broad-spectrum antiviral activity of rna interference against four genotypes of japanese encephalitis virus based on single microrna polycistrons广谱抗病毒活性rna干扰对四个基因型的基于单一microrna polycistrons日本脑炎病毒.pdf

Broad-Spectrum Antiviral Activity of RNA Interference against Four Genotypes of Japanese Encephalitis Virus Based on Single MicroRNA Polycistrons Zhiqiang Wu., Ying Xue., Bei Wang, Jiang Du, Qi Jin* State Key Laboratory for Molecular Virology and Genetic Engineering, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China Abstract Japanese encephalitis virus (JEV), a neurotropic mosquito-borne flavivirus, causes acute viral encephalitis and neurologic disease with a high fatality rate in humans and a range of animals. Small interfering RNA (siRNA) is a powerful antiviral agent able to inhibit JEV replication. However, the high rate of genetic variability between JEV strains (of four confirmed genotypes, genotypes I, II, III and IV) hampers the broad-spectrum application of siRNAs, and mutations within the targeted sequences could facilitate JEV escape from RNA interference (RNAi)-mediated antiviral therapy. To improve the broad- spectrum application of siRNAs and prevent the generation of escape mutants, multiple siRNAs targeting conserved viral sequences need to be combined. In this study, using a siRNA expression vector based on the miR-155 backbone and promoted by RNA polymerase II, we initially identified nine siRNAs targeting highly conserved regions of seven JEV genes among strains of the four genotypes of JEV to effectively block the replication of the JEV vaccine strain SA14-14-2. Then, we constructed single microRNA-like polycistrons to simultaneously express these effective siRNAs under a single RNA polymerase II promoter. Finally, these single siRNAs or multiple siRNAs from the microRNA-like polycistrons showed effective anti-virus activity in g