasmid vector. E2 protein was determined in PK15 cells transfected with purified pSCA1-E2.The stimulation index (SI) of T lymphoid cell of mice were significant different between the experimental groups and the control group at10d (p<0.05), 20d and 30d (p<0.01) after the second immunization. The anti-CSFV serum antibodies could be screened in experimental pigs at 21d after the second immunization with pSCA1-E2.4. The E0 and E2 genes of CSFV as well as a LacZ reporter gene were all inserted into a non essential gene of fowlpox virus (FV282) by homologous recombinant and the recombinant fowlpox virus FV282-E0-E2 was constructed. The experimental mice and pigs immunized with FV282-E0-E2 could induce a high anti-CSFV antibody level, also showed that the E0 and E2 genes were expressed in experimental animals. Partial protective immunity was observed in the experimental pigs immunized with FV282-E0-E2 when they challenged with the highly virulence CSFV, 75% (6/8) immunoprotection was observed.