Remdesivir as a potent antiviral against prototype and current epidemic Oropouche virus strains (BeAn19991 and PE-IAM4637)

• We generated a recombinant reporter OROV that expresses the eGFP fluorescent protein in infected cells. • We found that remdesivir efficiently inhibited the replication of Oropouche virus (OROV) using this reporter OROV. • We demonstrated strain-dependent differences in the replication efficiency of OROV. The Oropouche virus (OROV), an orthobunyavirus transmitted by biting midges, is the causative agent of Oropouche fever, which has caused multiple outbreaks in South and Central America. During the most recent epidemic in 2023–2025, more than 25,000 laboratory-confirmed cases were reported in Brazil, and no licensed antivirals have been reported to be effective date. In this study, we generated a recombinant OROV-expressing enhanced green fluorescent protein (rOROV/GFP) to facilitate rapid and sensitive antiviral evaluation. Growth kinetics demonstrated that rOROV/GFP replicated less efficiently than wild-type rOROV and that the historical prototype strain (BeAn19991) exhibited higher replication efficiency than the recent epidemic isolate (PE-IAM4637) in both Vero E6 and Huh7 cells. Using this system, we evaluated the antiviral activity of ribavirin, favipiravir (T-705), and remdesivir against OROV. All three compounds inhibited OROV replication in a dose-dependent manner, with remdesivir showing the greatest potency (IC₅₀ values of 0.31 µM). Taken together, our findings highlight remdesivir as a promising candidate for the treatment of Oropouche fever caused by OROV. Furthermore, we established rOROV/GFP as a powerful tool for antiviral drug screening.