Combinations of approved oral nucleoside analogues confer potent suppression of alphaviruses in vitro and in vivo
BackgroundAlphaviruses, including chikungunya virus (CHIKV), pose a significant global health threat, yet specific antiviral therapies remain unavailable. MethodsWe evaluated combinations of three oral directly acting antiviral drugs (sofosbuvir (SOF), molnupiravir (MPV), and favipiravir (FAV)), which are approved for other indications, against CHIKV, Semliki Forest virus (SFV), Sindbis virus (SINV), and Venezuelan Equine Encephalitis virus (VEEV) in vitro and in vivo. We assessed antiviral efficacy in human skin fibroblasts and liver cells, as well as in a mouse model of CHIKV-induced arthritis. FindingsIn human skin fibroblasts, synergistic antiviral effects were observed for combinations of MPV + SOF and FAV + SOF against CHIKV, and for FAV + SOF against SFV. In human liver cells, FAV + MPV conferred additive to synergistic activity against VEEV and SINV, while SOF synergized with FAV against SINV. In mice, MPV improved CHIKV-induced foot swelling and reduced systemic infectious virus titres. Combination treatment with MPV and SOF significantly reduced swelling and infectious virus titres compared to monotherapies of each drug. Sequencing of CHIKV RNA from joint tissue revealed that MPV caused dose- dependent increases in mutations in the CHIKV genome. Upon combination therapy of MPV with SOF, the number of mutations was significantly lower compared to monotherapy with several higher doses of MPV. InterpretationCombining these approved oral nucleoside analogues confers potent suppression of multiple alphaviruses in vitro and in vivo with enhanced control of viral genetic evolution in face of antiviral pressure. These drug combinations may ultimately lead to the development of potent combinations of pan-family alphavirus inhibitors. FundingThis work was supported by a PhD fellowship granted to S.V. by the Research Foundation - Flanders (FWO) (11D5923N). L.D.C. was also supported by Research Foundation - Flanders (FWO) PhD fellowship (11L1325N). Dr. Polyak and Schiffer are partially supported by R01AI121129. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSAlphaviruses such as chikungunya virus (CHIKV), Sindbis virus (SINV), and Venezuelan Equine Encephalitis virus (VEEV) are a major threat for global health. Alphaviruses are responsible for debilitating diseases with major public health implications, yet no antiviral drugs are currently approved for treating these virus infections. Existing treatment options are limited to supportive care and are unlikely to provide protection against future outbreaks of other alphaviruses. Previous studies have shown that oral approved nucleoside analogues such as favipiravir (FAV), molnupiravir (MPV), and sofosbuvir (SOF) have antiviral activity against certain RNA viruses, including alphaviruses. However, systematic in vivo evaluations of these drugs and testing of drug combinations in both in vitro and in vivo settings are limited. Added value of this studyThis study provides a comprehensive evaluation of combinations of FAV, MPV and SOF against multiple alphaviruses in two human cell lines and a CHIKV mouse model. We demonstrate that certain combinations of these drugs confer synergistic antiviral activity, effectively suppressing CHIKV, SFV, SINV, and VEEV replication in vitro. Moreover, in vivo, we show for the first time that MPV treatment results in reduced CHIKV-induced foot swelling and systemic virus replication. Combining MPV with SOF enhances antiviral activity in mice as compared to monotherapy. By sequencing the viral genome, we show that MPV increases the number of mutations in a dose-dependent manner. Combination therapy of MPV and SOF reduces the number of mutations compared to higher doses of MPV. These findings highlight the potential of nucleoside analogue combinations as a promising therapeutic strategy against alphavirus infections. Implications of all the available evidenceThe results of this study suggest that combination therapy with approved nucleoside analogues could provide an effective treatment strategy for alphavirus infections. The observed increased efficacy of drug combinations supports the potential for dose optimization to enhance efficacy while reducing toxicity and development of resistance. Future research should focus on clinical evaluation of these drug combinations, pharmacokinetic studies, and further exploration of their impact on viral evolution. Given the expanding geographical distribution of alphaviruses and the lack of available treatments, these findings provide a foundation for developing pan-alphavirus antiviral therapies that could improve patient outcomes and global outbreak preparedness.