Inhibition Of Proliferation Of Orf Virus By Small Interfeirng RNAs

Contagious ecthyma, also known as Orf, is an acute, highly contagious zoonosiscaused by orf virus. Because of the highly infectious and high incidence of thisdisease, it can cause great threat and huge economic losses for the world’s sheepcountry. At present there is no effective prophylactic vaccines of Orf, and theinoculated animals will be hurt in the process of vaccine inoculation. CBP of ORFVwhich caused the host immune response, block the production of acquired immunityis related to repeated infection of host. However, it is a host of pathogenic virulencefactor and immune regulatory factor. In the process of replication of ORFV, DNApolymerase is synthesised as a early protein, which has effects on catalytic synthesisof DNA and complementary activity, and plays an extremely important role in theprocess of viral replication. RNA interference （RNAi） is a kind of gene silencingphenomena induced by double-stranded RNA and have been widely used in virusdisease gene therapy and selection of drug target. Thus, in order to explore newmethod and thought for inhibiting viral replication of orfv, the ORFV CBP and DNApolymerase were chosed as the RNAi target genes for this study.The proliferation features of ORFV, which grew on sheep source of primaryOFTu cells, were analyzed by using indirect immunofluorescent assay (IFA), CPEanalysis, virus titration assay and real-time PCR assay to detect ORFV proteinexpression and virus genome DNA replication and assembly of matured virusparticles, respectively. The results showed that the cells appear primary focus at8hpost-infection, which were fully infected at72h post-infection.The virus genomeDNA synthesized rapidly at36h to48h. The degeneration of cells will be found, andthe pathological changes of the cells gradually were increased by CPE analysis alongwith the extension of time, and most cells were appeared in CPE at72h post-infection.The changes of the result of virus titration assay and Real time PCR assay are similarto each other.In this study the specific effect of RNAi on the replication of ORFV was explored. Three species of small interfering RNA (siRNA), targeting different regions of theCBP genes, were prepared by in vitro transcription. After transfection of OFTu cellswith each of the siRNAs followed by infection with ORFV, examination of CPEand IFA demonstrated that the three siRNAs were no capable of protecting cellsagainst ORFV invasion. In addition, transfection with siRNAs also no markedsuppressed the production of infectious virus as assessed by up to1.06-1.11-fold asassessed by TCID50and1.12-1.2-fold viral genome copy number. Furthermore, inorder to verify the specificity of siRNAs, CBP protein content was tested by westernblot. The results showed that siRNAs can effectively inhibit ORFV CBP proteinexpression, and suggested that siRNAs targeting the CBP gene were specificity,however the capability of them inhibiting ORFV proliferation effect was weak.At last three species of siRNAs, targeting different regions of the ORFVpolymerase genes, were prepared by in vitro transcription. After transfection of OFTucells with each of the siRNAs followed by infection with ORFV, examination ofcytopathic effects (CPE) demonstrated that the three siRNAs were capable ofprotecting cells against ORFV invasion with very high specificity and efficiency. Inaddition, viral proliferation within cells was examined by indirectimmunofluorescence microscopy. At48hours post-infection, only a fewsiRNA-treated cells were positive for viral antigen staining, whereas most untreatedvirus-infected cells were positive. Transfection with siRNAs also suppressed theproduction of infectious virus by up to59-199-fold as assessed by TCID50assay.Furthermore, treatment with siRNAs caused a84%，89%and73%reduction in viralgenome copy number as assessed by real time PCR, respectively. These resultssuggested that the three species of siRNAs can efficiently inhibit ORFV genomereplication and infectious virus production, but the effects were transient.Tocircumvent this problem, two siRNA expression plasmids (shR1and shR2) weregenerated to target two different coding regions of ORFV polymerase. The shRNAswere transiently transfected into OFTu cells, to determine whether these constructsinhibited ORFV production. Infected cells were evaluated for antiviral activity againstthe ORFV strain by indirect immunofluorescence microscopy (IFA), viral titrationand real-time PCR at48h post-infection. Transfection with siRNAs also suppressedthe production of infectious virus by up to58-207-fold as assessed by TCID50assay.And only a few siRNA-treated cells were positive for viral antigen staining, whereas most untreated virus-infected cells were positive. Furthermore, treatment withshRNAs caused a90.8%and76%reduction in viral genome copy number as assessedby real time PCR, respectively. Our results revealed that both shRNAs were highlycapable of inhibiting viral RNA genome replication.These results indicated that RNAi targeting of ORFV gene could facilitate studiesof the specific function of viral genes associated with ORFV replication and may havepotential therapeutic applications.