Beyond Members of the Flaviviridae Family, Sofosbuvir Also Inhibits Chikungunya Virus Replication

Abstract

Chikungunya virus (CHIKV) causes a febrile disease associated with chronic arthralgia, which may progress to neurological impairment. Chikungunya fever (CF) is an ongoing public health problem in tropical and subtropical regions of the world, where control of the CHIKV vector, Aedes mosquitos, has failed. As there is no vaccine or specific treatment for CHIKV, patients receive only palliative care to alleviate pain and arthralgia. Thus, drug repurposing is necessary to identify antivirals against CHIKV. CHIKV RNA polymerase is similar to the orthologue enzyme of other positive-sense RNA viruses, such as members of the Flaviviridae family. Among the Flaviviridae, not only is hepatitis C virus RNA polymerase susceptible to sofosbuvir, a clinically approved nucleotide analogue, but so is dengue, Zika, and yellow fever virus replication. Here, we found that sofosbuvir was three times more selective in inhibiting CHIKV production in human hepatoma cells than ribavirin, a pan-antiviral drug. Although CHIKV replication in human induced pluripotent stem cell-derived astrocytes was less susceptible to sofosbuvir than were hepatoma cells, sofosbuvir nevertheless impaired virus production and cell death in a multiplicity of infection-dependent manner. Sofosbuvir also exhibited antiviral activity in vivo by preventing CHIKV-induced paw edema in adult mice at a dose of 20 mg/kg of body weight/day and prevented mortality in a neonate mouse model at 40- and 80-mg/kg/day doses. Our data demonstrate that a prototypic alphavirus, CHIKV, is also susceptible to sofosbuvir. As sofosbuvir is a clinically approved drug, our findings could pave the way to it becoming a therapeutic option against CF.

Keywords: antiviral; arthralgia; chikungunya; chikungunya virus; drug; sofosbuvir.

FIG 1

Sofosbuvir triphosphate and NsP4 interactions. (A) Structural representation of the nsP4 model of CHIKV and its interaction with SFV and UTP. Hydrogen bonds and electrostatic interactions between SVF (B and C) and UTP (D and E) and nsP4 model of CHIKV. The interactions are represented by blue (H-bonds), green (attractive electrostatic interactions), and red (repulsive electrostatic interactions) interrupted lines. The nitrogen atoms are shown in blue, oxygen in red, fluor in pink, and the carbon chain in gray.

FIG 2

Inhibition of CHIKV replication in hepatoma cells. Huh-7 cells (10⁴ cells/well in 96-well plate [A] and 2.10⁵ cells/well in 24-well plate [B]) were infected with CHIKV at an MOI of 0.1 and exposed to various concentrations of sofosbuvir, ribavirin, or mycophenolic acid for 24 h. Supernatant was harvested, and virus content was determined by measuring RNA levels (A) or titers (B) in Vero cells. The data represent means ± standard errors of the means (SEM) from five independent experiments.

FIG 3

Sofosbuvir inhibits CHIKV-induced cell death and replication in iPSC-derived human astrocytes. (A and B) Astrocytes were infected at the indicated MOIs and treated with sofosbuvir at 10 μM. After 3 days, cells were labeled for activated caspase-3/7 and propidium iodide (A) and titers of virus in the supernatant were determined in Vero cells (B). (C and D) Astrocytes were infected at an MOI of 1.0 and treated with sofosbuvir or ribavirin at the indicated concentrations. After 3 days, cells death (C) and virus titers in Vero cells (D) were determined. The data represent means ± SEM from five independent experiments performed with five technical replicates. *, P < 0.05 for comparisons between infected, untreated (gray bars) and treated (black bars) groups (A and B) and for comparisons between groups infected/treated with sofosbuvir (open squares) and ribavirin (black squares) (C and D).

FIG 4

Sofosbuvir ameliorated CHIKV-induced paw edema. Male Swiss Webster mice (20 to 25 g) received RPMI medium (mock infected) or 2 × 10⁵ PFU CHIKV in 50 μl per paw in the ventral side of the right hind foot. Oral treatment with sofosbuvir (20 mg/kg/day) started 1 h prior to infection and continued thereafter daily. (A) Paw volume was measured in a hydropletismometer and normalized to the paw volume of each animal before injection. *, P < 0.05 by Tukey's multiple-comparison test (n = 8/group). (B) On the sixth day after infection, animals were euthanized and quantitative RT-PCR was performed with RNA from the serum or macerated paw. *, P < 0.05 by Student's t test (n = 3/group).

FIG 5

Representative CHIKV-associated paw edema and sofosbuvir protection. The hind paws of mock-infected, CHIKV-infected (untreated), and CHIKV-infected/treated animals at the 6th day after infection are displayed. Red arrows indicate the infected paw (A to C). Histopathology data representing H&E-stained sections of paws at 40× (D to F) and 100× (G to I) are presented. Panels A, D, and G represent the mock-infected group. Panels B, E, and H represent the CHIKV-infected and untreated group. Panels C, F, and I represent the CHIKV-infected group treated with sofosbuvir. Images are representative of at least 5 animals per group.

FIG 6

Sofosbuvir, at concentrations of 40 and 80 mg/kg/day, increased survival and inhibited weight loss of CHIKV-infected mice. Three-day-old Swiss mice were infected with CHIKV (2 × 10² PFU) and treated with sofosbuvir (SF) either 1 day before (pretreatment) or 2 days after infection (late treatment). Survival (A and C) and weight variation (B and D) were assessed during the course of treatment. (A and B) Experiments of both pretreatment and late treatment with sofosbuvir at 20 mg/kg/day. (C and D) Pretreatment with the indicated concentrations of sofosbuvir. 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 day was used to assess significance. Independent experiments were performed with at least 10 mice/group (n = 30). *, P < 0.01.

FIG 7

Sofosbuvir prevented neuromotor impairment of CHIKV-infected mice. Three-day-old Swiss mice were infected with CHIKV (2 × 10² PFU) and treated with sofosbuvir (SF) beginning at 1 day before infection (pretreatment) or 2 days after infection (late treatment). (A) Treatment was performed with a dose of 20 mg/kg/day. (B) Pretreatment was performed with the indicated concentrations. At the sixth day after infection, animals were turned on their backs and allowed up to 60 s to return upright. The results are presented as means ± SEM. This was a routine measure, and at least 10 animals per group were analyzed. Student's t test was used to compare untreated CHIKV-infected mice with other groups individually. *, P < 0.01 mock- versus CHIKV-infected animals. #, P < 0.01 untreated versus treated animals.