Chikungunya disease results from an infection with the arbovirus, chikungunya virus (CHIKV). Symptoms of CHIKV include fever and persistent, severe arthritis. In recent years, several antiviral drugs have been evaluated in clinical trials; however, no registered antivirals have been approved for clinical therapy. In this study, we established a high-throughput screening (HTS) system based on CHIKV 26S mediated insect cell fusion inhibition assay. Our screening system was able to search potential anti-CHIKV drugs in vitro. Using this system, four compounds (niclosamide, nitazoxanide, niflumic acid, tolfenamic acid) were identified. These compounds were then further analyzed using a microneutralization assay. We determined that niclosamide and nitazoxanide exhibit ability to against CHIKV-induced CPE. The anti-CHIKV abilities of these compounds were further confirmed by RT-qPCR and IFA. Moreover, niclosamide and nitazoxanide were found to (1) limit virus entry, (2) inhibit both viral release and cell-to-cell transmission, and (3) possess broad anti-alphavius activities, including against two clinical CHIKV isolates and two alphaviruses: Sindbis virus (SINV) and Semliki forest virus (SFV). In conclusion, our findings suggested that niclosamide and nitazoxanide were able to inhibit CHIKV entry and transmission, which might provide a basis for the development of novel human drug therapies against CHIKV and other alphavirus infections.

The mosquito-borne Chikungunya virus (CHIKV) is a profound global threat due to its high rate of contagion and the lack of vaccine or effective treatment. Suramin is a symmetric polyanionic naphthylurea that is widely used in the clinical treatment of parasite infections. Numerous studies have reported the broad antiviral activities of suramin; however, inhibition effects against CHIKV have not yet been demonstrated. The aim of this study was thus to investigate the antiviral effect of suramin on CHIKV infection and to elucidate the molecular mechanism underlying inhibition using plaque reduction assay, RT-qPCR, western blot analysis, and plaque assay. Microneutralization assay was used to determine the EC50 of suramin in the CHIKV-S27 strain as well as in three other clinical strains (0611aTw, 0810bTw and 0706aTw). Time-of-addition was used to reveal the anti-CHIKV mechanism of suramin. We also evaluated anti-CHIKV activity with regard to viral entry, virus release, and cell-to-cell transmission. Cytopathic effect, viral RNA, viral protein, and the virus yield of CHIKV infection were shown to diminish in the presence of suramin in a dose-dependent manner. Suramin was also shown the inhibitory activities of the three clinical isolates. Suramin inhibited the early progression of CHIKV infection, due perhaps to interference with virus fusion and binding, which subsequently prevented viral entry. Results of a molecular docking simulation indicate that suramin may embed within the cavity of the E1/E2 heterodimer to interfere with their function. Suramin was also shown to reduce viral release and cell-to-cell transmission of CHIKV. In conclusion, Suramin shows considerable potential as a novel anti-CHIKV agent targeting viral entry, extracellular transmission, and cell-to-cell transmission.

BackgroundChikungunya fever is a pandemic disease caused by the mosquito-borne Chikungunya virus (CHIKV). E1 glycoprotein mediation of viral membrane fusion during CHIKV infection is a crucial step in the release of viral genome into the host cytoplasm for replication. How the E1 structure determines membrane fusion and whether other CHIKV structural proteins participate in E1 fusion activity remain largely unexplored.MethodsA bicistronic baculovirus expression system to produce recombinant baculoviruses for cell-based assay was used. Sf21 insect cells infected by recombinant baculoviruses bearing wild type or single-amino-acid substitution of CHIKV E1 and EGFP (enhanced green fluorescence protein) were employed to investigate the roles of four E1 amino acid residues (G91, V178, A226, and H230) in membrane fusion activity.ResultsWestern blot analysis revealed that the E1 expression level and surface features in wild type and mutant substituted cells were similar. However, cell fusion assay found that those cells infected by CHIKV E1-H230A mutant baculovirus showed little fusion activity, and those bearing CHIKV E1-G91D mutant completely lost the ability to induce cell-cell fusion. Cells infected by recombinant baculoviruses of CHIKV E1-A226V and E1-V178A mutants exhibited the same membrane fusion capability as wild type. Although the E1 expression level of cells bearing monomeric-E1-based constructs (expressing E1 only) was greater than that of cells bearing 26S-based constructs (expressing all structural proteins), the sizes of syncytial cells induced by infection of baculoviruses containing 26S-based constructs were larger than those from infections having monomeric-E1 constructs, suggesting that other viral structure proteins participate or regulate E1 fusion activity. Furthermore, membrane fusion in cells infected by baculovirus bearing the A226V mutation constructs exhibited increased cholesterol-dependences and lower pH thresholds. Cells bearing the V178A mutation exhibited a slight decrease in cholesterol-dependence and a higher-pH threshold for fusion.ConclusionsCells expressing amino acid substitutions of conserved protein E1 residues of E1-G91 and E1-H230 lost most of the CHIKV E1-mediated membrane fusion activity. Cells expressing mutations of less-conserved amino acids, E1-V178A and E1-A226V, retained membrane fusion activity to levels similar to those expressing wild type E1, but their fusion properties of pH threshold and cholesterol dependence were slightly altered.

The chikungunya virus (CHIKV), an arthritogenic alphavirus, has caused explosive epidemics involving millions of cases. Globally expanding pandemics involving CHIKV and post-CHIKV rheumatic disorders are increasing public health concerns. However, no antiviral interventions or vaccines to control CHIKV infection have yet been approved. Although suramin has been possess anti-CHIKV activity in vitro, whether suramin has anti-CHIKV activity in vivo remains unknown. This study aimed to determine whether suramin treatment could ameliorate CHIKV-induced arthritis in a C57BL/6 mice model. C57BL/6 mice were infected with CHIKVs to evaluate anti-CHIKV activities of suramin in terms of histopathology, viral burden and disease score. Not only did suramin treatment substantially decrease viral loads, but it also significantly ameliorated acute foot lesions in mice. In addition, suramin treatment markedly restores cartilage integrity and reduces the number of IHC positive chondrocyte in mice infected with CHIKV strains 0810bTw and 0706aTw. This in vivo study highlights the potential ability of suramin to treat CHIKV infection in clinical settings.