Abstract Influenza outbreaks are associated with substantial morbidity, mortality and economic burden. Next generation antivirals are needed to treat seasonal infections and prepare against zoonotic spillover of avian influenza viruses with pandemic potential. Having previously identified oral efficacy of the nucleoside analog 4’-Fluorouridine (4’-FlU, EIDD-2749) against SARS-CoV-2 and respiratory syncytial virus, we explored activity of the compound against seasonal and highly pathogenic influenza (HPAI) viruses in cell culture, human airway epithelium organoids, and/or two animal models, ferrets and mice, that assess IAV transmission and lethal viral pneumonia, respectively. 4’-FlU inhibited a panel of relevant influenza A and B viruses with nanomolar potency in organoids. In vitro polymerase assays revealed immediate chain termination of IAV polymerase after 4’-FlU incorporation, in contrast to delayed chain termination of SARS-CoV-2 and RSV polymerase. Once-daily oral treatment of ferrets with 2 mg/kg 4’-FlU initiated 12 hours after infection rapidly stopped virus shedding and prevented direct-contact transmission to untreated sentinels. Treatment of mice infected with a lethal inoculum of pandemic A/CA/07/2009 (H1N1)pdm09 (Ca09) with 2 mg/kg 4’-FlU alleviated pneumonia. Three doses mediated complete survival when treatment was initiated up to 60 hours after infection, indicating an unusually broad window for effective intervention. Therapeutic oral 4’-FlU ensured survival of animals infected with HPAI A/VN/12/2003 (H5N1) and of immunocompromised mice infected with pandemic Ca09. Recoverees were fully protected against homologous reinfection. This study defines the mechanistic foundation for high sensitivity of influenza viruses to 4’-FlU and supports 4’-FlU as developmental candidate for the treatment of seasonal and pandemic influenza. Author Summary Next-generation antiviral therapeutics are needed to better mitigate seasonal influenza and prepare against zoonotic virus spillover from animal reservoirs. At greatest risk are the immunocompromised and patients infected with highly pathogenic influenza viruses. In this study, we have demonstrated efficacy of a broad-spectrum nucleoside analog, 4’-fluorouridine, against a representative panel of influenza viruses in cell culture, human organoids, and two animal models, ferrets and mice. Acting as an immediate chain terminator of the influenza virus polymerase, once-daily oral treatment protected against lethal infection with seasonal and highly pathogenic avian influenza viruses, prevented direct-contact transmission to untreated sentinels, and mitigated lethal infection of immunocompromised hosts. These results support the developmental potential of 4’-fluorouridine for treatment of vulnerable patient groups and mitigation of pandemic influenza, providing a much-needed additional therapeutic option for improved disease management.

4’-Fluorouridine mitigates lethal infection with pandemic human and highly pathogenic avian influenza viruses