Antiviral activity of four types of bioflavonoid against dengue virus type-2

Abstract

Background: Dengue is a major mosquito-borne disease currently with no effective antiviral or vaccine available. Effort to find antivirals for it has focused on bioflavonoids, a plant-derived polyphenolic compounds with many potential health benefits. In the present study, antiviral activity of four types of bioflavonoid against dengue virus type -2 (DENV-2) in Vero cell was evaluated. Anti-dengue activity of these compounds was determined at different stages of DENV-2 infection and replication cycle. DENV replication was measured by Foci Forming Unit Reduction Assay (FFURA) and quantitative RT-PCR. Selectivity Index value (SI) was determined as the ratio of cytotoxic concentration 50 (CC50) to inhibitory concentration 50 (IC50) for each compound.

Results: The half maximal inhibitory concentration (IC50) of quercetin against dengue virus was 35.7 μg mL-1 when it was used after virus adsorption to the cells. The IC50 decreased to 28.9 μg mL-1 when the cells were treated continuously for 5 h before virus infection and up to 4 days post-infection. The SI values for quercetin were 7.07 and 8.74 μg mL-1, respectively, the highest compared to all bioflavonoids studied. Naringin only exhibited anti-adsorption effects against DENV-2 with IC50 = 168.2 μg mL-1 and its related SI was 1.3. Daidzein showed a weak anti-dengue activity with IC50 = 142.6 μg mL-1 when the DENV-2 infected cells were treated after virus adsorption. The SI value for this compound was 1.03. Hesperetin did not exhibit any antiviral activity against DENV-2. The findings obtained from Foci Forming Unit Reduction Assay (FFURA) were corroborated by findings of the qRT-PCR assays. Quercetin and daidzein (50 μg mL-1) reduced DENV-2 RNA levels by 67% and 25%, respectively. There was no significant inhibition of DENV-2 RNA levels with naringin and hesperetin.

Conclusion: Results from the study suggest that only quercetin demonstrated significant anti-DENV-2 inhibitory activities. Other bioflavonoids, including daidzein, naringin and hesperetin showed minimal to no significant inhibition of DENV-2 virus replication. These findings, together with those previously reported suggest that select group of bioflavonoids including quercetin and fisetin, exhibited significant inhibitory activities against dengue virus. This group of flavonoids, flavonol, could be investigated further to discover the common mechanisms of inhibition of dengue virus replication.

Figure 1

Cytotoxicity of flavonoids against Vero cells. MTT assay was performed on Vero cells after 96 h treatment with increasing concentrations of the flavonoids. Results are presented as percentage of cell viability from triplicate assays.

Figure 2

Anti-viral assay of flavonoids against DENV-2 intracellular replication. Foci forming unit reduction assay (FFURA) was used to evaluate the in vitro anti-dengue virus activities of flavonoids after viral adsorption (a) and the respective DENV-2 RNA copies were quantified using qRT-PCR (b). The percentages of foci reduction (% RF) were calculated relative to the untreated controls maintained in parallel. Data from triplicate assays were plotted using Graph Pad Prism Version 5 (Graph Pad Software Inc., San Diego, CA. USA).

Figure 3

Anti-viral effects of continuous treatment with flavonoids against DENV-2 replication. Foci forming unit reduction assay (FFURA) was used to evaluate the in vitro anti-dengue virus activities of the flavonoids. Cells were treated with the flavonoids at 5 h before infection and continuously treated with fresh flavonoids upto 4 days post infection (a). The respective DENV-2 RNA copy numbers were quantified using qRT-PCR (b). The percentages of foci reduction (% RF) were obtained by comparing against untreated controls maintained in parallel. Data from triplicate experiments were plotted using Graph Pad Prism Version 5 (Graph Pad Software Inc., San Diego, CA).

Figure 4

Anti-adsorption activity of flavonoids against DENV-2. Flavonoids were added directly to virus inocula for 2 h at 37 C. The inocula were used to infect Vero cell monolayers in 24 wells cell culture microplates. The reduction in foci forming unit was calculated relative to the controls maintained in parallel (a) and the respective DENV-2 RNA copy numbers were quantified using qRT-PCR (b). Data from triplicate experiments were plotted using Graph Pad Prism Version 5 (Graph Pad Software Inc., San Diego, CA).

Figure 5

Foci size reduction in DENV-infected cell treated with quercetin. Foci forming unit reduction assay (FFURA) was used to evaluate the in vitro anti-dengue virus activity of quercetin after viral adsorption. Foci of the non-treated infected cells (a) compared with foci in cells treated with quercetin (50 μg/ml) postadsorption (b). Arrows indicate foci which are more diffused, less intensely stained and smaller than the foci in non-treated cells.