Abstract
Purpose: To evaluate the inhibition of Dengue virus 3 by synthetic siRNAs targeting the untranslated regions UTR and structural regions of DENV3 genome in Vero-81 cell line.Methods: Vero-81 cells transfected with synthetic siRNAs were challenged by DENV3. The effectiveness of siRNAs was confirmed by four established virus quantification procedures. Starting with focus assay, DENV3 was quantified using anti-E antibody (Envelope), in which DENV3 was quantified by counting the number of foci per well. Initial results were then confirmed by immuno-florescence assay (IFA) as the number of Vero-81 cells displaying DENV3 (Envelope) E antigen had a higher florescent intensity in comparison to cells lacking DENV3 replication . DENV3 RNA copy numbers were quantified by real-time quantative polymerase chain reaction RT-qPCR and in the final step supernatant of Vero-81 cells challenged with DENV3 was collected and protein analysis was performed to determine the presence of DENV3 E protein via western blot analysisResults: A marked decrease in virus titer of DENV3 in Vero cells was observed with DV3UTR3'siRNA2 targeting the 3'UTR. Focus assay data revealed more than 70 % reduction in DENV3 in Vero-81 cells treated with DV3UTR3'siRNA2. Images showing IFA of infected Vero-81 cells exhibited a major drop in DENV3 titer in the presence of DV3UTR3'siRNA2 and DV3UTR5'siRNA1. DENV3 RNA, quantified by qPCR, DV3UTR3'siRNA2 showed 80 % reduction in DENV3 RNA level in comparsion with positive control cells having higher titers of DENV3. Finally, a negligible level of DENV3 E protein was detected in the supernatant of Vero-81 cells containing DV3UTR3'siRNA2. These findings suggest that DV3UTR3'siRNA2 and DV3UTR5'siRNA1 can significantly inhibit DENV3 in mammalian cell line.Conclusion: Overall, the results demonstrate that DV3UTR3'siRNA2 and V3UTR5'siRNA1 can become a potential vital component of a therapeutic formulation for major anti-dengue therapy against DENV3.Keywords: Dengue virus, siRNA, anti-E antibody, Conserve regions, Vero-81 cells
Highlights
Dengue virus (DENV) belongs to the genus Flavivirus and are named arboviruses, denoting their transmission via mosquitos
Vero-81 cells that were transfected with siRNA were challenged with DENV3
Positive control wells had Vero-81 cells transfected with control siRNA that was not related with DENV, while the negative control well was without any virus infection
Summary
Dengue virus (DENV) belongs to the genus Flavivirus and are named arboviruses, denoting their transmission via mosquitos. DENV transmission involves two vectors Aedes aegypti and Aedes albopictus. As per the recent valuation, approximately 50 to 100 million people are infected with DENV annually and 0.5 million progress to DHF/DSS [1]. DENV has a strand of positive sense RNA with the genome size of 11kb. DENV genome encodes a poly-protein, which is later cleaved into ten proteins [2]. DENV has four distinct serotypes (DENV1-4), which are genetically similar yet diverse on the basis of antigenic variation. DENV serotypes are further divided into genotypes on the basis of 3 % variations within individual serotype [3]
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