Developing Synthetic Full-Length SARS-CoV-2 cDNAs and Reporter Viruses for High-Throughput Antiviral Drug Screening

Area of Science:

• Virology
• Molecular Biology
• Drug Discovery

Background:

• The ongoing spread of SARS-CoV-2 necessitates the development of effective antiviral therapies.
• Reverse genetics systems and reporter viruses are crucial for efficient antiviral screening and quantification of viral infections.

Purpose of the Study:

• To generate wild-type and Nluc (nanoluciferase) reporter full-length SARS-CoV-2 molecular clones and viruses.
• To establish a high-throughput platform for antiviral drug screening against SARS-CoV-2 variants.

Main Methods:

• Utilized cDNA chemical synthesis services to generate full-length SARS-CoV-2 clones (wild-type and Nluc reporter) for Delta and Omicron variants.
• Successfully rescued clone-derived viruses and validated their replication kinetics against patient isolates.
• Employed Nluc reporter viruses for antiviral assays, measuring replication inhibition by established antivirals.

Main Results:

• Nluc reporter viruses demonstrated stable luciferase expression correlating with viral titers, serving as reliable indicators of viral replication.
• Antiviral assays using Nluc expression yielded IC50 values and selectivity indices for Remdesivir, Molnupiravir, and Nirmatrelvir consistent with published data.
• Clone-derived Delta Nluc viruses successfully replicated in primary human bronchial epithelial cells, validating their use in relevant physiological models.

Conclusions:

• SARS-CoV-2 cDNA clones and Nluc reporter viruses generated via DNA synthesis offer a rapid and scalable reverse genetics platform.
• This platform facilitates the generation of new viruses and the development of assays for assessing antiviral compounds against current and emerging SARS-CoV-2 variants and future coronaviruses.