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

Yellow fever virus (YFV) is a member of the Flaviviridae family, that causes major mortality. In Brazil, YFV activity increased in the last years. It has been registered that sylvatic, instead of urban, yellow fever (YF) leads our contemporary public health concern. Low vaccinal coverage leaves the human population near the jangle vulnerable to the outbreak, making it necessary to identify therapeutic options. Repurposing of clinically approved antiviral drugs represents an alternative for such identification. Other Flaviviruses, such Zika (ZIKV) and dengue (DENV) viruses, are susceptible to Sofosbuvir, a clinically approved drug against hepatitis C virus (HCV). Moreover, sofosbuvir has a safety record on critically ill hepatic patients, making it an attractive option. Our data show that YFV RNA polymerase uses conserved amino acid resides for nucleotide binding to dock sofosbuvir. This drug inhibited YFV replication in different lineages of human hepatoma cells, Huh-7 and HepG2, with EC50 value of 4.8 µM. Sofosbuvir protected YFV-infected neonatal Swiss mice from mortality and weight loss. Our pre-clinical results indicate that sofosbuvir could represent an option against YFV.


-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 beginning of the 20 th century, when vaccination and vector control limited the urban virus circulation (1). Classically, sylvatic and urban cycles of YFV transmission occurs. Non-human primates are sylvatic reservoirs of jungle YFV and non-immunized humans entering the rain forest and those living at the ecotone (between preserved rain forest and urban area) are highly susceptible to severe YF, which may be transmitted by mosquitoes from Haemagogus and Sabeties genuses (2). The virus is usually brought to the urban setting by a viremic human who was infected in the jungle (2). The urban cycle involves transmission of the virus among humans by vectors like Aedes spp. mosquitoes (2). YFV causes massive lethality for new world monkeys and around 30 % to humans (5). Once displaying severe signs of severe infection such as bleeding, shock, liver function decay and jaundice, infected individuals are likely to progress to poor clinical outcomes. Nevertheless, progression to acute hepatic failure is a reality for these individuals. No specific treatment option to YFV exist and patients solely receive just intensive palliative care.. Therefore, antivirals with anti-flavivirus activity may represent an important alternative for drug repurposing in an attempt to block or mitigate liver lesions induced by YFV replication.
Developed in the 1930s, YF 17D live-attenuated vaccine confers long-lasting immunity to its recipients. Vaccination is recommended for individuals aged ≥9 months who are living in or travelling to areas at risk for YF. Contraindications include hypersensitivity to vaccine components, severe immunodeficiency and age under 6 months-old (6). Even though 17D is highly effective and one of the safest vaccines in history, rare severe adverse events have been reported. YF vaccine-associated neurologic disease (YEL-AND) and YF vaccine-associated viscerotropic disease (YEL-AVD) which is similar to classic disease caused by wild-type YFV. Reporting rates of YEL-AND and YEL-AVD are 0.8 and 0.4 cases per 100,000 doses distributed (8). Specific treatment would be of utmost importance for individuals with yellow fever vaccine associated diseases and for YFV-exposed people for whom vaccination is contraindicated.
Our group has recently shown that sofosbuvir, a clinically approved anti-hepatitis C virus (HCV) drug, is also endowed with anti-Zika virus (ZIKV) antiviral activity in vitro and in vivo models (9,10). Others have demonstrated that sofosbuvir also block dengue virus (DENV) replication (11). Sofosbuvir was approved by food and drug administration (FDA) in 2013. It has been used in therapy regimens thereafter in large 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 (12)(13)(14). Sofosbuvir in very effective against against HCV genotype 1, 2 and 3, safety doses may range from 400 to 1200 mg daily for up to 24 weeks (12)(13)(14).
In the intention to have an active antiviral compound to inhibit YFV replication and block the progress of this disease to fatal outcomes, we tested whether sofosbuvir may possess anti-YFV activity. We found by in silico analysis that SFV binds to conserved amino acid residues on the YFV RNA RNA polymerase (NS5), inhibiting virus replication in human hepatoma cells and diminishing mortality in neonatal Swiss mice.

-Material and Methods
Reagents. The antivirals sofosbuvir and ribavirin were donated by the BMK Consortium and Instituto de Tecnologia de Farmacos (Farmanguinhos, Fiocruz), respectively. Drugs were dissolved in 100 % dimethylsulfoxide (DMSO) and subsequently diluted at least 10 4 -fold in culture or reaction medium before each assay. The final DMSO concentrations showed no cytotoxicity. The materials for cell culture were purchased from Thermo Scientific Life Sciences (Grand Island, NY) unless otherwise mentioned.

Cells and Virus.
Human hepatoma cell lines (Huh-7 and HepG2) and African green monkey (Vero) cells were cultured in DMEM supplemented with 10 % fetal bovine serum (FBS; HyClone, Logan, Utah), 100 U/mL penicillin, and 100 µg/mL streptomycin (15,16). Cells were incubated at 37 °C in 5 % CO2.   (21), which employs spatial restriction techniques based on the 3D-template structure. The preliminary model was refined in the same software, using three 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 (22) (stereochemical quality analysis) and VERIFY 3D (23)  Treatments with sofosbuvir were carried out with sofosbuvir at 20 mg/kg/day administered intraperitoneally. Treatment started one day prior to infection (pretreatment). Animals were monitored daily for survival and weight gain.
If necessary to alleviate animal suffering, euthanasia was performed. The criteria were the following: i) differences in weight gain between infected and control groups >50%, ii) ataxia, iii) loss of gait reflex, iv) absence of righting reflex within 60 seconds, and v) separation, with no feeding, of moribund offspring by the female adult mouse.
Statistical analysis. All assays were performed and codified by one professional.
Subsequently, 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 least three independent times, including technical replicates in each assay. The dose-response curves used to calculate the EC50 and CC50 values were generated by Prism GraphPad software 7.0. The equations to fit the best curve were generated based on R 2 values ≥ 0.9. Fisher's exact and ANOVA tests were also used, with P values <0.05 considered statistically significant. The significance of survival curves was evaluated using the Log-rank (Mantel-Cox) test. P values of 0.05 or less were considered statistically significant.

3-Results.
Prediction of the complex between Sofosbuvir triphosphate and YFRP using comparative modeling.
We initially compared the disparities among region encoding the RDRP region of the contemporary Flaviviruses, DENV, ZIKV and YFV. The YFV RDRP shares the conserved domain for catalytic activity with the orthologues enzymes (Supplementary   File 1 and Table 1). Interestingly, these interactions are all described as relevant for incorporation of natural ribonucleotides (26) (Figure 1). Therefore, these results motivate further testing in biologically relevant models to YFV replication.

Sofosbuvir inhibits YFV replication in different models of human hepatocytes. YFV
primarily replicates in the liver, where sofosbuvir is majorly converted from prodrug to its active metabolite (12)(13)(14). Thus, we monitored YFV susceptibility to sofosbuvir using human hepatocellular carcinoma cells. YFV was yield in Huh-7 cells in the presence of sofosbuvir for 24 h, when supernatant was harvested and tittered in Vero cells. Indeed, sofosbuvir inhibited in dose-dependent manner YFV replication (Figure 2A). As a control, ribavirin, a broad spectrum antiviral, also inhibited YFV replication (Figure 2A).
Sofosbuvir`s and ribavirin`s potencies to inhibit YFV replication were quite comparable, with EC50 values equal to 4.8 ± 0.2 and 3.9 ± 0.3 µM (Table 1), respectively. Since sofosbuvir is around 25 % less cytotoxic than ribavirinits selectivity index (SI), the ratio between EC50 and CC50, was higher ( Table 2). In order to gain further knowledge about the antiviral properties of sofosbuvir in hepatic cells, viral infectivity was assessed by flow cytometry using the 4G2 antibody, a pan-flavivirus antibody raised against the envelope protein (Fig 3). Huh7 cells were infected with YFV at a MOI of 1 and then treated with sofosbuvir in concentrations ranging from 0.4 to 50 μM for 72 hours. Sofosbuvir dramatically reduced the number of YFV-infected cells in a dose-dependent manner. Doses above 2 µM totally abolished YFV antigen production in Huh7 infected cells (Fig 3A). Additionally, sofosbuvir treatment resulted in decreased production of infectious virus particles (Fig 3A) also in a dose-dependent manner. Treatment with 10 μM and 50 μM sofosbuvir led to a 4,034 and 561,142-fold reduction in the level of infectious particles. Similar results were obtained for HepG2 cells (Fig 3B). Sofosbuvir treatment resulted in a reduction in the number of YFV-infected cells and in the detection of infectious YFV (Fig 3B) compared to untreated cells. These results indicate that sofosbuvir is a good candidate against YF, deserving further in vivo testing.

Sofosbuvir enhances survival of YFV-infected neonatal 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(14). Sofosbuvir protected treated mice from YFV-induced mortality (Fig 4A). Infected mice died within two weeks after infection, whereas 70% of sofosbuvir-treated YFV-infected animals survived to lethal inoculum (Fig. 4A). We also evaluated the weight gain during the time course of our experiment in mice. YFV-infected animals had reduced postnatal development, whereas sofosbuvir-treated YFV-infected mice gained weight almost as much as the uninfected controls (Fig. 4B).

-Discussion
Brazil has been challenged in recent years by the (re)emergence of arboviruses.
More recently, YFV activity increased, affecting both non-human primates and humans without constituted immunity (3, 5). These facts clearly demonstrate that strategies of vector control failed and highlights the necessity of other ways to control or even mitigate the diseases provoked by the arboviruses.
In the context of this article, re-emergence of YFV also points out that poor vaccination coverage left human population living or entering 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 captains 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 in critical cases.
Several groups demonstrated that the clinically approved anti-HCV drug sofosbuvir is endowed with antiviral activity towards other flavivirus such as ZIKV (9, 10) and DENV (11). Considering that these agents belong to the same family of viruses, it was plausible to test sofosbuvir against YFV.Indeed, we observed that sofosbuvir predictively targets the YFV RNA RNA polymerase in silico and by dose response antiviral assays. In fact, the in vitro antiviral results display that sofosbuvir`s EC50% towards YFV is in micromolar range and comparable to what has been described for ZIKV and DENV (9)(10)(11). Sofosbuvir`s metabolism is complex and some facets are clearly undescribed. Although it was thought to be converted from prodrug to active compound only on liver cells, we found it also happens on human neural stem cells (10). Given this complexity, in vivo testing of sofosbuvir is pivotal to better comprehend its pharmacology and in vivo antiviral potency.
Our data showed that sofosbuvir inhibits YFV in hepatic cell lines. This is particularly relevant since YFV targets hepatocytes and the liver is the most affected organ in YF (32). The degree of liver damage measured by elevated aspartate aminotransferase and jaundice is associated with higher mortality (33). Massive apoptosis and necrosis of hepatocytes are reported in fatal cases (34). Additionally, impaired synthesis of clotting factors caused by YFV-induced liver injury is key to the pathogenesis of haemorrhagic manifestations in severe YF (35). Therefore, sofosbuvir treatment may improve liver function in YF patients and hopefully impact clinical outcome. In