Fig 1.
4’-Fluorouridine (4’-FIU) exposure in hamster following a single and multi-oral dose.
(A) Timeline illustration of the pharmacokinetics study conducted after 4’-FIU oral gavage (single dose). Female hamsters (n = 8) received either 2 or 10 mg/kg 4’-FIU. Blood samples were collected as indicated prior final blood and tissue harvest at 24 hours post dosing. (B-C) 4’-FIU concentration in plasma and brain tissues over time. Note that the bioactive triphosphate form of 4’-Fluorouridine (4’-FIU-TP) is detected in the tissue. (D) Timeline illustration of the 4’-FIU oral gavage multi dose treatment prior to the pharmacokinetics study. Hamsters (n = 8) received 5 doses of 10 mg/kg 4’-FIU treatment prior serial blood collection as in illustration A. (E) 4’-FIU concentration in plasma over time. Data are presented as median with error or mean with standard deviation. Panel A and D partially created with BioRender. Plemper, R. (2026) https://BioRender.com/6ri40jk.
Fig 2.
Efficacy of orally dosed 4’-FIU against an intraperitoneal rNiV-B Gluc challenge in the Syrian hamster model (Cohort 1).
Five groups of hamsters (n = 10 per group, male and female evenly distributed) received each a targeted 105 PFU dose of rNiV-B Gluc through the intraperitoneal route and were then either immediately or 8-10 hours later (delayed) treated daily and for 7 days with vehicle, or 10 to 25 mg/kg 4’-FIU. One additional group was used as negative control (A). Created in BioRender. Pearson, M. (2026) https://BioRender.com/983h0ga. All six groups were further monitored for 21 days to assess for (B) survival as well as (C, D) clinical signs of disease. Note that some data sets are either (B) aligned or (C, D) staggered for improving visualization of values from each subject over time. The yellow shade (B, C, D, and in legend) indicates days of treatment. The Log-rank test was applied for comparison of survival curves and the Chi-square test was used to compare mortality rates. Asterisks ****) indicate statistical differences of p < 0.0001 between groups.
Fig 3.
Efficacy of 4’-FIU at controlling rNiV-B Gluc shedding and dissemination in the Syrian hamster model (Cohort 1).
(A, B) Nasal and oral shedding of virus was assessed during treatment (day 2, 4, and 6) and up to day 10 post challenge through detection of luminescence from the Gaussia luciferase activity. (C) Viremia was assessed through the same method as in A and B. Data are shown as mean with replicates in A, B, and C. Open symbols in C correspond to values from survivors from the challenge.
Fig 4.
Effect of prolonged 4’-FIU treatment against an intraperitoneal rNiV-B Gluc challenge in the Syrian hamster model (Cohort 2).
Four groups of hamsters (n = 10 per group, male and female evenly distributed) received each a targeted 105 PFU dose of rNiV-B Gluc through the intraperitoneal route and were then dose similarly as in cohort 1 for 28 days total or 21 consecutive days with 10 mg/kg 4’-FIU. Two additional groups were used as virus and negative controls, similar to cohort 1 (A). Created in BioRender. Pearson, M. (2026) https://BioRender.com/983h0ga. Animals were monitored up to day 34 or 51 after challenge to assess for (B) survival and (C, D) clinical signs of disease. Note that some data sets are either (B) aligned or (C, D) staggered for improving visualization of values from each subject over time. The yellow shade (C, D, and legend) indicates days of treatment whereas arrowheads (B) indicate treatment cessation. The Log-rank test was applied for comparison of survival curves, and the Chi-square test was used to compare mortality rates. Asterisks (*, ****) indicate statistical differences of respectively p < 0.05, and 0.0001 between groups.
Fig 5.
Effect of prolonged 4’-FIU treatment on rNiV-B Gluc shedding and dissemination in the Syrian hamster model (Cohort 2).
(A, B) Nasal and oral shedding of virus was assessed during treatment (day 1, and 19 or 28) and at the end of study through detection of luminescence from the Gaussia luciferase activity. (C) Viremia was assessed through the same method as in A and B. Data are shown as mean with replicates in A, B, and C. Open symbols in A, B, and C correspond to values from subjects succumbing to disease. One way ANOVA followed by Tukey’s multiple comparisons test was used to compare shedding and viremia data between groups. Asterisks (*, **, ****) indicate statistical differences of respectively p < 0.05, 0.01, or 0.0001 between groups. “ns” for not significant.
Fig 6.
Virus nucleocapsid antigen distribution in the lung and associated lesions following rNiV-B Gluc challenge.
(A) Virus nucleocapsid staining (dark red signal) in the alveolar area including in type II pneumocytes and endothelial cells of the small vasculature. (B) No specific virus nucleocapsid staining is observed in the alveolar space of mock tissues. (C) Hematoxylin and eosin staining showing virus-induced lung lesions including syncytia formation, necrosis, and hemorrhages. (D) Hematoxylin and eosin staining of a mock-infected untreated animal showing no remarkable lung lesions.
Fig 7.
Virus nucleocapsid antigen distribution in the brain and associated lesions following rNiV-B Gluc challenge.
(A, B) Virus nucleocapsid staining (dark red signal) in the hypothalamus, thalamus, and pons including in neurons, and endothelial cells. Hematoxylin and eosin staining showing virus-induced brain lesions including (C) mononuclear cells infiltrations, (D) focal necrosis, and (E) meningitis in the cortex. (F) Hematoxylin and eosin staining of a negative control showing no remarkable brain lesions.
Fig 8.
Nonsynonymous single-nucleotide variations are in components of the viral replicase.
(A) Structural modeling depicting the viral replicase complex, consisting of the vRNA wrapped in N forming the vRNP, and displaced N where the RdRp, L, is threaded along the template vRNA; L is associated with a tetramer of P. PDB structures 7NT5 (NiV-N bound to RNA) and 9CGI (NiV-L and NiV-P) were manipulated in UCSF ChimeraX to generate these visualizations. NiV-N is depicted in light green, vRNA is depicted in black, NiV-P is depicted in blue, and NiV-L is depicted in yellow. (B) Visualization of the internal cavity of NiV-L with regions corresponding to the GDNE motif, priming loop, and intrusion loop indicated in pink, purple, and yellow, respectively. The T1341 residue identified in one virus as T1341I is highlighted in red. The PRNTase domain is depicted in green, whereas the vRdRp domain is depicted in light blue. (C) Amino acid alignment of the region of the intrusion loop from L proteins of NiV-B, NiV-M, HeV (Hendra virus), CedV (Cedar virus), GhV (Ghana virus), AngV (Angavokely virus), and MeV (Measles virus); T1341 and corresponding residues are highlighted in red, and the highly conserved HR motif is highlighted in orange. (D) Structural depiction of NiV-N bound to vRNA, with the T191 residue highlighted in red. Adjacent residue R192, which is involved in RNA binding, is shown in blue. (E) Amino acid alignment of the region of N containing T191 from NiV-B, NiV-M, HeV, CedV, GhV, AngV, and MeV. T191 and corresponding residues are highlighted in red. (F) Amino acid alignment of the region of P containing I286 from NiV-B, NiV-M, HeV, CedV, GhV, AngV, and MeV. I286 and corresponding residues are highlighted in red. For all sequence alignments, ClustalW was implemented in MEGA-X; phylogenetic trees depict topology only and were constructed for each alignment using the neighbor-joining method in MEGA-X. Conserved residues are shown with an * below each respective alignment. Numbering and mutants for each alignment refer to NiV Bangladesh sequences for each respective protein.
Fig 9.
In vitro efficacy of 4’-FIU against rNiV-B Gluc incorporating T191S (N gene), I286V (P gene), or T1341I (L gene) change and viral fitness in human astrocytes.
(A) Antiviral assays were concomitantly conducted in Vero CCL81 and on 4 individual 96-well plates with each mock and virus control (8 biological replicates). Four biological replicates were used per 4’-FIU concentration per plate. Determination of half-maximal effective concentration (EC50) against each virus at day 2 post-infection was done using the luminescence reporter on a Cytation 5 (Agilent). Values indicate the percentage of virus replication based on intensity of reporter gene expression from 4’-FIU- versus vehicle-treated infected cells. Results are expressed as the average of biological replicates, and error bars represent standard deviations. (B) Growth kinetic of rNiV-B Gluc and mutants in primary human astrocytes. Supernatants were collected 6 times between 1 and 48 hours post infection and were then tittered by plaque assay. Results are expressed as the average of biological triplicates, and error bars represent standard deviations. Horizontal dotted line shows the limit of detection.