In this study we have reported for the first time the in vivo detection of HEV replication using molecular beacons and inhibition of replication and translation of the Hepatitis E virus (HEV) genome by shorthair pin RNA (shRNAs). Methods Two molecular beacons complementary to the sequence of negative sense HEV RNA were designed using the software RNA structure and synthesized. A HepG2 stable cell line expressing RdRp-EGFP was transfected with the positive sense HEV transcripts (5679–7194 nt) to be detected along with each molecular beacon individually. Fluorescence was observed with confocal microscopy. Negative sense Firefly luciferase transcript transfected into HepG2 RdRp-EGFP stable cell line was used as non specific control for replication detection. For the RNA interference studies shRNAs targeted against Helicase, Replicase and 3’end region of HEV positive sense RNA were designed using the software siRNA converter, synthesized and cloned into pSilencer U6 1.0. The target consisted of the full length HEV replicon fused to Renilla luciferase (pSGHEV-rLuc). Firefly luciferase DNA (pCDNA3 fluc) was used for normalization of tranfection efficiency. shRNA, pSGHEV-rLuc and pCDNA3Fluc were transiently transfected into HepG2 cell line and inhibition was recorded in the form of relative decrease in renilla luciferase luminescence w.r.t. the positive control that lacked shRNAs. Results: Fluorescence characteristic of molecular beacon fluorophore was observed in Replicase-EGFP expressing cell line transiently transfected with HEV transcript (5679–7194 nt) No fluorescence was observed in case of the non specific control. In case of shRNAs designed against the HEV genome 30–70% relative decrease in Renilla luciferase luminescence was observed which indicates a decrease in the translatable positive sense HEV RNA. Since this positive sense RNA is the precursor for the negative sense replication intermediate its implies inhibition of HEV replication. Thus we have shown
