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

Oral 4’-fluorouridine completely suppresses epidemic Oropouche virus replication in mice and confers protection against virus-induced disease | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var 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Hendrickx , Kim Donckers , Thayara Morais Portal , Bert Vanmechelen , Joana Rocha-Pereira , Concetta Castilletti , Peter Mombaerts , Stéphane Bressanelli , Manon Laporte , Johan Neyts doi: https://doi.org/10.1101/2025.09.22.677733 Martin Ferrié 1 KU Leuven, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, Virology, Antiviral Drug and Vaccine Research Group , Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: martin.ferrie{at}kuleuven.be johan.neyts{at}kuleuven.be Maïlis Damuzey 1 KU Leuven, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, Virology, Antiviral Drug and Vaccine Research Group , Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Thibault Tubiana 2 Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay , CEA, CNRS, Gif-sur-Yvette, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mona Khan 3 Max Planck Research Unit for Neurogenetics , Frankfurt, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tania Roskams 4 KU Leuven, Department of Imaging and Pathology, Division of Translational Cell and Tissue Research , Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Birgit Weynand 4 KU Leuven, Department of Imaging and Pathology, Division of Translational Cell and Tissue Research , Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Niels Cremers 1 KU Leuven, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, Virology, Antiviral Drug and Vaccine Research Group , Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stijn Hendrickx 1 KU Leuven, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, Virology, Antiviral Drug and Vaccine Research Group , Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kim Donckers 1 KU Leuven, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, Virology, Antiviral Drug and Vaccine Research Group , Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Thayara Morais Portal 1 KU Leuven, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, Virology, Antiviral Drug and Vaccine Research Group , Leuven, Belgium 5 KU Leuven, VirusBank Platform , Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bert Vanmechelen 1 KU Leuven, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, Virology, Antiviral Drug and Vaccine Research Group , Leuven, Belgium 5 KU Leuven, VirusBank Platform , Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Joana Rocha-Pereira 6 KU Leuven, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, Virus-Host Interactions & Therapeutic Approaches Research Group , Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Concetta Castilletti 7 Department of Infectious-Tropical Diseases and Microbiology, IRCCS Sacro Cuore Don Calabria Hospital , Negrar Di Valpolicella, Verona, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Peter Mombaerts 3 Max Planck Research Unit for Neurogenetics , Frankfurt, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stéphane Bressanelli 2 Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay , CEA, CNRS, Gif-sur-Yvette, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Manon Laporte 1 KU Leuven, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, Virology, Antiviral Drug and Vaccine Research Group , Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Johan Neyts 1 KU Leuven, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, Virology, Antiviral Drug and Vaccine Research Group , Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: martin.ferrie{at}kuleuven.be johan.neyts{at}kuleuven.be Abstract Full Text Info/History Metrics Preview PDF ABSTRACT 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. INTRODUCTION The Oropouche virus (OROV, Orthobunyavirus genus, Peribunyaviridae family) is an arthropod-borne virus, transmitted mainly by the midge Culicoides paraensis ; and was isolated from humans for the first time in 1955 in the Oropouche region of the Caribbean island Trinidad 1 . OROV has emerged as a significant arboviral threat across Central/South America and caused recurrent outbreaks, with cumulative case numbers estimated in the hundreds of thousands 2 . Strikingly, Brazil has experienced a sharp rise in Oropouche fever cases, with >8,000 confirmed cases by August 2024, representing a substantial increase from the 831 cases reported in 2023 3 . Recently, OROV infections have soared in regions where the virus was not previously detected, including in Cuba and French Guyana 4 . The virus has also reached non-endemic areas such as North America and Europe through travelers 2 , 5 – 7 . OROV-associated symptoms in humans are rather vague (characterized by fever, headache, asthenia and myalgia), and are often mimicking dengue-like or Zika-like syndromes, making it difficult to assess the real burden of OROV 8 . Unlike previous outbreaks, which involved only asymptomatic or mild cases of OROV infection in humans, the 2024 outbreak in Central and South America, marked the first time that human fatalities were reported, with two deaths linked to the virus 9 . In July 2024, the Pan American Health Organization issued an epidemiological alert about the potential of vertical transmission of OROV 10 . In December 2024, Brazil reported three confirmed cases of vertical OROV transmission—resulting in stillbirths or congenital malformation 3 , 11 , 12 . Given the swift geographic expansion of OROV, its potential to cause severe clinical outcomes (deaths and vertical transmission) and the toll the current epidemic is already taking on healthcare systems, a strong and integrated global response, including medical countermeasures, is urgently needed 13 . The OROV genome consists of three segments of negative-sense RNA, called Small (S; encoding the nucleocapsid N and non-structural Ns protein), Medium (M; encoding the surface glycoproteins Gn/Gc and the non-structural protein NSm) and Large (L; encoding the RNA-dependent RNA-polymerase) 2 . The segmented nature of the OROV genome enables reassortment events, a process commonly observed among bunyaviruses 14 . Reassortment events have played a critical role in the emergence of current epidemic strains, resulting from the reassortment of the M segment from virus circulating between 2009 and 2018 in the eastern Amazon, and the L and S segments from viruses identified between 2008 and 2021 in Peru, Colombia and Ecuador 15 , 16 . These recent genetic exchanges, between different viral lineages, are considered a driving force behind the recent outbreaks and the expanding geographic range of OROV. Despite the geographical expansion of OROV and its heightened virulence in humans there are no antiviral drugs for the clinical management of OROV infections. Treatment remains supportive, and is limited to relieving symptoms and preventing complications. The broader-spectrum RNA virus inhibitor ribavirin lacks antiviral activity against OROV 17 . Human and mouse interferon-α are active in vitro and in vivo (prophylactic dosing), respectively, but lose their protective effects in a mouse model, when treatment is initiated after infection 18 . This underscores the pressing need for an effective and safe drug for the treatment of OROV infection. We therefore explored whether (i) a panel of drugs with known broader-spectrum activity against a number of RNA viruses and (ii) baloxavir [an influenza endonuclease inhibitor that exerts modest in vitro antiviral activity against Bunyamwera virus (another Orthobunyavirus ) 19 ] can inhibit the replication of the OROV both of an epidemic clinical isolate (OROV-IRCCS) 20 and a pre-epidemic reference OROV strain isolated in the 1960’s (OROV-TR9760). Only 4’-FlU [a molecule reported to exert antiviral activity against a number of viruses such as respiratory syncytial virus (RSV), Influenza A virus (IAV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Lassa virus (LASV), Nipah virus (NiV) and Chikungunya virus (CHIKV) 21 – 24 ] was found to markedly inhibit OROV replication. These results promoted us to study its efficacy in a mouse infection model for which we used the 2024 epidemic clinical isolate OROV-IRCCS. We demonstrate that oral administration of 4’-FlU completely blocked viral replication and conferred protection against virus-induced disease. RESULTS 4’-FlU is a potent inhibitor of OROV replication in cell culture We explored whether (i) a panel of molecules with reported antiviral activity against multiple RNA viruses [favipiravir, GS-441524 (the parent nucleoside of remdesivir), obeldesivir (an oral prodrug form of GS-441524), ribavirin, molnupiravir, galidesivir, sofosbuvir and 4′-Fluorouridine (4′FlU)], as well as (ii) baloxavir [an influenza endonuclease inhibitor with modest antiviral activity against Bunyamwera virus 19 ], can inhibit the in vitro replication of an epidemic clinical isolate (OROV-IRCCS; obtained from a patient who returned from Cuba to Italy in July 2024) 20 and a pre-epidemic reference OROV strain (isolated in the 1960’s; OROV-TR9760) in VeroE6, A549 and JEG3 cells ( Supplementary Fig. 1 ). None of the molecules, except for 4’FlU, resulted in a notable antiviral effect. 4’-FlU inhibited OROV-induced cytopathic effects (CPE) formation [EC₅₀ values ranging from 0.012 µM (VeroE6) to 0.048 µM (JEG3)] and reduced viral RNA levels in culture supernatant [EC₉₀ values of 0.17 µM (A549) and 0.24 µM (JEG-3), Fig. 1b ]. No detectable cytotoxicity was noted at the highest concentration tested (50µM) ( Fig. 1a ). Download figure Open in new tab Figure 1. Antiviral activity of 4’ FIU against OROV in cell culture. (a) 4’FIU inhibits the replication of OROV in three cell lines. Dose–response curves of the antiviral activity of 4’-FIU determined by MTS-based CPE reduction assay. Cytotoxicity was determined by MTS cell viability assay in mock-infected cells. Data points are the mean ± SD of n = 4 biologically independent experiments. (b) OROV viral load reduction in human A549 and JEG-3 cells based on the quantification of viral RNA in the supernatant by RT-qPCR 3 dpi. Data points are mean ± SD of n=3 biologically independent experiments. An OROV-IRCCS infection model in AG129 mice To assess whether the potent in vitro antiviral effect of 4’-FlU translates to efficacy in an OROV animal infection model, we first aimed to establish such a model using the epidemic strain OROV-IRCCS ( Supplementary Fig. 2 ). AG129 mice (which lack functional type I and type II IFN receptors) are known to be susceptible to infection with pre-2024 epidemic OROV strains 25 . In a pilot study, AG129 mice were subcutaneously inoculated in the footpad with 10⁵ TCID₅₀ of OROV-IRCCS. Infected mice developed a severe acute disease characterized by reduced activity, weakness, progressive weight loss and a decline in surface body temperature. Clinical deterioration progressed quickly, with some mice reaching humane endpoints as early as 3 days post-infection (dpi) and a cumulative lethality of 80% at day 7, the study endpoint ( Supplementary Fig. 2b-e ). At euthanasia, viral RNA and infectious virus titers were quantified in serum and in brain, lungs, liver and spleen ( Supplementary Fig. 2f-g ). High levels of viral RNA were detected in infected mice [mean of 2.16×10 10 RNA copies/mL in serum; 3.91×10 8 RNA copies/mg brain; 2.10×10 10 RNA copies/mg lung, 4.59×10 10 RNA copies/mg liver and 7.49×10 9 RNA copies/mg spleen] ( Supplementary Fig. 2f ). Likewise, infectious titers were very high [4.05×10 8 TCID 50 /mL serum, 2.32×10 6 TCID 50 /mg brain; 4.47×10 7 TCID50/mg lung; 4.41×10 9 TCID 50 /mg liver and 1.06×10 9 TCID50/mg spleen] ( Supplementary Fig. 2G ). Given the high level of viral replication, AG129 mice can be considered as highly susceptible to OROV-IRCCS, and thus represent a robust small-animal infection model to assess interventions against OROV. 4’-FlU protects AG129 mice against OROV-IRCCS infection, also with delayed treatment To assess the in vivo antiviral activity of 4’-FlU against OROV-IRCCS, AG129 mice were randomly assigned into five groups (n = 5 per group) and administered orally once daily, starting at 2h before infection and then for 7 consecutive days, with either vehicle or 4’-FlU at doses of 10 mg/kg, 5 mg/kg, 2 mg/kg, or 0.4 mg/kg. ( Fig. 2a ). The once-daily oral dosing schedule was inspired by the reported pharmacokinetic profile in mice with sustained plasma concentrations for over 12h after dosing 24 . Mice in the vehicle group exhibited progressive weight loss, a decline in surface body temperature and clinical signs including reduced activity and weakness, leading to the onset of humane endpoints beginning as soon as 3 dpi. All mice within this group were euthanized or dead by 6 dpi ( Fig. 2b, c ; Supplementary Fig. 3 ). In stark contrast, none of the treated mice -regardless of the dose received-showed clinical evidence of disease, except for some weight loss in the 2 mg/kg and 0.4 mg/kg groups ( Fig. 2b , 2c; Supplementary Fig. 3 ). At the endpoint (7 dpi), viral RNA in serum and organs from 4’-FlU– treated mice was generally undetectable or remained below the lower limit of quantification (LLOQ) of the RT-qPCR assay ( Fig. 2d ). In the brain, viral RNA levels were typically reduced by 5 to 6 log10; in some animals low levels of viral RNA were still detectable. In lung, liver and spleen, even at a dose as low as 0,4 mg/kg, viral RNA levels were reduced by 6 to 9 log 10 . Notably, no infectious virus could be recovered from serum or organs of any treated mouse, regardless of the dose ( Fig. 2e ), suggesting that the residual viral RNA detected in some of the treated mice likely represents non-infectious or degraded material. Download figure Open in new tab Figure 2. 4’-FlU protects against OROV-IRCCS infection in AG129 mice when dosed prophylactically. (a) Experimental design scheme. AG129 mice (n=5 per group) were infected subcutaneously in the left rear footpad with 10 5 TCID 50 of epidemic OROV-IRCCS strain and treated orally once daily with 4’-FlU (doses ranging from 10 mg/kg to 0.4 mg/kg) starting from 2h prior infection, and during 7 days. Mice were closely monitored until the study endpoint (7 dpi) or until reaching humane endpoints according to a clinical scoresheet. Uninfected mice (n=4) were included as controls. (b) Kaplan-Meier survival curves display the percentage of survival for each experimental group. Significance was calculated by log-rank (Mantel-Cox) test. (c) Weight change of the different experimental groups at time of euthanasia as a percentage, normalized to the body weight at the time of infection. Datapoints are mean ± SD. Significance was calculated by individual Mann-Whitney tests. (d and e) Viral RNA loads (d) or infectious titers (e) in serum and various organs measured at the time of euthanasia (humane endpoints or study endpoint) by RT-qPCR and cytopathic effects observation, respectively. Individual animals are presented as dots and error bars represent the SD. Significance was calculated by individual Mann-Whitney tests for non-normally distributed data and by Welch tests for normally distributed data (viral RNA-lung-0.4 mg/kg). Only statistically significant differences are shown. ND, not determined; LLOQ, lower limit of quantification. Histological analysis was performed on brain, lung, and liver sections collected at the time of euthanasia. In the infected vehicle-treated mice, clear pathological changes were noted, including intra-alveolar hemorrhage in the lungs and extensive coagulative necrosis in the liver ( Fig. 3a ). No pathological changes were detected in the brain ( Supplementary Fig. 4 ). In treated mice, lung and liver architecture appeared preserved across all dose groups. Gross examination of liver tissue from mice in the vehicle group animals revealed pronounced dark coloration, consistent with extensive necrosis. By contrast, livers from treated mice, regardless of the dose, appeared overall normal and comparable to those of uninfected controls ( Supplementary Fig. 5a ). Histopathological findings were corroborated by RNAscope in situ hybridization ( Fig. 3b ) The RNAscope probe was custom-designed to target the genomic strand of the viral nucleoprotein (NP) gene. The confocal images revealed abundant viral RNA localized within the brain, lung, and liver of mice in the vehicle group. In marked contrast, no viral RNA was detected in any organ of mice in the treated groups ( Fig. 3b ; Supplementary Fig. 6 ). Download figure Open in new tab Figure 3. 4’-FlU protects against OROV-induced disease when dosed prophylactically. (a) Representative hematoxylin and eosin-stained images of mice lungs and livers obtained at the time of euthanasia (HEP or study endpoint). Blue and green arrowheads indicate intra-alveolar hemorrhage (lungs) and coagulative necrosis (liver), respectively. Scale bar = 50 µm. (b) Confocal images of 5 µm sections through brains, lungs and liver samples, processed for in situ hybridization using the RNAscope platform, with a probe for the OROV S segment. DAPI serves as a nuclear stain. Scale bar = 20 µm. Next, the antiviral efficacy of 4’-FlU (10 mg/kg, orally, once daily) in OROV-IRCCS infected AG129 mice was also assessed in a therapeutic set-up ( Fig. 4a ). The start of treatment was delayed until 24, 48, or 72h post-infection (hpi) and continued daily until the study endpoint at 7 dpi ( Fig. 4a ). All mice in the vehicle group had died or needed to be euthanized by 5 dpi ( Fig. 4b ). In contrast, all treated mice, regardless of the timepoint at which treatment was initiated, survived until the study endpoint ( Fig. 4b ). All mice, also those in which treatment was initiated as late as 72 hpi, remained clinically healthy throughout the study, maintaining body weight, normal surface temperature without observable signs of disease ( Fig. 4c , Fig. S7 ). In mice in which treatment was initiated at 24 or 48 hpi, viral RNA levels in serum and organs were mostly either undetectable or below the lower limit of quantification (LLOQ). Even when treatment was started as late as 72 hpi, viral RNA levels at endpoint were 1 log 10 (brain) to 7 log 10 (liver) lower than in the vehicle group and no infectious virus was recovered from serum or organs ( Fig. 4e ). In stark contrast to the mice in the vehicle group, lung and liver architecture remained preserved across all treatment groups ( Fig. 5b ). Confocal imaging after RNAscope in situ hybridization, revealed abundant viral RNA localized within the brain, lung and liver of mice in the vehicle group, but no viral RNA in organs from mice in treated groups, with the exception of a sparse and punctate signal observed in the liver of mice in the 72 hpi group ( Fig. 5b ; Supplementary Fig. 8 ). Residual foci of genomic viral RNA, not associated to infectious material, likely persist in the liver of these mice. Taken together, these results demonstrate that oral administration of 4’-FlU completely blocked viral replication and conferred protection against virus-induced disease, also when dosing was initiated as late as 72 hpi. Download figure Open in new tab Figure 4. Therapeutic efficacy of 4’FlU against OROV-IRCCS in AG129 mice. (a) Experimental design scheme. AG129 mice (n=5 per group, except for the 72h group where n=4 due to one mouse reaching humane endpoints prior to treatment initiation and exclusion from the study) were infected subcutaneously in the left rear footpad with 10 5 TCID 50 of epidemic OROV-IRCCS strain and treated orally once daily with 4’-FlU (10 mg/kg) starting either at 24h, 48h or 72h post-infection and during 7 days. Mice were closely monitored until the study endpoint (7 dpi) or until reaching humane endpoints according to a clinical scoresheet. (b) Kaplan-Meier survival curves display the percentage of survival for each experimental group. Significance was calculated by log-rank (Mantel-Cox) test. (c) Weight change of the different experimental groups at time of euthanasia as a percentage, normalized to the body weight at the time of infection. Data points are mean ± SD. Significance was calculated by individual Mann-Whitney tests. (d and e) Viral RNA loads (d) or infectious titers (e) in serum and various organs measured at the time of euthanasia (HEP or study endpoint) by RT-qPCR and cytopathic effects observation, respectively. Individual animals are presented as dots and error bars represent the SD. The red triangle dataset corresponds to the prophylactic 10mg/kg dataset from Figure 2 and is shown here only for visual comparison. Significance was calculated by individual Mann-Whitney tests for non-normally distributed data and by Welch tests for normally distributed data (viral RNA-spleen-72hpi). Only statistically significant differences are shown. ND, not determined; LLOQ, lower limit of quantification. Download figure Open in new tab Figure 5. 4’-FlU protects against OROV-induced disease when dosed therapeutically. (a) Representative hematoxylin and eosin-stained images of mice lungs and livers obtained at the time of euthanasia (HEP or study endpoint). Blue and green arrowheads indicate intra-alveolar hemorrhage (lungs) and coagulative necrosis (liver), respectively. Scale bar = 50 µm. (b) Confocal images of 5 µm sections through brains, lungs and liver samples, processed for in situ hybridization using the RNAscope platform, with a probe for the OROV S segment. DAPI serves as a nuclear stain. Scale bar = 20 µm. 4’-FlU treatment is characterized by a high barrier to the emergence of resistance As its 5’-triphosphate form, 4’-FlU has been reported to target the viral polymerase 22 – 24 . Ideally, an antiviral drug should have a high barrier-to-resistance. Therefore, we attempted to select for 4’-FlU resistant OROV variant(s) in vitro by serially passaging OROV-IRCCS in the presence of suboptimal concentrations of the drug. After 24 days of passaging, the compound concentration could be increased 10-fold as compared to the starting concentration at passage 0, allowing for cytopathogenic effect formation. Whole viral genome sequencing from culture medium, was done using an in-house nanopore sequencing pipeline, did not reveal changes in the viral genome, which suggests a relatively high barrier to resistance. To elucidate the structural basis for this, we leveraged recent advances in protein structure prediction capable of atomic-level accuracy 26 , including for biomolecular complexes 27 . No experimental structures are currently available for the OROV L polymerase, but extensive structural data exist for related bunyavirus L proteins, particularly from the closely related La Crosse virus (LACV) 28 , providing a valuable framework for comparative analysis. We generated high-confidence structural models of the OROV L polymerase in complex with a template-primer RNA and incoming nucleotides, including the active triphosphate form of 4’-FlU (4’-FlU-TP). The models exhibited favorable quality metrics and closely matched the experimentally determined structures of LACV L protein ( Supplementary Fig. 9a and 9b ). The predicted domains architecture recapitulated the canonical organization: an N-terminal PA-like domain, a central PB1-like domain containing the catalytic core, and a C-terminal PB2-like domain 28 ( Fig. 6a ). Download figure Open in new tab Figure 6. Structural insights into 4’-FlU/OROV-IRCCS polymerase interaction. (a) Overall view of an AlphaFold 3 prediction of OROV-IRCCS polymerase in complex with a template/primer duplex of RNA (cartoon representation), two Mg 2+ ions (green spheres) and ATP (sticks). From N- to C-terminus the successive domains are PA-like (light pink), PB1-like (polymerase core, cyan) and PB2-like (light green). The tunnel through which shuttle incoming nucleotides (NTP) and analogs is indicated. (b) Magnification of the catalytic site of OROV-IRCCS polymerase complex with 4’-FlU-TP predicted with Boltz-2, with the same color code as in a . The three major contacts of the 4’-fluor are with conserved residues (displayed as sticks and labeled) and are indicated with green dotted lines. Focusing on the nucleotide-binding site within the PB1-like domain, our models closely aligned with the catalytic site architecture observed in LACV L polymerase, including conserved nucleotide positioning ( Supplementary Fig. 9a and 9b , right panels). The fluorine moiety of 4’-FlU-TP was accommodated within a defined pocket formed by residues W1051, N1136, and S1173 ( Fig. 6b ), with low predicted aligned errors (PAE) ranging from 2.3 Å to 2.8 Å, suggesting a stable interaction. In contrast, computational substitution of 4’-FlU-TP with the active triphosphate form of sofosbuvir, which lacked antiviral activity in our in vitro assays ( Supplementary Fig. 1 ), resulted in poor fitting within the active site. The model showed markedly higher PAE values (3.9 Å to 6.5 Å) for key interactions involving W1051, N1136, S1052, and W1130, with notable steric clashes involving the two tryptophan residues ( Supplementary Fig. 9c ). Taken together, our structural modeling reveals that a defined pocket within the OROV L polymerase active site can accommodate a 4′-fluoro substitution on the ribose ring of an incoming nucleotide, in place of the native 4′-hydrogen. This pocket appears well-suited to incorporate 4′-FlU-TP in direct competition with UTP. Incorporation of 4′-FlU-TP is predicted to disrupt RNA synthesis via chain termination or RNA corruption, consistent with known mechanisms of action for ribose-modified nucleotide analogs 29 . The three residues making favorable contacts with the 4‘-Fluor ( Fig. 6a ) are an integral part of the active site, notably positioning canonical motifs A and C that are involved in shuttling nucleotides and magnesium cofactors to close the active site of viral RNA-dependent RNA polymerases 30 , 31 . These observations provide a rationale for the observed relatively high genetic barrier to resistance of 4’-FlU-TP. DISCUSSION OROV has been circulating for decades in Central and South America, causing recurrent sporadic outbreaks with asymptomatic to mild symptomatic cases. The 2024 OROV outbreak was unprecedented by its magnitude as well as by the severe clinical outcomes. For the first time, human deaths associated with OROV infection were reported and the vertical transmission of OROV was identified. OROV has now considerable epidemic potential in Latin America and beyond 32 . There are no vaccines or antivirals drugs for the prevention or management of OROV infections 33 and care is limited to managing the symptoms. In an effort to identify potential antiviral drugs that can be repurposed for the treatment of infections with OROV, we evaluated a selection of molecules with reported activity against other RNA viruses (obeldesivir, galidesivir, GS-441524, sofosbuvir, ribavirin, favipiravir and molnupiravir). Since OROV encodes, alike influenza viruses, an endonuclease, we explored also whether baloxavir, a molecule with reported modest antiviral activity against Bunyamwera virus 19 , can inhibit the replication of OROV. Only 4’FlU resulted in noticeable activity against in vitro viral replication. 4’-FlU (also known as EIDD-2749) is a potent broader-spectrum ribonucleoside analog in phase 1 studies, with reported antiviral activity against several RNA viruses [including RSV, SARS-CoV-2, IAV, LASV, NiV and CHIKV 21 – 24 ]. To assess whether the potent in vitro activity against OROV also translates in protection in an animal infection model, we developed a robust mouse infection model. The interferon (IFN) response has previously been identified as a key determinant of host control over OROV replication 34 . AG129 mice, which carry knockouts of type I and II IFN receptor genes, are highly susceptible to multiple viral infections 35 – 37 . We here observed that these mice are highly susceptible to the epidemic strain OROV-IRCCS. Infected mice develop a severe acute disease that progressed rapidly, characterized by reduced activity, weakness, progressive weight loss and a decline in surface body temperature, with some mice reaching humane endpoints as early as 3 dpi. Prophylactic administration of 4’-FlU, even at a very low dose (0.4 mg/kg/day), conferred complete protection against virus-induced disease, accompanied by preservation of the tissue architecture and clearance of infectious virus. The potent antiviral activity was corroborated by a reduction of viral RNA synthesis in organs, as shown by RNAscope in situ hybridization staining. Remarkably, such observations were also made when the treatment was initiated as late as 72 hpi, a time at which several infected untreated mice already showed signs of disease. Notably, one mouse in the study already reached humane endpoints at 3 dpi. Thus, 4’-FlU is sufficiently potent to curb an aggressive ongoing infection. These results represent the first demonstration of antiviral efficacy by a clinical-stage compound in an animal model of OROV infection. Antiviral drugs have ideally a high genetic barrier-to-resistance. We passaged OROV-IRCCS for more than 3 consecutive weeks in vitro in the presence of suboptimal concentrations of 4’-FlU and did not identify adaptive mutations. Structural modeling of the OROV L polymerase revealed a conserved binding pocket accommodating 4’-FlU-triphosphate within the active site, rationalizing its selective incorporation and antiviral mechanism. Residues forming contacts with the fluorine moiety of 4′-FlU-TP, the active form of 4’-FlU, are conserved in bunyaviruses and across a broader range of negative-sense RNA viruses, including influenza viruses 24 . This conservation supports the extended antiviral activity of 4′-FlU-TP, well beyond the bunyavirus group, and also helps to explain the consistently high barrier to resistance observed in multiple viral systems. 4′-FlU is able to select for some in vitro resistance-associated mutations in the influenza A virus genome 24 , 38 . These mutations reduce however the replication fitness of the virus 38 . Mechanistically, 4′-FlU-TP functions as a non-obligate chain terminator 29 , capable of causing premature termination of viral RNA synthesis (despite the presence of a 3’-hydroxyl group) once incorporated into the nascent strand 38 . Given the mechanistic diversity in genome replication and transcription strategies both amongst bunyaviruses on the one hand and between bunyaviruses and other negative-sense RNA viruses 28 on the other hand, the potent antiviral activity of 4′-FlU observed in this study may reflect its ability to disrupt multiple critical steps in OROV genome replication and transcription. Further studies are needed for which also the purified OROV polymerase (alike the LACV L polymerase 39 ), is needed. It remains of course to be explored how to use antiviral drugs (such as 4’-FlU) in the management of OROV infections. According the US CDC, disease typically starts with the abrupt onset of fever (38-40°C) with (often severe) headache, chills, myalgia and arthralgia; symptoms may also include photophobia, eye pain, vomiting and maculopapular rash. These symptoms typically last less than a week (2–7 days) but may relapse in up to 60% of patients. Up to 4% of patients will develop neurologic symptoms after their initial febrile illness. Peak viral RNA levels occur around day 2 of illness, with rapid decline by the end of the first week 40 . Hence, the therapeutic window for the use of antiviral drugs will likely be within the first 3 to 5 days of symptom onset. Since most patients seek care during the febrile phase, this should be clinically feasible, but rapid diagnosis will obviously be essential. Age above 65 and comorbidities such as immunosuppression, hypertension, diabetes, and cardiovascular disease seem to increase the risk for severe disease 40 . Early initiation of treatment in those with increased risk of severe disease may be considered. It will need to be explored whether antiviral treatment can stop disease evolution in those with severe or relapsing disease. During epidemic situations, prophylactic treatment of high risk patients might also be considered, as long as an effective vaccine is not available. This study identifies 4’-FlU as a promising therapeutic candidate for the treatment of OROV infections. Beyond its efficacy against OROV, our findings also further support the continued development of 4′-FlU as a broader-spectrum antiviral, given its previously demonstrated activity against a range of bunyaviruses—including LASV, Crimean-Congo hemorrhagic fever virus (CCHFV), and Sin Nombre virus (SNV) 23 —as well as viruses from more phylogenetically divergent families 21 , 22 , 24 . Finally, our study highlights the strategic value of targeting conserved viral structural elements for the development of robust and broad(er) active antiviral agents. METHODS Cells and viruses VeroE6 cells (Rhesus monkey kidney, ATCC, CRL-1586) and A549 cells (human lung adenocarcinoma, ATCC, CCL-185) were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone) and 1% Non-Essential Amino Acids (NEAA, Gibco). JEG-3 cells (human choriocarcinoma, ATCC, HTB-36) were maintained in Minimum Essential Medium (MEM) supplemented with 10% FBS, 1% sodium pyruvate (Gibco), 1% HEPES (Gibco), 1% NEAA (Gibco) and 1% L-glutamine (Gibco). VeroE6-H2B-miRFP670 cells constitutively expressing near infrared fluorescent protein miRFP-670 were generated by lentiviral transduction and monoclonal selection as described in 41 and were maintained in DMEM. Endpoint titrations were performed on VeroE6 cells using medium containing 2% fetal bovine serum, 1% NEAA, 100 U/mL penicillin-streptomycin and 2.50µg/mL amphotericin B (Gibco). For the antiviral experiments with VeroE6-H2B-miRFP670, A549 and JEG3 cells, the respective cell culture medium was used but with 2% FBS instead of 10% FBS. Cell culture incubations were done in humidified incubators at 37°C and 5% CO 2 . Cell lines tested negative for mycoplasma contamination. The epidemic OROV-IRCCS strain, designated OROV IRCCS-SCDC_1/2024 , was isolated from a serum sample of a patient returning from Cuba and admitted to the IRCCS Sacro Cuore Don Calabria Hospital in Negrar di Valpolicella (Italy) in June 2024 20 . The whole genome sequence is available in GenBank (BankIt2843215; S segment: PP952117 , M segment: PP952118 , L segment: PP952119 ) 5 . The isolate was propagated for 2-3 passages in VeroE6 cells to generate the virus stock used for inoculation, which has a titer of 2.81×10 7 TCID 50 /mL. The pre-epidemic OROV-TR9760 strain, designated OROV-IP/OROV/TR9760/12/08/2009, was obtained from EVAg (Ref. 014V-02948), and passaged on VeroE6 cells for virus stock production. Molecules For in vitro studies, favipiravir, ribavirin and remdesivir were purchased from Carbosynth. GS-441524, molnupiravir, sofosbuvir and obeldesivir were purchased from Excenen Pharmatech Co. Baloxavir marboxil, galidesivir and 4’-fluorouridine were purchased from MedChemExpress. For animal studies, 4’-Fluorouridine was purchased from Excenen Pharmatech Co. and formulated as follows: 0.5% Tween80 (Sigma) in 10 mM sodium citrate (Sigma) dissolved in water. Compound solutions were prepared daily, one to two hours prior dosing. In vitro antiviral and cytotoxicity assays VeroE6-H2B-miRFP670 (25,000 cells/well), A549 (15,000 cells/well) and JEG-3 cells (8,000 cells/well) were seeded in 96-well plates one day before compound treatment and virus infection. The next day (day 0), cells were treated with three-fold serial dilutions of the compounds and infected with OROV at a multiplicity of infection (MOI) of 0.006 (A549 and VeroE6) or 0.15 (JEG-3) median TCID 50 per cell. Three days post infection, the cell viability was determined using staining with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (CellTiter 96 ® Aqueous MTS reagent, Ref. G1111, Promega). The percentage of antiviral activity was calculated by subtracting the background and normalizing to the untreated − uninfected control wells, and the EC 50 was determined using logarithmic interpolation. The potential toxicity of compounds was assessed in a similar set-up in treated − uninfected cultures, in which metabolic activity was quantified at day 3 using the MTS assay. The 50% cytotoxic concentration (CC 50 , the concentration at which cell viability reduced to 50%) was calculated by logarithmic interpolation. OROV infection in mice AG129 mice (129/Sv mice with knockouts in both IFNα/β and IFNγ receptor genes) were bred in house. Breeding pairs of AG129 mice had been purchased from Marshall BioResources. The specific pathogen-free status of the mice was regularly checked at the KU Leuven animal facility of the Rega Institute. OROV inoculation of AG129 mice was performed in 6-12 week-old female and male animals. There was no preference for the sex of the mice used in all experiments; the choice of sex depended on their availability. Mice were housed in individually ventilated isolator cages (IsoCage N Biocontainment System, Tecniplast) at a temperature of 21°C, humidity of 55%, and subjected to 12:12 dark/light cycles. Mice had access to food and water ad libitum, and their cages were enriched with cotton and cardboard play tunnels. Animal work was performed under Biosafety Level 3 conditions. Housing conditions and experimental procedures were approved by the Animal Ethics Committee of KU Leuven (approval numbers 202/2024, 036/2025, and M003/2025) following institutional guidelines approved by the Federation of European Laboratory Animal Science Associations (FELASA). Mice were anesthetized by inhalation with isoflurane before infection. Virus suspension consisting of 10 5 TCID 50 , prepared in Opti-MEM medium, was injected subcutaneously in the rear of the left footpad. At humane endpoint or study endpoint, animals were euthanized by administration of Dolethal before sample collection. Animal body weight, signs of disease and surface body temperature (using a Gentle Temp 720 device, Omron) were monitored at least twice daily. Mice were euthanized according to the following clinical criteria: huddling (2 points), hunch back, ruffled fur, eye recession, hyperactivity, 30°> surface body T°C surface body temperature <30°C, weight loss 10% of baseline (5 points each) and bleeding, paralysis, moribund, unresponsive, surface body temperature < 28°C, weight 20% of baseline (10 points each). The humane endpoint was defined when the score reached ≥ 10. Treatment regiment In prophylactic setting experiments, AG129 mice were administered orally either vehicle (0.5% Tween80 in 10 mM sodium citrate dissolved in water) (n=5), 10 mg/kg (n=5), 5 mg/kg (n=5), 2 mg/kg (n=5) or 0.4 mg/kg (n=5) 4’-FlU, once daily from two hours prior infection to study endpoint. Uninfected mice were used as controls (n=4). In therapeutic setting experiments, AG129 mice were administered orally either vehicle (n=5) or 10 mg/kg (n=5) 4-FlU, once daily, 24hpi, 48hpi or 72hpi to study endpoint. In the “72h” group, one animal reached humane endpoint before the initiation of the compound treatment and was therefore excluded from the dataset (n=4 for this group). Uninfected mice were used as controls (n=4). Endpoint/HEP virus titration assay Serum was obtained from total blood by centrifugation at 10.000x g for 5 min. Organs were homogenized using bead disruption in 400 µL of DMEM medium and centrifuged (10.000x g, 5 min) to pellet cell debris. To quantify infectious OROV particles, endpoint titrations were performed on VeroE6 cells (10 4 cells/well) infected with 10-fold sample serial dilutions, in 96-well plates. After seven days, viral titers were determined by the Reed-Muench method using the Lindenbach calculator and were expressed as 50% tissue culture infectious dose (TCID 50 ) per mL of serum or g of tissue. The limit of quantification (LOQ) of the titration assay was set based on the lowest measured virus titer taking tissue weight into account. For statistical analysis, negative samples were assigned the value of the LOQ (1.78×10 2 TCID 50 /mL or /g). RNA extraction and RT-qPCR Serum was obtained as described above and RNA was extracted using the Nucleospin ® RNA virus kit (Macherey-Nagel) according to the manufacturer’s instructions. Organs were collected and homogenized using bead disruption (Precellys) in 700 µL of TRK lysis buffer (E.Z.N.A. ® Total RNA kit, Omega Bio-Tek) and centrifuged (15.000x g, 10 min) to pellet cell debris. RNA was extracted according to the manufacturer’s instructions. RT-qPCR was conducted on a QuantStudio5 platform (Applied Biosystems) using the iTaq Universal probe One-Step RT-qPCR kit (Ref. 1725141, Bio-Rad). The primers targeting the genomic M segment of OROV-IRCCS were: forward-5’-ACGCAGACTAACCAACTTCC-3’ and reverse-5’-CAGTTCCGATAGTTGACCCATTA-3’. The probe was 5’-TGCAGTGCGGCTCAATCCAAGTAA-3’. Primers and probes were purchased from Integrated DNA Technologies, Inc (IDT). Standards of OROV-IRCCS cDNA (IDT) were used to express viral genome copies per mL of serum or mg of tissue. The LLOQ was set based on the lowest measured titers of quantified standards. For statical analysis, samples showing values below the LOD (determined as 9.77 RNA copies/mL), were assigned the LOD value. Histology Brain, lungs and liver samples were fixed overnight in 4% paraformaldehyde (ThermoScientific) and embedded in paraffin. Tissue sections (5 µm) were stained with hematoxylin and eosin and analyzed blindly by expert pathologists. RNAscope in situ hybridization Probe V-OROV-S-O5-sense-C1 (Advanced Cell Diagnostics, Ref. 1811921-C1) was custom-designed against the S segment nucleoprotein gene, reverse-complement of nt 10-937 of the Genbank PP952117.3 sequence 5 . This probe is publicly available. Manual RNAscope assays were performed 5 µm FFPE sections of brain, lung and livers using the Multiplex Fluorescent Detection Kit v2 (Advanced Cell Diagnostics, Ref. 323110) according to the manufacturer’s protocol and as previously described 36 . Briefly, slides were baked at 60°C for 1 h, followed by deparaffinization in xylene (Leica Biosystems, Ref. 3803665EG) and 100% ethanol wash, and incubation with hydrogen peroxide for 15 min at room temperature. Tissue pretreatment was performed by steaming in target retrieval reagent (Advanced Cell Diagnostics, Ref. 322000) for 15 min followed by incubation in Protease Plus (Advanced Cell Diagnostics, Ref. 322330) at 40°C for 20 min. Signal amplification was followed by development with the fluorophore dye Opal 520 (Akoya Biosciences, Ref. FP1487001KT). DAPI (Thermo Fisher Scientific, Ref. D1306) served as nuclear stain. Slides were mounted in Mount Solid antifade (Abberior, Ref. MM-2011-2X15ML). Confocal images were taken on a Zeiss LSM 980. Resistance mutation selection and Oxford Nanopore sequencing OROV-IRCCS was passaged in A549 cells in the presence of increasing concentrations of 4’-FlU. Selection was initiated at 10 nM and, for each passage, the virus was cultured at the same concentration, and at 3×, 10× and 30× higher concentrations. The culture with the highest compound concentration that still showed virus breakthrough, as observed by a significant cytopathic effect, was then used for the next passage. At passage 10 (day 34), vRNA in the cell-culture medium of all lineages was extracted as described above. Following extraction, purified viral RNA was converted to cDNA by sequence-independent, single-primer amplification as previously described 42 . Amplified cDNA was sequenced on an Oxford Nanopore Technologies GridION device (MinKNOW v25.03.7) using real-time super-accurate basecalling (Dorado v7.8.3). SISPA adapters were removed from the obtained reads using Porechop ( https://github.com/rrwick/Porechop ), after which variant calling was performed using an in-house script that performs reference mapping with minimap2 43 , followed by BAM file inspection with bam-readcount 44 to obtain a detailed overview of nucleotide variation at each position. Only sites with >20x coverage were included in the analysis and only variants with >10% prevalence and >10x variant-specific coverage were assessed. Structure predictions The OROV L protein sequence (UniPRot A0A0B5CUJ1) was used together with short RNA sequences as input for either AlphaFold3 (AF3) 27 through the AF3 server at https://alphafoldserver.com/ or for a local implementation of Boltz 45 , for modeling with nucleotide analogues including 4’-FlU-TP, which were input as SMILES strings. Results were visualized and analyzed with Pymol (The PyMOL Molecular Graphics System, Version 3.0 Schrödinger, LLC) and ChimeraX 46 . Comparisons with La Crosse virus polymerase used Protein Data Bank entries 7ORI to 7ORM 47 . In Supplementary Fig. 9b , 7ORK, which harbors an ATP at the catalytic site, is used. Graphs and statistical analyzes All graphs and statistical analyses were performed using GraphPad Prism version 10.4.1. Normality of the data distribution was assessed using the Shapiro-Wilk test. Survival analyses were conducted using the log-rank (Mantel-Cox) test. For comparisons involving groups with non-normally distributed data, unpaired Mann-Whitney tests were used. For comparisons involving groups with normally distributed data, Welch tests were used. SUPPLEMENTARY INFORMATIONS Download figure Open in new tab Supplementary Figure 1: In vitro antiviral activity of broad-spectrum antiviral molecules against OROV. (a) Table summarizing the antiviral activity and cytotoxicity of eight broad-spectrum antivirals against OROV-TR9760 and OROV-IRCCS compared to 4’-FIU. NT= not tested. (b) Dose–response curves of the antiviral activity against OROV-IRCCS determined by MTS-based CPE reduction assay. Cytotoxicity was determined by MTS cell viability assay in mock-infected cells. Data points are the mean ± SD of n = 2-4 biologically independent experiments. Download figure Open in new tab Supplementary Figure 2: The epidemic OROV-IRCCS strain replicates efficiently in AG129 mice and induces an acute severe disease. (a) Experimental design scheme. AG129 mice (n=5 per group) were infected subcutaneously in the left rear footpad with 10 5 TCID 50 of epidemic OROV-IRCCS strain. Mice were closely monitored until the study endpoint (7 dpi) or until reaching humane endpoints according to a clinical scoresheet. (b) Kaplan-Meier survival curves display the percentage of survival for each experimental group. Significance was calculated by log-rank (Mantel-Cox) test. (c) Weight change of the different experimental groups at time of euthanasia as a percentage, normalized to the body weight at the time of infection. Mean ± SD are shown. Significance was calculated by individual Mann-Whitney tests. (d) Daily surface body temperature measured on the abdomen of animals from each experimental group, using an infrared thermometer. Mean ± SD are shown. (e) Daily individual clinical scores for each experimental group, assessed using a standardized clinical scoresheet. Grey shading indicates the cessation of monitoring due to euthanasia or humane endpoints. (f and g) Viral RNA loads (f) or infectious titers (g) in serum and various organs measured at the time of euthanasia (humane endpoints or study endpoint) by RT-qPCR and cytopathic effects observation, respectively. Due to technical reasons, blood samples could not be collected from two mice in the OROV-infected group. Individual mice are presented as dots and error bars represent the SD. Significance was calculated by individual Mann-Whitney tests. ND, not determined; LLOQ, lower limit of quantification. Download figure Open in new tab Supplementary Figure 3: Surface body temperature and clinical scores of AG129 mice infected with the epidemic OROV-IRCCS strain and administered prophylactically with 4’-FlU. (a) Daily surface body temperature measured on the abdomen of mice from each experimental group, using an infrared thermometer. Mean ± SD are shown. (b) Daily individual clinical scores for each experimental group, assessed using a standardized clinical scoresheet. Grey shading indicates the cessation of monitoring due to euthanasia/HEP. Download figure Open in new tab Supplementary Figure 4: Absence of brain pathology in OROV-IRCCS infected AG129 mice, prophylactically or therapeutically administered with 4’-FlU. Representative haematoxylin and eosin-stained images of mice brains obtained at the time of euthanasia (HEP or study endpoint). ( a ) Prophylactic study. ( b ) Therapeutic study. Scale bar = 50 µm Download figure Open in new tab Supplementary Figure 5: Liver gross pathology of AG129 mice infected with the epidemic OROV-IRCCS strain and prophylactically or therapeutically administered with 4’-FlU. Representative paraformaldehyde-fixed liver samples pictures from OROV-IRCCS-infected mice ( a ) prophylactically or ( b ) therapeutically administered with either vehicle or different doses of 4’-FlU. Scale bar = 1 cm. Download figure Open in new tab Supplementary Figure 6: Additional RNAscope images of organs sections from AG129 mice infected with the epidemic OROV-IRCCS strain and prophylactically treated with 4’-FlU. Additional confocal images of 5µm sections through brains, lungs and liver samples, processed for in situ hybridization using the RNAscope platform, with a probe for the OROV S segment. DAPI serves as a nuclear stain. This figure complements and is associated with the main Figure 3 . Scale bar = 20 µm. Download figure Open in new tab Supplementary Figure 7: Surface body temperature and clinical scores of AG129 mice infected with the epidemic OROV-IRCCS strain and therapeutically administered with 4’-FlU. (A) Daily surface body temperature measured on the abdomen of mice from each experimental group, using an infrared thermometer. Mean ± SD are shown. (B) Daily individual clinical scores for each experimental group, assessed using a standardized clinical scoresheet. Grey shading indicates the cessation of monitoring due to euthanasia/HEP. Download figure Open in new tab Supplementary Figure 8: Additional RNAscope images of organs sections from AG129 mice infected with the epidemic OROV-IRCCS strain and therapeutically administered with 4’-FlU. Additional confocal images of 5µm sections through brains, lungs and liver samples, processed for in situ hybridization using the RNAscope platform, with a probe for the OROV S segment. DAPI serves as a nuclear stain. This figure complements and is associated with the main Figure 5 . Scale bar = 20 µm. Download figure Open in new tab Supplementary Figure 9: OROV AND LACV L proteins models comparison and predicted complexes with different nucleotide analogues. ( a ) The same OROV L – primer/template – ATP predicted complex as in Fig. 6a . Magnification on the right: residues involved in contacts with the fluors of nucleotide analogues in (c) and (d) are displayed as sticks and labeled. ( b ) LACV L experimental complex with ATP. The LACV L counterparts of the residues in A, right are indicated. ( c ) Predicted complex of OROV L with the active metabolite of sofosbuvir. The four major contacts of the 2’-F are indicated with orange dotted lines to signal that their PAE are high. ( d ) The same complex with 4’-FlU-TP as in Fig. 6b . AUTHORS CONTRIBUTION MF, MD, ML: Project design, design and execution of the experiments, data analysis and manuscript writing. TB and SB: Design, execution and analysis of the structural prediction studies. MK : Design and execution of the RNAcope experiments. PM: Design of RNAscope experiments. TMP and BV: Execution and analysis of the sequencing work. TR, BW : analysis of histopathology data. NC, SH: Execution of experiments. KD: Execution of in vitro antiviral assays and resistance selection experiments. JRP: Access to OROV-TRVL9760 strain. CC: Access to OROV-IRCCS strain. ML, JN: Funding acquisition, project design and manuscript writing. All authors reviewed the manuscript, provided feedback, and approved the final version for submission. COMPETING INTERESTS The authors declare no competing interests. DATA AND MATERIALS AVAILABILITY All data generated and analyzed in this paper are included in this article. Additional imaging data related to the RNAscope analysis are available from the corresponding authors upon request. Source data are provided with this paper. ACKNOWLEDGEMENTS We thank Tina Van Buyten for growing the OROV-TR9760 stock and initial antiviral assays. We thank Steven De Jonghe and Wim Smet for compound ordering, Thibault Francken for culturing the VeroE6-H2B-miRFP670 cells and the colleagues at the Rega animal facility at KU Leuven for technical assistance. This work was funded by European project DURABLE (Delivering a Unified Research Alliance of Biomedical and public health Laboratories against Epidemics, to JN) and by the ANRS-Maladies Infectieuses Emergentes (grant ANRS0380 to SB). The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication. REFERENCES 1. ↵ Anderson , C. R. , Spence , L. , Downs , W. G. & Aitken , T. H. G. Oropouche Virus: a New Human Disease Agent from Trinidad, West Indies . Am. J. Trop. Med. Hyg . 10 , 574 – 578 ( 1961 ). OpenUrl Abstract / FREE Full Text 2. ↵ Bai , F. , Denyoh , P. M. D. , Urquhart , C. , Shrestha , S. & Yee , D. A. A Comprehensive Review of the Neglected and Emerging Oropouche Virus . Viruses 17 , 439 ( 2025 ). OpenUrl PubMed 3. ↵ Ceccarelli , G. et al. Oropouche virus infection: Differential clinical outcomes and emerging global concerns of vertical transmission and fatal cases . Int. J. Infect. Dis . 150 , 107295 ( 2025 ). 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Share Oral 4’-fluorouridine completely suppresses epidemic Oropouche virus replication in mice and confers protection against virus-induced disease Martin Ferrié , Maïlis Damuzey , Thibault Tubiana , Mona Khan , Tania Roskams , Birgit Weynand , Niels Cremers , Stijn Hendrickx , Kim Donckers , Thayara Morais Portal , Bert Vanmechelen , Joana Rocha-Pereira , Concetta Castilletti , Peter Mombaerts , Stéphane Bressanelli , Manon Laporte , Johan Neyts bioRxiv 2025.09.22.677733; doi: https://doi.org/10.1101/2025.09.22.677733 Share This Article: Copy Citation Tools Oral 4’-fluorouridine completely suppresses epidemic Oropouche virus replication in mice and confers protection against virus-induced disease Martin Ferrié , Maïlis Damuzey , Thibault Tubiana , Mona Khan , Tania Roskams , Birgit Weynand , Niels Cremers , Stijn Hendrickx , Kim Donckers , Thayara Morais Portal , Bert Vanmechelen , Joana Rocha-Pereira , Concetta Castilletti , Peter Mombaerts , Stéphane Bressanelli , Manon Laporte , Johan Neyts bioRxiv 2025.09.22.677733; doi: https://doi.org/10.1101/2025.09.22.677733 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7618) Biochemistry (17635) Bioengineering (13859) Bioinformatics (41846) Biophysics (21401) Cancer Biology (18534) Cell Biology (25423) Clinical Trials (138) Developmental Biology (13352) Ecology (19860) Epidemiology (2067) Evolutionary Biology (24286) Genetics (15582) Genomics (22463) Immunology (17700) Microbiology (40298) Molecular Biology (17141) Neuroscience (88429) Paleontology (666) Pathology (2825) Pharmacology and Toxicology (4813) Physiology (7633) Plant Biology (15107) Scientific Communication and Education (2042) Synthetic Biology (4284) Systems Biology (9808) Zoology (2267)