Antiviral Effects Of Single And Double SiRNA Expressing Vector Against Coxsackievirus B4 RNA

There are so many hypotheses to interpret the etiology of theinsulin dependent diabetes mellitus(IDDM),such as autoimmunity,some medicines, virus infections and genetic factors. Virus infections,especially the destruct of β -cells induced by Coxsackieviralinfections is one important factor. So many epidemical, basic andclinical experimental study show CVB is the important pathogen ofIDDM. CVB induce the destruction of the β-cell by direct cytocidaleffect, inflammation response and autoimmunity caused by mimicry.Now effective measurements to prevent and treat Coxsackieviralinfection are still not available. There are some searches forcontrolling Coxsackieviral myocarditis by RNA interference.We construct the single siRNA expressing vector with U6promoter and GFP marker targeting to P1A, P2A, P2B, and P3D geneof Coxsackievirus B4, and named them as pGCsi-U6/Neo/C1A,pGCsi-U6/Neo/C2A,pGCsi-U6/Neo/C2B,pGCsi-U6/Neo/C3D-2,and transfect them into Hela cells, respectively. The CoxsackievirusB4 was infected after different transfection time as 5h, 24h, 48h and72h. Then the inhibition effects were test by cytopathogenic effect,the TCID50 and the expressing level of viral mRNA and proteins. It isshow that the single expressing vector pGCsi-U6/Neo/C1A,pGCsi-U6/Neo/C2A and pGCsi-U6/Neo/C2B can inhibit thereplication of Coxsackievirus B4 effectively. And the inhibition effectof single siRNA expressing vector is strongest after 24h transfection.We construct the double siRNA expressing vector pU6/d-siRNA /neo/GFP/C1A-2A targeting to P1A and P2A gene ofCoxsackievirus B4, it can effectively inhibit the replication ofCoxsackievirus B4 infected after 24h siRNA expressing vectortransfection. And the single and double siRNA expressing vector canoccur more than 15d in cell line.Coxsackievirus B4 was injected into mice abdominal cavityafter 24h double siRNA expressing vector injection by tail veins. Allof experimental and control mice were sacrificed after 3 days, 1 week,3 weeks, 6 weeks and 8 weeks after viral infection. The weight, theblood glucose level were tested and the pancreas were taken forhistological test. The results show that the mean weights of doublesiRNA expressing vector protecting groups are close to those ofnormal control groups, and the mean weight of control double siRNAexpressing vector transfecting groups are lower than that of normalcontrol groups. The blood glucose level of double siRNA expressingvector protecting group are higher than normal control after 72 hoursto 1 weeks viral infection, and it return to the level close to that ofnormal control, but the blood glucose lever of control vector group islower than that of normal control after 72 hours infection, and thenthey are persistently higher than normal about 1~2 mmol/L after 1week, 3 weeks, 6 weeks and 8 weeks after viral infection. Thehistological tests of pancreas also show that the viral antigen level ofdouble siRNA expressing vector protecting groups are lower than thatof no protecting control vector groups. And the pathogenic change ofdouble vector protecting group are severer than that of no protectingcontrol vector groups.So we draw following conclusions: 1) the single and doublesiRNA expressing vectors we constructed can inhibit the replicationof Coxsackievirus B4 in vitro, and both of them can occur more than15 days in cell line;2) the inhibition effect of double siRNAexpressing vector are stronger than that of single siRNA expressingvector;3) the double siRNA expressing vector can alleviate thesymptom or inhibit the happening of IDDM of mice induced byCoxsakievirus infection.The innovations of this experiment are summarized as follows:constructing four single siRNA expressing vector and testing theprotecting of them to Coxsackievirus B4 infection, and constructingthe double siRNA expressing vectors to inhibit Coxsackievirus B4replication in vitro and testing the protection of it in IDDM miceinduced by Coxsackievirus B4 for the first time, and the doublesiRNA expressing vector can alleviate symptom of IDDM mice orblock the happening of IDDM.