Inhibiting The Replication Of Canine Distemper Virus(CDV)in Vero Cells By SiRNA Targeting The Nucleocapsid Protein Gene And The Hemagglutinin Protein Gene

Canine distemper is an acute and highly contagious disease among canine caused by canine distemper virus. Animal protection and economical canine farming have been badly damaged due to the world-wide range, strong infectivity and high death rate of the virus. It additionally has the potential to endanger human. Precaution by vaccine is the major method for this disease, an effective therapy has been needing for long. What worse is vaccine has danger as well. The nucleocapsid protein is a helical protein which is out of RNA and is the most conservative region in CDV gene. The presence of the nucleocapsid protein ensure the integrality of viral RNA. There are 9 bps which can express T cell receptor in nucleocapsid protein gene. The hemagglutinin protein is a protein locate at the surface of bursa of canine distemper virus, which has primary responsibility to mediate virus bound to the host cells. Host cells getting infected is of vital importance for the whole process. Induced by the double-stranded RNA, RNAi is a kind of gene silencing phenomena widely applied to virus-caused disease gene therapy and selection of drug targeted gene. Therefore, to figure out new approach and think of inhibition viral replication of CDV, two kinds of protein constituting the virus named nucleocapsid and hemagglutinin were chosen as the RNAi targeted gene for this research.The proliferation feature of CDV affecting Vero cells was detected and analyzed by various kinds of approaches such as IFA, CPE, virus titration assay and real-time PCR assay which aims to inspect CDV protein expression, virus genome RNA replication and assembly of matured virus particles, individually. The parameters indicate that cells that were fully infected at 72 h post-infection appear main focus at 12 h post-infection. The whole process of the virus genome RNA synthesis accelerates from 36 h to 48 h. And the CPE analysis witnesses the cytopathy of Vero cells. The results of the different testing approaches mentioned above were in accordance with each other.Six pairs of RNAs(si RNA), named si N1、si N2、si N3 and si H1、si H2、si H3 and targeting nucleocapsid protein gene(N gene) and the hemagglutinin protein gene(H gene) respectively, were prepared by in vitro transcription. Then, each of them was transfected with vero cells taken from an African green monkey before infected with CDV. AT 48 hours post-infection, these infected cells were evaluated for their antiviral activities against the CDV strain through cytopathic effect(CPE), indirect immuno-fluorescence microscopy(IFA), TCID50, and realtime fluores-cence quantitative PCR. Results demonstrated that there was obviously less green fluorescent in the interfered si RNAs, compared with the control groups. The virus titers in the comparisons between the interfered groups and the control groups were 1/293, 1/11, 1/29 1/83, 1/302 and 1/90. The number of CDV genes copied in the infected groups was 12.96%, 57.75%, 23.47%, 31.82%, 12.82% and 15.58% of the control groups. As is seen from the above, the six pairs of si RNAs had different effects on inhibiting the copy of CDV in vero cells. si H2 and si N1 had the best effect among H gene targeted si RNAs and N gene targeted si RNAs respectively.In order to make the differences obvious,si H2 and si N1 were chosen in the experiments. Three groups had been designed: First, transfect si RNAs into cells before infect CDV; Second, transfect si RNAs into cells before infectious CDV appear; Finally, transfect si RNAs into cells after infectious CDV appear. The results would be detected by IFA assay, CPE assay, virus titration assay, Real-time PCR assay and western blot assay. Transfection with si RNAs before infection of CDV had better effect among there groups(si H2 and si N1),while other groups did not have remarkable difference compared with control group. Fluorescence of first group less than other groups. Similar result happened in IFA assay, CPE assay, virus titration assay, Real-time PCR assay and western blot assay.These results indicated that RNAi targeting of CDV gene could facilitate studies of the specific function of viral genes associated with CDV replication and may have potential to be new therapy for canine distemper...