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Enterovirus 70 (EV70) is recognized as the main causative agent of acute hemorrhagic conjunctivitis (AHC), a highly contagious viral infection of the eye. Currently, there is no available treatment for EV70 infections. In this study, we developed a potential intervention strategy using RNA interference (RNAi) against EV70 infection in an in vitro system. Two synthetic 19-mer siRNAs, si-3D1 and si-3D2, were designed to target the 3Dpol region of the EV70 genome. Significant dosage dependent inhibition of EV70 in rhabdomyosarcoma cell line, as shown by reduction of viral RNA and VP1 production, was observed. Both siRNAs prevented EV70 replication in RD cells when transfected into these cells 48 hr prior to virus infection. Introduction of these siRNAs into RD cells 1-3 hr after infection with EV70 reduced production of viral RNA by approximately 60%. Thus, RNAi is a promising strategy to prevent EV70 infections and may have therapeutic potential.