Suppression of Replication of Rabies Virus by Short Hairpin RNAs Expressed by Plasmid

Targeting the N gene of rabies virus (RV), four shRNA expression plasmids were designed and constructed based on the vector pRNATU6.3-Hygro that expresses fusion protein with GFP as a reporter gene. Four cell strains (N1, N2, N3, N4) expressing the short hairpin RNAs (shRNA) were obtained after the plasmids were transfected into BHK-21 cells and screened under the pressure of Hygromycin B (300 μg/mL). These cell strains were infected with 100× the TCID 50 of rabies virus CVS-11 strain, and the viral replication was quantified at 24, 48, 72 and 96 hours by directed immunofluorescence assay (DFA), real-time PCR, and the 50% tissue culture infective dose (TCID 50 ). The results showed variable inhibition of viral replication, with BHK-N2 being the most effective strain (99% inhibition). There was close correspondence between results using the three methods of evaluation. The shRNA-mediated inhibition persisted to at least 96 hours after infection. Effective inhibition of replication of RV in BHK-21 cells was achieved by siRNA targeting the N gene, with N 2 , aimed at the region starting at position 701 of the gene, being the most potent.

Targeting the N gene of rabies virus （RV）, four shRNA expression plasmids were designed and constructed based on the vector pRNATU6.3-Hygro that expresses fusion protein with GFP as a reporter gene. Four cell strains （NI, N2, N3, N4） expressing the short hairpin RNAs （shRNA） were obtained after the plasmids were transfected into BHK-21 cells and screened under the pressure of Hygromycin B （300 ~tg/mL）. These cell strains were infected with 100x the TCIDs0 of rabies virus CVS-11 strain, and the viral replication was quantified at 24, 48, 72 and 96 hours by directed immunofluoreseence assay （DFA）, real- time PCR, and the 50% tissue culture infective dose （TCIDs0）. The results showed variable inhibition of viral replication, with BHK-N2 being the most effective strain （99% inhibition）. There was close correspondence between results using the three methods of evaluation. The shRNA-mediated inhibition persisted to at least 96 hours after infection. Effective inhibition of replication of RV in BHK-21 cells was achieved by siRNA targeting the N gene, with N2, aimed at the region starting at position 701 of the gene, being the most potent.