Tizoxanide Antiviral Activity on Dengue Virus Replication

Abstract

Dengue virus is an important circulating arbovirus in Brazil responsible for high morbidity and mortality worldwide, representing a huge economic and social burden, in addition to affecting public health. In this study, the biological activity, toxicity, and antiviral activity against dengue virus type 2 (DENV-2) of tizoxanide (TIZ) was evaluated in Vero cell culture. TIZ has a broad spectrum of action in inhibiting different pathogens, including bacteria, protozoa, and viruses. Cells were infected for 1 h with DENV-2 and then treated for 24 h with different concentrations of the drug. The quantification of viral production indicated the antiviral activity of TIZ. The protein profiles in infected Vero cells treated and not treated with TIZ were analyzed using the label-free quantitative proteomic approach. TIZ was able to inhibit virus replication mainly intracellularly after DENV-2 penetration and before the complete replication of the viral genome. Additionally, the study of the protein profile of infected not-treated and infected-treated Vero cells showed that TIZ interferes with cellular processes such as intracellular trafficking and vesicle-mediated transport and post-translational modifications when added after infection. Our results also point to the activation of immune response genes that would eventually lead to a decrease of DENV-2 production. TIZ is a promising therapeutic molecule for the treatment of DENV-2 infections.

Keywords: antiviral; dengue virus; tizoxanide; virucidal activity.

Figure 1

TIZ dose-response inhibition curve on DENV-2 replication in Vero cells. Different concentrations of TIZ were added to Vero cells for 72 h, right after viral infection. Then, the viral yield was titrated by plaque assay and the values were converted to %VI. The experiment shows the mean of three determinations of viral inhibition in plaque assay, compared to non-treated cell controls ± SD. The SD bars are obscured by the dot. Chemical structure of TIZ in the insert.

Figure 2

DENV-2 stock was treated with different concentrations of TIZ for 1 h at 37 °C for subsequent titration by plaque assay for virucidal test and viral release assays. The experiment shows the mean of three determinations of viral inhibition + SD in plaque assay: virus released in supernatant (SN), virus released from cell monolayer (ML), and virucidal activity.