Aug 02, 2025
Live Virus EV-A71 - RD - Antiviral Screening Assay
- Briana McGovern1,2
- 1Icahn School of Medicine at Mount Sinai;
- 2ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Briana McGovern 2025. Live Virus EV-A71 - RD - Antiviral Screening Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov12j7kgr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 20, 2025
Last Modified: August 02, 2025
Protocol Integer ID: 124745
Keywords: antiviral, live virus, screening, drug discovery, cytotoxicity, EV-A71, antiviral screening against ev, antiviral screening assay, antiviral screening assay the following, antiviral screening, live virus ev, antiviral candidate, assay in uninfected cell, protocol for live virus, screening assay, ev, uninfected cell, matching cytotoxicity, immunofluorescent staining, analysis of immunofluorescent staining, assay, a71
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
Disclaimer
Assay is performed under biosafety level 3 conditions.
Abstract
The following is a protocol for live virus antiviral screening against EV-A71 in vitro. Cells are prophylactically treated with antiviral candidates that have been serially diluted to investigate activity, via IC50 values generated via analysis of immunofluorescent staining, against EV-A71.
Each live virus screening assay is accompanied by a matching cytotoxicity screening assay in uninfected cells to investigate drug toxicity.
Materials
10% Media
2% Media
96-well plates
96-well deep well plates
12-channel 200/300ul multichannel pipette
200/300ul filtered tips
Compounds of interest
Virus(es) of interest
PPE
reservoir
Protocol materials
Deep Well Plate (96 well)Thermo FisherCatalog #10045
Troubleshooting
Safety warnings
Please be sure to wear proper Personal Protective Equipment (PPE) while performing this experiment.
Before start
Always wear appropriate PPE for this protocol
Refer to Material Safety Data Sheets for additional safety and handling information.
Seeding
The day before your assay, seed 96-well plates with RD cells at 5,000 cells/well using standard RD growth media
You will need to make one pair of plates for every 3 compounds. One plate is the antiviral plate that will be infected, and the other is the cytoxicity plate that will measure the toxicity of the antiviral candidates.
For each pair you can test 3 compounds (in triplicate, 8 dilutions per compound), 2 DMSO controls, and 1 uninfected control in the following layout:
Treatment layout
Setting up the conditions
Create a screening priority list ahead of time. This list will have the compounds of interest, what maximum concentration you'd like to screen at, and where the compounds are located. We recommend organizing the compounds the day before.
The serial dilutions will follow different protocols depending on if you will do them by hand or by using a
Equipment
Tecan D300e
NAME
Liquid Handler
TYPE
Tecan
BRAND
30100152
SKU
https://lifesciences.tecan.com/products/liquid_handling_and_automation/tecan_d300e_digital_dispenser
LINK
Before the assay, you should prepare enough 2% media supplemented with DMSO for your screening. DMSO supplementation will correspond to the highest amount of drug stock used.
Manual12 steps
Performing the serial dilutions manually.
If your lab does not have a Tecan D300e machine you will need to do the dilutions by hand.
Remove your compounds from -20 to thaw while you set up.
Prepare the Deep Well Plate (96 well)Thermo FisherCatalog #10045 according to this diagram:
Deep well set up for manual serial dilutions
The first row will be the [max] of your compounds. This row will contain 1100ul of 2% media without DMSO. The media for this row does not receive any DMSO since you will add drug dissolved in DMSO directly to it.
The remaining rows of the plate will be filled with 750ul 2% media + supplemented with DMSO.
These volumes are enough to treat both the antiviral and cytotoxicity plates.
Perform the serial dilutions:
Add the compounds of interest to the first row of the deep well (containing 1100ul media).
mix well and move 375ul from row 1 to the row 2. This is your first 1:3 dilution.
Schematic of the manual serial dilutions
Repeat until the entire deep well has been done.
You now have 8 concentrations of your compounds of interest.
Remove the growth media from the plates. Work one pair of plates at a time to prevent drying of the cells.
Add 100ul of the serial dilutions to the wells in the following set up: You will perform this for both the infection plate and the matching cytotoxicity plate
Treatment layout of the HeLa-ACE2 plates.
Notice how the inner columns of the plate receive experimental treatment, while the outer columns are the controls.
You should always have the plates in the correct orientation (top left corner should be A1) so that you are sure where the highest and lowest [compound] are.
Incubate the cells at 37 °C 5% CO2 for 02:00:00 .
This pretreatment time frame could differ depending on what sort of compounds you'd like to test.
2h
Prepping for infection
1h 30m
Dilute EV-A71 in 2% media to MOI 0.5
perform the mock infection on the cytoxicity plates. We want both plates to match so that we can compare them later.
Add 50ul 2% media to the wells.
Infection
1d
Infect the wells with 50ul
Incubate at 37 °C for 7 days
1d
MTS
Instead of fixing with formaldehyde, use the MTS protocol at the end of the cytoxicity assay for both the antiviral and cytotoxicity plates