Inhibition of Semliki Forest virus replication with long double-stranded RNA in Aedes albopictus cells

Arthropod-borne viruses represent an increasing threat to the global health system, requiring the development of novel and sustainable control strategies to reduce the risk of arboviral infections. RNA interference (RNAi) offers a potential approach to directly prevent viral replication within vectors due to its specificity in gene silencing. In this study, we evaluated the efficacy of long double-stranded RNAs (dsRNAs) targeting six regions of the Semliki Forest virus (SFV) genome in Aedes albopictus U4.4 cells. The antiviral efficiency of dsRNA alone is low, therefore we evaluated its use after complexing with the K4 Transfection System (K4). A cytotoxicity assay based on ATP quantification showed that both uncomplexed and complexed dsRNA had no cytotoxic effects on U4.4 cells at a concentration up to 2 ng/µL. Complexed dsRNA achieved higher antiviral efficacy, significantly reducing viral replication compared to uncomplexed dsRNA. We found that complexed dsRNA retained its antiviral activity when challenged with SFV up to 72 h post-transfection. Among our synthesized dsRNA constructs, nsP4-dsRNA in complex with K4 led to an 80 % reduction in viral replication at 72 h post-infection at 0.5 ng/µL. Using RT-qPCR, we confirmed a significant 32.2 % reduction of nsP4 mRNA after transfection of complexed nsP4-dsRNA. Dose response assays showed that complexed dsRNAs with a concentration of 0.5 ng/µL are effective for viral reduction. Our results highlight the importance of efficient dsRNA delivery and selection of critical viral targets, such as nsP4, for successful RNAi-mediated viral suppression. This work elucidates the potential of dsRNAs to target Semliki Forest virus replication, highlighting viral gene targeting as a viable strategy for RNAi-based suppression of arboviral replication.