Inhibition Of Proliferation Of Classical Swine Fever Virus By Small Interfering RNAs

RNA interference (RNAi) refers to the phenomenon of post-translational silencingof gene expression that occurs in response to the introduction of double-stranded RNAinto a cell. This phenomenon can result in highly specific suppression of geneexpression, which is mediated by 21-to 23-nucleotide small interfering RNA (siRNA)that is homologous in sequence to the silenced gene. RNAi can shut down or regulatesgene expression in mammalian cells and confers as a cellular defense mechanismagainst gene invaders such as transposons and viruses. siRNA has showed its potentialantiviral abilities in cultured mammalian cells and animals, and has been used as anew powerful tool for gene-specific therapeutics for viral disease.Classical swine fever (CSF) is a highly contagious disease of pigs, which leads toimportant economic losses worldwide. The etiological agent of CSF is classical swinefever virus (CSFV), which is a member of the genus Pestivirus within the familyFlaviviridae. The current preventive and control measures of CSF epidemic arevaccine inoculation or slaughter the infected pigs. Although this has prevented thedisease efficiently, failure of immunization and persistent infection of CSFV occuroccasionally. Thus, the development of new antiviral methods is necessary in order toprevent the prevalence of CSF. Here we report that in vitro transcribed siRNAs by useof T7 RNA polymerase and a DNA vector-based RNAi technology specificallysuppresses CSFV replication in PK-15 cells and in rabbits.The features of CSFV, which grown on porcine kidney PK-15 cell lines, wereanalyzed by using indirect immunofluorescent assay (IFA) and real-time PCR assayand virus titration assay to detect CSFV protein expression and virus genome RNAreplication and assembly of matured virus particles, respectively. The results showedthat the cells appear primary focus at 8 h post-infection (p.i.), which were fullyinfected at 72 h p.i. The virus genome RNA synthesized rapidly p.i. , which reach itspeak at 72 h p.i. , while the TCID50 can be detected at 8 h p.i., which indicated theeclipse period of CSFV, and peaked at 48 h p.i..Based on the results mentioned above, PK-15 cells were transfected with CSFV-specific siRNAs, which were obtained by in vitro transcriptional methods, target onNpro and NS5B region of CSFV genome, named as siN1,siN2 , si5B1 and the controlsiRNA (siCtrl) were obtained by using T7 RNA polymerase and in vitro transcription.siN1,siN2 , si5B1 and siCtrl were transfected into PK-15 cells and infected with CSFVstrain of Shimen. IFA results showed that CSFV-siRNAs specifically suppress CSFVgene expression. Further more, the TCID50 of CSFV of CSFV-specific siRNAtransfected cell were significantly reduced, in contrast, the TCID50 were 1/468, 1/31.6and 1/56.2 of siN1,N2 and si5B1 less than that of control , respectively.34 CSFV-specific-shRNAs, which were expressed by using DNA-vector-basedRNAi technology, target on all genes of CSFV genome, and two control shRNAexpressing plasmids were constructed and obtained. All of these plasmids weretransfected into PK-15cell lines followed screening by antibiotics G-418 andHygyomycin B, respectively. The results demonstrated that CSFV-specific-shRNAsinhibited the syntheses of virus protein, which lead to deficiency of replication of virusgenome RNA, and assembly of virus particles, subsequently. Of all these...