Antiviral Effects of Novel 2-Benzoxyl-Phenylpyridine Derivatives

Figures

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  Figure 1. Antiviral activities of novel 2-benzoxyl-phenylpyridine derivatives against CVB3 and ADV7. (a) The compounds inhibited virus-induced cytopathic effects (CPEs). Hep-2 or HeLa cells were infected with 100 TCID50 of CVB3 or ADV7 for 1.5 h at 37 °C. Then, the inocula were aspirated and the cells were incubated with various concentrations of test compounds for 48 h. Cell viability was determined using the MTT assay. (b) Images showing the morphology of Hep-2 and HeLa cells treated with 120 μM W-9, 160 μM W-13, or 160 μM W-15 (magnification, 20×).

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  Figure 2. The effects of W9, W13, and W15 on progeny virus production. Hep-2 cells infected with 100 TCID50 of CVB3 (a), or HeLa cells infected with 100 TCID50 of ADV7(b), were incubated in the absence (virus control) or presence of 120 μM W-9, 160 μM W-13, 160 μM W-15, or 160 μM ribavirin for 36 h, and the culture media and cell lysates then harvested for virus titration. Viral titers are presented as Log10 TCID50/mL. * p < 0.05; ** p < 0.01, compared with the virus control group.

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  Figure 3. Analysis of the modes of action of W9, W13, and W15 against CVB3. (a) Analysis of effective stage. The antiviral effects of test compounds against CVB3 were detected by measuring cell viability after 48 h of infection when cells were treated with W9, W13, or W15 before, simultaneously, or after inoculation with CVB3 (100 TCID50). (b) Analysis of the effects on CVB3 adsorption. Mock-, 120 μM W-9-, 160 μM W-13-, 160 μM W-15-, or 160 μM ribavirin-treated CVB3 (106 TCID50) was inoculated onto Hep-2 cells and adsorbed for 2 h. Infected cells were then harvested and subjected to virus titration, using the TCID50 method. (c) The effects of test compounds on CVB3 release from Hep-2 cells. Hep-2 cells infected with 100 TCID50 of CVB3 were incubated with test compounds for 12 h, then cells and culture supernatants (intra- and extracellular) analyzed for virus yield, separately or together. Mock, no infection; VC, virus control. Values represent the means ± SDs of three independent experiments. * p < 0.05; ** p < 0.01, compared with the virus control group.

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  Figure 4. Time-of-addition assay. 120 μM W-9, or 160 μM W-13, W-15, or ribavirin, were added to Hep-2 cells for different time periods after CVB3 infection. Progeny virus yield was determined at 12 h post-infection ((−1)–0 h, viral infection period; 0–10 h, period of virus proliferation in cells). Values are presented as means ± SDs. * p < 0.05; ** p < 0.01, compared with the virus control group.

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  Figure 5. Effect of W13 on CVB3 replication in Hep-2 cells. Hep-2 cells infected with 100 TCID50 CVB3 were incubated in the absence or presence of 160 µM W13 and harvested at the indicated times post-infection. (a) Progeny virus yields were determined. (b) Viral RNA levels were measured by analysis of total RNA. GAPDH was amplified and the resulting data was used for normalization. Values represent the means ± SDs of three independent experiments. * p < 0.05; ** p < 0.01, compared with the virus control group. (c) CVB3 protein levels were determined by indirect immunofluorescence, using the mouse anti-coxsackie virus B3 monoclonal antibody, MAB948, and an alexa-fluor-488-conjugated affiniPure goat anti-mouse IgG (H + L), after incubation with W13 for 24 h. Nuclei were stained with DAPI and green foci indicate the presence of CVB3 protein.