The 2′-α-Fluoro,2′-β-Bromo uridine phosphoramidate prodrug (2-BFU) reduces dengue virus replication and attenuates infection-induced thrombocytopenia In Vivo

TL;DR

A new drug called 2-BFU was found to reduce dengue virus levels and improve platelet counts in mice, suggesting potential as a treatment.

Contribution

2-BFU is a novel prodrug that shows antiviral efficacy and reduces thrombocytopenia in a murine model of dengue.

Findings

2-BFU significantly reduced DENV-2 viral titers in plasma, spleen, and liver.

2-BFU attenuated DENV-2-induced thrombocytopenia at 72 and 120 hours post-infection.

2-BFU did not affect inflammatory mediators or prevent weight loss and mortality.

Abstract

Dengue virus (DENV) infection remains a major global health concern, with clinical manifestations ranging from mild febrile illness to severe, life-threatening complications. In the absence of specific antiviral therapies, the development of novel treatment strategies is crucial. This study evaluates the in vivo antiviral efficacy of 2-BFU, a 2′-α-fluoro,2′-β-bromouridine monophosphate prodrug, in a murine model of DENV Serotype 2 (DENV-2) infection. Adult A129 mice were infected subcutaneously with DENV-2 and treated intraperitoneally with 2-BFU at 15 mg/kg, starting 12 h post-infection. Treatment with 2-BFU significantly reduced viral titers in plasma, spleen, and liver, demonstrating potent antiviral activity. Moreover, 2-BFU effectively attenuated DENV-2 -induced thrombocytopenia at 72- and 120-h post-infection. However, the treatment did not significantly affect the production of inflammatory mediators, nor did it prevent infection-associated weight loss or mortality. These findings suggest that 2-BFU holds promise as an antiviral candidate by lowering viral burden and ameliorating thrombocytopenia, although it may require adjunctive anti-inflammatory strategies to improve overall clinical outcomes. Further investigation is warranted to optimize its therapeutic potential for dengue.

The online version contains supplementary material available at 10.1007/s00430-026-00868-0.

Figures

The nucleoside analog 2-BFU reduces viral titers in DENV-2-targeted tissues. a Adult A129 mice were subcutaneously infected with 2 × 10² PFU of DENV-2 in the right paw. Mice received intraperitoneal injections of 2-BFU at doses of 5 or 15 mg/kg, administered twice daily starting 12 h post-infection, and continued for 72 h. After 3 days of treatment, the mice were euthanized, and blood and tissue samples were collected for analysis. Each experimental group included six mice, with equal distribution of males and females. Viral titers in plasma (b), spleen (c), liver (d), and brain (e) were quant

The nucleoside analog 2-BFU reverses thrombocytopenia in DENV-2-infected mice. Adult A129 mice were subcutaneously infected (intraplantar) with 2 × 10² PFU of DENV-2. Mice were treated with intraperitoneal injections of 2-BFU (5 or 15 mg/kg) twice daily, starting 12 h post-infection and continuing until 72—or 120-h post-infection (a). Each group consisted of six mice, with equal distribution of males and females. Mice were monitored daily for weight loss (b). After 3 or 5 days of treatment, blood samples were collected to assess platelet counts (c). Lymphocyte and neutrophil counts were measur

Treatment with 2-BFU did not affect weight loss or lethality in dengue virus-infected mice. Adult A129 mice were infected via the intraplantar subcutaneous route with 2 × 10² PFU of DENV-2. Following infection, mice received intraperitoneal injections of 2-BFU (15 mg/kg) twice daily, starting 12 h post-infection and continuing until 120 h post-infection. Mice were euthanized after 3 or 5 days of treatment, and blood and tissues were collected and processed for further analysis (A). Each group consisted of six mice, with equal distribution of males and females. Mice were monitored daily for wei

Full text

Introduction

Dengue is a systemic disease with a wide range of clinical manifestations, caused by one of four serotypes of the Dengue virus (DENV1-4), which belongs to the Orthoflavivirus genus. It is transmitted to humans through the bite of infected female mosquitoes from the Aedes genus [1–3]. Recent estimates suggest that around 4 million people are affected by DENV infections globally each year [4].

Most DENV-infected individuals (~ 75%) remain asymptomatic or develop a mild form of the disease, known as dengue without warning signs. This form is characterized by flu-like symptoms such as headache, myalgia, arthralgia, and rash. However, a subset of patients progresses to more severe clinical forms, classified as dengue with warning signs or severe dengue. These forms are associated with increased vascular permeability, exacerbated systemic inflammation, respiratory distress, severe bleeding, and liver dysfunction [5]. Although vaccines like CYD-TDV (Dengvaxia^®^) and TAK-003 (QDENGA) have been approved in certain countries, their use remains limited due to age restrictions (e.g., individuals over 59 years) and an elevated risk of severe disease in some populations [6]. Currently, no antiviral drugs are approved for the treatment of DENV infection, and clinical management remains restricted to supportive care.

Non-structural protein 5 (NS5) is a highly conserved and multifunctional protein essential for flavivirus replication [7, 8]. It comprises two key domains: an N-terminal methyltransferase domain, involved in RNA capping and immune evasion, and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. Given its crucial role in viral replication, NS5 has emerged as a promising target for antiviral drug development [8, 9]. Nucleoside analogs, a class of molecules that mimic natural nucleosides, have long been used to alter cellular metabolism in cancer and to inhibit the replication of various viruses [10–14]. Once inside the host cell, these analogs are phosphorylated by cellular kinases into nucleoside triphosphates (NTPs), which are erroneously incorporated into the viral RNA by RdRp, disrupting chain elongation and halting RNA synthesis [11, 15].

Previous studies have identified several nucleoside analogs with selective antiviral activity against flaviviruses such as DENV and Japanese encephalitis virus (JEV), in both in vitro and in vivo models. Among these, the phosphoramidate prodrug 2′-α-fluoro,2′-β-bromo uridine (2-BFU) - originally developed as a hepatitis C virus (HCV) inhibitor—has shown potent antiviral activity against yellow fever virus (YFV) in primary human macrophages, hepatocyte spheroids, and A129 mice [16, 17]. In this study, we evaluated the antiviral efficacy of 2-BFU in a murine model of DENV-2 infection that replicates key features of human disease, including thrombocytopenia, systemic inflammation, and widespread viral dissemination. Our findings demonstrate that 2-BFU significantly reduces viral titers in the blood, liver, and spleen, and improves thrombocytopenia during DENV-2 infection, supporting its potential as a antiviral candidate.

Materials and methods

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-2

Virus

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.

Results

The nucleoside analog 2-BFU exhibits Anti-DENV-2 activity in vivo

We assessed the antiviral efficacy of the nucleoside analog 2-BFU using an A129 immunocompromised mouse model of DENV-2 infection. Mice were inoculated subcutaneously with a clinical isolate of DENV-2 (2 × 10² PFU), and treatment with 2-BFU was initiated 12 h post-infection, administered intraperitoneally twice daily at doses of 5 and 15 mg/kg (Fig. 1a). In vehicle-treated animals, DENV-2 infection led to high viral titers in plasma, spleen, liver, and brain (Fig. 1b–e). Although plasma viral titers in the 5 mg/kg group showed variability and did not differ significantly from vehicle, treatment with 2-BFU resulted in reduced viral titers in the liver at both doses (Figure S1). Notably, the higher dose (15 mg/kg) reduced viral titers in several tissues Fig. 1b–e). These findings demonstrate that 2-BFU possesses dose-dependent antiviral activity in vivo against DENV-2, effectively reducing viral burden systemically and supporting its potential as a therapeutic candidate for dengue virus infection. Finally, to determine whether the antiviral activity observed against DENV-2 also extended to other serotypes, VERO CCL-81 cells were infected with each of the four DENV serotypes (DENV-1–4). Treatment with 2-BFU significantly reduced viral titers across all serotypes (Fig. S2). These findings confirm that 2-BFU exhibits broad, pan-serotype antiviral activity against DENV.

Fig. 1. The nucleoside analog 2-BFU reduces viral titers in DENV-2-targeted tissues. a Adult A129 mice were subcutaneously infected with 2 × 10² PFU of DENV-2 in the right paw. Mice received intraperitoneal injections of 2-BFU at doses of 5 or 15 mg/kg, administered twice daily starting 12 h post-infection, and continued for 72 h. After 3 days of treatment, the mice were euthanized, and blood and tissue samples were collected for analysis. Each experimental group included six mice, with equal distribution of males and females. Viral titers in plasma (b), spleen (c), liver (d), and brain (e) were quantified by plaque assays. Results are expressed as PFU per mL of plasma or per mg of tissue. Significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test: *p < 0.05, **p < 0.01, ***p < 0.001, and **p < 0.0001, treated-infected versus untreated-infected groups. Data is representative of two independent experiments.

2-BFU prevents thrombocytopenia and partially ameliorates liver inflammation induced by DENV-2 infection in mice

We next evaluated the effects of 2-BFU on clinical, hematological, and inflammatory parameters to determine whether treatment at 15 mg/kg mitigates DENV-2-induced morbidity and tissue damage in A129 mice. Following DENV-2 infection, mice were treated with 2-BFU and euthanized at 72- and 120-h post-infection for assessment of body weight, platelet and leukocyte counts, and liver pathology (Fig. 2a). As expected, DENV-2 infection led to significant body weight loss and thrombocytopenia in A129 mice. Although 2-BFU treatment did not significantly alter body weight loss (Fig. 2b), it markedly attenuated thrombocytopenia at both 72- and 120-h post-infection (Fig. 2c, S3). DENV-2 infection also induced an increased neutrophil-to-lymphocyte ratio (NLR), a recognized marker of disease severity. However, 2-BFU treatment did not reduce the NLR at either time point (Fig. 2d, S3). Histopathological analysis of liver tissue at 120 hpi (peak of hepatic inflammation in DENV-2-infected A129 mice) revealed substantial leukocyte infiltration, dilated sinusoidal capillaries, and vascular congestion (Fig. 2e and f). Treatment with 2-BFU resulted in a modest but consistent reduction in hepatic inflammation, as evidenced by lower inflammatory scores and reduced leukocyte infiltration. Together, these results indicate that 2-BFU can ameliorate key hematological manifestations of DENV-2 infection—most notably thrombocytopenia—and offers partial attenuation against liver inflammation.

Fig. 2. The nucleoside analog 2-BFU reverses thrombocytopenia in DENV-2-infected mice. Adult A129 mice were subcutaneously infected (intraplantar) with 2 × 10² PFU of DENV-2. Mice were treated with intraperitoneal injections of 2-BFU (5 or 15 mg/kg) twice daily, starting 12 h post-infection and continuing until 72—or 120-h post-infection (a). Each group consisted of six mice, with equal distribution of males and females. Mice were monitored daily for weight loss (b). After 3 or 5 days of treatment, blood samples were collected to assess platelet counts (c). Lymphocyte and neutrophil counts were measured and expressed as the ratio between the two leukocyte types (d). Liver samples were collected for histological analysis to evaluate tissue damage and inflammatory infiltrate, with results expressed as the percentage of mice exhibiting damage (e), and representative images for each group are shown (f). Significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test: *p < 0.05, **p < 0.01, ***p < 0.001, and **p < 0.0001, treated-infected versus untreated-infected groups. Data is representative of two independent experiments.

The nucleoside analog 2-BFU does not reduce the concentration of inflammatory mediators in the plasma of infected mice

The effect of 2-BFU treatment on circulating inflammatory mediators was assessed in the plasma of DENV-2–infected A129 mice. Administration of 2-BFU to mock-infected animals did not affect basal plasma levels of IFN-γ, IL-6, CXCL1, or CCL2 (Fig. 3a–d). DENV-2 infection was associated with increased plasma concentrations of IFN-γ at 72 and 120 h post-infection and of IL-6 at 120 h post-infection, whereas CXCL1 and CCL2 levels remained unchanged compared with mock controls (Fig. 3a–d). No significant differences in the levels of these mediators were observed between vehicle- and 2-BFU–treated DENV-2–infected mice at either 72–120 h post-infection.

Fig. 32-BFU treatment does not alter systemic cytokine and chemokine levels in DENV-2–infected mice. Adult A129 mice were infected via the intraplantar route with 2 × 10² PFU of DENV-2 and treated intraperitoneally with 2-BFU (5 or 15 mg/kg) twice daily, starting 12 h post-infection and continuing until euthanasia at 72–120 h post-infection (hpi) (A). Each experimental group consisted of six mice, with equal numbers of males and females. Plasma concentrations of interferon-γ (IFN-γ) (a), interleukin-6 (IL-6) (b), chemokine (C-X-C motif) ligand 1 (CXCL1) (c), and chemokine (C-C motif) ligand 2 (CCL2) (d) were quantified by enzyme-linked immunosorbent assay (ELISA) in infected and mock-infected mice, treated or not with 2-BFU. Treatment with 2-BFU did not significantly modify the circulating levels of these cytokines and chemokines compared with infected, untreated controls at either time point. Statistical significance was assessed by one-way ANOVA followed by Tukey’s multiple-comparisons test. P values are shown for the following comparisons: Mock vs. Mock + 2-BFU; Mock vs. DENV-2 (72 hpi); Mock vs. DENV-2 (120 hpi); DENV-2 (72 hpi) vs. DENV-2 + 2-BFU (72 hpi); and DENV-2 (120 hpi) vs. DENV-2 + 2-BFU (120 hpi)

Therapeutic administration of 2-BFU does not prevent body weight loss or lethality in A129 mice infected with DENV-2

To assess the protective effects of 2-BFU against DENV-2-induced weight loss and mortality, we administered 2-BFU starting 12 h post-inoculation, continuing twice daily until 120 h post-infection (Fig. 4). Mice were monitored for body weight changes and lethality over 14 days following DENV-2 inoculation. By day 9 post-infection (dpi), nearly 100% of the infected mice had succumbed to the infection. Importantly, 2-BFU treatment did not prevent the body weight loss or lethality associated with DENV-2 infection.

Fig. 4. Treatment with 2-BFU did not affect weight loss or lethality in dengue virus-infected mice. Adult A129 mice were infected via the intraplantar subcutaneous route with 2 × 10² PFU of DENV-2. Following infection, mice received intraperitoneal injections of 2-BFU (15 mg/kg) twice daily, starting 12 h post-infection and continuing until 120 h post-infection. Mice were euthanized after 3 or 5 days of treatment, and blood and tissues were collected and processed for further analysis (A). Each group consisted of six mice, with equal distribution of males and females. Mice were monitored daily for weight loss (a) and mortality (b) until day 14 post-infection. Significance was determined using two-way ANOVA followed by Dunnett’s multiple comparison test (*p < 0.05; n = 6). For the survival curves of 2-BFU–treated DENV-2–infected mice compared with mock and vehicle controls, significance was assessed using Kaplan–Meier survival analysis (*p < 0.05; n = 6). Data is representative of two independent experiments.

Discussion

Dengue is a mosquito-borne viral infection prevalent in subtropical and tropical regions worldwide, affecting millions of people each year. The severity of dengue is often associated with either excessive viral replication or an overactive immune response. Currently, no antiviral drugs are approved for the treatment of DENV infections, highlighting the urgent need for novel therapeutic strategies. In this study, we evaluated the uridine nucleoside analog 2-BFU in a mouse model of DENV-2 infection. Our data indicate that: (1) therapeutic administration of 2-BFU at 15 mg/kg resulted in a significant reduction in viral load in the mouse model; (2) 2-BFU attenuated thrombocytopenia induced by DENV-2 infection in A129 mice; and (3) 2-BFU treatment did not affect the concentration of inflammatory mediators, nor did it prevent lethality or body weight loss. These results suggest that while 2-BFU demonstrates antiviral effects against DENV-2 infection, lethality rates remain unchanged, likely due to its lack of impact on inflammatory mediator levels.

Several nucleoside analogs, including sofosbuvir, remdesivir, and molnupiravir, have been evaluated as potential therapeutic agents against flaviviruses [13, 15, 17]. Dengue infection is typically characterized by viremia and viral replication in target organs such as the spleen and liver. Our findings highlight the promising anti-DENV-2 activity of 2-BFU, as evidenced by reduced viral titers in the blood, spleen, and liver of infected A129 mice.

In this study, 2-BFU administration reversed thrombocytopenia at 72- and 120-h post-infection. Dengue infections are often accompanied by hematological complications, including thrombocytopenia, though the underlying mechanisms remain unclear [19, 20]. DENV infects megakaryocytes, the cells responsible for platelet production, via the FcγRII receptor, disrupting platelet biogenesis. Additionally, DENV directly interacts with platelets, inducing the expression of surface proteins such as P-selectin and the αIIbβ3 complex [21–23]. These proteins trigger platelet activation, aggregation, and apoptosis. Thus, the attenuation of thrombocytopenia in 2-BFU-treated A129 mice may be linked to a reduction in viremia and viral titers in target organs. The development of novel anti-DENV therapies is challenging and requires a complex balance between controlling viral load, preventing hematologic changes, and modulating the inflammatory response. Despite effectively reducing viral load and attenuating thrombocytopenia, 2-BFU failed to prevent mortality, mitigate weight loss, leukocytosis, or alleviate liver damage in DENV-2-infected mice.

Multiple studies have demonstrated that DENV infection in humans activates host pattern recognition receptors (PRRs), leading to the production of inflammatory mediators such as IFN-γ, IL-6, CXCL-1, and CCL2 [24–27]. While these mediators are crucial for the immune response against DENV, excessive levels have been associated with increased severity of dengue complications, including hepatic and vascular dysfunction [28]. This suggests a delicate balance between the protective and detrimental effects of these inflammatory mediators during dengue infection, making the control of chemokine and cytokine levels an attractive target for novel therapeutic interventions. Treatment with 2-BFU in A129 mice infected with DENV-2 did not alter the concentration of inflammatory mediators in plasma. In contrast, therapeutic administration of 2-BFU in YFV-infected A129 mice protected the mice from liver damage and inflammation. Additionally, previous studies have demonstrated the antiviral efficacy of 7-deaza-7-fluoro-2’-C-methyladenosine (DFMA) against ZIKV and SARS-CoV-2 in mouse models [29]. DFMA administration to ZIKV-infected A129 mice (10 mg/kg) delayed mortality and prevented viral replication in the central nervous system, as well as brain damage induced by ZIKV infection. Del Sarto et al. (2020) also reported that DFMA prevented neuroinflammation, as evidenced by reduced levels of CCL5, decreased neutrophil recruitment, and reduced microgliosis (Iba1-positive cells) [30].

The development of novel anti-DENV-2 therapies requires a multifaceted approach that addresses viral load control, hematologic disorders, and the modulation of inflammation. The present study demonstrates that administering 2-BFU shortly after infection exhibits significant antiviral activity and attenuates thrombocytopenia induced by DENV-2 infection. Combining 2-BFU with anti-inflammatory molecules may represent a promising strategy for treating severe dengue disease.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1

Supplementary Material 1

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