Oct 26, 2025
A549-ACE2 SARS-CoV-1 antiviral effect screening assay
- Nick Lynch1,2
- 1Curlew Research;
- 2ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Nick Lynch 2025. A549-ACE2 SARS-CoV-1 antiviral effect screening assay . protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrw6z2lmk/v1
Manuscript citation:
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: August 02, 2025
Last Modified: October 26, 2025
Protocol Integer ID: 223934
Keywords: SARS-CoV-1, antiviral screening, A549 cells, cellular, drug discovery, antiviral efficacy, antiviral efficacy of compound, antiviral effect, a549 human lung adenocarcinoma cell, ace2 receptor, human lung adenocarcinoma cell, ace2 sar, remdesivir viral replication, ace2 cell line model, gp inhibitor
Abstract
To evaluate the antiviral efficacy of compounds against SARS-CoV-1 infection using an engineered A549-ACE2 cell line model. A549 human lung adenocarcinoma cells stably transfected to express ACE2 receptors were used as the host cell system. Cells were pre-treated with a range of test compounds prior to SARS-CoV-1 infection at a multiplicity of infection levels.
The regular P-GP inhibitor used was Elacridar and positive control was Remdesivir
Viral replication was assessed at post-infection using detection methods. Control compounds/vehicle controls were included for comparison.
Materials
2uM Elacridar
Positive control = 50uM Remdesivir
Troubleshooting
Safety warnings
Always wear appropriate PPE for this protocol
Samples contain BSL-3 level high risk agents (viral) with infectious aerosol transmission potential.
Before start
Always wear appropriate PPE for this protocol
Refer to Material Safety Data Sheets for additional safety and handling information.
Summary
SARS-CoV-1 utilizes the angiotensin-converting enzyme 2 (ACE2) receptor for cellular entry, making ACE2-expressing cell lines valuable models for antiviral drug screening. The A549-ACE2 system provides a standardized platform for evaluating compound efficacy against coronavirus infection.
To screen potential antiviral compounds for inhibitory activity against SARS-CoV-1 replication using engineered A549 lung epithelial cells expressing ACE2 receptors.
A549-ACE2 cells were treated with test compounds in the presence of 2 μM elacridar (efflux pump inhibitor) to enhance intracellular drug accumulation. Following compound treatment, cells were infected with SARS-CoV-1 and viral replication was monitored. Remdesivir (50 μM) served as the positive control for antiviral activity.
Viral replication inhibition was measured through cytopathic effect (CPE) reduction.
Cell viability and viral inhibition were assessed to determine compound efficacy and cytotoxicity profiles.
Analysis
All statistical tests were executed using GraphPad Prism. The EC50 values were defined as the concentration at which there was a 50% decrease in viral replication relative to vehicle alone (0% inhibition) and for and EC90 which there was a 90% decrease in viral replication relative to vehicle alone. Curves were fitted based on four parameter non-linear regression analysis.
Protocol references
A Newly Engineered A549 Cell Line Expressing ACE2 and TMPRSS2 Is Highly Permissive to SARS-CoV-2, Including the Delta and Omicron Variants
Cell culture model system utilizing engineered A549 cells to express high levels of 1 ACE2 and TMPRSS2 for investigating SARS-CoV-2 infection and antivirals
A novel cellular tool for screening human pan-coronavirus antivirals
Remdesivir Inhibits SARS-CoV-2 in Human Lung Cells and Chimeric SARS-CoV Expressing the SARS-CoV-2 RNA Polymerase in Mice
A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors
Acknowledgements
We thank Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA for details of this assay