Fig 1.
Flow chart of the steps of the EBOV drug screen assay.
Cells and media are prepared in 100 μl/well cell plates and incubated overnight. Drugs in 50 μl/well are transferred from drug dilution plates to cell plates using a 96-well manual benchtop pipettor for 1 h of contact. In the biosafety level-4 (BSL4) laboratory, EBOV in 50 μl/ well is transferred to the cell/drug plates using a 96-well manual benchtop pipettor for a final volume of 200 μl/well. At specific assay endpoints, cells are fixed and transferred to the BSL-2. Immunostaining was performed with a EBOV-specific antibody against VP40 and a fluorescent or chemiluminescent secondary antibody using a plate washer/Dispenser. Fluorescence is quantified on a plate reader. The HCI system (Operetta) is used to detect EBOV-positive cells and count cells with a nuclei stain (Hoechst 33342). In parallel, cytotoxicity assays (CellTiter Glo) with mock infected cells are performed at BSL-2. Luminescence is read on the Infinite® M1000 Tecan plate reader. Data are analyzed using GraphPad Prism and/or Columbus software (Operetta).
Fig 2.
Susceptibility of different cell types to infection with EBOV.
Vero E6 (A, B), Huh 7 (C, D), and MDM (E, F) cells were infected with EBOV/Mak at a range of MOIs. The assay endpoints were 24, 48, 72 or 96 hpi. Following staining with a VP40-antibody, HCI was performed to determine the percentage of EBOV-positive cells. Corresponding HCI images for EBOV-positive cells and nuclei (B, D, F) are shown. (G) Cell layer viability. Vero E6 cells were infected with EBOV/Mak at an MOI of 0.33 for 96 h. Two representative well images are shown; one image (well C4) has an intact cell layer, while the other image (well C6) shows the cell layer peeling off the well surface. Top panels show cells stained with the anti-EBOV VP40 antibody, bottom panels show the nuclei stain. Values are the average of triplicate wells (mean ± SD; n = 3). Abbreviations: EBOV, Ebola virus; hpi, hours post-inoculation; HCI, high-content imaging; Mak, Makona; MOI, multiplicity of infection; SD, standard deviation; VP40, EBOV VP40 matrix protein.
Table 1.
Effect of time postinoculation and virus inoculum on the fluorescent EBOV drug screen assay on Vero E6 cells.
Table 2.
Effect of time postinoculation and virus inoculum on the fluorescent EBOV drug screen assay on Huh 7 cells.
Fig 3.
Impact of cell passaging on EBOV infection.
Vero E6 (A) and Huh 7 (B) cells were passaged and infected with EBOV/Mak at an MOI of 1 at 48 hpi, the plates were fixed, stained, and the percentage of EBOV-positive cells was determined by HCI. Each point is the mean of 3 replicate wells and represents an independent experiment. For each passage, the median value of all experiments was determined. Abbreviations: EBOV, Ebola virus; HCI, high content imaging; hpi, hours post-inoculation; Mak, Makona.
Fig 4.
Infectivity of EBOV at an early and late cell passage of Vero E6 and Huh 7 cells.
The signal-to-noise ratio (Tecan plate reader) was determined for Vero E6 (A) cells at passage 6 and 28 and for Huh 7 (B) cells at passage 7 and 30. The cells were infected at different MOIs of EBOV/Mak for 48 h, then fixed and stained. The percentage of EBOV-positive cells (C, D) by HCI was determined in parallel. The S/N ratios were determined from the mean values (± SD, n = 3) of triplicate signal and noise wells. Values for % EBOV-positive cells were determined from triplicate wells (mean ± SD, n = 3). The data were derived from one individual experiment. Two-tailed paired t test was performed to compare the values for % of EBOV-positive cells between early and late cell passage in variable MOIs, which is significantly higher in early passage of than late passage in Vero E6 cells only. Abbreviations: EBOV, Ebola virus; HCI, high content imaging; Mak, Makona; MOI, multiplicity of infection; S/N, signal-to-noise.
Fig 5.
Impact of exposure time and virus input on efficacy of toremifene citrate.
(A) Huh 7 cells were infected at an MOI of 1, and antiviral activity of toremifene citrate was evaluated at indicated time points. (B) Huh 7 cells were infected at indicated MOIs and antiviral activity of toremifene citrate was evaluated at 72 hpi. (C, D) EC50s with corresponding assay endpoints or MOIs are shown for comparison. The experiment was performed twice using the fluorescent assay. Representative graphs are shown. Abbreviations:EC50, half maximal effective concentration; hpi, hours post-inoculation; MOI, multiplicity of infection.
Fig 6.
Anti-EBOV activity of toremifene citrate in Vero E6 and Huh 7 cells under different conditions.
(A) Vero E6 cells and (B) Huh 7 cells were infected at varying MOIs with different assay end points and treated with toremifene citrate. EC50s were determined from 8-point dose response curves using the fluorescent assay. (C, D) EC50s of toremifene citrate with corresponding assay end points or MOIs are shown for comparison. Representative graphs from 1 to 4 independent experiments are shown. Abbreviations: EBOV, Ebola virus; EC50, half maximal effective concentration; h, hour; MOI, multiplicity of infection; N.A., not applicable.
Table 3.
Effect of virus input and assay endpoint on the performance of EBOV drug screen assays.
Table 4.
Comparison of anti-EBOV efficacy of toremifene citrate under different assay conditions.
Fig 7.
Assay parameters and toremifene activity in the CELIA.
The antiviral activity of toremifene citrate was evaluated at an MOI of 0.5 (EBOV/Mak) with a 48 hpi endpoint in Vero E6, Huh 7 and MDM cells using the CELIA. (A) The signal-to-noise ratio and Z' factor were determined in multiple independent experiments. (B) EC50s were determined. Data were collected from 4 to over 10 experiments and the median value of all experiments was determined. Ordinary one-way ANOVA following Turkey’s post Multiple Comparison in Graphpad Prism 7.0 were performed to compare the differences of signal-to-noise ratio, Z' factor and EC 50S among three cell lines. Abbreviation: EBOV, Ebola virus; EC50, half maximal effective concentration; Mak, Makona; MDM, monocyte-derived macrophages.