Tropical Journal of Pharmaceutical Research

Original Research Article | OPEN ACCESS

Inhibition of Dengue Virus 3 in Mammalian Cell Culture by Synthetic Small Interfering RNAs Targeting Highly Conserved Sequences

Ummar Raheel , Najam Us Sahar Sadaf Zaidi

Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology (NUST), H-12 Sector, Islamabad, Pakistan;

For correspondence:- Ummar Raheel Email: ummarraheel@gmail.com Tel:+923325416727

Received: 28 June 2014 Accepted: 13 September 2014 Published: 19 October 2014

Citation: Raheel U, Zaidi NU. Inhibition of Dengue Virus 3 in Mammalian Cell Culture by Synthetic Small Interfering RNAs Targeting Highly Conserved Sequences. Trop J Pharm Res 2014; 13(10):1621-1627 doi: 10.4314/tjpr.v13i10.8

© 2014 The authors.
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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 analysis.

Results: 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 DV3UTR5'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