Viral infections are highly dynamic, and traditional endpoint assays in virology have a limitation that a chosen endpoint can lead to missed information. The main advantage of this technique is that with cell-based electrical impedance, it is possible to follow a viral infection and the activity of antiviral compounds in real time. Demonstrating this procedure will be Eef Meyen, a lab technician from our laboratory.
To begin, prepare the CEI plate by taking an interdigitated electrode-based 96 well plate. Add 100 microliters of growth culture medium to each well and fill the spaces between the wells with DPBS. Then, incubate the plate at 37 degrees Celsius and 5%carbon dioxide for four hours.
To seed the A549 cells, detach them from the culture flask as described in the manuscript and resuspend them in fresh growth medium. Transfer the cell suspension to a 50-milliliter tube. Count the viable cells and dilute them in fresh growth medium to obtain the desired cell density.
After incubation, remove the growth culture medium with a multi-channel pipette from the 96 well plate. Dispense 100 microliters of cell suspension per well. Once the cells are equilibrated, place the plate in the CEI device to set up and conduct the CEI assay.
Open the device's software, and in the Collect Data pane, click Setup to set a new experiment. Connect a laptop to the ECIS device. Configure the plate by checking the wells'impedances as indicated by a green color.
In the Well Configuration pane, select array type according to the used cultureware and click on Multiple Frequency time mode. Then, start the measurement by pressing Start. Keep the compound on ice.
Make the 10-times-concentrated dilution with an equilibrated assay medium in a five-milliliter polypropylene tube and then add the compound. In the Data Collection Setup pane, click Pause, and after completing the current time point, take out the 96 well plate. Observe the adherence and monolayer formation of the cells under a light microscope.
Next, aspirate 20 microliters of medium from each well using a multichannel pipette, and add 20 microliters of compound solution or vehicle in the desired wells. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 15 minutes. To prepare the virus solution, thaw a vial of virus stock.
After making dilutions at the desired MOI with assay medium in a 15-milliliter polypropylene tube, add virus stock. Once virus dilution is added in the assigned wells, add 100 microliters of assay medium to the cell control wells. Then, decontaminate the employed virus containers.
Place the plate back in the CEI device and click on Resume to continue the measurements for six consecutive days. For analyzing the data, click Finish to end the experiment and add Enter Experiment Summary. Once data collection is completed, select OK.Select the desired frequency in the Graph pane.
Ensure all wells are selected in the Well Configuration pane and click on File, then Export Data, followed by Graph Data to export the data in a spreadsheet format. The CEI assay showed that the infection kinetics were highly dependent on the cell type, and U87 cells were only susceptible to Zika virus infection at high MOIs. The CEI assays also demonstrated a fully-grown cell monolayer at high cell seeding and cannot further spread across the CEI electrodes, leading to a slight difference between the cell control and virus control.
For U87, a larger cell type, seeding cells too densely might have led to the complete detachment of the cell monolayer. A representative Zika virus strain of the African MR766 in the Asian lineage PRVABC59 exhibited similar CEI patterns. However, PRVABC59 has slightly slower cytopathic-effect-inducing properties, also reflected by the CIT50 values.
The impedance profiles of three different MOIs of both Zika virus strains demonstrated that the particular compound concentration delayed the impedance drop caused by Zika virus infection. The CEI assay revealed that the antiviral activity was dependent on the MOI and AUCN calculations determined IC50 values. The evaluated CIT50 values demonstrated compound potencies in the kinetics of cell growth and infection.
CEI is a powerful technology to evaluate and characterize compounds against Zika virus replication in a real-time, label-free, and non-invasive manner.
