Oral 4-fluorouridine completely suppresses epidemic Oropouche virus replication in mice and confers protection against virus-induced disease.

Oropouche virus (OROV) is an orthobunyavirus that causes increasingly frequent and severe outbreaks in Central and South America. We report that 4-fluorouridine (4-FlU) inhibits the in vitro replication of epidemic and pre-epidemic OROV strains in multiple cell lines. 4-FlU is known to inhibit the replication of various RNA viruses by targeting -as its 5-triphospate- viral polymerases. Following 24 days of consecutive in vitro passages of OROV in the presence of suboptimal concentrations of 4-FlU, no viral variants with drug associated mutations were identified in the viral polymerase. Analysis of high-confidence structural models of the OROV polymerase in complex with a template-primer RNA and incoming nucleotides provides a structural rationale for the relatively high barrier to resistance. In a stringent, acute OROV-infection model in AG129 mice, oral administration of 4-FlU completely blocked viral replication and virus-induced disease, also when administration was delayed until 72 hours after infection. Our findings support exploring the potential of 4-FlU for the management of OROV infections in humans.