Like the Yellow Fever Virus, the Dengue Virus Is a Single-stranded Rna Virus in The___ Family

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Yellow fever virus is susceptible to sofosbuvir both in vitro and in vivo

• Luiza Grand. Higa ,
• Carolina Q. Sacramento,
• André C. Ferreira,
• Patrícia A. Reis,
• Rodrigo Delvecchio,
• Fabio L. Monteiro,
• Giselle Barbosa-Lima,
• Harrison James Westgarth,
• Yasmine Rangel Vieira,
• Mayara Mattos,
• Natasha Rocha,
• Lucas Villas Bôas Hoelz,
• Rennan Papaleo Paes Leme,
• Mônica Thou. Bastos,
• Gisele Olinto L. Rodrigues,
• Carla Elizabeth One thousand. Lopes,
• Celso Martins Queiroz-Junior,
• Cristiano X. Lima,
• Vivian V. Costa,
• Mauro M. Teixeira,
• Fernando A. Bozza,
• Patrícia T. Bozza,
• Nubia Boechat,
• Amilcar Tanuri,
•  [ ... ],
• Thiago Moreno Fifty. Souza
• [ view all ]
• [ view less ]

Yellow fever virus is susceptible to sofosbuvir both in vitro and in vivo

• Caroline S. de Freitas,
• Luiza Thou. Higa,
• Carolina Q. Sacramento,
• André C. Ferreira,
• Patrícia A. Reis,
• Rodrigo Delvecchio,
• Fabio L. Monteiro,
• Giselle Barbosa-Lima,
• Harrison James Westgarth,
• Yasmine Rangel Vieira

x

• Published: January 30, 2019
• https://doi.org/ten.1371/periodical.pntd.0007072

Figures

Abstract

Yellow fever virus (YFV) is a member of the Flaviviridae family unit. In Brazil, yellow fever (YF) cases have increased dramatically in sylvatic areas neighboring urban zones in the concluding few years. Because of the loftier lethality rates associated with infection and absenteeism of whatsoever antiviral treatments, it is essential to identify therapeutic options to respond to YFV outbreaks. Repurposing of clinically approved drugs represents the fastest culling to notice antivirals for public health emergencies. Other Flaviviruses, such equally Zika (ZIKV) and dengue (DENV) viruses, are susceptible to sofosbuvir, a clinically approved drug confronting hepatitis C virus (HCV). Our data showed that sofosbuvir docks onto YFV RNA polymerase using conserved amino acrid residues for nucleotide binding. This drug inhibited the replication of both vaccine and wild-type strains of YFV on human hepatoma cells, with EC₅₀ values effectually 5 μM. Sofosbuvir protected YFV-infected neonatal Swiss mice and adult blazon I interferon receptor knockout mice (A129^-/-) from mortality and weight loss. Because of its rubber profile in humans and significant antiviral effects in vitro and in mice, Sofosbuvir may represent a novel therapeutic option for the treatment of YF. Key-words: Yellow fever virus; Yellow fever, antiviral; sofosbuvir

Author summary

Xanthous fever virus is transmitted past mosquitoes and its infection may be asymptomatic or lead to a broad clinical spectrum ranging from a mild febrile illness to a potentially lethal viral hemorrhagic fever characterized by liver impairment. Although a xanthous fever vaccine is available, low coverage allows 80,000–200,000 cases and 30,000–60,000 deaths annually worldwide. There are no specific therapy and handling relies on supportive intendance, reinforcing an urgent demand for antiviral repourposing. Hither, we showed that sofosbuvir, clinically approved against hepatitis C, inhibits yellow fever virus replication in liver prison cell lines and animal models. In vitro, sofosbuvir inhibits viral RNA replication, decreases the number of infected cells and the production of infectious virus particles. These data is particularly relevante since the liver is the primary target of yellow fever infection. Sofosbuvir also protected infected animals from mortality, weight loss and liver injury, especially prophylatically. Our pre-clinical results supports a second use of sofosbuvir confronting xanthous fever.

Citation: de Freitas CS, Higa LM, Sacramento CQ, Ferreira AC, Reis PA, Delvecchio R, et al. (2019) Yellowish fever virus is susceptible to sofosbuvir both in vitro and in vivo. PLoS Negl Trop Dis 13(1): e0007072. https://doi.org/10.1371/journal.pntd.0007072

Editor: Samuel 5. Scarpino, Northeastern University, UNITED STATES

Received: March 25, 2018; Accepted: December 12, 2018; Published: January 30, 2019

Copyright: © 2019 de Freitas et al. This is an open access article distributed nether the terms of the Creative Eatables Attribution License, which permits unrestricted utilize, distribution, and reproduction in whatsoever medium, provided the original author and source are credited.

Data Availability: All relevant data are within the newspaper and its Supporting Information files.

Funding: The financial support was provided by Fundação de Amparo à Pesquisa practice Estado do Rio de Janeiro (FAPERJ - http://world wide web.faperj.br/ - Grant Number Due east-26/201.573/2014) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - http://cnpq.br/ - Grant Numbers 306389/2014-two and 425636/2016-0). TMLS received the funds. This work has received financial support from the National Establish of Science and Technology in Dengue (INCT dengue), a scheme funded by the Brazilian National Science Council (CNPq, Brazil) and Minas Gerais Foundation for Science (FAPEMIG, Brazil). Funding was likewise provided by National Quango for Scientific and Technological Development (CNPq), Ministry of Scientific discipline, Engineering, Information and Communications (no. 465313/2014-0); Ministry of Education/CAPES (no. 465313/2014-0); Research Foundation of the State of Rio de Janeiro/FAPERJ (no. 465313/2014-0) and Oswaldo Cruz Foundation/FIOCRUZ to National Institute for Science and Engineering on Innovation on Diseases of Neglected Populations (INCT/IDPN), Centre for Technological Development in Wellness (CDTS), Fiocruz, Rio de Janeiro, RJ, Brazil. This study was financed in role by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Yellow fever virus (YFV) is a single-strand positive-sense RNA virus which belongs to the Flaviviridae family. Yellow fever (YF) outbreaks were very common throughout the tropical world until the outset of the twenty^thursday century, when vaccination and vector command limited the urban virus apportionment [ane]. Classically, sylvatic and urban cycles of YFV transmission occur. Non-human primates are sylvatic reservoirs of jungle YFV and non-immunized humans inbound the rain forest and those living in the ecotone (between preserved rain forest and urban surface area) are highly susceptible to YFV, which is transmitted by mosquitoes from Haemagogus and Sabethes genera [ii]. The virus is usually brought to urban settings by viremic humans infected in the jungle [2]. The urban cycle involves manual of the virus among humans by vectors like Aedes spp. mosquitoes [two].

Brazil, an endemic land for YF, failed to vaccinate a large proportion of the susceptible population. This scenario of low homo vaccinal coverage along with increased sylvatic YFV activity in primates has been occurring in Brazil since 2016, leading to bursts of man cases of YF. For instance, between the second semester of 2017 and March 2018, 4,847 epizootics were reported and 920 human being cases were confirmed. In that location were 300 deaths associated with this outbreak [3, four]. In fact, cases of YF increased one.viii-times compared to the previous 35 years [3]. Birthday, these data besides show that YFV spread from Brazilian rain forests to the outskirt of major cities in the Southeastern region of the country. Despite the detection of YFV in some urban areas in humans and primates during this recent reemergence, the Brazilian Ministry of Health (MoH) has argued this was a sylvatic cycle with no urban autochthonous transmission. Indeed, virtually of the contempo activity of YFV was observed in areas adjacent to the Atlantic wood, where the genotype I was introduced 2 times in 2005 (95% interval: 2002–2007) and 2016 (95% interval: 2012–2017), spilling over from the Amazon forest [5].

YFV causes massive lethality in new world monkeys and around 30% in humans [six]. Once displaying signs of severe infection, such as bleeding, shock, liver function decay and jaundice, infected individuals are likely to progress to poor clinical outcomes. Most often, astute hepatic failure occurs rapidly. No specific treatment options to YFV exist and patients solely receive intensive palliative intendance. Therefore, antivirals with anti-flavivirus action may stand for an important culling for drug repurposing in an attempt to improve patient outcome.

Developed in the 1930s, YF 17D live-attenuated vaccine confers long-lasting immunity to its recipients. Vaccination is recommended for individuals aged ≥ ix months who are living in or travelling to areas at risk for YF. Contraindications include hypersensitivity to vaccine components, severe immunodeficiency, and age nether ≤6 months [7]. Even though 17D is highly effective and one of the safest vaccines in history, rare severe adverse events accept been reported. YF vaccine-associated neurological (YEL-AND) and viscerotropic (YEL-AVD) diseases are like to classic YF caused past wild-type (WT) virus. Reporting rates of YEL-AND and YEL-AVD are 0.8 and 0.four cases per 100,000 doses distributed [8]. Specific treatment would be of utmost importance for individuals with YF vaccine-associated diseases and for YFV-exposed people for whom vaccination is contraindicated.

Our group and others have recently shown that sofosbuvir, a clinically approved anti-hepatitis C virus (HCV) drug, is besides endowed with anti-Zika virus (ZIKV) antiviral activity in vitro and in vivo [9–11]. It has also been shown that sofosbuvir also blocks dengue virus (DENV) replication [12]. Creature model studies of sofosbuvir on Flaviviruses reveal that this drug is more effective when used prophylactically or equally early as possible. Sofosbuvir was approved by the Food and Drug Administration (FDA) in 2013. Information technology has been used in therapy regimens thereafter in big worldwide scale to treat HCV-infected individuals, with infrequent registers of toxicity and adverse effects even for complex patients, such as those co-infected with both HIV/HCV or with substantial liver damage provoked past HCV [xiii–fifteen]. Sofosbuvir is very effective confronting HCV genotype 1/two/three, and condom doses may range from 400 to 1200 mg daily for up to 24 weeks [xiii–15]. When compared to pan-antiviral drugs such as ribavirin, sofosbuvir is considered safer for pregnant woman [16, 17].

In the intention to have a safe and an active antiviral compound to inhibit YFV replication, we tested whether the virus was susceptible to sofosbuvir. We found that sofosbuvir binds to conserved amino acid residues on the YFV RNA polymerase (NS5), inhibiting virus replication in man hepatoma cells, diminishing YFV-associated bloodshed and improving the hepatic condition in animal models.

Materials and methods

Reagents

The antiviral sofosbuvir was kindly donated past Dr. Jaime Rabi (Microbiológica Química e Farmacêutica, Brazil; part of the BMK Consortium). Ribavirin and AZT were provided by Instituto de Tecnologia de Farmacos (Farmanguinhos, Fiocruz). Drugs were dissolved in 100% dimethylsulfoxide (DMSO) and subsequently diluted at least 10⁴-fold in culture medium earlier each assay. The last DMSO concentration showed no cytotoxicity. The materials for cell culture were purchased from Thermo Scientific Life Sciences (Yard Island, NY) unless otherwise mentioned.

Cells and virus

Human hepatoma cell lines (Huh-seven and HepG2) and African green monkey (Vero) cells were cultured in DMEM supplemented with ten% fetal bovine serum (FBS; HyClone, Logan, Utah), 100 U/mL penicillin, and 100 μg/mL streptomycin [18, nineteen]. Cells were incubated at 37°C in 5% CO_ii. Aedes albopictus C6/36 cells were cultured at 28°C in Leibovitz L15 medium supplemented with two mM L-glutamine, 0.75 g/50 sodium bicarbonate, 0.3% tryptose phosphate broth, non-essential amino acids and 5% FBS.YFV vaccinal and WT strains were passaged at an multiplicity of infection (MOI) of 0.01 in either Vero (for 24–72 h at 37°C) or C6/36 (for 6 days at 28°C) cells. Virus titers were also determined in Vero jail cell cultures by TCID_l/mL [20] or plaque forming units (PFU)/mL (described below). The vaccine strain 17D was donated by the Reference Laboratory for Flavivirus, Fiocruz, Brazilian Ministry of Health, whereas the WT strain was isolated in Vero cells from the serum of a symptomatic patient with confirmed RT-PCR result for YFV (GenBank accession #MH018072) [21].

Cytotoxicity analysis

Monolayers of 1.5 x 10⁴ hepatoma cells in 96-well plates were treated for five days with various concentrations of sofosbuvir or ribavirin equally a control. And so, 5 mg/ml 2,3-bis-(two-methoxy-4-nitro-v-sulfophenyl)-2H-tetrazolium-five-carboxanilide (XTT) in DMEM was added to the cells in the presence of 0.01% of North-methyl dibenzopyrazine methyl sulfate (PMS). After incubating for 4 h at 37°C, the plates were read in a spectrophotometer at 492 nm and 620 nm [22]. The 50% cytotoxic concentration (CC_l) was calculated by a not-linear regression analysis of the dose-response curves.

Yield-reduction assay

Monolayers of 5.v x x⁶ Huh-7 cells in half-dozen-well plates were infected with YFV at an MOI of 0.1 for one h at 37°C. The cells were washed with PBS to remove residual viruses, and various concentrations of sofosbuvir, or ribavirin, in DMEM with i% FBS were added. After 24 h, the cells were lysed, the cellular droppings was cleared by centrifugation, and the virus titers in the supernatant were determined. A not-linear regression analysis of the dose-response curves was performed to summate the concentration at which each drug inhibited the plaque-forming action of YFV past l% (EC₅₀).

Immunofluorescence analysis

Huh-7 and HepG2 cells were seeded on black 96-well microplates with clear lesser (Greiner Bio-One, Kremsmünster, Republic of austria) and infected with YFV at an MOI of i. After ane hour, the viral inoculum was removed and cells were incubated with growth medium containing sofosbuvir, Ribavirin or AZT for 2 days. Cells were then fixed with iv% paraformaldehyde in PBS for 20 min at room temperature. The fixative was removed and cells monolayers were washed with PBS. Blocking of unspecific binding of the antibody and permeabilization were performed with 3% bovine serum albumin (BSA, Sigma Aldrich) and 0.1% Triton 10-100 in PBS for 20 min at room temperature. SCICONS J2 antibody (Scicons, Hungary), which recognizes double-stranded RNA, was diluted ane:yard in PBS and incubated for 1 h at room temperature. The master antibody was removed and cell monolayer was washed twice with PBS. Secondary antibiotic Ass anti-mouse IgG coupled to AlexaFluor488 fluorochrome (Thermo Fisher Scientific) was diluted 1:1000 in PBS and incubated for 40 min at room temperature. Later washing cells with PBS, nucleus staining with DAPI diluted 1:10,000 in PBS was performed at room temperature for x min and then done with PBS. Cells were imaged using a Nikon TE300 (Tokyo, Nippon) inverted microscope coupled to a Leica DFC310FX camera (Leica Biosystems, Wetzlar, Germany). Images referent to AlexaFluor488 and DAPI signals were merged using the microscope software.

Menstruation cytometry

Huh-7 and HepG2 cells were seeded in half-dozen-well plates at density of vi x10⁴ cells/well and 2.5 x 10⁵ cells/well, respectively. For infection, the growth medium was replaced by serum-free medium containing the virus at an MOI of 1. Mock-infected cells were incubated with conditioned medium from uninfected cells prepared exactly equally performed for viral propagation. Later on 1 60 minutes, inoculum was removed and replaced by growth medium containing either the vehicle or the antivirals at different concentrations and incubated for 48 h (for HepG2) or 72 h (for Huh-7) cells. After that, cells were harvested by treatment with a 0.25% trypsin solution. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) in phosphate buffered saline (PBS) for 15 min at room temperature and washed with PBS. Cells were permeabilized with 0.ane% Triton X-100 (Sigma Aldrich) in PBS, washed with PBS, and blocked with PBS with 5% FBS. Cells were incubated with 4G2, a pan-flavivirus antibody raised against the envelope E protein produced in 4G2-iv-fifteen hybridoma cells (ATCC), diluted i:10 in PBS with 5% FBS. Cells were labeled with donkey anti-mouse Alexa Fluor 488 antibody (Thermo Scientific,Waltham, MA, United states) diluted 1:1000 in PBS with five% FBS, and were analyzed by flow cytometry in a BD Accuri C6 (Becton, Dickinson and Company, Franklin Lakes, NJ, United states) for YFV infection. The gate strategy to clinch accuracy in the analysis is displayed every bit S1 Fig.

Plaque forming analysis

Virus titers were determined as PFU on Vero cells. Supernatants containing virus were serially diluted and incubated over confluent monolayers. After 1 h, cells were overlaid with semisolid medium, alpha-MEM (GIBCO) containing 1.4% carboxymethyl cellulose (Sigma-Aldrich) and ane% FBS (GIBCO). Cells were farther incubated for 4 to five days. Cells were fixed in 4% formaldehyde and stained with 1% crystal violet in 20% ethanol for plaque visualization.

Sequence comparisons

The sequences encoding the C-terminal portion of the RNA polymerase from Flaviviruses were acquired from the consummate sequences deposited in GenBank. An alignment was performed using the ClustalW algorithm in the Mega 6.0 software. The sequences were analyzed past disparity alphabetize per site. Compared regions are displayed in the S1 Tabular array.

Comparative modeling

The amino acid sequence encoding YFV RNA polymerase (UniProtKB code: P03314) was obtained from the EXPASY proteomic portal [23] (http://ca.expasy.org/). The template search was performed using the Blast server (http://nail.ncbi.nlm.nih.gov/Nail.cgi) with the Protein Data Bank [24] (PDB; http://www.pdb.org/pdb/home/domicile.do) as the database and the default options. The T-COFFEE algorithm was used to generate the alignment between the amino acrid sequences of the template proteins and YFV RNA polymerase. Subsequently, the construction of the YFV RNA polymerase complex was performed using MODELLER 9.19 software [25], which employs spatial restriction techniques based on the 3D-template structure. The preliminary model was refined in the same software, using iii cycles of the default optimization protocol. The structural evaluation of the model was then performed using two independent algorithms in the SAVES server (http://nihserver.mbi.ucla.edu/SAVES_3/): PROCHECK software [26] (stereochemical quality assay) and VERIFY 3D [27] (compatibility analysis between the 3D model and its own amino acid sequence by assigning a structural class based on its location and environment and by comparing the results with those of crystal structures).

Animals

The procedures described in this written report were in accordance with the ethical and beast experiments regulations of the Brazilian Government (Law 11794/2008), guidelines published at the Brazilian participant Institutions and National Institutes of Health guide for the care and use of laboratory animals. The study is reported in accord with the Go far guidelines for reporting experiments involving animals [28].

Neonate mouse model

Swiss albino mice (Mus musculus) (pathogen-free) from the Oswaldo Cruz Foundation convenance unit (Instituto de Ciência e Tecnologia em Biomodelos; ICTB/Fiocruz) were used for these studies. The animals were kept at a abiding temperature (25°C) with free access to chow and water in a 12-h low-cal/dark cycle. The experimental laboratory received significant mice (at approximately the 14th gestational day) from the convenance unit. Pregnant mice were observed daily until delivery to accurately determine the postnatal 24-hour interval. We established a litter size of 10 animals for all experimental replicates. The Animal Welfare Committee of the Oswaldo Cruz Foundation (CEUA/FIOCRUZ) approved and covered (license number L-016/2016) the experiments in this study.

Developed mouse model

In parallel, some experiments were conducted using type I interferon receptor deficient mice (A129^−/−), SV129 background, obtained from Bioterio de Matrizes da Universidade de Sao Paulo (USP). Adult male and female A129^−/− mice were bred and kept at Immunopharmacology Laboratory of the Universidade Federal de Minas Gerais (UFMG) nether specific pathogen-gratis weather condition. Mice were kept at a constant temperature (25°C) with costless access to chow and water in a 12-h low-cal/dark bike. Experimental protocol was approved by the Committee on Animal Ideals of the UFMG (CEUA/UFMG, Permit Protocol Number 84/2018).

Experimental infection and treatment.

As proof-of-principle, sofosbuvir treatment was initially performed in three-day-old Swiss mice infected intraperitoneally with different doses of vaccinal virus. To monitor drug efficiency in a highly aggressive system, adult (vii–nine week-old) A129^-/- (type I interferon receptor knockout) mice were infected with different inoculums of YFV by the intravenous (i.v.) route (tail vein). Both male and female mice were used in the experiments. Treatments with sofosbuvir were performed at 20 mg/kg/solar day administered intraperitoneally for Swiss newborn and orally (gavage) for A129^-/- mice. Regimen was administered daily showtime 1 twenty-four hours prior to infection or one day after infection until command group (animals infected and untreated) death. Animals were monitored daily for survival and weight variation. Of note, both male and female mice were used in the experiments.

If necessary, euthanasia was performed to alleviate animal suffering. The criteria were the following: i) differences in weight between infected and control groups [> l% for Swiss newborn (variation in weight gain) and > 20% for A129^-/- (variation in weight loss)], 2) ataxia, iii) loss of gait reflex, iv) absenteeism of righting reflex within 60 seconds, and five) separation, with no feeding, of moribund offspring by the female person adult mouse (for Swiss newborn mice).

Histopathological liver assay.

Liver samples from adult euthanized mice at day three post-YFV inoculation were obtained. After, they were immediately fixed in 10% buffered formalin for 24 h and embedded in methane series. Tissue sections (iv mm thicknesses) were stained with hematoxylin and eosin (H&E) and evaluated under a microscope, Axioskop 40 (Carl Zeiss, Göttingen, Frg) adapted to a digital camera (PowerShot A620, Catechism, Tokyo, Japan). Histopathology score was performed according to a prepare of custom designed criteria modified from evaluating cellular infiltration, hepatocyte swelling and degeneration and then added to reach a four-points score (0, absent-minded; 1, slight; 2, moderate; 3, marked; and 4, astringent) in each analysis [29]. For easy interpretation, the overall score was taken into business relationship and all the parameters summed for a maximum possible score of 8 points. A total of 2 sections for each animal were examined and results were plotted as the media of damage values in each mouse.

Statistical analysis.

All assays were performed and codification by one professional. Afterwards, a different professional analyzed the results before the identification of the experimental groups. This approach was used to keep the pharmacological assays blind. All experiments were carried out at to the lowest degree three independent times, including technical replicates in each assay. The dose-response curves used to calculate the EC_l and CC_l values were generated by Prism GraphPad software 7.0. The equations to fit the all-time curve were generated based on R² values ≥ 0.9. Fisher's exact and ANOVA tests were too used, with P values <0.05 considered statistically significant. For flow cytometry and viral titer analysis, data were analyzed by ANOVA. When ANOVA revealed a significant effect, data were farther analyzed with Dunnet post hoc test to correct for multiple comparisons and to determine specific group differences. The significance of survival curves was evaluated using the Log-rank (Mantel-Cox) exam. P values of ≤0.05 were considered statistically significant.

Results

Prediction of the complex betwixt Sofosbuvir triphosphate and YFV RNA polymerase using comparative modeling

We initially compared the disparities among regions encoding the RNA-dependent RNA polymerase (RDRP) region of the contemporary Flaviviruses DENV, ZIKV and YFV (Table 1). The YFV RNA polymerase shares a conserved domain for catalytic activeness with the orthologous enzymes (S1 Tabular array).

Next, the crystal structures of DENV NS5 complexed with viral RNA (PDB code: 5DTO) [30],HCV NS5B in complex with Sofosbuvir diphosphate (PDB code: 4WTG) [31], and complexed to UTP (PDB lawmaking: 1GX6) [32] were selected and used in a comparative modeling procedure, covering 100% of the YFV RNA polymerase sequence considered here (residues Thr252-Ile878). These iii template proteins represent orthologous viral RNA polymerases from the Flaviviridae family. Consequently, the resulting 3D model of YFV RNA polymerase in complex with Sofosbuvir triphosphate showed expert structural quality.

The analysis of the YFV RNA polymerase model suggests that sofosbuvir triphosphate binds between the palm and the fingers regions, making hydrogen bonds with Gly538, Trp539, Ser603, and Lys 693 residues and common salt bridge interactions with Lys359 and two Mg²⁺ ions. Interestingly, these interactions are all described as relevant for incorporation of natural ribonucleotides [31] (Fig 1). Therefore, these results motivate further testing in biologically relevant models to YFV replication.

Fig 1. 3D model of YFV RNA polymerase in circuitous with sofosbuvir triphosphate.

(A) Drawing representation of YFV RNA polymerase 3D model. The sofosbuvir triphosphate construction is represented past sticks and colored co-ordinate to atom type (red, oxygen; blue, nitrogen; orange, carbon; xanthous, phosphorus) and the two catalytic Mg²⁺ ions are shown every bit orange spheres. (B) Schematic representation of the interaction between amino acrid residues of the enzyme and the sofosbuvir triphosphate structure, where the salt bridges are shown in orange and hydrogen bonds are in green.

https://doi.org/10.1371/journal.pntd.0007072.g001

Sofosbuvir inhibits YFV replication in different models of human hepatoma cells.

YFV primarily replicates in the liver, where sofosbuvir is majorly converted from prodrug to its agile metabolite [xiii–15]. Thus, we monitored YFV susceptibility to sofosbuvir using human hepatocellular carcinoma cells. YFV was yield in Huh-vii cells in the presence of sofosbuvir for 24 h, then jail cell lysates were titered in Vero cells. Indeed, sofosbuvir inhibited both vaccine and WT strains of YFV replication similarly, in dose-dependent style (Fig 2). As a positive control to inhibit virus replication, we used ribavirin, a broad spectrum antiviral which was previously shown to inhibit YFV replication in vitro and in vivo [33–35] (Fig 2). Sofosbuvir'south and ribavirin's potencies over YFV were quite comparable, with EC₅₀ values varying 12% (Table ii). Of annotation, AZT, used as a negative command, did not touch virus replication (Fig 2).

Fig two. Pharmacology of sofosbuvir against YFV.

Huh-7 were infected with YFV at an MOI of 0.1 and exposed to diverse concentrations of the antivirals for 24 h. Supernatants were harvested and titered in Vero cells past TCID₅₀/mL. The data represent means ± SEM of five independent experiments.

https://doi.org/10.1371/journal.pntd.0007072.g002

Since sofosbuvir is effectually 25% less cytotoxic than ribavirin, its selectivity alphabetize (SI; ratio between EC_fifty and CC₅₀) was also college (Tabular array two). Sofosbuvir displayed improved SI values (when compared to ribavirin) past ten and 50% with respect to the vaccine and WT strains, respectively (Tabular array 2).

We further expanded whether YFV susceptibility to sofosbuvir was similar in dissimilar lineages of hepatoma cells, Huh-seven and HepG2. To do so, titers were determined by PFU, for more than authentic comparisons, with respect to differences in levels of virus replication. Consistently, sofosbuvir handling decreased the product of infectious virus particles (Fig 3) in a dose-dependent manner in Huh-seven (Fig 3A and 3B) and HepG2 cells (Fig 3C and 3D). Ribavirin likewise inhibited viral replication, whereas AZT was ineffective (Fig 3). Sofosbuvir at doses of 10 and 50 μM inhibited, respectively, 3- and 5-log₁₀ the production of infectious particles past infected Huh-seven cells (Fig 3A and 3B). Sofosbuvir was at least 100 times more constructive than ribavirin at inhibiting YFV infectivity (regardless of strain) in infected Huh-7 cells (Fig 3A and 3B). YFV susceptibility to the tested antiviral drugs in HepG2 cells was lesser when compared to Huh-7 jail cell (Fig 3A, 3B, 3C and 3D). Sofosbuvir treatment reduced the product of infectious YFV in HepG2 cells by 1- and two-log₁₀ at doses of x and 50 μM, respectively (Fig 3C and 3D). In HepG2 cells, ribavirin's and sofosbuvir'due south effects over virus replication were comparable (Fig 3C and 3D).

Fig 3. Sofosbuvir reduces the production of YFV viral particles.

Huh-vii cells were infected with vaccine strain YFV17D (A) or WT-YFV (B) at an MOI of i. HepG2 cells were infected with vaccine strain YFV17D (C) or WT-YFV (D) at an MOI of 1. YFV-infected were exposed to the indicated concentrations of AZT, ribavirin, or sofosbuvir. Supernatants were analyzed by plaque assay to determine viral titers. Data represent mean ± SEM of at to the lowest degree four independent experiments. Comparison between untreated and treated YFV-infected cells was performed using ANOVA followed by Dunnet post hoc test. Statistical significance compared to untreated YFV-infected cells is indicated by asterisks (* P < 0.05, ** P < 0.01 and *** P <0.001).

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Knowing the pharmacological activity and putative target, we used a cell-based analysis to demonstrate that sofosbuvir inhibits YFV RNA polymerase. During YFV replication, an anti-genomic negative-sense RNA strand is synthetized, creating an intermediate double-stranded (ds) RNA with the positive-sense virus genome. Viral genome replication was thus assessed by immunodetection of dsRNA. HepG2 and Huh-7 hepatoma cells lines were infected with an MOI of i and treated with sofosbuvir (0.4 to 50 μM) for 48 h. Ribavirin and AZT were used as positive and negative controls, respectively. Sofosbuvir reduced the genome replication of both vaccine and WT strains of YFV in Huh-7 and HepG2 cells in a dose dependent manner (Fig iv). Again, YFV replication was more susceptible to sofosbuvir in Huh-7 (Fig 4A and 4B) than HepG2 (Fig 4C and 4D) cells. Although ribavirin showed activity both against YFV 17D and WT YFV, total inhibition was only achieved at higher concentrations (when compared to sofosbuvir) (Fig 4). In Huh-seven cells treated with 10 μM of the drugs, nosotros observe less foci of dsRNA in sofosbuvir- than in ribavirin-treated cells (Fig 4A and 4B). AZT was inefficient in blocking either vaccine or WT YFV replication at the tested concentrations (Fig 4).

Fig iv. Sofosbuvir inhibits viral dsRNA, an intermediate in viral genome replication.

Huh-seven cells were infected with either vaccine strain, YFV-17D (A) or WT-YFV (B); HepG2 cells were infected with YFV-17D (C) or WT-YFV (D). Infected cells were treated with AZT, ribavirin (RBV), or sofosbuvir (SOF) at the indicated concentrations for 48 h. Viral replication was analyzed by immunofluorescence using J2 antibiotic to detect viral replication intermediate dsRNA (light-green) and DAPI to stain jail cell nuclei (blue). White scale bars indicate 50 μm. Images are representative of four contained experiments.

https://doi.org/ten.1371/journal.pntd.0007072.g004

Nosotros adjacent determined antigen production by flow cytometry using the 4G2 antibiotic, a pan-flavivirus antibody raised confronting the envelope protein (Fig five and S2 Fig). Huh-seven cells were infected with vaccine (Fig 5A) or WT (Fig 5B) YFV strain at an MOI of 1 and and then treated with ribavirin or AZT at ten and 50 μM or with sofosbuvir in concentrations ranging from 0.4 to 50 μM for 72 h. Menstruation cytometry assay showed a pronounced reduction in the number of YFV-infected Huh-7 cells treated with l μM ribavirin and ten–50 μM sofosbuvir compared to untreated YFV-infected cells (Fig 5A and 5B). AZT treatment did not present a pregnant anti-YFV event. Untreated WT YFV-infected cells presented with 89.75% of 4G2+ cells whereas sofosbuvir handling at ten and 50 μM resulted in 0.36 and 0.06% of antigen positive cells, respectively. Treatment with 10 and fifty μM ribavirin led to 65.43 and ane.79% of YFV-infected cells, indicating that sofosbuvir was more efficient than ribavirin in reducing the number of infected cells. HepG2 cells were also infected with vaccine (Fig 5C) or WT (Fig 5D) YFV and treated with AZT, ribavirin, or sofosbuvir for 48 h and analyzed past flow cytometry. Similar to the results observed in Huh-vii, ribavirin and sofosbuvir treatment led to a reduction in the number of YFV-infected HepG2 cells. Noteworthy, sofosbuvir and ribavirin-induced reduction of YFV-infected cells was more pronounced in Huh-7 cells than in HepG2 cells (Fig 5). This result is consistent with our observations from immunodetection of viral dsRNA (Fig 4). Sofosbuvir treatment reduced the number of YFV-infected cells in a dose-dependent manner, both in Huh-7 and HepG2 cells (Fig 5). We did non observe differences in sofosbuvir susceptibility betwixt vaccine and WT YFV strain, with respect to antigenic product.

Fig v. Sofosbuvir reduces the number of YFV-infected cells.

Huh-7 cells were infected with vaccine strain YFV17D (A) or wild-type YFV (B) at an MOI of 1. HepG2 cells were infected with vaccine strain YFV17D (C) or wild-type YFV (D) at an MOI of 1. YFV-infected cells were exposed to the indicated concentrations of AZT, ribavirin, or sofosbuvir. Anti-pan-flavivirus antibody was used for menses cytometry analysis. The data stand for mean ± SEM of at least iii independent experiments. Differences between untreated and treated YFV-infected cells were assessed using ANOVA followed by Dunnet post hoc test. Statistical significance compared to untreated YFV-infected cells is indicated by asterisks (* P < 0.05, ** P < 0.01 and *** P <0.001).

https://doi.org/ten.1371/journal.pntd.0007072.g005

Altogether, sofosbuvir demonstrated higher potency in the reduction of viral genome replication, protein synthesis, and infectious viral particle production than ribavirin in both hepatoma cell lines tested. These results betoken that sofosbuvir is a good candidate against YFV and deserving of farther in vivo testing.

Sofosbuvir enhances survival of YFV-infected mice.

To test sofosbuvir in vivo, we used the dose of 20 mg/kg/day in newborn Swiss outbred mice. This dose is consistent with its pre-clinical/clinical studies for drug approval [15]. Since in vitro experiments had shown that vaccine and WT strains of YFV are equally susceptible to sofosbuvir, we initially used the vaccine strain because it required easier handling and biosafety containment. Three-days-old Swiss mice were infected intraperitoneally with 1.0 x10⁴ PFU of vaccine virus. These animals began to receive treatment 1 day prior to infection (pre-handling) or 1 day post-infection (late-treatment). Sofosbuvir significantly enhanced the survival of YFV-infected mice who received pre-treatment, pointing out to safe activity and a possible narrow time frame for antiviral intervention (Fig 6A). Variations in body weight among groups were marginal (Fig 6B). Despite the fact that bloodshed starts to occur but 7 days afterwards infection, late-handling did not statistically increment animal survival (Fig 6A).

Fig half dozen. Early on/Condom treatment with sofosbuvir increases survival of YFV-infected mice.

Iii-twenty-four hours-erstwhile Swiss mice were infected with YFV-17D (i 10 10^iv PFU) and treated with sofosbuvir either ane 24-hour interval before (pre) or subsequently (late) infection. Survival (A) and weight variation (B) were assessed during the grade of treatment. Survival was statistically assessed by Log-rank (Mentel-Cox) test. Differences in weight are displayed as the means ± SEM. At least three independent experiments were performed with 10 mice/group. * P < 0.05.

https://doi.org/10.1371/journal.pntd.0007072.g006

Next, we repeated the same model of three-days-old Swiss mouse infection, but with a higher dose of vaccine virus (1 x ten^half-dozen PFU) to achieve higher bloodshed. Since late-treatment did non previously enhance survival in this model, the efficacy of sofosbuvir was accessed only with the pre-handling status. Sofosbuvir protected pre-treated mice from YFV-induced mortality (Fig 7A). Infected mice died within two weeks afterwards infection, whereas 70% of sofosbuvir pre-treated YFV-infected animals survived to lethal inoculum (Fig 7A). We too evaluated the weight gain during the fourth dimension course of our experiment in mice. YFV-infected animals had reduced postnatal development, whereas sofosbuvir-treated YFV-infected mice gained weight nigh as much as the uninfected controls (Fig 7B). We monitored through the grade of infection the levels of alanine aminotransferase (ALT), an of import biomarker of liver integrity. At day 12 post-infection, when untreated animals experience the highest mortality (Fig 7A), ALT levels became two-times higher in the infected/untreated mice than in those treated with sofosbuvir (Fig 7C). Consistent with the increment in this biomarker of liver injury, untreated animals displayed viral replication near 3-times higher in blood plasma and liver than treated mice (Fig 7D).

Fig 7. Pre-treatment with sofosbuvir increases survival and inhibits weight loss of YFV-infected mice.

Three-day-one-time Swiss mice were pre-treated with sofosbuvir get-go one mean solar day earlier infection with YFV 17D (1 x x^{half dozen} PFU). Survival (A) and weight variation (B) were assessed during the course of handling. Survival was statistically assessed by Log-rank (Mentel-Cox) exam. Differences in weight are displayed every bit the means ± SEM. Three-contained experiments were performed with 10 mice/group. Throughout the course of the experiment, a subset of animals (3 mice/group) were euthanized to monitor plasma ALT (C) and viral RNA levels (D). Data are presented as means ± SEM. For panels B, C and D, two-way ANOVA for each day was used to appraise the significance * P < 0.05.

https://doi.org/10.1371/journal.pntd.0007072.g007

Although these initial in vivo results are exciting, documented information of sofosbuvir protection in a highly virulent in vivo system is noteworthy. Mice with impaired innate immune response, due to the lack of blazon I interferon receptor (A129^-/-), were infected with WT YFV and treated with sofosbuvir at 20 mg/kg/day, again kickoff one day before (pre-treatment) or after (post-treatment) infection. Results show that inoculation with iv x 10⁴ and 4 x 10³ PFUs caused 100% bloodshed within one week along with massive loss in body weight (Fig 8). At these high virus inputs, pre-treatment with sofosbuvir enhanced the mean fourth dimension of survival by 57%, although mortality was the last upshot for all mice (Fig 8A and 8B). At a virus dose able to crusade 50% of bloodshed, four ten 10² PFU, pre-handling with sofosbuvir enhanced survival significantly (Fig 8C).

Fig eight. Treatment with sofosbuvir increases survival and inhibits weight loss of developed YFV-infected A129^-/- mice.

7–nine week-old A129^-/- mice were inoculated with 4x10⁴, 4x10³ or 4x10ⁱⁱ PFU of a WT strain of YFV i.v. A, C and E) Trunk weight was measured daily until twenty-four hours 14^th and expressed as % of body weight loss from day 0 before YFV inoculation. B, D and F) Survival rates were analysed every 12 hours and expressed as % of survival mice until mean solar day xiv^th postal service-YFV inoculation. Dashed lines are representative of MOCK-infected group. Survival was statistically assessed by Log-rank (Mentel-Cox) test. Differences in weight are displayed as the means ± SEM, and two-way ANOVA for each solar day was used to assess the significance. Three contained experiments were performed with at least iv mice/grouping. * P < 0.05.

https://doi.org/10.1371/journal.pntd.0007072.g008

Taking this concluding condition as reference, infectious viral titers were measured in different organs, forth with serum ALT levels, liver histopathology, and white cell counts. WT YFV replicated in different anatomical compartments of the A129^-/-, like peripheral claret, liver, spleen and kidney (Fig 9A–9D). In parallel to viscerotropic replication, animals rapidly progress to loftier virus titers in the brain (Fig 9E). Pre-handling with sofosbuvir reduced the virus levels in the encephalon of the infected mice by two-log (Fig 9E), along with leucocytosis (Fig 9F). Hepatic condition was improved in sofosbuvir-treated mice, regardless of the regimen, indicated past measuring ALT levels (Fig 9G) and liver injury (Fig 9H and 9I).

Fig nine. Treatment with Sofosbuvir ameliorates disease outcomes of adult YFV-infected A129^-/- mice.

A129^-/- mice were infected with 4x10² PFU of low passage clinical isolate of YFV past intravenous route. A-E) Viral loads from serum (A), liver (B), spleen (C), kidneys (D), and encephalon (E) assessed by plaque analysis. Results are shown as median f Log₁₀ PFU equivalents per mL or chiliad. F) Total and differential cell counts in blood were represented as number of differential cell counts (leukocytes, mononuclear cells and neutrophils) normalized to % of total cells counts. 1000) Hepatic transaminase levels of ALT were measured in serum of MOCK and YFV-infected mice at days 3 and 6 p.i. Results are shown as ALT (U/Fifty) of serum. H) Histopathological score of liver tissue sections. I) Representative of hepatic damage and Hematoxylin & Eosin staining of liver sections of command and YFV-infected mice 3 days after infection (Calibration Bar—100 μm). The images presented are representative animals on the third day of infection. Four animals per grouping were assayed. * for p<0.05 when compared to MOCK. # for p< 0.05 when compared to YFV.

https://doi.org/x.1371/journal.pntd.0007072.g009

Although pre-handling was to be better than late-treatment to raise survival, hepatic histology and ALT levels show that benefits from late treatment may exist (Fig 9G–9I). Histopathological analysis revealed mild alterations in the liver of vehicle-treated mice, including detached and focal neutrophil inflammatory infiltrate and hepatocyte degeneration. Interestingly, pre-treatment with sofosbuvir abrogated such alterations while mail-treatment partially reverted the observed liver lesions. This concluding effect prove that, although limited in efficacy, late treatment may be better than leaving the animals untreated. Our in vivo data reinforces the repurposing of sofosbuvir towards YF, with limited timing opportunity for intervention.

Discussion

Brazil has been challenged in contempo years past the (re)emergence of arboviruses. Massive cases of microcephaly associated with ZIKV circulation were registered in 2015/16 [36]. Two different genotypes of chikungunya virus (CHIKV) started to co-circulate from 2014 onward [37, 38]. The four DENV serotypes are hyperendemic throughout the country [39]. More recently, YFV activity increased, affecting both non-human primates and humans without constituted immunity [3, vi]. These facts clearly demonstrate that strategies of vector control failed and highlights the necessity of alternative strategies to control or fifty-fifty mitigate the diseases provoked by the arboviruses.

In the context of this commodity, re-emergence of YFV also points out that poor vaccination coverage left human population living in or inbound the ecotone susceptible to this virus. Unvaccinated individuals may quickly progress to severe YF, with hepatic and even neurological impairment [1]. In addition, due to massive YF vaccine campaigns, a fair amount of vaccine-related severe adverse events may be expected. Thus, finding antiviral drugs to treat YFV-infected individuals is critical for medical intervention over cases provoked by WT or even vaccine strains.

Several groups demonstrated that the clinically approved anti-HCV drug sofosbuvir is endowed with antiviral activity towards other flaviviruses, such as ZIKV [10, 40] and DENV [12]. Because that these agents belong to the same family unit, it was plausible to test sofosbuvir confronting YFV. Indeed, we observed that sofosbuvir targets the YFV RNA polymerase in silico and inhibits YFV RNA replication and infectious virus production. The in vitro antiviral results brandish that sofosbuvir'due south EC_fifty towards YFV is in micromolar range and comparable to what has been described for ZIKV and DENV [ten, 12, 40].

Our data showed that sofosbuvir inhibits YFV in hepatoma cell lines. This is particularly relevant since YFV targets hepatocytes and the liver is the almost affected organ in YF [41]. The degree of liver damage measured by elevated aminotransferase levels and jaundice is associated with higher mortality [42]. Our Swiss mouse neonate model of virus infection reproduced the deleterious association among increased ALT, viral loads, and mortality. Past inhibiting virus replication, sofosbuvir attacked this deleterious loop towards the liver protection and enhancement of the survival. In humans, massive apoptosis and necrosis of hepatocytes are reported in fatal cases [43]. Additionally, impaired synthesis of clotting factors caused by YFV-induced liver injury is primal to the pathogenesis of haemorrhagic manifestations in severe YF [44]. Experimental cases of liver transplant after YFV infection have been conducted during contempo outbreak in Brazil [4, 45, 46] with l% survival charge per unit. We envision that sofosbuvir may improve liver function in YF patients, and hopefully enhance the survival rates of transplanted individuals.

In the highly virulent infection mouse model, A129^-/- mice infected with WT YFV, sofosbuvir besides macerated virus replication in the brain and improved liver condition. Both viscerotropic and neurological replication injuries are hallmarks of YFV pathogenesis [eight, 47, 48]. Our data point to a possible benefit of early handling with sofosbuvir for patients that may progress to complications at tardily stages in the natural history of infection. Interestingly, although sofosbuvir was more efficient when used prophylactically, it was able to improve the hepatic condition of the infected animals receiving a post-infection handling.

Sofosbuvir reduced the YFV-induced mortality and lack of weight gain in mice models. Considering our information and the safety history of sofosbuvir in hepatitis, it is not a hyperbolic conclusion to consider sofosbuvir in clinical utilize for YF infection in humans. Primarily, sofosbuvir could be worthwhile for acutely infected individuals and those displaying neurotropic and viscerotropic diseases provoked by virus replication. Since vaccine is not recommended for special groups of individuals at higher risk of severe adverse events, such as elderly and those with immunodeficiencies, sofosbuvir could be used prophylactically.

Most normally, we used ribavirin, a pan-antiviral drug with anti-YFV activity demonstrated both in vitro and in vivo [33–35], as a positive control to inhibit YFV replication and to compare with sofosbuvir. Our in vitro data showed that sofosbuvir was more efficient than ribavirin in reducing viral genome replication, number of infected cells, and production of infectious viral particles in Huh-seven cells. Moreover, nosotros thus empathise that sofosbuvir possesses advantages over ribavirin in terms of safe and efficacy. Ribavirin is more toxic than sofosbuvir, especially for critically ill hepatic and renal patients [49], such as those with YF. According to FDA categorization of risk during pregnancy; sofosbuvir is class B (drugs with the second to lowest risk of causing malformations), whereas ribavirin is class X (forbidden, even for men having intimate contact with women at gestational age). Thus, ribavirin would accept a more express telescopic of utilize. Indeed, when ribavirin was used in a clinical trial confronting a flavivirus, the Japanese encephalitis virus, it failed to be effective [l].

Although our results are translational, information technology is important to cite that further studies are necessary to precisely decide sofosbuvir's mechanism(south) of action towards YFV life bike and the dose adjustment to treat patients with YF. YFV RNA polymerase is the likely target, based on docking onto this enzyme and reduction of viral dsRNA levels. As with other RNA polymerases from positive-sense RNA viruses, well-conserved motifs are found the YFV and HCV RNA polymerase, such as D-ten(four,5)-D and GDD, which are spatially juxtaposed, wherein Asp binds Mg²⁺ and Asn selects ribonucleotide triphosphates over dNTPs, determining RNA synthesis [9]. Information technology would not exist surprising to run across sofosbuvir triphosphate bound to these critical residues for catalysis, because even other positive-sense RNA virus across members of the Flaviviridae family unit are susceptible to sofosbuvir [51]. Sofosbuvir inhibits HCV RNA polymerase as chain terminator [31]. Nevertheless, this drug is considered a not-obligate chain terminator, due to the presence of 3'-OH moiety. Indeed, sofosbuvir inhibited ZIKV replication by targeting its RNA polymerase directly and provoking A-to-Grand mutation in the virus genome [40]. Whether sofosbuvir acts directly on YFV RNA polymerase, induces an error-prone virus replication by its incorporation in the virus genome or inhibition inosine monophosphate dehydrogenase and/or independent mechanisms–it remains to be elucidated. We also observed that sofosbuvir inhibits 100- to1000-times more potently HCV than YFV production/replication [13, 31, 52]. These differences in potencies demand to exist interpreted in light of the sofosbuvir's concentrations constitute in anatomical compartments and its long-half life [16], which may already be enough to compensate the limitation in the in vitro pharmacological authority. Sofosbuvir'due south chemical structure allows its vectorization to the liver, where it is establish at 77 μM when patients are treated with the reference dose of 400 mg/day [53]. YFV product was reduced upwardly to 3-log_x when hepatoma cells where treated with sofosbuvir at 50 μM. Since sofosbuvir has been used clinically at doses up to 1200 mg/day [16], information technology is possible to have enough sofosbuvir in the liver to inhibit or reduce YFV replication in humans remains. Only clinical trials will reveal the best regimen and posology for sofosbuvir towards YF. In the our mouse models, sofosbuvir efficacy was observed at the reference pre-clinical dose previously studied for HCV, 20 mg/kg/mean solar day [15].

The results described hither demonstrate for the get-go time the antiviral activity of sofosbuvir to YFV, which caused a recent outbreak in Brazil, providing primary scientific show for a new potential use of a clinically approved antiviral drug and reinforcing that its chemical structure may be used to generate selective anti-YFV specific drugs.

Supporting information

S1 Fig. Gate strategy from menses cytometry assay.

Menses cytometry events were gated on a dot plot FSC-A 10 FSC-H to exclude doublets (A-C). Cells were identified on FSC-A x SSC-A dotplots and gated to eliminate droppings from analysis (D-F) and so evaluate percentage of 4G2-positive cells (Chiliad-I). Panels are representative of five independent experiments.

https://doi.org/10.1371/journal.pntd.0007072.s001

(PDF)

S2 Fig. Dot plots from flow cytometry assay.

4G2 positive cells quantified from Huh-7 (A-Due east) and HepG2 (F-J) cells infected with YFV and treated with sofosbuvir at indicated concentrations. Representative of at least five independent experiments.

https://doi.org/10.1371/journal.pntd.0007072.s002

(PDF)

S1 Table. Alignment of RNA polymerases from members of the Flaviviridae family unit.

C-final region of the RNA polymerase from Zika, Dengue, hepatitis C and yellowish fever viruses. Conserved amino acrid residues are highlighted in xanthous. Critical amino acid residues are highlighted in scarlet.

https://doi.org/10.1371/periodical.pntd.0007072.s003

(PDF)

Acknowledgments

Thanks are due to Dr. Karin Brüning and Dr. Jaime Rabi, from the BMK Consortium (Blanver Farmoquímica Ltda; Microbiológica Química east FarmacêuticaLtda; Karin Bruning & Cia. Ltda), for donating sofosbuvir, and to Dr. Ana M. B. de Filippis, from Laboratório de Flavivírus, Instituto Oswaldo Cruz, Fiocruz, for kindly providing the YFV strain. We thank Ilma Marcal and Tania Colina for technical assistance. We also thank the Liver Center at Federal Academy of Minas Gerais for technical support.

References

1. 1. Beasley DW, McAuley AJ, Bente DA. Yellow fever virus: genetic and phenotypic diversity and implications for detection, prevention and therapy. Antiviral Res. 2015;115:48–70. pmid:25545072
   • View Article
   • PubMed/NCBI
   • Google Scholar
2. 2. Butler D. Fears rise over yellow fever's next move. Nature. 2016;532(7598):155–6. pmid:27075072
   • View Commodity
   • PubMed/NCBI
   • Google Scholar
3. iii. @NewsfromScience. When will yellow fever strike Brazil over again? Monkeys and mosquitoes hold clues | Science | AAAS. 2017. Bachelor from: https:// https://www.sciencemag.org/news/2017/08/when-volition-yellow-fever-strike-brazil-again-monkeys-and-mosquitoes-hold-clues
4. 4. Villegoureix I. PAHO/WHO | 20 March 2018: Yellow Fever—Epidemiological Update 2018 [updated 2018-03-20 19:57:54. Bachelor from: https://www.paho.org/hq/index.php?choice=com_content&view=article&id=14207:20-march-2018-yellow-fever-epidemiological-update&Itemid=42346〈=en.
5. v. Mir D, Delatorre East, Bonaldo M, Lourenço-de-Oliveira R, Vicente AC, Bello One thousand. Phylodynamics of Yellow Fever Virus in the Americas: new insights into the origin of the 2017 Brazilian outbreak. Scientific Reports. 2017;7(1):7385. pmid:28785067
   • View Article
   • PubMed/NCBI
   • Google Scholar
6. 6. Amarela CF. MONITORAMENTO DOS CASOS E ÓBITOS DE FEBRE AMARELA NO BRASIL, INFORME–Nº 43/2017 2017 [Bachelor from: http://portalarquivos2.saude.gov.br/images/pdf/2017/junho/02/COES-FEBRE-AMARELA—INFORME-43—Atualiza—-o-em-31maio2017.pdf.
7. vii. Staples JE, Gershman M, Fischer M, (CDC) CfDCaP. Yellowish fever vaccine: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep. 2010;59(RR-vii):1–27.
   • View Article
   • Google Scholar
8. viii. Lindsey NP, Schroeder BA, Miller ER, Braun MM, Hinckley AF, Marano N, et al. Adverse event reports following yellow fever vaccination. Vaccine. 2008;26(48):6077–82. pmid:18809449
   • View Article
   • PubMed/NCBI
   • Google Scholar
9. 9. Sacramento CQ, de Melo GR, de Freitas CS, Rocha Northward, Hoelz LVB, et al. The clinically approved antiviral drug sofosbuvir inhibits Zika virus replication. Scientific Reports. 2017;7:40920. pmid:28098253
   • View Article
   • PubMed/NCBI
   • Google Scholar
10. 10. Ferreira Air conditioning, Zaverucha-do-Valle C, Reis PA, Barbosa-Lima M, Vieira YR, Mattos M, et al. Sofosbuvir protects Zika virus-infected mice from mortality, preventing brusk- and long-term sequelae. Scientific Reports. 2017;seven(1):9409. pmid:28842610
    • View Article
    • PubMed/NCBI
    • Google Scholar
11. 11. Bullard-Feibelman KM, Govero J, Zhu Z, Salazar V, Veselinovic Thou, Diamond MS, et al. The FDA-approved drug sofosbuvir inhibits Zika virus infection. Antiviral Res. 2017;137:134–40. pmid:27902933
    • View Article
    • PubMed/NCBI
    • Google Scholar
12. 12. Xu H-T, Colby-Germinario SP, Hassounah SA, Fogarty C, Osman Northward, Palanisamy North, et al. Evaluation of Sofosbuvir (β-D-2′-deoxy-2′-α-fluoro-two′-β-C-methyluridine) as an inhibitor of Dengue virus replication. Scientific Reports. 2017;seven(1):6345. pmid:28740124
    • View Article
    • PubMed/NCBI
    • Google Scholar
13. 13. Bhatia HK, Singh H, Grewal Northward, Natt NK. Sofosbuvir: A novel handling option for chronic hepatitis C infection. J Pharmacol Pharmacother. 2014;5(4):278–84. pmid:25422576
    • View Article
    • PubMed/NCBI
    • Google Scholar
14. 14. Gilead. Production Monograph Pr SOVALDI (sofosbuvir) Tablets 400 mg sofosbuvir Antiviral Amanuensis 2015 [Bachelor from: http://world wide web.gilead.ca/pdf/ca/sovaldi_pm_english.pdf.
15. 15. European Medicines Agency, EMA. Sovaldi 2013 [Available from: http://world wide web.ema.europa.eu/docs/en_GB/document_library/EPAR__Public_assessment_report/human/002798/WC500160600.pdf.
16. 16. Gilead Sciences Canada I. Production Monograph SOVALDI (sofosbuvir) Tablets 400 mg sofosbuvir Antiviral Agent 2015 [Available from: http://www.gilead.ca/application/files/3915/2589/0723/Sovaldi_English_PM_e162880-GS-007.pdf.
17. 17. FDA FaDA. Copegus (ribavirin) tablets 2015 [Available from: http://www.fda.gov/Safety/MedWatch/SafetyInformation/ucm218877.htm.
18. 18. Souza TML, De Souza M, Ferreira VF, Canuto C, Marques IP, Fontes CFL, et al. Inhibition of HSV-1 replication and HSV Dna polymerase by the chloroxoquinolinic ribonucleoside 6-chloro-i,4-dihydro-4-oxo-1-(beta-D-ribofuranosyl) quinoline-three-carboxylic acid and its aglycone. Antiviral Enquiry. 2008;77(i):20–7. pmid:17931712
    • View Commodity
    • PubMed/NCBI
    • Google Scholar
19. 19. Hottz ED, Lopes JF, Freitas C, Valls-de-Souza R, Oliveira MF, Bozza MT, et al. Platelets mediate increased endothelium permeability in dengue through NLRP3-inflammasome activation. Blood. 2013;122(20):3405–xiv. pmid:24009231
    • View Article
    • PubMed/NCBI
    • Google Scholar
20. xx. Reed LJ, & Muench H. A elementary method of estimating fifty percent endpoints. Am J Hygiene. 1938(27).
    • View Article
    • Google Scholar
21. 21. Faria NR, Kraemer MUG, Hill SC, Goes de Jesus J, Aguiar RS, Iani FCM, et al. Genomic and epidemiological monitoring of yellow fever virus transmission potential. Science. 2018;361(6405):894–9. pmid:30139911
    • View Article
    • PubMed/NCBI
    • Google Scholar
22. 22. Lu J, Pan Q, Rong L, He W, Liu SL, Liang C. The IFITM proteins inhibit HIV-ane infection. J Virol. 2011;85(5):2126–37. pmid:21177806
    • View Article
    • PubMed/NCBI
    • Google Scholar
23. 23. Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A. ExPASy: The proteomics server for in-depth protein cognition and analysis. Nucleic Acids Res. 2003;31(13):3784–viii. pmid:12824418
    • View Commodity
    • PubMed/NCBI
    • Google Scholar
24. 24. Dutta Due south, Burkhardt K, Young J, Swaminathan GJ, Matsuura T, Henrick K, et al. Information degradation and annotation at the worldwide protein data banking company. Mol Biotechnol. 2009;42(i):1–13. pmid:19082769
    • View Article
    • PubMed/NCBI
    • Google Scholar
25. 25. Sali A, Blundell TL. Comparative poly peptide modelling by satisfaction of spatial restraints. J Mol Biol. 1993;234(iii):779–815. pmid:8254673
    • View Article
    • PubMed/NCBI
    • Google Scholar
26. 26. Laskowski MWM R. A., Moss D. S. and Thornton J. M., TI. PROCHECK: a programme to check the stereochemical quality of protein structures. Journal of Practical Crystallography. 1993;26(2):283–91.
    • View Article
    • Google Scholar
27. 27. Eisenberg D, Lüthy R, Bowie JU. VERIFY3D: assessment of protein models with iii-dimensional profiles. Methods Enzymol. 1997;277:396–404. pmid:9379925
    • View Article
    • PubMed/NCBI
    • Google Scholar
28. 28. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the Make it guidelines for reporting animal inquiry. Osteoarth Cartil. 2012;20(4):256–threescore.
    • View Commodity
    • Google Scholar
29. 29. Costa VV, Fagundes CT, Valadão DF, Cisalpino D, Dias ACF, Silveira KD, et al. A Model of DENV-3 Infection That Recapitulates Severe Affliction and Highlights the Importance of IFN-γ in Host Resistance to Infection. PLoS Negl Trop Dis. 62012.
    • View Article
    • Google Scholar
30. xxx. Zhao Y, Soh TS, Lim SP, Chung KY, Swaminathan M, Vasudevan SG, et al. Molecular footing for specific viral RNA recognition and 2'-O-ribose methylation by the dengue virus nonstructural protein v (NS5). Proc Natl Acad Sci U Due south A. 2015;112(48):14834–nine. pmid:26578813
    • View Article
    • PubMed/NCBI
    • Google Scholar
31. 31. Appleby TC, Perry JK, Murakami E, Barauskas O, Feng J, Cho A, et al. Viral replication. Structural basis for RNA replication by the hepatitis C virus polymerase. Science. 2015;347(6223):771–5. pmid:25678663
    • View Article
    • PubMed/NCBI
    • Google Scholar
32. 32. Bressanelli S, Tomei L, Rey FA, De Francesco R. Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides. J Virol. 2002;76(7):3482–92. pmid:11884572
    • View Commodity
    • PubMed/NCBI
    • Google Scholar
33. 33. Buckwold VE, Wei J, Wenzel-Mathers M, Russell J. Synergistic in vitro interactions betwixt alpha interferon and ribavirin against bovine viral diarrhea virus and yellowish fever virus every bit surrogate models of hepatitis C virus replication. Antimicrob Agents Chemother. 2003;47(seven):2293–viii. pmid:12821481
    • View Article
    • PubMed/NCBI
    • Google Scholar
34. 34. Julander JG, Morrey JD, Blatt LM, Shafer M, Sidwell RW. Comparison of the inhibitory effects of interferon alfacon-1 and ribavirin on yellow fever virus infection in a hamster model. Antiviral Res. 2007;73(2):140–six. pmid:17049380
    • View Article
    • PubMed/NCBI
    • Google Scholar
35. 35. Leyssen P, De Clercq E, Neyts J. The anti-yellow fever virus activity of ribavirin is independent of error-prone replication. Mol Pharmacol. 2006;69(4):1461–7. pmid:16421290
    • View Article
    • PubMed/NCBI
    • Google Scholar
36. 36. Metsky HC, Matranga CB, Wohl S, Schaffner SF, Freije CA, Winnicki SM, et al. Zika virus evolution and spread in the Americas. Nature. 2017;546(7658):411–5. pmid:28538734
    • View Article
    • PubMed/NCBI
    • Google Scholar
37. 37. Zanluca C, Melo VC, Mosimann AL, Santos GI, Santos CN, Luz K. Start report of autochthonous manual of Zika virus in Brazil. Mem Inst Oswaldo Cruz. 2015;110(4):569–72. pmid:26061233
    • View Article
    • PubMed/NCBI
    • Google Scholar
38. 38. de Santana F. Secretaria Municipal de Saúde. Boletim da Febre practice Chikungunya. Situação epidemiológica dos casos de chikungunya. 2014; one to 11 2014 [Available from: http://www.feiradesantana.ba.gov.br/sms/arq/Chikungunya_Feira.pdf.
    • View Article
    • Google Scholar
39. 39. Teixeira MG, Costa MaC, Barreto F, Barreto ML. Dengue: xx-v years since reemergence in Brazil. Cad Saude Publica. 2009;25 Suppl 1:S7–xviii.
    • View Commodity
    • Google Scholar
40. 40. Sacramento CQ, de Melo GR, de Freitas CS, Rocha N, Hoelz LV, Miranda M, et al. The clinically approved antiviral drug sofosbuvir inhibits Zika virus replication. Sci Rep. 2017;7:40920. pmid:28098253
    • View Article
    • PubMed/NCBI
    • Google Scholar
41. 41. Monath TP, Vasconcelos PF. Yellow fever. J Clin Virol. 2015;64:160–73. pmid:25453327
    • View Commodity
    • PubMed/NCBI
    • Google Scholar
42. 42. Tuboi SH, Costa ZG, da Costa Vasconcelos PF, Hatch D. Clinical and epidemiological characteristics of yellow fever in Brazil: analysis of reported cases 1998–2002. Trans R Soc Trop Med Hyg. 2007;101(2):169–75. pmid:16814821
    • View Article
    • PubMed/NCBI
    • Google Scholar
43. 43. Quaresma JA, Barros VL, Pagliari C, Fernandes ER, Andrade HF, Vasconcelos PF, et al. Hepatocyte lesions and cellular immune response in yellow fever infection. Trans R Soc Trop Med Hyg. 2007;101(2):161–8. pmid:16872652
    • View Article
    • PubMed/NCBI
    • Google Scholar
44. 44. Paessler S, Walker DH. Pathogenesis of the viral hemorrhagic fevers. Annu Rev Pathol. 2013;8:411–40. pmid:23121052
    • View Article
    • PubMed/NCBI
    • Google Scholar
45. 45. Song ATW, Abdala East, de Martino RB, Malbouisson LMS, Tanigawa RY, Andrade GM, et al. Liver transplantation for fulminant hepatitis due to yellow fever. Hepatology. 2018.
    • View Article
    • Google Scholar
46. 46. Paulo F-FdMdUdS. The kickoff liver transplant due to Yellow Fever patient will be discharged from the hospital his week 2018 [Bachelor from: http://www.fm.usp.br/en/news/the-commencement-liver-transplant-due-to-yellow-fever-patient-will-be-discharged-from-the-hospital-his-week.
47. 47. Barnett ED, Maxwell Finland Laboratory for Infectious Diseases BMC, Boston, Massachusetts. Yellow Fever: Epidemiology and Prevention. Clinical Infectious Diseases. 2018;44(6):850–6.
    • View Article
    • Google Scholar
48. 48. Meier KC, Gardner CL, Khoretonenko MV, Klimstra WB, Ryman KD. A mouse model for studying viscerotropic disease acquired by yellow fever virus infection. PLoS Pathog. 2009;v(10):e1000614. pmid:19816561
    • View Article
    • PubMed/NCBI
    • Google Scholar
49. 49. Smolders EJ, de Kanter C, van Hoek B, Arends JE, Drenth JPH, Burger DM. Pharmacokinetics, Efficacy, and Condom of Hepatitis C Virus Drugs in Patients with Liver and/or Renal Impairment. Drug Saf. 2016;39:589–611. pmid:27098247
    • View Commodity
    • PubMed/NCBI
    • Google Scholar
50. 50. Kumar R, Tripathi P, Baranwal Grand, Singh S, Tripathi S, Banerjee Thou. Randomized, controlled trial of oral ribavirin for Japanese encephalitis in children in Uttar Pradesh, India. Clin Infect Dis. 2009;48(iv):400–vi. pmid:19143532
    • View Article
    • PubMed/NCBI
    • Google Scholar
51. 51. Ferreira Air conditioning, Reis PA, de Freitas CS, Sacramento CQ, Villas Boas Hoelz L, Bastos MM, et al. Beyond members of the Flaviviridae family unit, sofosbuvir besides inhibits chikungunya virus replication. Antimicrob Agents Chemother. 2018.
    • View Article
    • Google Scholar
52. 52. Sofia MJ, Bao D, Chang W, Du J, Nagarathnam D, Rachakonda Southward, et al. Discovery of a β-d-2'-deoxy-2'-α-fluoro-2'-β-C-methyluridine nucleotide prodrug (PSI-7977) for the treatment of hepatitis C virus. J Med Chem. 2010;53(nineteen):7202–18. pmid:20845908
    • View Commodity
    • PubMed/NCBI
    • Google Scholar
53. 53. Babusis D, Curry MP, Kirby B, Park Y, Murakami Due east, Wang T, et al. Sofosbuvir and Ribavirin Liver Pharmacokinetics in Patients Infected with Hepatitis C Virus. Antimicrob Agents Chemother. 2018;62(5).
    • View Article
    • Google Scholar

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