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Tropical Journal of Pharmaceutical Research
Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, Nigeria
ISSN: 1596-5996
EISSN: 1596-5996
Vol. 13, No. 10, 2014, pp. 1621-1627
Bioline Code: pr14223
Full paper language: English
Document type: Research Article
Document available free of charge

Tropical Journal of Pharmaceutical Research, Vol. 13, No. 10, 2014, pp. 1621-1627

 en Inhibition of Dengue Virus 3 in Mammalian Cell Culture by Synthetic Small Interfering RNAs Targeting Highly Conserved Sequences
Raheel, Ummar & Zaidi, Najam Us Sahar Sadaf


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.

Dengue virus; siRNA,; anti-E antibody; Conserve regions; Vero-81 cells

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