Combinations of approved oral nucleoside analogues confer potent suppression of alphaviruses in vitro and in vivo

Alphaviruses, including chikungunya virus (CHIKV), pose a significant global health threat, yet specific antiviral therapies remain unavailable. We 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. In 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 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. Combining 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.