Aug 04, 2025
Live Virus Zika - Vero-TMPRSS2 + PGP Inhibitor - 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 Zika - Vero-TMPRSS2 + PGP Inhibitor - Antiviral Screening Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v92xj1l3e/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: September 25, 2024
Last Modified: August 04, 2025
Protocol Integer ID: 108370
Keywords: antiviral, live virus, screening, drug discovery, cytotoxicity, ZIKV, Zika virus, antiviral screening against zika, live virus zika, antiviral screening assay, antiviral screening assay the following, antiviral screening, pgp inhibitor, screening assay, antiviral candidate, zika, protocol for live virus, live virus, matching cytotoxicity, screening, tmprss2
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
Disclaimer
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Abstract
The following is a protocol for live virus antiviral screening against Zika 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 Zika.
You may choose to omit PgP inhibitor.
Each live virus screening assay is accompanied by a matching cytotoxicity screening assay 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
Formaldehyde
Troubleshooting
Safety warnings
Please be sure to wear proper Personal Protective Equipment (PPE) while performing this experiment.
Before start
Assay is performed under biosafety level 3 conditions.
Seeding
The day before your assay, seed 96-well plates with Vero-TMPRSS2 cells at 4,000 cells/well using standard 10% 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 3uM elacridar (PgP inhibitor) and DMSO for your screening. DMSO supplementation will correspond to the highest amount of drug stock used.
You may omit PgP inhibitor if desired.
Manual18 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.
Calculate how much media you will need for the entire experiment using the following values in step 5.1
For this assay, the total volume of media that you make will need to be supplemented with 2uM PGP Inhibitor Elacridar. Elacridar should also be present in the media used for the actual infection so take that volume into account as well.
You may choose to omit PgP inhibitor.
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 ZIKV stock in 2% media with 2uM elacridar (or omit if desired) for infection to an MOI of approximately 0.05.
MOI is chosen to facilitate about 30% infection in the DMSO control wells.
Perform the mock infection on the cytotoxicity plates.
Add 50ul 2% media with elacridar (or omit) to the wells.
Infection
1d
Add 50uL of diluted virus to infected wells of antiviral plate
Incubate at 37 °C for 24:00:00
1d
Fixing
1d
After 24 hours, add 100ul 10 % (v/v) Formaldehyde to all wells on the plate.
Incubate for 24:00:00 at room temperature inside the bags
1d
In parallel, process the cytotoxicity plates.
Staining
Follow the ZIKA Antiviral Staining Protocol
Imaging
Image the infected plates on a plate reader. We use a Cytation1
Equipment
Cytation1
NAME
Imager
TYPE
Biotek
BRAND
n/a
SKU
LINK
Analysis