Synthesis of compound

The nucleoside analog 2-BFU was synthesized at the Laboratory of Biochemical Pharmacology, Emory University School of Medicine, following previously established protocols [17]. The compound’s purity exceeded 98%, as confirmed by nuclear magnetic resonance (NMR) and liquid chromatography–mass spectrometry (LC-MS) analysis.

Infection of A129 mice with DENV-2Virus

The DENV-2 strain used in this study was kindly provided by the Duke-NUS Medical School, Singapore [18]. The virus was propagated in Aedes albopictus C6/36 cells, obtained from the Rio de Janeiro Cell Bank (BCRJ – 0343). Cells were incubated at 37 °C in Leibovitz’s L-15 culture medium supplemented with 1.5% HEPES, 1% antibiotics, 1% L-glutamine, 1% non-essential amino acids, and 10% fetal bovine serum for 5–7 days. After incubation, the culture supernatant was centrifuged at 600 × g for 10 min to remove cellular debris, then concentrated using a Vivacell 1000 centrifugal concentrator (2000 × g for 10 min). The concentrated viral stock was aliquoted and stored at − 80 °C. Viral titers were determined by plaque-forming unit (PFU) assay as described in Sect. 2.2.5.

Ethics statement

All animal procedures were performed in accordance with Brazilian regulations (Law 11.794/2008) governing the ethical use of animals in research. The experimental protocol was approved by the Animal Ethics Committee of the Federal University of Minas Gerais (CEUA/UFMG) under protocol number 234/2019.

Mouse infection

In vivo experiments were conducted using type I interferon receptor-deficient A129 mice on an SV129/Ev background. Male and female mice (8–10 weeks old) were housed under specific pathogen-free conditions at 23 °C with a 12-h light/dark cycle and ad libitum access to food and water at the Immunopharmacology Laboratory, ICB/UFMG.

Mice were infected via subcutaneous (intraplantar) injection with 2 × 10² PFU of DENV-2 in 30 µL of phosphate-buffered saline (PBS). Treatment with 2-BFU (5 or 15 mg/kg in 200 µL) was administered intraperitoneally 12 h post-infection and repeated every 12 h until day 3 or day 5, depending on the planned endpoint. Control mice received 200 µL of saline (0.9% NaCl) at the same intervals. Clinical signs and body weight were monitored daily. Mice were euthanized on day 3 or day 5 post-infection for sample collection. Blood, spleen, liver, and brain were harvested for viral load quantification, inflammatory mediator analysis, and histopathological examination. All procedures were performed under ketamine/xylazine anesthesia. Each group included six mice (three males and three females).

Hematological analysis

To evaluate thrombocytopenia, blood was collected from the cava vein using heparinized syringes (final concentration: 50 U/mL). Platelet counts were performed using a Neubauer chamber and expressed as platelets per microliter of blood. Plasma, spleen, liver, and brain samples were used for viral titration. Blood samples were centrifuged at 3000 × g for 15 min at room temperature, and plasma was stored at − 80 °C until analysis.

Viral titers

Spleen, liver, and brain tissues were aseptically collected on day 3 post-infection and stored at − 80 °C. Tissues were weighed, ground with a mortar and pestle, and homogenized to 10% (w/v) suspensions in RPMI 1640 medium (without fetal bovine serum). Viral loads in plasma and tissue homogenates were determined by direct plaque assay using Vero CCL-81 cells, following standard protocols. Results were expressed as PFU per 100 mg of tissue or per mL of plasma. The detection limit was 100 PFU/g of tissue or 100 PFU/mL of plasma.

Cytokine and chemokine analysis

Plasma samples collected on days 3 and 5 post-infection were analyzed for pro-inflammatory cytokines and chemokines, including IFN-γ, IL-6, CXCL1, and CCL2. Quantification was performed using commercial DuoSet ELISA kits (R&D Systems), following the manufacturer’s instructions. Cytokine concentrations were expressed as pg/mL of plasma.

Histopathological analysis

Liver samples were collected on day 5 post-infection, fixed in 10% neutral-buffered formalin for 24 h, and embedded in paraffin. Section (4 μm thick) were stained with hematoxylin and eosin (H&E) and examined using an Axioskop 40 microscope (Carl Zeiss, Göttingen, Germany) equipped with a PowerShot A620 digital camera (Canon, Tokyo, Japan). Inflammatory cell infiltration, blood vessel dilation, hepatocyte degeneration, necrosis, and hemorrhage parameters were scored using a five-point scale: 0 (absent – 0%), 1 (minimal – 1–20%), 2 (slight – 21–40%), 3 (moderate – 41–60%), 4 (marked – 61–80%), and 5 (severe – 81–100%), in accordance with extension and intensity of the alterations, as previously described [18]. Two sections per animal were analyzed, and results were expressed as the percentage of mice exhibiting each infiltration score.

In vitro infection with DENV-1–4 and treatment with 2-BFU

VERO CCL-81 cells (code 0245) were obtained from Banco de Células do Rio de Janeiro (BCRJ) repository and cultured in RPMI 1640 medium (Cultilab). For in vitro experiments, low passage human clinical isolates of DENV serotypes DENV-1 (EDEN 2402), DENV-2 (EDEN 3295), DENV-3 (EDEN 863), and DENV-4 (EDEN 2270) were propagated in Aedes albopictus C6/36 cells, and the supernatants of infected cells were harvested, filtered, concentrated, tittered by plaque assay, and stored at − 80 °C until Vero cells were seeded in 96-well plates at a density of 1 × 10^5 cells per well. After 24 h, cells were infected with one of the four dengue virus serotypes (DENV-1, DENV-2, DENV-3, or DENV-4) at a multiplicity of infection (MOI) of 0.01 for 1 h at 37 °C. Following adsorption, the inoculum was removed, and cells were washed once with PBS. Fresh culture medium alone or medium containing RS2850 (5 µM) was then added. Supernatants were collected 48 h post-infection, as previously established [16]. Viral titers were determined by plaque assay, and results were expressed as log10 PFU/mL.