Spiramycin and azithromycin, safe for administration to children, exert antiviral activity against enterovirus A71 in vitro and in vivo

•Some macrolide antibiotics possess antiviral activity against enterovirus.•Spiramycin inhibits EV-A71 replication through interfering with viral RNA replication.•The mutations of spiramycin-resistant EV-A71 variants were reproducibly mapped to VP1 and 2A.•Azithromycin significantly increased survival rate of mice severely infected with EV-A71. Hand-foot-mouth disease (HFMD) is a common viral disease in young children, mainly caused by enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16). Specific antiviral agents are not commercially available yet. Here we report that the macrolide antibiotics spiramycin (SPM) and azithromycin (AZM) possess antiviral activities against EV-A71 and CV-A16. SPM significantly reduced EV-A71 RNA and protein levels, most likely through interfering with viral RNA replication. The SPM-resistant EV-A71 variants showed similar resistance to AZM, indicating a similar anti-EV-A71 mechanism by which these two drugs exert their functions. The mutations of these variants were reproducibly mapped to VP1 and 2A, which were confirmed to confer resistance to SPM. Animal experiments showed that AZM possesses stronger anti-infection efficacy than SPM, greatly alleviated the disease symptoms and increased the survival rate in a mouse model severely infected with EV-A71. In all, our work suggests that AZM is a potential treatment option for EV-A71-induced HFMD, whose proved safety for infants and children makes it even more promising.