Oct 26, 2025
Live Virus EV-D68 - on RD cells - Antiviral Screening Assay
- Nick Lynch1,2
- 1Curlew Research;
- 2ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Nick Lynch 2025. Live Virus EV-D68 - on RD cells - Antiviral Screening Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.261gek27og47/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 30, 2025
Last Modified: October 26, 2025
Protocol Integer ID: 228530
Keywords: EV-D68, drug discovery, cytotoxicity, antiviral, Enterovirus, antiviral screening assay, antiviral screening assay the following, antiviral screening, antiviral screening against ev, assay in uninfected cell, rd cell, antiviral candidate, protocol for live virus, live virus ev, live virus, screening assay, rhabdomyosarcoma, uninfected cell, matching cytotoxicity, drug toxicity
Disclaimer
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgement, advice, diagnosis, or treatment. Any action you take or refrain from taking, using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held
Abstract
The following is a protocol for live virus antiviral screening against EV-D68 in vitro on Rhabdomyosarcoma (RD) cells. RD Cells are prophylactically treated with antiviral candidates that have been serially diluted to investigate activity. IC50 values are generated via analysis of MTT/MTS data as a readout of CPE (cytopathic effect).
Each live virus screening assay is accompanied by a matching cytotoxicity screening assay in uninfected cells to investigate drug toxicity.
Guidelines
4. Adding Virus:
a. Prepare virus to appropriate dilution in ASSAY MEDIUM
a. EV-D68: Prepare a 100 TCID50/mL (adding 100 µL/well results in 10 TCID50/well and thus with 20,000 cells and MOI = 0.0004 TCID50/cell
i. For the current screening stock which is 1.00x10^8 TCID50/mL a 1/5x10^5 predilution is needed.
b. Add 100 µL of virus preparation to C1-10
5. Incubate plates (35°C / 5% CO2)
6. On day 4 determine cell viability using MTS (see Jochmans D et al. J Virol Methods. 2012 Aug;183(2):176-9. doi: 10.1016/j.jviromet.2012.04.011).
For toxicity measurement the same protocol is applied but without virus.
Materials
- DMEM (gibco cat no 41965-039)
- Heat-inactivated FCS
- Pen-Strep (10,000 U/mL) (Gibco cat no 15140-148)
- Trypsin 0.25%/trypsine/EDTA
- DPBS
- T150 bottle
- 96-well plate (example Greiner Bio One 655090 or Falcon)
Troubleshooting
Safety warnings
Please be sure to wear proper Personal Protective Equipment (PPE) while performing this experiment.
Note
This protocol can be used for anti virus assay and the equivalent Cytotoxicity assay where no virus is added.
For the cytotoxicity assay, do not add the virus in the later stages
Cell Culture (1/10 split, twice each week)
RD Cells are propagated in GROWTH MEDIUM which is prepared by supplementing DMEM (gibco cat no 41965-039) with 2% v/v heat-inactivated FCS and 1% Pen-Strep (10,000 U/mL)(Gibco cat no 15140-148). Cells are cultured at 37°C, 5%CO2 in T150 bottle in 35 mL GROWTH MEDIUM and split 1/10 twice a week. Detachment is performed by washing the monolayer 1x with DPBS, cover cell layer 1 minute with 5 mL trypsine 0.25%trypsine/EDTA at room temperature, remove 4.5 mL and incubate 3 minute at 37°C.
Antiviral Assay
ASSAY MEDIUM is prepared by supplementing DMEM (gibco cat no 41965-039) with 2% v/v heat-inactivated FCS, 1% Pen-Strep (10,000 U/mL)(Gibco cat no 15140-148).
Prepare cell suspension:
start with confluent T150 bottle
wash monolayer with DPBS
add 5 mL trypsine 0.25%trypsine/EDTA, cover the cell layer by placing the bottle horizontally, incubate for 1 min at room temperature, remove 4.5 mL of the trypsine solution
incubate 15 minutes at 37°C
resuspended in 10 mL ASSAY MEDIUM
count
resuspend at 25,000 cells/100 µL in ASSAY MEDIUM
seed 100 µL of cell suspension to each well of a 96w plate (example Greiner Bio One 655090 or Falcon)
in case of insufficient cells: medium instead of cells can be added to border wells
Incubate plates overnight (37°C / 5%CO2)
Adding Compound:
Add 100 µL ASSAY MEDIUM to C11 and C12
Add 50 µL ASSAY MEDIUM to C2
Add compound to C2 row B-G:
For stock 10 mM to 100 µM final start conc. you need to add 3 µL. With stock 10 mM and 10 µM final start conc. you prepare a 1/10 predilution in ASSAY MEDIUM (for example mix 10 µL 10 mM with 90 µL in C1 and add 3 µL to C2).
Dilute 1/3 over the plate (transfer 50 µL) from C2-9 – change tips in C4 and C7
Adding Virus:
Prepare virus to appropriate dilution in ASSAY MEDIUM
EV-D68: Prepare a 100 TCID50/mL (adding 100 µL/well results in 10 TCID50/well and thus with 20,000 cells and MOI = 0.0004 TCID50/cell
For the current screening stock which is 1.00x108 TCID50/mL a 1/5x105 predilution is needed.
Add 100 µL of virus preparation to C1-10
Incubate plates (35°C / 5% CO2)
On day 4 determine cell viability using MTS (see Jochmans D et al. J Virol Methods. 2012 Aug;183(2):176-9. doi: 10.1016/j.jviromet.2012.04.011).
For toxicity measurement the same protocol is applied but without virus.
Protocol references
A novel method for high-throughput screening to quantify antiviral activity against viruses that induce limited CPE
Jochmans D et al. J Virol Methods. 2012 Aug;183(2):176-9. doi: 10.1016/j.jviromet.2012.04.011.
Acknowledgements
We thank KU Leuven Laboratory of Virology & Antiviral Research for providing the protocol details