Study On Inhibition Of Japanese Encephalitis Virus Replication By Small Interfering RNAs And Suicidal DNA Vaccine Of Japanese Encephalitis Virus

Japanese encephalitis virus (JEV), a serious mosquitoborne flavivirus, causes an acute infection of the central system resulting in encephalitis of humans and many kinds of animals. In humans, a high proportion of the survivors exhibit neurogical and psychiatric sequelae. JEV is also a veterinary problem, from which the pig industry suffered serious losses because of the reproduce failure in sows and death in piglets. JEV is a major public health problem facing the world today. With no specific and effective treatment available for Japanese encephalitis, new strategies are needed to control these diseases. RNA interference (RNAi) is a recently discovered ancient natural antiviral mechanism in plant and animal. To investigate new method for fighting JEV, the plasmid-derived siRNAs targeting non-structure gene (NS1) of JEV was constructed and the inhibition effect of these siRNAs was measured. In the present, vaccination is the most effective prophylactic method to prevent and control these diseases. However, there are problems in both inactivated and attenuated vaccines that are used currently. Based on the reasons mentioned above, suicidal DNA vaccine encoding the main immunogenic gene NS1 or E of JEV was constructed, the immunoadjuvant effect of BHV-1 VP22 was evaluated, and the immune efficacy of suicidal DNA vaccine and conventional DNA vaccine encoding NS1 or E protein of JEV were compared. The main projects are:1. Inhibition of JEV NS1 expression by siRNAsNS1 is one of important non-structural proteins, which was found to be associated with viral RNA replication. To inhibit NS1 expression, four small interfering RNAs (siRNAs) expression plasmids (pS-NS1A, pS-NS1B, pS-NS1C and pS-NS1D) were generated to target four different coding regions of the NS1 gene, and were separately co-transfected into Vero cells with NS1-EGFP fusion expression plasmid pNS1-EGFP. NS1 expression was evaluated by fluorescence microscope, flow cytometry assay, Western blot and RT-PCR. The results revealed that pS-NS1B, pS-NS1C and pS-NS1D could effectively and specifically inhibit NS1 expression in Vero cells. Among those effective siRNAs, the inhibition effects of pS-NS1C and pS-NS1D were 80%-90%o2. Inhibition of JEV replication by siRNAsThe effects of these siRNAs on the infection and replication of JEV were investigated with indirect immunofluorescence, plaque forming, Western blotting, and RT- PCR assays. The results of these experiments showed that three of four siRNAs (pS-NS1B, pS-NS1C and pS-NS1D) could effectively inhibit JEV RNA replication and protein expression in BHK-21 cell. Among those effective siRNAs, the inhibition effects of pS-NS1C and pS-NS1D were 79%-86%. Our study suggested RNAi would be a feasibility treatment for JEV infection.3.Comparison of immune efficacy of suicidal DNA vaccine and conventional DNA vaccine encoding NS1 or E protein of JEV in mice...