Related Experiment Video
Updated: Apr 17, 2026
An Improved and High Throughput Respiratory Syncytial Virus RSV Micro-neutralization Assay
Published on: January 26, 2019
High-Throughput Hit Screening Cascade to Identify Respiratory Syncytial Virus (RSV) Inhibitors
Helen Plant1, Clare Stacey2, Choi-Lai Tiong-Yip3
1Discovery Sciences, AstraZeneca, Macclesfield, Cheshire, UK helen.plant@astrazeneca.com.
Insights
Researchers developed a new high-throughput screening method to find drugs that stop respiratory syncytial virus (RSV) replication. This robust assay successfully identified potential RSV inhibitors from a large compound library.
Area of Science:
- Virology
- Drug Discovery
- Biochemistry
Background:
- Respiratory syncytial virus (RSV) is a major cause of severe respiratory infections and hospitalizations in infants.
- Developing effective RSV therapeutics is challenging due to the absence of suitable high-throughput screening (HTS) assays.
- RSV infects nearly all children by age two, highlighting the need for novel treatments.
Purpose of the Study:
- To establish a robust HTS cascade for identifying inhibitors of RSV replication.
- To validate a novel RSV replicon luminescence-reporter assay for primary screening.
- To discover novel small molecules targeting RSV replication.
Main Methods:
- Development and validation of an RSV replicon luminescence-reporter assay for HTS.
- Screening of the AstraZeneca compound library (1 million compounds) at 10 µM.
- Hit confirmation through secondary assays, including cell toxicity and live viral inhibition assays.
Main Results:
- The primary HTS assay demonstrated consistent performance with a Z' of 0.55 ± 0.08 and signal-to-background ratios >40.
- The screening cascade successfully identified hit molecules specifically targeting RSV replication.
- Confirmed hits were validated in live viral inhibition assays, demonstrating physiological relevance.
Conclusions:
- A robust and scalable HTS cascade was successfully developed for RSV drug discovery.
- The developed assay enables efficient identification of compounds that inhibit RSV replication.
- This approach provides a foundation for the development of novel RSV therapeutics.
Abstract:
Respiratory syncytial virus (RSV) infects 99% of children by age 2 years and is a leading cause of serious lower respiratory tract infection (LRTI) and infant hospitalization in the United Kingdom. Identification of efficacious RSV therapeutics has been hindered by the lack of a robust and appropriate primary assay for high-throughput screening (HTS). Here we report an HTS cascade that identified inhibitors of RSV replication using a robust RSV replicon luminescence-reporter assay for the primary campaign. The performance of the assay was consistent and reliable at scale, with Z' of 0.55 ± 0.08 across 150 assay plates and signal-to-background ratios >40. The HTS assay was used to screen the AstraZeneca compound library of 1 million compounds at a single concentration of 10 µM. Hits specifically targeting the RSV replicon were determined using a series of hit generation assays. Compounds nonspecifically causing cell toxicity were removed, and hits were confirmed in live viral inhibition assays exhibiting greater physiological relevance than the primary assay. In summary, we developed a robust screening cascade that identified hit molecules that specifically targeted RSV replication.
