Aug 03, 2025
In Vitro Antiviral Screening assay Against Viruses of Pandemic Potential
- Nick Lynch1,2
- 1Curlew Research;
- 2ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Nick Lynch 2025. In Vitro Antiviral Screening assay Against Viruses of Pandemic Potential . protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo9jddv4o/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 03, 2025
Last Modified: August 03, 2025
Protocol Integer ID: 124240
Keywords: antiviral, cytotoxicity, protease, screening, drug discovery, panel, vitro antiviral screening assay, vitro antiviral screening assay against virus, evaluating antiviral compound efficacy, antiviral compound efficacy, antiviral activity, compounds with antiviral activity, vero 76 cell monolayer, virus inoculation, virus control, test compound, pandemic potential, virus, cell monolayer, compound, reduction assay, known active drug
Abstract
This protocol details a primary cytopathic effect (CPE) reduction assay for evaluating antiviral compound efficacy. Vero 76 cell monolayers are prepared in 96-well plates and exposed to eight serial half-log10 concentrations of test compounds, along with infected and uninfected controls, and a known active drug. After virus inoculation and incubation until >80% CPE is observed in virus controls, cell viability is quantified using neutral red staining and spectrophotometric analysis at 540 nm. The 50% effective (EC50) and 50% cytotoxic (CC50) concentrations are determined by regression analysis, and the selectivity index (SI50) is calculated to identify compounds with antiviral activity (EC50 <10 μM, SI50 > 5).
Guidelines
Compounds need should be pre-weighed into tubes clearly marked with the compound name, exact weight, molecular weight, and Compound number.
This information can also be included as a table. Starting solutions 10 millimolar (mM) or 3.2 millimolar (mM) solutions if they are stable in solution. If 10 mM is used, half the volume may be sent.
Figure 1. Antiviral Compound Assay Schematic
| A | B | C | D | E | F | G | H | I | J | K | L | M | N | |
| Drug [µM] | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | Drug [µM] | |
| 32 | D1 | D1 | D1 V | D1 V | D1 V | C | C | DP V | DP V | DP V | DP | DP | 32 | |
| 10 | D1 | D1 | D1 V | D1 V | D1 V | C | C | DP V | DP V | DP V | DP | DP | 10 | |
| 3.2 | D1 | D1 | D1 V | D1 V | D1 V | C | C | DP V | DP V | DP V | DP | DP | 3.2 | |
| 1.0 | D1 | D1 | D1 V | D1 V | D1 V | Blank | Blank | DP V | DP V | DP V | DP | DP | 1.0 | |
| 0.32 | D1 | D1 | D1 V | D1 V | D1 V | Blank | Blank | DP V | DP V | DP V | DP | DP | 0.32 | |
| 0.1 | D1 | D1 | D1 V | D1 V | D1 V | V | V | DP V | DP V | DP V | DP | DP | 0.1 | |
| 0.032 | D1 | D1 | D1 V | D1 V | D1 V | V | V | DP V | DP V | DP V | DP | DP | 0.032 | |
| 0.01 | D1 | D1 | D1 V | D1 V | D1 V | V | V | DP V | DP V | DP V | DP | DP | 0.01 |
D1 = DRUG #1
DP = POSITIVE CONTROL DRUG
C = Cell controls, cells fed with MEM + 2% FBS and 50 ug/mL gentamicin MEM + 10 U/mL trypsin & 1 ug/mL EDTA for influenza
V = Virus stock (≤100 CCID50 for most viruses)
Table 1. Procedural details for in vitro screening with various viruses. Highlighted viruses are representatives that may be used for a broad-screen of multiple virus groups.
Table 2. Cell lines that are validated for antiviral testing with the proposed viruses
aIdentification and validation of additional cell lines is in process, especially seeking
human cell lines.
bDose-response tests are done by VYR assay because this virus does not produce
measurable CPE in this cell line.
cAlternate cell line validated for virus yield reduction (secondary assay) only due to lack
of CPE produced by the virus.
*Cell origins. Human: Caco2, A549, HeLa, HeLa-Ohio, HEK, HEL 299, HEp2, Huh-7,
RD, Calu- 3, dNHBE; Monkey: BS-C-1, CV-1, LLC-MK2, MA-104, Vero, Vero e6, Vero
76; Canine: MDCK;
Hamster: BHK-21
Materials
Compound Quantities:
Minimum amount needed for the number of assays
| A | B | C | |
| # Assays | mg Powder | µL of a 3.2 mM solution* | |
| 1 | 0.5 | 42 | |
| 2 | 0.6 | 74 | |
| 3 | 0.7 | 106 | |
| 4 | 0.8 | 138 | |
| 5 | 0.9 | 170 | |
| 6 | 1.0 | 202 | |
| 7 | 1.1 | 234 | |
| 8 | 1.2 | 266 | |
| 9 | 1.3 | 298 | |
| 10 | 1.4 | 330 | |
| 11 | 1.5 | 346 | |
| 12 | 1.6 | 362 | |
| 13 | 1.7 | 378 | |
| 14 | 1.8 | 394 | |
| 15 | 1.9 | 410 | |
| 16 | 2.1 | 426 | |
| 17 | 2.2 | 442 | |
| 18 | 2.3 | 458 | |
| 19 | 2.4 | 474 | |
| 20 | 2.5 | 490 | |
| 21 | 2.6 | 506 |
Troubleshooting
Reduction of virus-induced cytopathic effect (Primary CPE assay)
2h 30m
Prepare the confluent or near-confluent cell culture monolayers of Vero 76 cells (or another appropriate cell line) in 96-well disposable microplates the day before testing.
Maintain the cells in MEM supplement with 5% FBS.
For antiviral assays, use the same medium but with FBS reduced to 2% and supplement with 50 µL gentamicin.
Dissolve the compounds in DMSO, saline, or the diluent requested by the submitter. Vortex, heat, and/or sonicate the less soluble compounds and test if they still do not go into solution as colloidal suspensions.
Prepare the test compound is at eight serial half-log10 concentrations, usually 32 micromolar (µM) , 10 micromolar (µM) , 3.2 micromolar (µM) , 1.0 micromolar (µM) , 0.32 micromolar (µM) , 0.1 micromolar (µM) , 0.032 micromolar (µM) and 0.01 micromolar (µM) (or per sponsor preference).
Use the lower concentrations when insufficient compound is supplied or when a lower starting concentration is requested.
Use the five microwells per dilution: three for infected cultures and two for uninfected toxicity cultures.
Controls for the experiment consist of six microwells that are infected and not treated (virus controls) and six that are untreated and uninfected (cell controls) on every plate.
Test a known active drug in parallel as a positive control drug using the same method as is applied for test compounds. Test the positive control with every test run.
On the testing day, remove the growth media from the cells and the test compound is applied in 0.1 ml volume to wells at 2X concentration.
Virus, normally at a titer that will cause >80% CPE (usually an MOI <0.003), in 0.1 mL volume is added to the wells designated for virus infection.
Place the medium devoid of virus in toxicity control wells and cell control wells.
Incubate the plates at 37 °C with 5% CO2 until marked CPE (>80% CPE for most virus strains) is observed in virus control wells.
Then stain the plates with 0.011% neutral red for approximately 02:00:00 at 37 °C in a 5% CO2 incubator.
2h
Remove the neutral red medium by complete aspiration, and may rinse the cells 1X with phosphate-buffered solution (PBS) to remove the residual dye.
Remove the PBS completely, and elute the incorporated neutral red with 50% Sorensen’s citrate buffer/50% ethanol for at least 00:30:00 .
30m
Neutral red dye penetrates into living cells, thus, the more intense the red color, the larger the number of viable cells present in the wells.
Quantify the dye content in each well using a spectrophotometer at 540 nm wavelength.
Convert the dye content in each set of wells to a percentage of dye present in untreated control wells using a Microsoft Excel-based spreadsheet and normalized based on the virus control.
Then calculate the 50% effective (EC50, virus-inhibitory) concentrations and 50% cytotoxic (CC50, cell-inhibitory) concentrations by regression analysis.
Divide the quotient of CC50 by EC50 gives the 50% selectivity index (SI50) value. Compounds showing EC50 <10 micromolar (µM) and SI50 values > 5 are considered minimally active.
Reduction of virus yield (VYR assay)
2h 30m
Test the active compounds further in a confirmatory VYR assay. This assay is run for compounds that have an EC50 < 10 micromolar (µM) and SI50 ≥ 5.
After sufficient virus replication occurs (generally 3 days for many viruses), a sample of supernatant is taken from each infected well (replicate wells are pooled) and held frozen at -80 °C for later virus titer determination.
After maximum CPE is observed, stain the viable plates with neutral red dye. Quantify the incorporated dye content as described above to generate the EC50 and CC50 values.
The VYR test is a direct determination of how much test compound is required to inhibit 90% virus replication.
Titrate the virus yielded in the presence of the test compound and compare to virus titers from the untreated virus controls.
Perform the titration of the viral samples (collected as described in the paragraph above) by endpoint dilution (Reed and Muench).
Make the serial 10-fold dilutions of supernatant and plate into 4 replicate wells containing fresh cell monolayers of Vero 76 cells.
Incubate the plates, and score the cells for the presence or absence of the virus after distinct CPE is observed, and calculate the CCID50 using the Reed-Muench method (24).
Calculate the 90% (one log10) effective concentration (EC90) by regression analysis by plotting the log10 of the inhibitor concentration versus log10 of the virus produced at each concentration. Dividing EC90 by the CC50 gives the SI90 value for this test.
Summary of virus panel
Antiviral Summary
Protocol references
Reed, L.J., and H. Muench. "A Simple Method of Estimating Fifty Percent Endpoints." Am J Hyg 27 (1938): 493-98. Print.
Acknowledgements
We thank Institute for Antiviral Research Utah State University for sharing the protocol of this assay