Combating yellow fever virus with 7-deaza-7-fluoro-2'-C-methyladenosine

Abstract

Yellow fever virus (YFV) is a deadly zoonotic flavivirus endemic in tropical/sub-tropical Africa and South America transmitted by mosquito vector (Aedes aegypti; Haemagogus leucocelaenus) to humans and non-human primates. There are no approved antiviral agents for YFV. We previously identified 7-deaza-7-fluoro-2'-C-methyladenosine (DFA) with anti-YFV activity. Interestingly, DFA exhibits pan-activity in vitro against flaviviruses, such as dengue, Japanese encephalitis, Zika, and hepatitis C. This study aimed to expand DFA's anti-flavivirus profile. DFA exhibited potent sub-micromolar anti-YFV activity in vitro against both the vaccine strain (YFV-17D) and a viscerotropic clinical YFV isolate (DakH1279) concomitantly with low cellular cytotoxicity and no notable mitochondrial toxicity. In vivo, efficacy was assessed against both YFV-17DD and a human clinical isolate in A129 and AG129 mouse flavivirus infection models, respectively. DFA significantly reduced virus replication in the livers of YFV-infected mice and the hallmarks of YFV-induced liver damage, including alanine transaminase levels and indocyanine green clearance. Collectively, this work identifies DFA as a potent YFV inhibitor and lays the groundwork for further therapeutic development as a YFV and, potentially, pan-flavivirus therapeutic.

Keywords: 2-FBU; DFA; antiviral agents; flavivirus; nucleoside analogs; yellow fever virus.

Fig 1

Chemical structures of compounds studied herein: A) DFA, B) 2-FBU, and C) sofosbuvir.

Fig 2

Molecular model predicting the binding of DFA-DP in the YFV RdRp catalytic pocket. Active site residues predicted to be involved in DFA-DP binding are highlighted. Shown residues: F atom of the DFA halogen bonds (dashed line) with E460 of the active site. Manganese co-factors (purple) bind residues D667 and N612 of the active site.

Fig 3

DFA reduces levels of infectious YFV yields in Huh-7 cells. (A, B) Huh-7 cells were infected with 260 FFU of YFV-17D (A) or 500 FFU YFV-Dak1279 (B) in a 96-well plate at 90% confluency. (A) After a 2 h incubation, the virus was removed, and cells were treated with twofold serially diluted compound (0–10 µM) in 1% methyl cellulose. After 3 days, cells were fixed, and foci were detected by ELISpot assay. (B) After a 2 h incubation, the virus was removed, and the cells were treated with twofold serially diluted compound (0–10 µM). After 2 days, infected cell supernatants were transferred to a new 96-well plate of Huh-7 cells to quantify infectious virus via foci-forming assay. (A, B) Foci were enumerated using an ImmunoSpot CTL reader, and EC50/EC90 values were calculated by linear regression on GraphPad Prism. n = 6. Shown mean ± standard error of the mean.

Fig 4

DFA’s anti-YFV-17DD activity in A129 mice. Equal numbers of male and female A129 mice aged 8–10 weeks (six mice in total) were infected i.p. with 10⁶ PFU of YFV-17DD in the presence or absence of 10 mg/kg of DFA, injected i.v. 1 h before infection, and every 24 h for 3 days thereafter. Control mock and mock + DFA groups had five mice (three males, two females; n = 5 per group). A) Liver alanine aminotransferase (ALT) levels measured by colorimetric assay from murine sera, expressed as U/mL. Statistical differences among groups were assayed by one-way ANOVA plus Dunnett’s multiple-comparison test. B) Indocyanine green (ICG) clearance on day 3 before euthanasia. Mice were administered 20 mg/kg ICG 20 m before euthanasia. After euthanasia, blood was collected from the vena cava; serum was isolated; and ICG levels were quantified by spectrophotometry. A, B) Statistical differences between groups were determined by one-way ANOVA plus Dunnett’s multiple-comparison test. Shown mean ± standard error of the mean (SEM); ****P > 0.0001. C) qRT-PCR to assess viral load in the murine liver tissue 3 dpi, following euthanasia. Statistical significance between groups assessed by two-tailed unpaired Student t-test. Shown mean ± SEM; *P = 0.0128.

Fig 5

DFA’s anti-wt-YFV activity in AG129 mice. Equal numbers of male and female AG129 mice (n = 6) aged 8–9 weeks were infected i.p. with 10³ PFU of clinical YFV isolate WT306. At 1 hpi and every 24 h thereafter, mice were administered i.v. 10 mg/kg of DFA or saline (vehicle control) for 5 days after which mice were sacrificed, and blood and tissues were collected and analyzed. An uninfected mock group with three males and two female mice was used as a negative control (n = 5). (A) Quantification of the total circulating leukocytes. Leukocytes were isolated from the murine blood collected from the vena cava and counted. Mean counts from the YFV-infected group were compared with mean counts from mock or YFV + DFA groups by one-way ANOVA plus Šídák’s multiple-comparison test. Shown, mean ± standard error of the mean (SEM); *P = 0.019 (mock vs YFV + vehicle), *P = 0.03 (YFV + vehicle vs YFV + DFA). (B) Liver alanine aminotransferase (ALT) levels measured by colorimetric assay from murine sera, expressed as U/mL. Statistical differences were assayed by one-way ANOVA plus Kruskal–Wallis multiple-comparison test. Shown mean ± SEM; *P = 0.03 (mock vs YFV + vehicle). (C) Indocyanine green (ICG) clearance on day 5 before euthanasia. Mice were administered 20 mg/kg ICG 20 min before euthanasia. After euthanasia, blood was collected from the vena cava; serum was isolated; and ICG levels were quantified by spectrophotometry. Statistical differences between groups were determined by one-way ANOVA plus Dunnett’s multiple-comparison test. Shown mean ± SEM; **P = 0.036 (mock vs YFV + vehicle), **P = 0.0075 (YFV + vehicle vs YFV + DFA). (D) qRT-PCR to assess viral load in liver tissue. Statistical significance between groups assessed by two-tailed unpaired Student’s t-test. Shown mean ± SEM; *P = 0.02. (E) Quantification of infectious viral particles in liver tissues via plaque assay on Vero cells, expressed as PFU/mL. Statistical significance between groups assessed by two-tailed unpaired Student’s t-test. Shown mean ± SEM; ***P = 0.0004. (F) Histological grading of inflammatory infiltrates in murine livers. Statistical differences between groups were determined by one-way ANOVA plus Dunnett’s multiple-comparison test. Shown mean ± SEM; ***P = 0.0006 (mock vs YFV + vehicle), **P = 0.0081 (YFV + vehicle vs YFV + DFA). G–I) H&E-stained liver slice micrographs of (G) mock, (H) YFV + vehicle, and (I) YFV + DFA. Scale bar: 50 µM.