Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice

Following the largest Ebola virus disease outbreak from 2013 to 2016, viral RNA has been detected in survivors from semen and breast milk long after disease recovery. However, as there have been few cases of sexual transmission, it is unclear whether every RNA positive fluid sample contains infectious virus. Virus isolation, typically using cell culture or animal models, can serve as a tool to determine the infectivity of patient samples. However, the sensitivity of these methods has not been assessed for the Ebola virus isolate, Makona. Described here is an efficiency comparison of Ebola virus Makona isolation using Vero E6, Huh-7, monocyte-derived macrophage cells, and suckling laboratory mice. Isolation sensitivity was similar in all methods tested. Laboratory mice and Huh-7 cells were less affected by toxicity from breast milk than Vero E6 and MDM cells. However, the advantages associated with isolation in Huh-7 cells over laboratory mice, including cost effectiveness, sample volume preservation, and a reduction in animal use, make Huh-7 cells the preferred substrate tested for Ebola virus Makona isolation

Testing therapeutics in cell-based assays: Factors that influence the apparent potency of drugs.

Identifying effective antivirals for treating Ebola virus disease (EVD) and minimizing transmission of such disease is critical. A variety of cell-based assays have been developed for evaluating compounds for activity against Ebola virus. However, very few reports discuss the variable assay conditions that can affect the results obtained from these drug screens. Here, we describe variable conditions tested during the development of our cell-based drug screen assays designed to identify compounds with anti-Ebola virus activity using established cell lines and human primary cells. The effect of multiple assay readouts and variable assay conditions, including virus input, time of infection, and the cell passage number, were compared, and the impact on the effective concentration for 50% and/ or 90% inhibition (EC50, EC90) was evaluated using the FDA-approved compound, toremifene citrate. In these studies, we show that altering cell-based assay conditions can have an impact on apparent drug potency as measured by the EC50. These results further support the importance of developing standard operating procedures for generating reliable and reproducible in vitro data sets for potential antivirals

Simian Hemorrhagic Fever Virus Cell Entry Is Dependent on CD163 and Uses a Clathrin-Mediated Endocytosis-Like Pathway

Simian hemorrhagic fever virus (SHFV) causes a severe and almost uniformly fatal viral hemorrhagic fever in Asian macaques but is thought to be nonpathogenic for humans. To date, the SHFV life cycle is almost completely uncharacterized on the molecular level. Here, we describe the first steps of the SHFV life cycle. Our experiments indicate that SHFV enters target cells by low-pH-dependent endocytosis. Dynamin inhibitors, chlorpromazine, methyl-β-cyclodextrin, chloroquine, and concanamycin A dramatically reduced SHFV entry efficiency, whereas the macropinocytosis inhibitors EIPA, blebbistatin, and wortmannin and the caveolin-mediated endocytosis inhibitors nystatin and filipin III had no effect. Furthermore, overexpression and knockout study and electron microscopy results indicate that SHFV entry occurs by a dynamin-dependent clathrin-mediated endocytosis-like pathway. Experiments utilizing latrunculin B, cytochalasin B, and cytochalasin D indicate that SHFV does not hijack the actin polymerization pathway. Treatment of target cells with proteases (proteinase K, papain, α-chymotrypsin, and trypsin) abrogated entry, indicating that the SHFV cell surface receptor is a protein. Phospholipases A2 and D had no effect on SHFV entry. Finally, treatment of cells with antibodies targeting CD163, a cell surface molecule identified as an entry factor for the SHFV-related porcine reproductive and respiratory syndrome virus, diminished SHFV replication, identifying CD163 as an important SHFV entry component

Effect of virus input and assay endpoint on the performance of EBOV drug screen assays.

<p>Effect of virus input and assay endpoint on the performance of EBOV drug screen assays.</p

Anti-EBOV activity of toremifene citrate in Vero E6 and Huh 7 cells under different conditions.

<p>(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.</p

Impact of cell passaging on EBOV infection.

<p>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.</p

Impact of exposure time and virus input on efficacy of toremifene citrate.

<p>(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.</p

Flow chart of the steps of the EBOV drug screen assay.

<p>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).</p

Infectivity of EBOV at an early and late cell passage of Vero E6 and Huh 7 cells.

<p>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.</p