Vero 76 Cell Reduction of the Virus-Induced Cytopathic Effect (Primary CPE Assay)

Confluent or near-confluent cell culture monolayers of Vero 76 cells are prepared in 96-well disposable microplates the day before testing. Cells are maintained in the minimum essential medium (MEM) supplemented with 5% fetal bovine serum (FBS). For antiviral assays, the same medium is used but with FBS reduced to 2% and supplemented with 50 μg/mL gentamicin. Compounds are dissolved in DMSO, saline, or the diluent requested by the submitter. Less soluble compounds are vortexed, heated, and sonicated, and if they still do not go into the solution, they are tested as colloidal suspensions. The test compound is prepared at four serial log₁₀ concentrations, usually 0.1, 1.0, 10, and 100 μg/mL or μM (as per the sponsor’s preference). Lower concentrations are used when an insufficient compound is supplied. Five microwells are used per dilution: three for infected cultures and two for uninfected toxicity cultures. Controls for the experiment consist of six microwells that are infected and not treated (virus controls) and six that are untreated and uninfected (cell controls) on every plate. A known active drug is tested in parallel as a positive control drug using the same method as is applied for test compounds. The positive control is tested with every test run.

Growth media are removed from the cells, and the test compound is applied in a 0.1 mL volume to wells at a 2× concentration. The virus, normally at an ∼60 CCID₅₀ (50% cell culture infectious dose) in a 0.1 mL volume is added to the wells designated for virus infection. The medium devoid of the virus is placed in toxicity control wells and cell control wells. Plates are incubated at 37 °C with 5% CO₂ until marked CPE (>80% CPE for most virus strains) is observed in virus control wells. The plates are then stained with 0.011% neutral red for approximately 2 h at 37 °C in a 5% CO₂ incubator. The neutral red medium is removed by complete aspiration, and the cells may be rinsed 1× with phosphate-buffered solution (PBS) to remove the residual dye. PBS is completely removed, and the incorporated neutral red is eluted with 50% Sorensen’s citrate buffer/50% ethanol for at least 30 min. The neutral red dye penetrates living cells; thus, the more intense the red color, the larger the number of viable cells present in the wells. The dye content in each well is quantified using a spectrophotometer at a 540 nm wavelength. The dye content in each set of wells is converted to a percentage of the dye present in untreated control wells using a Microsoft Excel computer-based spreadsheet and normalized based on the virus control. The 50% effective (EC₅₀, virus-inhibitory) concentrations and 50% cytotoxic (CC₅₀, cell-inhibitory) concentrations are then calculated by regression analysis. The quotient of CC₅₀ divided by EC₅₀ gives the SI value. Compounds showing SI values ≥ 10 are considered active.