The search for an antiviral lead molecule to combat the neglected emerging Oropouche virus

Abstract

Oropouche virus (OROV) is a member of the Peribunyaviridae family and the causative agent of a dengue-like febrile illness transmitted by mosquitoes. Although mild symptoms generally occur, complications such as encephalitis and meningitis may develop. A lack of proper diagnosis, makes it a potential candidate for new epidemics and outbreaks like other known arboviruses such as Dengue, Yellow Fever and Zika virus. The study of natural molecules as potential antiviral compounds is a promising alternative for antiviral therapies. Wedelolactone (WDL) has been demonstrated to inhibit some viral proteins and virus replication, making it useful to target a wide range of viruses. In this study, we report the in silico effects of WDL on the OROV N-terminal polymerase and its potential inhibitory effects on several steps of viral infection in mammalian cells in vitro, which revealed that WDL indeed acts as a potential inhibitor molecule against OROV infection.

Keywords: Antiviral; Oropouche virus; Wedelolactone.

Graphical abstract

Fig. 1

Wedelolactone chemical structure. Generated by ChemDraw.

Fig. 2

Inhibition effect of WDL over Endo-Nter domain of OROV followed by dose response curve for IC₅₀ determination and inhibition mode of the molecule. (a) Normalised inhibition effect of WDL, (b) the normalised response [%] of Endo-Nter is plotted against the Log of the inhibitor concentration (c) Lineweaver-Burk plot for WDL inhibition of Endo-Nter. Values are shown as the mean ± standard error obtained from three independent experiments.

Fig. 3

MTT viability assay for WDL in Vero cells analysed 48 h post-treatment. The y-axis represents cell viability in percentages, and the x-axis represents the used WDL concentrations ranging from 50 to 400 µM. Values are shown as the mean ± standard error obtained from three independent experiments. Generated by GraphPad Prism.

Fig. 4

WDL effect on viral yield is concentration-dependent. OROV production was measured in the presence of several dilutions of the tested compound in Vero cells, with an initial inoculum of MOI 0.1 under post-treatment. PFU infectivity titration of OROV is shown in the left vertical axis. Values are the mean ± standard error obtained from three independent experiments. Asterisks indicate statistical significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett's test (*, p < 0.05/ **, p < 0.001/ ****, p < 0.0001). Generated by GraphPad Prism.

Fig. 5

WDL affects viral progeny production. Antiviral effects of WDL (40 µM) against OROV (MOI = 0.1) infection. Vero cells were infected with OROV and treated with WDL. Culture supernatants were harvested at 12, 24, 48 and 72 hpi and OROV progeny yields were measured through plaque-forming assays. Values are the mean ± standard error obtained from three independent experiments. Asterisks indicate statistical significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett's test (ns, not significant/ *, p < 0.05/ **, p < 0.001/ ***, p < 0.0005). MOCK: cells treated only with DMSO; Viral control: OROV infected cells; WDL: cells infected with OROV and treated with Wedelolactone. Generated by GraphPad Prism.

Fig. 6

Ensemble docking with the 2000 frames of each replicates using Vina software. (a) Replicate 1 followed by the 3D model representing the best (purple ribbon) and the worst (pink ribbon) Vina score. (b) Replicate 2 followed by the 3D model representing the best (dark blue ribbon) and the worst (light blue ribbon) Vina score. (c) Replicate 3 followed by the 3D model representing the best (red ribbon) and worst (orange ribbon) Vina score. Mn⁺² is shown as a green sphere and the WDL molecule as a yellow stick.

Fig. 7

Per residue decomposition of the binding energy of the Endo-Nter/WDL complex of three independent replicates with the best Vina score (left panel). The 3D ligand binding representation (< 5 Å) is shown in the middle panel (residues are represented as a grey stick of the WDL molecule as a pink, blue, and yellow stick, respectively). The right panel shows the 2D interaction. (a) replicate one, (b) replicate two, and (c) replicate three. Residues are numbered according to the protein sequence, and Mn²⁺ is shown as a green sphere.

Fig. 8

Per residue decomposition of the binding energy of the Endo-Nter/WDL complex of three independent replicates with the worst Vina score (left panel). The 3D ligand binding representation (< 5 Å) is shown in the centre panel (residues are represented as a grey stick of the WDL molecule as a pink, blue, and yellow stick, respectively). The right panel shows the 2D interaction. (a) Replicate one, (b) replicate two, and (c) replicate three. Residues are numbered according to the protein sequence, and Mn2+ is shown as a green sphere.