Oral 4′-fluorouridine rescues nonhuman primates from advanced Lassa fever

Cross, Robert W.; Turcinovic, Jacquelyn; Prasad, Abhishek N.; Borisevich, Viktoriya; Agans, Krystle N.; Deer, Daniel J.; O’Toole, Rachel; Dobias, Natalie S.; Woolsey, Courtney; Fenton, Karla A.; Geisbert, Thomas W.

doi:10.1038/s41586-025-09906-y

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Published: 07 January 2026

Oral 4′-fluorouridine rescues nonhuman primates from advanced Lassa fever

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Abstract

There are no approved treatments for Lassa fever, which is estimated to cause 100,000 to 300,000 infections and 5,000 deaths annually in West Africa^1,2. Recently, it was shown that 4′-fluorouridine (also known as EIDD-2749), an orally available ribonucleoside analogue, protected guinea pigs from lethal challenge with the lineage IV prototype Josiah strain of Lassa virus when treatment was delayed beyond the onset of clinical signs of disease³. Here we assessed the therapeutic efficacy of 4′-fluorouridine in an African green monkey model of Lassa fever using the contemporary and apparently more pathogenic lineage VII Togo strain of Lassa virus. Daily treatment with 4′-fluorouridine beginning 6 days after Lassa virus infection, when the monkeys were viraemic and clinically ill, resulted in rapid and complete clearance of infectious virus in 4 out of 5 monkeys, and all treated monkeys survived to the pre-determined study end-point. Targeted transcriptomics showed that cellular responses and control of cytokinaemia contributed to the development of immunity. Our findings support the further development of 4′-fluorouridine both as a post-exposure prophylaxis to control outbreaks and as a therapeutic agent to treat symptomatic patients.

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Main

Lassa virus (LASV), the causative agent of Lassa fever, is an Old World mammarenavirus in the Arenaviridae family that can cause fatal haemorrhagic fever. LASV is endemic in West Africa, causing thousands of deaths each year^1,2. Periodic importation of LASV into non-endemic areas, most recently to the UK and the USA, underscores its potential for global spread^4,5,6. One study using ecological niche modelling suggested that LASV could spread more widely because of population growth and changes in land use and that up to 600 million people could be at risk of LASV infection by 2050⁷. LASV is included on the World Health Organization 2018 list of priority pathogens⁸, as well as the Coalition for Epidemic Preparedness Innovations list of priority diseases (https://cepi.net/priority-pathogens).

There are currently no licensed vaccines or therapeutic agents for combating Lassa fever. Treatment of Lassa fever is primarily supportive and includes the use of intravenous fluids and electrolytes to maintain hydration, oxygen therapy, dialysis, and blood transfusions in cases with excess or uncontrolled bleeding. Ribavirin has been used off label for almost 40 years to treat Lassa fever; however, its efficacy is contested^9,10,11, and it is contraindicated in pregnancy owing to its known teratogenicity¹². Substantial progress has been made in developing medical countermeasures that can provide efficacy in preclinical animal models of human Lassa fever^13,14,15. These include several preventive single-injection vaccines that can completely protect nonhuman primates (NHPs) against lethal exposure to LASV^{16,17,18,19,20,21,22}. Notably, one of these vaccine candidates, based on recombinant vesicular stomatitis virus and having strong protective efficacy in NHPs^16,22, became the first Lassa fever vaccine to enter phase 2 trials last year²³.

Regarding post-exposure treatments and therapies in preclinical animal models of Lassa fever, the broad-spectrum antiviral drug favipiravir has shown promising results and completely protected NHPs against lethal Lassa fever when intravenous treatment was initiated early in the disease course, beginning at day 4 after LASV exposure²⁴. A randomized controlled open label phase 2 clinical trial of favipiravir in 40 patients with Lassa fever was conducted in Nigeria in 2021 and 2022, but the results have not been reported²⁵. The most promising treatments to date in preclinical NHP models are human monoclonal antibodies directed against the LASV surface glycoprotein complex (GPC), with a cocktail known as Arevirumab-3 conferring substantial protection against lethal disease in NHPs when given beginning as late as eight days after exposure to LASV, when the monkeys are at an advanced stage of illness^26,27,28,29. Although these results with human monoclonal antibody-based therapies are encouraging, similar to ribavirin and favipiravir, the monoclonal antibodies must be given intravenously, which presents logistical challenges and additional costs, especially in disadvantaged areas where LASV is endemic. There is a clear need for oral antivirals against Lassa fever. Recently, we showed that the broad-spectrum oral antiviral obeldesivir can provide substantial protection when given to NHPs after lethal challenge with the Ebola, Sudan or Marburg filoviruses^30,31,32; however, there is no evidence that obeldesivir has strong activity against LASV.

Regarding oral antivirals against LASV, a recent study showed that 4′-fluorouridine (4′-FlU, also known as EIDD-2749)—a ribonucleoside analogue currently in pre-clinical development—can protect guinea pigs against arenaviruses including LASV when administered late in the disease course³. It has broad-spectrum activity against several families of negative-strand and positive-strand RNA viruses. In addition to having activity against arenaviruses³, 4′-FIU can inhibit the replication of respiratory syncytial virus and other paramyxoviruses³³, SARS-CoV-2³³ and influenza A viruses^34,35. The drug inhibits RNA-dependent RNA polymerase (RdRp) function and causes delayed stalling of both respiratory syncytial virus and SARS-CoV-2 polymerases³³, and incorporation of 4′-FIU-triphosphate triggered immediate chain termination of the influenza A virus RdRp complex³⁵.

Although results showing that 4′-FIU can protect guinea pigs against lethal exposure to LASV are encouraging, guinea pigs do not exhibit features of human Lassa fever as accurately as the gold-standard NHP models^36,37,38. In addition, serial adaption of LASV and/or inbred guinea pigs are often needed to achieve lethality and, importantly, guinea pig models are available for only a small number of strains of LASV, which do not represent a broad range of genetically diverse LASV isolates, including the apparently most pathogenic lineage VII variants such as the Togo strain^29,39. In vitro, 4′-FIU was shown to have antiviral activity against several genetically distinct LASV strains and, notably, the lowest activity was against the Togo strain³. We previously developed a cynomolgus monkey model of LASV Togo and showed that Arevirumab-3 was less efficacious in this model than against other LASV strains²⁹. Given the worldwide shortage of cynomolgus macaques caused by the COVID-19 pandemic, we recently developed an African green monkey (Chlorocebus aethiops) model of Lassa fever using a contemporary lineage II strain from Nigeria that reflects the human condition equally well as or better than the cynomolgus macaque model⁴⁰.

We first developed a uniformly lethal African green monkey (AGM) model for a lineage VII strain of LASV from Togo. We then used this model to determine whether 4′-FIU could provide protection when administered beginning at an advanced stage of Lassa fever to mimic a real-world scenario, in which viraemic and symptomatic patients present to a clinic at an advance stage of disease.

AGM model of LASV lineage VII Togo

To assess the pathogenic potential of a recent lineage VII LASV isolate, we challenged five AGMs by intramuscular injection with 1,000 plaque-forming units (PFU) of strain Germany ex Togo/2016/7082 (LASV Togo). All five monkeys (C1 to C5) showed disease features including anorexia, lethargy, lymphopenia, thrombocytopenia and increased circulating levels of liver-associated enzymes and C-reactive protein, and four out of five monkeys showed evidence of central nervous system disorders consistent with previous Lassa fever studies^{18,24,26,27,28,29,36,39,41} (Supplementary Table 1). The five AGMs succumbed to Lassa fever on 9, 10, 11, 11 and 13 days post-infection (DPI), respectively (mean time to death (MTD) = 10.8 ± 1.3 DPI) (Fig. 1a). To determine the congruence in the disease course of the LASV Togo AGM model with the more established cynomolgus macaque model of LASV infection, we used survival data from five historical control cynomolgus macaques that were challenged with the same seed stock of LASV Togo²⁹ and succumbed to lethal disease at 11–12 DPI (MTD = 11.2 ± 0.4 DPI). There was no statistical difference in the survival curves between species (P = 0.776; Mantel–Cox log-rank test). For the LASV Togo-infected AGMs, circulating viraemia levels up to 11.26 log₁₀ genome equivalents (GE) per millilitre and 6.30 log₁₀ PFU ml⁻¹ were observed (Fig. 1b,c), and tissue loads up to 13.06 log₁₀ GE per g tissue were noted (Fig. 1d).

**Fig. 1: Survival analysis and circulating viral burden of AGMs challenged with LASV Togo.**

Efficacy of 4′-FIU against LASV Togo

To determine whether 4′-FIU can be effective as a therapeutic treatment against LASV exposure, we challenged six AGMs were challenged with 1,000 PFU of LASV lineage VII strain Togo by intramuscular injection (Fig. 2). Beginning at 6 DPI, 5 mg kg⁻¹ doses of 4′-FIU were administered orally to the experimental group (n = 5) once daily for a total of 10 consecutive days. Treatment was withheld from the in-study positive control monkey (C6). All six monkeys displayed clinical signs consistent with LASV disease before treatment was initiated. These signs varied from monkey to monkey and included decreased appetite (6 out of 6), fever (5 out of 6), lymphopenia (5 out of 6), basopenia (5 out of 6); thrombocytopenia (5 out of 6), and increased circulating levels of C-reactive protein (5 out of 6) (Supplementary Table 2). Also, all 6 monkeys were viraemic at 6 DPI before treatment, with circulating viral loads that ranged from 7.05 to 8.02 log₁₀ GE ml⁻¹ and 2.97 to 4.26 log₁₀ PFU ml⁻¹ (Fig. 2b,c). Notably, all monkeys challenged with LASV Togo and treated with 4′-FIU survived to the 35 DPI end-point of the study (Fig. 2a), whereas the untreated positive control monkey reached a clinical score requiring euthanasia on 11 DPI (Fig. 2a). For statistical comparison of survival, the in-study positive control was pooled with the five untreated AGMs from the pilot study (n = 6; MTD = 10.8 ± 1.2 DPI). There was a significant difference in the survival curves between the 4′-FIU-treated cohort and the pooled untreated positive controls (Fig. 2a, P = 0.002, Mantel–Cox log-rank test). All 4′-FIU-treated monkeys had decreased appetites and/or were anorexic during the treatment period, but appetites returned to normal between 15 and 29 DPI and there were no overt clinical signs of Lassa fever in any of the 4′-FIU-treated AGMs at the 35 DPI study end-point (Supplementary Table 2). Notably, fevers in 4 out of 5 AGMs on 6 DPI just prior to the 4′-FIU treatment were absent on 7 DPI and throughout the study. Mild perturbations in clinical pathology parameters were noted throughout the study in all 4′-FIU-treated AGMs, some of which persisted to the 35 DPI study end-point in 3 monkeys (Tx1, Tx4 and Tx5), but overall trended toward baseline levels by the 35 DPI study end-point (Supplementary Table 2). Of note, all 4′-FIU-treated AGM were free of overt signs of clinical illness at the conclusion of the study.

**Fig. 2: Survival analysis and circulating viral burden of AGMs challenged with LASV Togo and treated with 4′-FIU.**

Reduction of viral load

Circulating viral genomic RNA (vRNA) and infectious LASV were cleared by the pre-determined 35 DPI study end-point in 4 out of 5 of the 4′-FIU-treated AGMs (Fig. 2b,c), and one 4′-FIU-treated monkey (Tx4) had moderate levels of circulating vRNA (5.97 log₁₀ GE ml⁻¹) and low infectious LASV (1.88 log₁₀ PFU ml⁻¹) that had not quite cleared by the 35 DPI study end-point, although vRNA and infectious LASV titres appeared to be decreasing. By contrast, the untreated LASV Togo-positive control monkey (C6) had circulating vRNA and infectious LASV levels ranging from 7.76 to 10.72 GE ml⁻¹ and 3.02 to 6.68 PFU ml⁻¹, respectively, when the monkey succumbed at 11 DPI, consistent with levels observed in the LASV Togo-infected AGMs used to establish the model (C1–C5). There were significant differences in the peak load of circulating LASV RNA in the 4′-FIU-treated cohort compared with the untreated positive control cohort and peak levels of circulating infectious virus in monkeys treated with 4′-FIU compared with untreated controls (Extended Data Fig. 1a,b, P = 0.008 for both comparisons, Mann–Whitney U-test), as well as the day on which each measure was detected (P = 0.016 and P = 0.008, respectively; Mann–Whitney U-test). Tissue viral loads up to 13.05 log₁₀ GE per g tissue were detected in the experimental positive control AGM (C6), also consistent with the AGMs used to develop the LASV Togo model (C1–C5). In comparison, levels of LASV RNA were much lower or undetectable in tissues from the 4′-FIU-treated monkeys, which all survived to the 35 DPI study end-point (Fig. 2d). As several 4′-FIU-treated AGMs had higher levels of LASV RNA in several tissues, and as there has been concern about virus persistence in immune-privileged tissues, we attempted to detect infectious LASV in those tissues. These tissues included spleen of all 5 treated AGMs (Tx1–Tx5), testis of Tx1, ovary of Tx3 and brain of Tx4. Despite levels of LASV RNA as high as 10.00 log₁₀ GE per g tissue in the spleen of Tx4 and 10.62 log₁₀ GE per g tissue in the spleen of Tx1, low levels (2.70 log₁₀ PFU per g tissue) of infectious LASV were detected only in the spleen of Tx4, and all other tested tissues contained no infectious LASV.

Assessment of antibody titres by ELISA

We assessed the presence of LASV GPC-specific IgG by enzyme-linked immunosorbent assay (ELISA). All surviving monkeys developed moderate end-point binding IgG titres (reciprocal dilution 400–800) at 12–21 DPI, which peaked at 1,600–3,200 by the study end-point (35 DPI) (Fig. 3a). By contrast, the in-study positive control monkey (C6), which succumbed to disease at 11 DPI, did not develop detectable GPC-specific IgG (Fig. 3a). To determine the neutralizing capacity of anti-LASV antibodies, we performed plaque reduction neutralization tests (PRNTs) on serum collected immediately prior to challenge (0 DPI), at 14 DPI (or 11 DPI for C6), and at the pre-determined study end-point (35 DPI). As expected, all monkeys lacked neutralizing antibodies to LASV on the day of challenge (Fig. 3b), and only three (Tx1, Tx2 and Tx4) exhibited neutralization activity at or above 50% by 14 DPI, with low reciprocal dilution end-point titres of 10–40 (Fig. 3c). More robust, albeit overall low to moderate, neutralization was observed at 35 DPI, with all 4′-FIU-treated monkeys exhibiting reciprocal dilution end-point titres ranging from 20 to 160 (Fig. 3d).

**Fig. 3: Humoral responses from LASV Togo-infected AGMs treated with 4′-FIU.**

Pathology

All five AGMs from the pilot study and the positive control from the 4′-FIU treatment study that succumbed to disease displayed gross lymphadenomegaly, splenomegaly, meningeal congestion, multifocal to coalescing regions of hepatic pallor consistent with necrotizing hepatitis (Fig. 4a), and patchy red and pink lung lobes consistent with interstitial pneumonia. Other lesions noted in at least one AGM included haemorrhagic adrenomegaly (C1, C3 and C4) and pleural effusion (C1, C2 and C3). No appreciable gross lesions were noted in any of the five 4′-FIU-treated AGMs (Fig. 4c).

Histological lesions and immunohistochemistry (IHC) with anti-LASV antibodies support the described gross lesions for all five pilot study AGMs and the untreated positive control AGM. Representative histopathologic lesions are necroinflammatory and often vasocentric. Necrotizing hepatitis composed of mixed inflammatory cells occupied sinusoidal spaces, and swollen and necrotic hepatocytes were noted as multifocal random aggregates of karyorrhectic debris admixed with eosinophilic material. Anti-LASV IHC was associated with hepatic lesions (Fig. 4b). Positive IHC labelling of thickened alveolar septal walls, alveolar macrophages and endothelium of medium-calibre vessels was noted in sections of lung (Fig. 4e). Necrotizing splenitis presented as disruption of normal white pulp structures with loss of lymphocytes and accumulation of cellular debris and fibrin. Mesothelial lining cells, endothelium and mononuclear cells of the red and white pulp were also labelled extensively (Fig. 4f). Similar germinal centre disruption and expansion of the subcapsular sinuses with infiltrating mononuclear cells were noted in lymph nodes.

Haemorrhagic necrosis of all layers of the adrenal gland were noted in severe cases of the LASV Togo-infected, untreated control AGMs. Diffuse cytoplasmic immunolabelling of cells within the adrenal cortex was present in most of the examined sections (Fig. 4i). Minimal interstitial lymphohistiocytic infiltrates were noted in the kidney along with IHC-positive endothelium within the glomerular tuft and interstitial vessels of the medulla (Fig. 4j,w). Lymphohistiocytic cystitis was noted in some untreated AGMs, with colocalized IHC labelling of endothelial cells, mononuclear cells and occasionally the overlying epithelium (Fig. 4m). IHC positivity within the brain, cervical spinal cord and meninges was largely associated with the endothelium of vessels and scattered mononuclear cells. Rare mononuclear perivascular cuffing with minimal IHC positivity and IHC-positive neurons within the cerebrum were noted in the most severe cases (Fig. 4n). Multifocal clusters of mononuclear cells within the neurohypophysis of the pituitary gland were IHC-positive in three untreated control AGMs (C3, C4 and C6). Positive IHC labelling for LASV antigen in reproductive organs included thecal cells and granulosa cells of the ovary (Fig. 4r), and in testicular tissues, IHC labelling was noted in the endothelium and the epithelium lining the epididymal tubules (Fig. 4q). Endothelium, uterine stromal cells and—rarely—the overlying epithelium were IHC-positive in examined sections of uterus from all untreated female AGMs (C2, C4, C5 and C6). Endothelium and/or small clusters of islet cells were IHC-positive in the pancreas of all positive control AGMs. Sections of palpebra exhibited IHC labelling of endothelium, scattered mononuclear cells within the subepithelial stroma, and rarely in the epithelium of the hair papilla (C2, C3, C4 and C6). Ocular lesions and IHC labelling were present in three AGMs, consistent with minimal anterior uveitis with associated IHC labelling of endothelium and mononuclear cells within the iris leaflet, ciliary body and drainage angle (C3, C4 and C6). One AGM had IHC labelling of the retina (C6).

Three of the 4′-FIU-treated AGMs (Tx2, Tx3 and Tx5) lacked IHC labelling for LASV antigen in all examined tissue sections (Fig. 4d,g,h,k,l,o,p,s,t). Three 4′-FIU-treated animals exhibited at least one of the following lesions: minimal multifocal necrotizing hepatitis (Tx5), locally extensive lymphohistiocytic hypercellularity of the red pulp in the spleen (Tx1 and Tx3), and minimal perivascular cuffing in the temporal lobe of the brain (Tx1) with no associated IHC labelling for LASV antigen. One 4′-FIU-treated AGM (Tx1) exhibited perivasculitis and/or vasculitis of medium-calibre vessels with colocalized IHC-positive smooth muscle of the vessel wall in the inguinal lymph node, kidney and testis (Fig. 4u). One 4′-FIU-treated AGM (Tx4) had choroid plexitis with IHC-positive ependymal cells (Fig. 4x), vacuolar degeneration of renal medulla with IHC-positive endothelium (Fig. 4v), and perivasculitis and/or vasculitis of medium-calibre vessels with colocalized IHC-positive smooth muscle of the vessel wall of the lymph nodes (axillary and inguinal), kidney, lung and brain. However, as noted above, we were unable to detect infectious LASV from the brain of this monkey. Inflammatory lesions were lacking in the spleen, adrenal gland and pancreas; however, IHC-positive smooth muscle was present in Tx4. Although Tx4 had elevated hepatic enzymes (for example, alanine aminotransferase, aspartate aminotransferase and γ-glutamyltransferase) at the study end-point, no apparent liver lesions were present upon gross or histologic examination.

Transcriptomics

We performed targeted transcriptomic profiling on serially sampled blood from positive control and 4′-FIU-treated AGMs to investigate how treatment affected the circulating host immune response. Naive clustering analysis via principal component analysis (PCA) (Fig. 5a and Extended Data Fig. 2a) suggested that whereas transcriptomic profiles between treatment groups were similar at baseline (0 DPI) and middle disease (6–7 DPI), the host response diverged following treatment. Late disease samples from positive controls (≥9 DPI) clustered distinctly from time-comparable samples from treated monkeys (9 and 12 DPI). These treated time points were distinct from middle disease, late disease and baseline; we therefore labelled them as a ‘transitional’ disease stage. Later time points in treated monkeys (≥15 DPI) clustered with baseline samples, indicating recovery. These disease stages were supported by comparing each group and treatment to their respective baseline samples (Fig. 5b and Extended Data Fig. 2c,d). The number of differentially expressed genes (DEGs) peaked in middle disease in the positive control and treated groups and remained high throughout late disease in positive control monkeys. By contrast, the magnitude of the host immune response diminished to less than half of its peak at transitional time points in treated monkeys. Of note, the host immune response did not fully resolve to baseline during recovery time points in the 4′-FIU-treated monkeys, with around 50 genes remaining significantly up- or downregulated relative to baseline, suggestive of ongoing antiviral response or repair.

**Fig. 5: The transcriptomic host response in control and 4′-FIU-treated AGMs challenged with LASV Togo.**

We directly compared baseline, middle disease and late and transitional samples between positive control and treated monkeys to quantify the differences between the host response in treatment groups. Several DEGs were identified at baseline (Fig. 5c and Extended Data Fig. 2b); however, this may be explained by the outbred nature of the AGMs. No statistically significant differences were observed between groups in middle disease. However, following treatment, the circulating host response diverged, as we observed a robust set of DEGs when comparing late disease in positive controls with transitional time points in treated monkeys (Fig. 5d). Transcripts with higher expression in positive control AGMs during late disease include those associated with neutrophilia (such as CEBPB), the acute phase response (S100A8/9), endothelial activation (VCAM1), and an interferon-driven innate immune antiviral response (IFNB1 and IFNG). By contrast, treated monkeys showed higher expression of mRNAs associated with platelets and the coagulation cascade (CD9, PPBP and F13A1), monocytes (CSF1R), natural killer cells (KLRG1 and KLRC3) and T cells (CD5 and CD6) during transitional time points.

We performed cell-type deconvolution (Fig. 5e and Extended Data Fig. 2e) and pathway enrichment analysis (Fig. 5f) to examine the functional impacts of disease course divergence following 4′-FIU treatment. We calculated mean log₂-transformed counts per million (CPM) using the marker genes listed in Supplementary Data 1. Comparing treated monkeys at transitional time points to positive control monkeys during late disease showed that the treated monkeys had higher scores for mast cells, monocytes and T helper 1 (T_H1) cells and lower scores for dendritic cells and neutrophils. There was no significant difference in B cell, T cell, cytotoxic or natural killer (NK) cell scores. Pathway enrichment analysis (Fig. 5f) showed that many processes involved in end-stage Lassa fever were enriched in late positive control samples (for example, neutrophil degranulation, inflammatory response and pathogen-induced cytokine storm) but not enriched or even depleted in transitional treated samples. Other pathways, such as IFNα and IFNβ signalling, were enriched in both groups but to a greater extent in positive controls. Finally, several pathways and upstream regulators were enriched in transitional treated samples but not in late positive controls. These included dendritic cell apoptosis, kidney damage and the upstream regulators CCR2 and ZEB2 (a regulator of monocyte and CD8 T cell development⁴²).

Discussion

The current outbreak of LASV in Nigeria, which has caused 122 deaths as of the end of March 2025⁴³, and the recent importation of the disease to other countries emphasize the constant threat that LASV poses regionally in Africa as well as globally. Promising oral antivirals offer distinct advantages for rapidly responding to and containing outbreaks, and thus their development is of critical importance for future outbreak preparedness. Moreover, antivirals that exhibit broad effectiveness across genetically diverse families and strains of viruses are needed to mitigate the possibility of resistance mutations that may arise with the use of more narrowly targeted monoclonal antibodies or preventive vaccines.

Here we show that the antiviral ribonucleoside analogue 4′-FIU protects AGMs from lethal Lassa fever when treatment is started at an advanced stage of systemic infection, with all treated monkeys surviving to the study end-point, and 4 out of 5 (80%) monkeys exhibiting a rapid elimination of detectable circulating infectious virus. Monkeys treated with 4′-FIU mounted detectable binding and neutralizing humoral immune responses, moderated granulocyte responses and transcriptomic evidence of cellular (T cell, NK cell and B cell) contributions to development of immunity as well as controlled cytokinaemia, suggesting that the rapid reduction in viral load following 4′-FIU treatment probably allowed the development of a protective host immune response. Despite development of circulating immunity, persistence of infectious LASV in tissues (for example, immune-restricted locations) is a potential problem with any post-exposure therapy. In humans, LASV sequelae may appear as any of several reported morbidities, including verbal deficits, sensorineural hearing loss, ophthalmological anomalies, alopecia, ataxia and cerebellar ataxia⁴⁴. Although neutralizing antibody titres were relatively low in 4′-FIU-treated monkeys, even by 35 DPI, it is well-established that cell-mediated immunity has an important role in long-term immunity to LASV in both humans⁴⁵ and NHPs⁴⁶, and this comports with our previous findings in AGMs⁴⁰. Specifically, whereas both LASV-binding IgM and IgG antibodies are produced in response to infection in humans, neutralizing antibodies are often detected in only low quantities post-convalescence, sometimes up to several months afterwards, if they are detected at all^47,48,49. Although most surviving monkeys had low to moderate amounts of LASV vRNA in lymphoid and other tissues at the study end-point, all tissues from the surviving monkeys lacked gross pathology lesions and most tissues lacked IHC reactivity. Attempts to isolate infectious LASV from several tissues from all but one of the 4′-FIU-treated monkeys were unsuccessful. In previous LASV vaccine and treatment studies we were unable to recover infectious virus from surviving monkeys with levels of LASV RNA as high as 10.40 log₁₀ GE per g of tissue^18,26,27,29. This finding is not unusual or unique to LASV, as residual vRNA in the absence of infectious virus has been reported in Ebola and Marburg filovirus treatment studies^30,31,32,50, as well as for a number of different virus infection models⁵¹, and is probably due to ongoing immune clearance mechanisms (for example, neutralizing antibodies or cellular immunity), which may correspond to the residual inflammation-related transcriptomic signals detected in this work at the study end-point. The persistence of circulating viraemia and infectious LASV in tissues up to the study end-point in Tx4 is of concern, but may be due to the slower development of cellular immunity in this individual following treatment cessation, allowing rebounding viraemia. Housing of NHPs in high-containment biosafety level 4 (BSL-4) laboratories for extended periods of time presents many logistical challenges (such as limited availability of space and high cost); consequently, the ability to assess long-term sequelae that may occur has been constrained in most studies, including the current one. Although our study in AGMs was extended by 7 to 14 days longer than most BSL-4 animal studies, efforts to house surviving NHPs for even longer durations may be necessary to allow complete resolution of disease.

A limitation of our 4′-FIU study is that the AGMs were treated beginning six days after exposure to LASV Togo, which, although an advanced stage of disease, was a day earlier than we previously treated LASV Togo-infected macaques with Arevirumab-3²⁹. Further studies are needed to determine whether 4′-FIU can still provide protection against lethal Lassa fever if treatment is initiated seven days after exposure to LASV Togo. Notably, although Arevirumab-3 provided complete protection to macaques exposed to the prototype lineage IV Josiah strain, the lineage II 0043/LV/14 strain or the lineage III Ojoko strain^26,27,28 when treatment was initiated at advanced disease beginning on day 8 after LASV exposure, treatment of LASV Togo-infected macaques beginning at the same day 8 time point provided only partial protection²⁹. Combination therapy using 4′-FIU with antibodies such as Arevirumab-3 offers the possibility of extending the therapeutic window, as we have shown when combining remdesivir with antibodies for the treatment of filovirus disease^52,53, and could mitigate against the possibility of recurrence following the cessation of treatment with 4′-FIU. LASV variants from different lineages can exhibit notable differences in disease course, infection kinetics, mortality rate and tissue or cell tropism in NHP models of infection^{29,39,41,54,55,56}, but the likely mode of action targeting the L polymerase, which is relatively well conserved (compared to the genome overall), particularly in the enzymatic regions across lineages, and its in vitro and in vivo antiviral activity against Junin virus³ suggest that 4′-FIU treatment is likely to be similarly effective, or even more so, across lineages. Thus the therapeutic window of efficacy may be wider against less pathogenic isolates than the lineage VII LASV that we used. Further studies are needed to determine whether the ten-day regimen of 4′-FIU can be reduced and whether lower doses can still provide protection against LASV.

Pre-clinical Lassa fever treatment studies utilizing NHPs have almost exclusively used intramuscular injection with high doses of LASV to simulate a needlestick injury, which is a hazard related primarily to medical and research personnel, and results in a rapid disease course and very high mortality rates in NHPs^26,27,29. However, we recently reported that experimental infection of cynomolgus monkeys and AGM with LASV via mucosal exposure, which better represents most naturally acquired infections in humans, results in decreased mortality and an extended disease course that more accurately replicates human cases of Lassa fever^28,40. Thus it is likely that the window of effective intervention is widened for treatments such as 4′-FIU in humans with Lassa fever compared with preclinical animal models. Conversely, exposure to small-particle aerosol (mimicking an intentional release) results in the deposition of virus particles in the lower respiratory tract⁵⁷, and may hasten disease progression and severity and thus shorten the effective treatment window. The assessment of both modalities of respiratory exposure to LASV and subsequent treatment with 4′-FIU may therefore be warranted to determine the breadth of efficacy of this compound across disparate exposure routes.

Our results support the further development of 4′-FIU for the post-exposure prophylaxis and therapeutic treatment of LASV infections. The ease of supply, storage, distribution and especially administration of oral antivirals versus drugs delivered by injection or infusion would simplify the rapid deployment of both easily scalable post-exposure prophylaxis and early disease intervention. These strategies could also help with alleviating resistance to more invasive, time- and resource-consuming forms of treatment, leading to better support of medicinal interventions by local populations. Along with the use of efficacious preventive vaccines, oral antivirals such as 4′-FIU offer potential to further improve the management of future LASV outbreaks.

Methods

Virus and 4′-FlU

A lineage VII LASV isolate Germany ex Togo/2016/7082 (Genbank accessions KU961971 and KU961972) originated from serum of a LASV-infected patient who was medically evacuated to Germany from Togo⁵⁹. The study challenge material was from the third passage of Vero 76 cells (ATCC CRL-1587) exposed to this serum. Authentication of Vero 76 cells was not performed beyond that performed by the providing repository (American Type Culture Collection (ATCC)). The passage two isolate was from the European Virus Archive and was obtained from T. Rieger and S. Gunther and passaged once at University of Texas Medical Branch (UTMB). The cell supernatants were stored at −80 °C as aliquots of ~1 ml. No mycoplasma or endotoxin was detected (<0.5 endotoxin units (EU) ml⁻¹). 4′-FlU was obtained from MedChemExpress (HY-146246) and prepared in DMSO as 60 mM working stocks and frozen at −80 °C until use.

Study oversight

All study protocols described were approved by the UTMB Institutional Animal Care and Use Committee (IACUC) which were compliant with UTMB Institutional Biosafety Committee (IBC) guidelines under BSL-4 containment. UTMB animal facilities used in this work are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and adhere to principles specified in the eighth edition of the Guide for the Care and Use of Laboratory Animals, National Research Council.

NHP challenge and treatment

Prior to conducting the 4′-FIU study, a power analysis was performed to determine the minimum cohort size for the study. For LASV, assuming a one-tailed alpha of 0.05, sample sizes of 5 per group will provide >80% power to detect a difference in the proportion of surviving animals between the treatment group (100% survival rate) and the control group (0% survival rate), using a Fisher’s exact test. For the 4′-FIU treatment study, animals were assigned to treatment or control groups using a random number generator in Microsoft Excel. All animals were anaesthetized via intramuscular injection with ketamine (10 mg kg⁻¹) prior to procedures (for example, blood collection, weight and rectal temperature measurement). An initial study of 5 (2 male, 3 female) healthy adult AGMs (C. aethiops, PreLabs) weighing ~3.3 to 6.1 kg was performed to determine the pathogenic potential of LASV Togo in AGMs. All five monkeys were challenged by intramuscular injection in the left quadricep with a target dose of 1,000 PFU of LASV Togo (actual dose 840 PFU). As all five of these LASV Togo-infected AGMs succumbed to Lassa fever, a single control animal was used to confirm lethality of the challenge material used in the treatment study in which clinical parameters were compared with the five AGMs derived from the initial model experiment using the identical challenge material (that is, same virus passage). This allows for an ethical reduction in the number of animals used for data with highly predictable, lethal outcomes.

For the 4′-FIU treatment study, 6 (2 male, 4 female) healthy adult AGMs (PreLabs) weighing ~2.9–6.3 kg were challenged by intramuscular injection in the left quadricep with target dose of 1,000 PFU of LASV Togo (actual dose 1,312 PFU). Assignment to the treatment group or untreated positive control group was determined prior to challenge by randomization by Excel. Blinding was not performed for this study. Five monkeys were treated by oral gavage with 5 mg kg⁻¹ 4′-FIU (as a 1:10 suspension of 4′-FIU in DMSO to 1% methyl cellulose in water vehicle) beginning 6 days after LASV-Togo exposure. These 5 monkeys received daily doses of 4′-FIU for 10 days (6–15 DPI). The LASV Togo positive control monkey was not treated. The duration of the study was 35 days. This study was not blinded. All six AGMs were monitored daily and scored for disease progression with an internal LASV humane end-point scoring sheet approved by the UTMB Institutional Animal Care and Use Committee. The scoring changes measured from baseline included posture and activity level, attitude and behaviour, food intake, respiration, and disease manifestations, such as visible rash, haemorrhage, ecchymosis or flushed skin. A score of ≥9 indicated that an animal met the criteria for euthanasia.

Haematology and serum biochemistry

Total white blood cell counts, white blood cell differentials, red blood cell counts, platelet counts, haematocrit values, total haemoglobin concentrations, mean cell volumes, mean corpuscular volumes, and mean corpuscular haemoglobin concentrations were analysed from blood collected in tubes containing EDTA using a Vetscan HM5 laser-based haematologic analyser (Zoetis). Serum samples from blood collected in serum-separating tubes were tested for concentrations of albumin, amylase, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, blood urea nitrogen, calcium, creatinine, C-reactive protein, γ-glutamyltransferase, glucose, total protein and uric acid by using a Piccolo point-of-care analyser and Biochemistry Panel Plus analyser discs (Abaxis).

RNA isolation from LASV-infected AGM

On procedure days, 100 μl of blood from K2-EDTA collection tubes was collected prior to centrifugation and was added to 600 μl of AVL viral lysis buffer with 6 μl carrier RNA (Qiagen) for RNA extraction. For tissues, approximately 100 mg was stored in 1 ml RNAlater (Qiagen) for at least 24 h for stabilization. RNAlater was completely removed, and tissues were homogenized in 600 μl RLT buffer and 1% β-mercaptoethanol (Qiagen) in a 2 ml cryovial using a tissue lyser (Qiagen) and 1.4 mm ceramic beads. The tissues sampled included axillary and inguinal lymph nodes, liver, spleen, kidney, adrenal gland, lung, brain, pancreas, urinary bladder, ovary or testis, uterus or prostate, conjunctiva and eye. All blood samples were inactivated in AVL viral lysis buffer, and tissue samples were homogenized and inactivated in RLT buffer prior to removal from the BSL-4 laboratory. Subsequently, RNA was isolated from blood using the QIAamp viral RNA kit (Qiagen), and from tissues using the RNeasy minikit (Qiagen) according to the manufacturer’s instructions supplied with each kit.

Quantification of viral load

Primers and a probe targeting the N gene of LASV were used for real-time quantitative PCR (RT–qPCR) with the following primers and probe for LASV Togo: forward: 5′-ACAGTTGCAAATGGTGTGCT-3′; reverse: 5′-TGGCAGTGATCTTCCCATGT-3′; Probe: 6-carboxyfluorescein (FAM)–5′-TGCCTCTCCCAGAGTCAAGTGCA-3′–6 carboxytetramethylrhodamine (TAMRA). Viral RNA was detected using the CFX96 detection system (Bio-Rad) with one-step probe RT–qPCR kits (Qiagen) with the following cycle conditions: 50 °C for 10 min, 95 °C for 10 s, and 45 cycles of 95 °C for 10 s and 55.7 °C for 30 s. Threshold cycle (C_t) values representing viral genomes were analysed with CFX Maestro v.5.3.022.1030 software, and the data are presented as genome equivalents. To create the GE standard, RNA from viral stocks was extracted, and the number of strain-specific genomes was calculated using Avogadro’s number and the molecular weight of each viral genome.

Plaque titration of infectious LASV

Virus titration was performed by plaque assay using Vero 76 cells (ATCC CRL-1587) from all plasma or tissue samples as previously described²⁶. In brief, increasing tenfold dilutions of the samples were adsorbed to Vero 76 cell monolayers in duplicate wells (200 μl) and overlaid with 0.8% agarose in 1× Eagle’s minimum essentials medium (MEM) with 5% fetal bovine serum and 1% penicillin-streptomycin. After 5 days incubation at 37 °C, 5% CO₂, neutral red stain was added, and plaques were counted after 48 h of incubation. The limit of detection for this assay is 25 PFU ml⁻¹ for plasma and 250 PFU per g for tissues. Independent validation of the Vero 76 cell line was not performed outside of any authentication performed by ATCC. Cells were tested for mycoplasma contamination. No detectable mycoplasma or endotoxin levels were measured (<0.5 EU ml⁻¹).

ELISA

Sera collected at the indicated time points were tested for total anti-LASV IgG antibodies by ELISA using MaxiSorp (44204 ThermoFisher) uncoated and ReLASV Pf-GP Lineage VII IgG (Zalgen) antigen-absorbed 96-well plates. Sera were initially diluted 1:200 in duplicate and then two-fold through 1:25,600 in ELISA diluent (2% BSA in 1× PBS, and 0.2% Tween-20). After 1 h incubation, cells were washed 4 times with wash buffer (1× PBS with 0.2% Tween-20) and incubated for 1 h with a dilution of horseradish peroxidase (HRP)-conjugated anti-monkey IgG (1:10,000; Fitzgerald Industries). O-phenylenediamine (OPD) substrate tablet (Thermo Scientific; 34006) and stable peroxide buffer (Thermo Scientific; 34062) were added to the wells after four additional washes to develop the colorimetric reaction. The reaction was stopped with 2.5 M sulfuric acid for about 10 min after OPD addition and absorbance values were measured at a wavelength of 492 nm on a spectrophotometer (BioTek Cytation 5). Absorbance values were normalized by subtracting uncoated from antigen-coated wells at the corresponding serum dilution. End-point titres were defined as the reciprocal of the last adjusted serum dilution with a value ≥0.2.

Plaque reduction neutralization test

Neutralization titres were calculated by determining the dilution of serum that reduced 50% of plaques (PRNT₅₀) as previously described¹⁶.

Histopathology and immunohistochemistry

Tissue sections were deparaffinized and rehydrated through xylene and graded ethanols. The tissue sections were processed for IHC using the Thermo Autostainer 360 (ThermoFisher). Slides were treated with Proteinase K for 5 min to unmask antigens (Dako). Specific anti-LASV CLD4 NP immunoreactivity was detected using an anti-LASV CLD4 NP primary antibody (Zalgen Labs) at a 1:1,000 dilution for 60 min. The secondary antibody used was biotinylated goat anti-rabbit IgG (Vector Laboratories, BA-1000) at 1:200 for 30 min followed by Vector Streptavidin Alkaline Phosphatase at a dilution of 1:200 for 15 min (Vector Laboratories, SA-5100). Slides were developed with ImmPact Red Substrate Kit (Vector Laboratories SK-5105) for 20 min and counterstained with haematoxylin for 30 s.

Transcriptional analysis

Targeted transcriptomic analysis of the expression of 770 host mRNAs was quantified via the Nanostring NHP Immunology v.2 panel. RNA from whole blood was extracted via the QIAamp Viral RNA Mini Kit (Qiagen) and run on the nCounter SPRINT Profiler according to the manufacturer’s instructions. Output files were loaded into nSolver v.4.0, and background thresholding was performed using the default parameters. Samples that did not pass the nSolver internal quality checks were removed from downstream analysis. Thresholded count matrices were exported from nSolver and analysed with limma v.3.62.1 (edgeR v.4.4.1) in R v.4.4.2^58,60; scripts are available on GitHub (https://github.com/geisbert-lab/lasv-togo-4fiu).

Naive clustering with PCA and k-means was used to bin samples into six disease states (Fig. 5a and Extended Data Fig. 2a): baseline (0 DPI), early (3–4 DPI, sampled in control animals), middle (7 DPI), late (>7 DPI in control animals), transitional (9–12 DPI in treated animals), and recovered (≥15 DPI in treated animals). The disease course in positive control and 4′-FIU-treated animals was quantified by comparing each post-infection disease state to baseline samples (Fig. 5b and Extended Data Fig. 2c,d). Disease course differences between positive control and treated animals were quantified by comparing baseline and middle disease states (Fig. 5c and Extended Data Fig. 2b). Transitional samples in treated animals and late samples in positive control animals were also compared to capture where disease course diverged (Fig. 5d). For all comparisons, genes with a false discovery rate-adjusted P value <0.05 and a log₂ fold change >1 or <−1 were considered significantly differentially expressed.

Cell-type deconvolution (Fig. 5e and Extended Data Fig. 2e) was performed by calculating a cell-type score per sample, which was defined as the mean log₂ counts per million of the cell-type marker genes defined by Nanostring. Canonical signalling pathways, functions and upstream regulators were identified via Ingenuity Pathway Analysis⁶¹ using the differentially expressed genes from controls at late time points compared to baseline and treated at transitional time points compared to baseline (Fig. 5f).

Statistics and reproducibility

Data collection and analysis were not performed blinded to the conditions of the experiments. No animals or data points were excluded from analysis in this work. Owing to the small numbers of animals used in this study, determination of effects of sex to treatment success were not possible; however, we did attempt to ensure that close to even distribution of each sex was represented in the experimental groups. Description of a priori power analysis to determine group size is described in ‘NHP challenge and treatment’. With regard to histopathological analysis of photomicrographs, representative photomicrographs were qualitatively considered to display lesions that were nominally or ordinally measured by masking of the pathologist post-examination and ranking lesions to satisfy the study objectives. Additionally, thorough examinations of multiple slides of the target tissues multiple times (at least two times per tissue) were performed in a timely manner to maintain interpretation consistency, which comports with established criteria⁶². Additional data analysis and plotting was performed in Microsoft Excel (current Office 360 version) and/or GraphPad Prism v. 9.3.1 and/or GraphPad Prism v. 10.5.0.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All study data are included in the main text or the supplementary materials. Nanostring transcriptomic data are available via NCBI Gene Expression Omnibus (GEO) under accession GSE294797. Requests for materials should be addressed to T.W.G. Lassa virus is a BSL-4 pathogen that requires permits from the US Centers for Disease Control and Prevention. Source data are provided with this paper.

Code availability

Bioinformatic code is available on GitHub (https://github.com/geisbert-lab/lasv-togo-4fiu) and via Zenodo (https://doi.org/10.5281/zenodo.15230970 (ref. ⁶³)).

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Acknowledgements

This study was supported in part by the Department of Health and Human Services, National Institutes of Health (NIH) grant U19AI181930 to T.W.G., in part by UTMB Department of Microbiology and Immunology funds to T.W.G., and by NIH grant UC7AI094660 for BSL-4 operations support of the Galveston National Laboratory. The authors thank the UTMB Animal Resource Center for husbandry support of laboratory animals, J. Geisbert for preparation of clinical pathology data and M. Harrison for assistance with animal studies.

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Galveston National Laboratory, University of Texas Medical Branch, Galveston, TX, USA
Robert W. Cross, Jacquelyn Turcinovic, Abhishek N. Prasad, Viktoriya Borisevich, Krystle N. Agans, Daniel J. Deer, Rachel O’Toole, Natalie S. Dobias, Courtney Woolsey, Karla A. Fenton & Thomas W. Geisbert
Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA
Robert W. Cross, Jacquelyn Turcinovic, Abhishek N. Prasad, Viktoriya Borisevich, Krystle N. Agans, Daniel J. Deer, Rachel O’Toole, Natalie S. Dobias, Courtney Woolsey, Karla A. Fenton & Thomas W. Geisbert

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Robert W. Cross
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Contributions

R.W.C. and T.W.G. conceived and designed the experiments. D.J.D. performed the challenges and treatments. R.W.C., A.N.P., D.J.D. and C.W. performed procedures and conducted clinical observations. J.T. performed the gene expression assays and transcriptomic analyses. K.N.A. and V.B. performed clinical pathology. K.N.A. performed the PCR assays. V.B. performed the LASV plaque assays and neutralization assays. R.O. performed the ELISAs. N.S.D. performed the IHC assays. K.A.F. performed gross pathologic, histologic and IHC analysis of the data. All authors analysed the data. T.W.G. wrote the paper with additions from J.T., A.N.P. and K.A.F. A.N.P. prepared the Figures. A.N.P. and T.W.G. prepared the tables. R.W.C. and A.N.P. edited the manuscript. All authors had access to the data and approved the final version of the manuscript.

Corresponding author

Correspondence to Thomas W. Geisbert.

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The authors declare no competing interests.

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Nature thanks Juan Carlos de la Torre and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer review reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Comparison of peak viral load and day of detection in LASV-challenged AGMs treated with 4′FIU beginning 6 days p.i.

The peak viral load and the day the peak viral load was detected was assessed by RT-qPCR of LASV vRNA isolated from whole blood (a) or plaque titration of infectious virus from plasma (b). For statistical comparison, the in-study control (C-6) was grouped with four positive control animals from the pilot study for a total n = 5. Data from subject C-1 was omitted from this analysis as blood was not sampled from this subject at the terminal timepoint. The horizontal dashed line in the leftmost panels of (a) and (b) denotes the LOQ for the assay. Statistical significance was determined using the non-parametric Mann-Whitney U-test. Reported p-values are two-tailed.

Extended Data Fig. 2 Targeted transcriptomics of circulating blood during LASV infection.

(a) Timeline of all samples subject to Nanostring transcriptomic analysis in untreated positive control and 4′-FIU-treated animals colored by disease state grouping. (b) Comparing baseline positive control animals to baseline treated animals identified 1 gene with significantly higher expression in positive control animals and 10 genes with significantly higher expression in 4′-FIU-treated animals. (c-d) Volcano plots showing differential gene expression in positive control animals (c) and 4′-FIU-treated animals (d) relative to baseline in each postinfection disease state. (e) Cell type deconvolution across baseline, mid-disease, late disease, -and transitional time points.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2.

Reporting Summary

Supplementary Data

Supplementary Data 1, containing Nanostring cell-type marker genes used for deconvolution of cell types.

Peer Review File

Source data

Source Data Figs. 1–3 and 5

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Cross, R.W., Turcinovic, J., Prasad, A.N. et al. Oral 4′-fluorouridine rescues nonhuman primates from advanced Lassa fever. Nature (2026). https://doi.org/10.1038/s41586-025-09906-y

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Received: 15 April 2025
Accepted: 12 November 2025
Published: 07 January 2026
Version of record: 07 January 2026
DOI: https://doi.org/10.1038/s41586-025-09906-y