Aug 02, 2025

Public workspaceLive Virus EV-D68 - RD - Antiviral Screening Assay

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvmywybv3p/v1
  • Rebecca Pearl1,2,
  • Randy Albrecht1,2
  • 1Icahn School of Medicine at Mount Sinai;
  • 2ASAP Discovery Consortium
  • ASAP Discovery
Nick Lynch
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvmywybv3p/v1
Protocol Citation: Rebecca Pearl, Randy Albrecht 2025. Live Virus EV-D68 - RD - Antiviral Screening Assay . protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmywybv3p/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: April 18, 2025
Last Modified: August 02, 2025
Protocol Integer ID: 130984
Keywords: EV-D68, live virus, antiviral, drug discovery, cytotoxicity, enterovirus, antiviral screening assay, antiviral screening assay the following, antiviral screening, antiviral screening against ev, assay in uninfected cell, protocol for live virus, antiviral candidate, live virus ev, using rhabdomyosarcoma, live virus, screening assay, pgp inhibitor in the assay, rd cell, uninfected cell, matching cytotoxicity, pgp inhibitor, assay, immunofluorescent staining, drug toxicity, analysis of immunofluorescent staining, d68
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 EV-D68 in vitro using Rhabdomyosarcoma (RD) cells. RD 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-D68.
You may opt to include PgP inhibitor in the assay by adding 2uM into the infection media.
Each live virus screening assay is accompanied by a matching cytotoxicity screening assay in uninfected cells to investigate drug toxicity.
Materials
1. D300e
- D300e program on computer
- D300e machine
- D4 cassettes
- T8 cassettes
2. Media
- Fresh DMSO
- Infection media:
a) 500 mL DMEM
b) 10 mL FBS for 2% FBS
c) 5 mL P/S
d) 5 mL NEAA
e) 5 mL HEPES
3. Compound
- Stock concentration
- Working concentration
- ***Rupintrivir as reference compound***
4. Materials
- 1x 96-well flat bottom Greiner plate (treated with Poly L-Lysine, black)
- 1x 96-well flat bottom Greiner plate (treated with Poly L-Lysine, clear)
- Deep 96-well plate
- Reservoirs
- Multichannel Pipette
- Automatic multichannel pipette
5. Cells
- RD cells (passage should be low ~15)
6. Virus
- EVD68 MO/14 virus stock 20180911 EV-D68 Virus stock (MO/14-18347) 20180911 (Titer: 6.25E5 PFU/mL)
- EVA71 Strain H LLCMK2 7DPI 5/16/22 (Titer: 7.5E5 PFU/mL)
Protocol materials
ReagentCorning® Fetal Bovine SerumCorningCatalog #35-010-CV
ReagentHEPES (1M)Gibco - Thermo Fisher ScientificCatalog #15630080
ReagentMEM Non-Essential Amino Acids Solution (100X)Gibco - Thermo Fisher ScientificCatalog #11140050
ReagentCorning® 500 mL DMEM (Dulbecco’s Modified Eagle’s Medium)CorningCatalog #10-017-CV
ReagentPenicillin-Streptomycin (10,000 U/mL)Gibco - Thermo Fisher ScientificCatalog #15140122
ReagentDimethyl sulfoxide 100mL Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2650-100ML
ReagentEnterovirus D68 VP1 antibodyGenetexCatalog #GTX132313
ReagentDAPI and Hoechst Nucleic Acid StainsInvitrogen - Thermo FisherCatalog #H1399
ReagentGoat anti-Rabbit IgG (H L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488InvitrogenCatalog #A-11034
ReagentBovine serum albumin (BSA) Gemini 700-101PGemini Bio-ProductsCatalog #700-101P
ReagentTriton X-100Fisher Scientific
Reagent37% FormaldehydeFisher ScientificCatalog #F79-1
ReagentPhosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 ± 0.1CorningCatalog #21-040-CV
ReagentMTT Reagent AMerck MilliporeSigma (Sigma-Aldrich)Catalog #CT01-5
ReagentIsopropanolFisher ScientificCatalog #BP2618500
ReagentCorning® 96 well platesCorningCatalog #CLS9102
ReagentClear 96-well flat-bottom microplateCorningCatalog #353072
ReagentFalcon® 175cm² Rectangular Straight Neck Cell Culture Flask with Vented CapCorningCatalog #353112
ReagentCorning® Costar® reagent reservoirsCorningCatalog #CLS4873
Reagent2.0ml 96 Well Deep Well Plates Square WellNEST BiotechnologyCatalog #503001/503501
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.
Summary
RD cells (2k per well) were seeded in 96 Well Black/Clear Bottom Plates and incubated overnight. On the following day, cell density was determined using Trypan Blue. Infection media containing DMSO was prepared, and deep 96-well plates were filled with 800 μL per well, except for column 12. Diluted compounds were added to the designated wells. After a 2.5-hour incubation at 37°C with 5% CO2, the test plate was infected with EV-D68 (strain US/MO/14-18949, ATCC/BEI Resources) at an MOI of 1. After 24 hours of infection, media was aspirated, and cells were fixed with 4% Formaldehyde + PBS.
Following fixation, formaldehyde was carefully removed and the monolayers were washed once with PBS. PBS was discarded, and cells were incubated with 0.1% Triton X-100 for 20 minutes. The monolayer was washed three times with PBS. Cells were then incubated with 0.3% BSA/PBS for 30 minutes. The primary antibody, Anti-Enterovirus D68 VP1 (GTX132313), was diluted in 0.3% BSA/PBS and added to the wells. Following a 1-hour incubation, cells were washed three times with PBS. The secondary antibody, goat antirabbit IgG (H+L) Alexa Fluor 488 (ab150077), diluted in 0.3% BSA/PBS, was added to the cells and incubated for an additional hour, protected from light. The monolayer was washed three times with PBS and counterstained with DAPI. The final PBS wash was left on the cells for imaging.

Imaging was performed using the Cytation 1 system, Viral infection and cell viability were assessed using fluorescence imaging to quantify Alexa Fluor 488 and DAPI signals. Images were analyzed using Gen5 software to calculate infection rates and determine antiviral
efficacy.

Rupintrivir was used as a positive antiviral control. RD cell cytotoxicity was determined by MTT assays on uninfected cells.
Materials
Plates:
  1. 96- Well Deep Well plate Reagent2.0ml 96 Well Deep Well Plates Square WellNEST BiotechnologyCatalog #503001/503501
  2. Black 96-well Flat Bottom plate with Clear Wells ReagentCorning® 96 well platesCorningCatalog #CLS9102
  3. Clear 96-well Flat Bottom plate ReagentClear 96-well flat-bottom microplateCorningCatalog #353072

Flask:
ReagentFalcon® 175cm² Rectangular Straight Neck Cell Culture Flask with Vented CapCorningCatalog #353112

Reservoirs:
ReagentCorning® Costar® reagent reservoirsCorningCatalog #CLS4873
Media:
Cell Culture Media:
Amount500 mL ReagentCorning® 500 mL DMEM (Dulbecco’s Modified Eagle’s Medium)CorningCatalog #10-017-CV
  1. Amount50 mL ReagentCorning® Fetal Bovine SerumCorningCatalog #35-010-CV
  2. Amount5 mL ReagentPenicillin-Streptomycin (10,000 U/mL)Gibco - Thermo Fisher ScientificCatalog #15140122
  3. Amount5 mL ReagentHEPES (1M)Gibco - Thermo Fisher ScientificCatalog #15630080 ReagentHEPES (1M)Gibco - Thermo Fisher ScientificCatalog #15630080
Infection Media:
  1. Amount500 mL ReagentCorning® 500 mL DMEM (Dulbecco’s Modified Eagle’s Medium)CorningCatalog #10-017-CV
  2. Amount10 mL ReagentCorning® Fetal Bovine SerumCorningCatalog #35-010-CV
  3. Amount5 mL ReagentPenicillin-Streptomycin (10,000 U/mL)Gibco - Thermo Fisher ScientificCatalog #15140122
  4. Amount5 mL ReagentMEM Non-Essential Amino Acids Solution (100X)Gibco - Thermo Fisher ScientificCatalog #11140050
  5. Amount5 mL ReagentHEPES (1M)Gibco - Thermo Fisher ScientificCatalog #15630080 ReagentHEPES (1M)Gibco - Thermo Fisher ScientificCatalog #15630080
DMSO + Infection Media:
  1. Amount50 mL of Infection Media (2.2)
  2. Amount150 µL ReagentDimethyl sulfoxide 100mL Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2650-100ML
Immunofluorescence Staining:
Antibodies:
  1. ReagentEnterovirus D68 VP1 antibodyGenetexCatalog #GTX132313
  2. ReagentDAPI and Hoechst Nucleic Acid StainsInvitrogen - Thermo FisherCatalog #H1399
  3. ReagentGoat anti-Rabbit IgG (H L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488InvitrogenCatalog #A-11034
Solutions:
  1. 0.3% ReagentBovine serum albumin (BSA) Gemini 700-101PGemini Bio-ProductsCatalog #700-101P
  2. 0.1% ReagentTriton X-100Fisher Scientific
  3. 4% Reagent37% FormaldehydeFisher ScientificCatalog #F79-1
  4. ReagentPhosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 ± 0.1CorningCatalog #21-040-CV
Cytotoxicity Testing:
  1. ReagentMTT Reagent AMerck MilliporeSigma (Sigma-Aldrich)Catalog #CT01-5
  2. ReagentIsopropanolFisher ScientificCatalog #BP2618500
Tecan D300e:


Equipment
Tecan D300e digital dispenser
NAME
Tecan
BRAND
30100152
SKU

Equipment
T8 Dispensehead Cassettes
NAME
Tecan
BRAND
30097370
SKU

Cytation 1:

Equipment
Agilent BioTek Cytation 1 Cell Imaging Multi-Mode Reader
NAME
Aligent BioTek
BRAND
BTCYT1FAV
SKU

Seeding (Day 1)
  1. 24 hours prior to treatment, using 96-well plates, seed 2,000 Rhabdomyosarcoma (RD) cells per well (Figure 1.) using Cell Culture Media (2.1).
  2. Place seeded cells in incubator at Temperature37 °C in 5 % overnight.
Figure 1. Cell Seeding Layout. Plate A. Clear flat bottom 96- well plate. Plate B. Black flat bottom 96-well plate with clear wells. Both plates consist of roughly 2,000 Rhabdomyosarcoma (RD) cells per well 24 hours before treatment.



Incubation
Overnight
Two types of flat bottom 96-well plates are used: Clear 96-well Flat Bottom plates, and Black 96-well Flat Bottom plates with Clear Wells. Each are paired for the assay. One plate is used for antiviral screening and the other for cytotoxicity screening.
Three compounds in three replicates are used per each plate pair (Figure 2.)
Figure 2. Layout of plates for antiviral and cytotoxicity screening. Plate A. Clear flat bottom 96- well plate used for Cytotoxicity testing of compounds. Plate B. Black flat bottom 96-well plate with clear wells used for Antiviral testing of compounds. Both consist of roughly 2,000 Rhabdomyosarcoma (RD) cells per well 24 hours prior to treatment.

Compound Dilution Preparation (Day 2)
Under the Biosafety Cabinet, use a Deep 96-well plate to prepare the compound dilutions for treatment. One compound will be assigned to one column in the deep 96-well plate (Figure 3.)
  1. Pipette Amount1000 µL of Infection Media (2.2) per well for row A (Figure 3.)
  2. Pipette Amount1000 µL of DMSO + Infection Media mixture (2.3) into the remaining wells (rows B-H) (Figure 3.)

Now you do NOT have to normalize the plates to DMSO when using the Tecan D300e digital dispenser.

Figure 3. Deep 96-well plate Layout with DMSO + Infection Media. Row A wells containing 1000µL of Infection Media, Row B-H wells containing 1000µL Infection Medica with DMSO.


Compound Dilutions Using the Tecan D300e Digital Dispenser
Preparing the Compounds:
  1. Thaw the compounds to be tested always including Rupintrivir as a control and organize the samples according to the compound list. Ensure that the order of the compounds matches the list exactly.
  2. Use a USB and copy and paste the list of compounds to be tested onto it.


Preparing the Tecan D300e Digital Dispenser:
Once samples are thawed, turn on the hood blower and the Tecan D300e Digital Dispenser (5). Power on the connecting computer and open the Tecan D300e software.
Plug in your USB with the list of compounds saved on it. Copy the list exactly and paste the copied list into the fluids section of the Tecan D300e software page. All of the compounds should appear as their own "tabs" on the left-hand tab of the page.
Click on "Quick Plate".
Under "Plate" Tab:
  1. Plate Type: 96 Well
  2. Additional Volume: Amount1000 µL
  3. DMSO limit (%): 1

Click "Next"
Under "Titration" Tab:
  1. Titration Levels: 8
  2. Specify titration using: Highest concentration
  3. Highest Concentration: (Highest desired Concentration) x (1.5) = input concentration i.e. : (Concentration100 micromolar (µM) ) x (1.5)= Concentration150 micromolar (µM) All compounds as of 2025 should have a starting concentration of Concentration100 micromolar (µM) Rupintrivir should have a starting concentration of Concentration1 micromolar (µM)
  4. Distribution: 1:3 (33%)
  5. Replicates per level: 1
  6. Order: High to Low

Click "Next"
Under "Layout" Tab:
  1. Layout: Layout by columns
  2. Interleave fluids: Do not interleave
  3. Choose the third display option

Click "Next"
Under "Summary" Tab:

Click "Finish"
All of the compounds listed under Fluids should now be seen in the Plate Layout (Figure 4.).


Click "Run" at the top left side of the software page.
Diluting the Deep 96-well Plate:
After preparing the protocol for the D300e program, take your prepared deep 96-well plate and place it on the plate holder of the Tecan D300e machine.
Place a T8 cassette (5) (8x well cassette on top of the D300e machine) and place on the cassette holder.
Click “loaded”.
Add in indicated compound amounts into each well of the cassette (1 compound/cassette well).
Repeat filling in the remaining compounds until finished.
Click “all filled” and allow the machine to add and dilute the compounds into the deep 96-well plate.
Once all compounds are added and properly diluted into the deep 96-well plate(s), make sure to cover the deep well plates to maintain sterility.
Treating and infecting the cells
  1. Retrieve cell plates from the incubator.
  2. Aspirate media from cells of columns 1 and 11 for plate B and plate C (Figure 5.).
  3. Add in 100 µL of diluted compounds from the deep 96-well plate. Each compound should be added around the same time for both plates.

Figure 5. Layout of plates for antiviral and cytotoxicity screening. Plate A. Deep 96-well plate with diluted Compounds and DMSO + Infection Media. Plate B. Clear flat bottom 96- well plate used for Cytotoxicity testing of compounds. Plate C. Black flat bottom 96-well plate with clear wells used for Antiviral testing of compounds. Plate C is infected with EVD68-MO/1418947 at an MOI of 1 two hours post treatment. Plates B. and C. both consist of roughly 4,000 Rhabdomyosarcoma (RD) cells per well on day of treatment.


Repeat steps until all the diluted compound is added to the cells.
**The 12th column is used for untreated cells - you do not need to aspirate this column**.
  1. Place cells back in incubator at Temperature37 °C in 5 % for roughly 2.5 hours, then infect plate(s) C (Antiviral Plates) with Amount50 µL of EVD68-MO/1418947 in Infection Media at an MOI of 1 two hours post treatment.
  2. Place plates back in incubator at Temperature37 °C in 5 %

Cytotoxicity Screening and Antiviral Screening Fixing
Cytotoxicity Testing and Fixing: Perform an MTT for plate(s) B around the same time plate(s) C are fixed.
Cytotoxicity Testing and Fixing: Perform an MTT for plate(s) B around the same time plate(s) C are fixed.
  1. Cytotoxicity Testing: MTT Reagent A powder is dissolved in 10mL of 1x PBS for a stock solution. Store the working stock at Temperature4 °C
  2. Before you begin, prepare a working stock of MTT Reagent A by diluting the working stock 1:10 in 1x PBS (i.e. 1mL stock + 9mL PBS). This is light sensitive, perform with hood light turned off.

Cytotoxicity Screening and Antiviral Screening Fixing
Gently aspirate off media from cells taking care not to disturb them.
Add 50uL MTT reagent A per well (GENTLY)
Incubate treated cells in incubator at Temperature37 °C in 5 % for 3 hours. Cells should appear dark/purple as crystals form in the cells (either by eye or under microscope)
  1. Solubilize with Amount150 µL 100% Isopropanol at TemperatureRoom temperature for 40 minutes. Make sure to pipette up and down to dissolve crystals/break membranes when adding the Isopropanol. Remember this is light sensitive, wrap in foil to ensure accurate read out.
  2. Make sure crystals are fully dissolved.

Cytation 1 Read out for Cytoxicity Screening: (23)
Fixing: Roughly 24 hours after infecting, aspirate the compound and virus from Plate C (Use a multichannel pipette and Quatricide to discard media waste) and add in 100-150 µL of 4% Formaldehyde + PBS/well to fix the cells for at least 20 minutes. Leave at TemperatureRoom temperature .

Immunostaining (21)

Immunostaining (Day 3/4)
Immunostaining: Immunostain the fixed Plate(s) C.
Day 1:
  1. Carefully remove the formaldehyde fixative and wash the monolayers once with PBS
  2. Discard the PBS and add Amount100 µL of 0.1% Triton X-100 (3.2) to each well.
  3. Incubate the cells in 0.1% Triton X-100 for 20 minutes at TemperatureRoom temperature
  4.  Carefully wash the monolayer 3x with 1x PBS
  5. Discard wash and incubate to block with 0.3% BSA/PBS for an hour (3.2).
  6. Dilute the primary antibody EVD68 VPI Rabbit (3.1) at a 1:800 dilation in 0.3% BSA/PBS, add 50uL per well. Incubate for at least 2 hours TemperatureRoom temperature or preferably overnight at Temperature4 °C

Day 2:
At this point, be careful with light as the assay will be light sensitive
  1. Carefully wash the monolayer 3x with 1x PBS
  2. Discard wash and dilute the secondary antibody goat anti-Rabbit FITC (alexa- flour) (3.1) at a 1:2000 dilation + DAPI in 0.3% BSA/PBS, add 50uL per well and incubate for at least 1 hour atTemperatureRoom temperature .
  3. Carefully wash the monolayer 3x with 1x PBS
  4. Add Amount100 µL of fresh 1x PBS to each well for read out.
  5. Cytation 1 Read out for Antiviral Screening: (24)


Cytation 1 Read out (Day 4/5)
Cytation 1: Read out with Cytation 1 machine.
Cytation 1, Cytotoxicity Screening: This is done the same day the Cytotoxicity assay begins. Read absorbance of Plate(s) B using the Cytation 1 machine.
  1. Power on the Cytation 1 Imaging Reader and the computer attached to it.
  2. Open up the Gen 5 3.15 Software on the computer
  3. Read absorbance at 630 and 570nm of Plate(s) B using the Cytation 1 machine.
Cytation 1, Antiviral Screening: Read GFP and DAPI stained cells of Plate(s) C using the Cytation 1 machine.
  1. Power on the Cytation 1 Imaging Reader and the computer attached to it.
  2. Open up the Gen 5 3.15 Software on the computer
  3. Read each individually stained GFP and DAPI stained cell for each well using the untreated as a control for GFP detection of Plate(s) C using the Cytation 1 machine.
  4. Data Reduction "Ratio": DS1S Cell Count 2 (GFP) / DS2S Cell Count 1 (DAPI). Set factor to 100 and label as a % Cell Count.
  5. "Normalize" % Cell Count to DMSO and express as a ratio.
CDD Formatting Information
Export all of the processed data from the Cytation 1 Machine to excel using software Add-ins to convert all information to Collaborative Drug Discovery CDD vault formatting.

Cytotoxicity Screening CDD Format:
Plate ID: EDRD#
Well: Filled
Well ID: Compound name, DMSO, Untreated, Rupintrivir
Name: ASAP#
Concentration/ Dilution: Filled
570: Replicates
630: Replicates
630/570: Replicates
Antiviral Screening CDD Format:
Plate ID: EDRD#
Well: Filled
Well ID: Compound name, DMSO, Untreated, Rupintrivir
Name: ASAP#
Concentration/ Dilution: Filled
Cell Count 2: Replicates
Cell Count 1: Replicates
% Cell Count: Replicates