A rational multi-target RNA interference approach for effective suppression of Japanese encephalitis virus replication

Japanese encephalitis virus (JEV), a major cause of viral encephalitis in the Asia–Pacific region, remains a significant public health concern due to suboptimal vaccine coverage and the lack of approved antiviral therapies. RNA interference (RNAi) provides a sequence-specific approach for suppressing viral gene expression and represents a promising antiviral strategy. Here, we designed small interfering RNAs (siRNAs) targeting selected regions of essential JEV genes, including prM, NS1, NS3, and NS5, and evaluated their antiviral efficacy in HeLa cells. Individual siRNAs reduced viral protein expression and infectious virus production while maintaining more than 85% cell viability. A multi-target siRNA cocktail reduced viral replication to approximately 5–6% of the scrambled control level and preserved cell viability after JEV infection. Neither individual siRNAs nor the cocktail produced a detectable increase in protein kinase R phosphorylation relative to the scrambled control under the conditions tested. These findings support the feasibility of multi-target RNAi-mediated suppression of JEV replication in vitro and provide a basis for further evaluation of RNAi-based antiviral approaches against related flaviviruses.