A New Weapon in the Fight Against Coronaviruses: The A549-ADT Cell

The global threat of coronaviruses, including the persistent SARS-CoV-2 and the highly pathogenic MERS-CoV, continues to challenge healthcare systems and economies worldwide. The emergence of new variants and ongoing outbreaks underscores the critical need for broad-spectrum antivirals capable of targeting a range of these deadly viruses. Researchers are constantly seeking robust and versatile tools to accelerate the discovery and validation of such treatments.

         Traditionally, drug development efforts for coronaviruses have relied on cell lines like African green monkey kidney Vero or VeroE6 cells due to their efficient support of viral replication. However, these non-human cells have significant drawbacks: they are unable to produce interferon, an important immune response, and express high levels of P-glycoprotein, an efflux transporter that can lead to an underestimation of a compound's efficacy. While human cell lines like Calu-3 exist, they are often difficult to handle, slow-growing, and not well-suited for high-throughput screening. This highlights a critical gap in standardized, permissive human cell models for studying diverse lethal coronaviruses.

         To address these challenges, researchers led by Ching-Wen Chang and David Perlin at The Center for Discovery & Innovation have developed a novel human cell model: the A549-Ace2-Dpp4-Tmprss2 (A549-ADT) cell line. This engineered cell line is a significant advancement in virology and antiviral research.

            The A549-ADT cell line was developed by genetically modifying human alveolar A549 cells, which are commonly used to study respiratory virus infections but typically express minimal levels of key viral entry receptors. The process involved:

• Starting with A549 cells engineered to express the SARS-CoV-2 receptor, angiotensin converting enzyme 2 (Ace2), and transmembrane serine protease 2 (Tmprss2), which primes the viral spike protein.

• Introducing the dipeptidyl peptidase 4 receptor (Dpp4) gene, which MERS-CoV relies on for cellular entry, into these cells using lentiviral transduction.

• Fine-tuning the expression levels of each receptor through cell sorting and clone selection to optimize viral entry and infectivity.

The selected A549-ADT cells, particularly clone 21, offer multiple benefits over previous models:

• They are easy to culture and handle, similar to Vero E6 cells.

• They have a morphology conducive to imaging and cytopathic effect-based high-throughput screening assays.

• As human lung cells, they provide a more physiologically relevant model for studying coronavirus infections.

• Unlike Vero E6 cells, A549-ADT cells can produce interferon.

• They allow direct testing of antiviral compound activity without needing P-glycoprotein efflux pump inhibitor co-treatment, which is often required with VeroE6 cells.

         A549-ADT cells demonstrate high susceptibility to a wide range of human coronaviruses, including:

• SARS-CoV-2 (including Omicron subvariants like JN.1 and EG5.1, as well as the original WT and Delta strains).

• MERS-CoV.

• Seasonal human coronaviruses like OC43-CoV and 229E-CoV.

This broad susceptibility makes them an indispensable tool for pan-coronavirus research.

         The A549-ADT cell model proved valuable in screening and identifying antiviral drugs.  Nirmatrelvir was found to be superior to Pomotrelvir in terms of pan-coronavirus antiviral activity. Both compounds showed significant efficacy against Omicron JN.1, MERS-CoV, and OC43-CoV, and exhibited potent antiviral activity against 229E-CoV, an alpha-coronavirus. This consistency with other studies validated the model's effectiveness.

            Furthermore, the A549-ADT model facilitated the screening of 13 known antimalarial drugs for potential antiviral activity against coronaviruses leading to a significant discovery.  Halofantrine, an antimalarial drug, was identified as having antiviral activity against SARS-CoV-2, specifically the JN.1, WT, and Delta strains. This finding opens new possibilities for repurposing existing drugs to combat coronavirus infections. While effective against SARS-CoV-2, halofantrine did not show broad-spectrum activity against MERS-CoV.

            The development and characterization of the A549-ADT human cell line, along with the pan-coronavirus antiviral assays it enables, represent a valuable and versatile cellular tool. Its ability to serve as a single cell line for investigating multiple antiviral targets, coupled with its suitability for high-throughput drug screening, significantly accelerates the development of broad-spectrum therapeutics. This innovation is crucial for mitigating the impact of current and future coronavirus pandemics.

Chang CW, Oswal N, Murugan M, Goldgirsh K, Tsao W, Park S, Perlin DS. A novel cellular tool for screening human pan-coronavirus antivirals. Antiviral Res. 2025 Aug;240:106212. doi: 10.1016/j.antiviral.2025.106212. Epub 2025 Jun 10. PMID: 40505777.

https://www.sciencedirect.com/science/article/pii/S016635422500138X?via%3Dihub