Related Experiment Video
Updated: Jun 27, 2026
Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies
Published on: August 29, 2017
Reverse genetics systems for contemporary isolates of respiratory syncytial virus enable rapid evaluation of antibody
Wendy K Jo1, Alina Schadenhofer1, Andre Habierski1
1Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, 30559 Hannover, Germany.
Insights
New reverse genetics systems for human respiratory syncytial virus (RSV) clinical isolates enable studying viral pathogenesis and developing interventions. These systems help investigate mutations that allow RSV to evade palivizumab neutralization.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Human respiratory syncytial virus (RSV) is a major cause of pediatric respiratory infections.
- Current reverse genetics systems use lab-adapted strains, not reflecting circulating genotypes.
- Limited therapeutic options exist for RSV, necessitating better research tools.
Purpose of the Study:
- To develop novel reverse genetics systems for clinical RSV isolates.
- To investigate the impact of specific F protein mutations on palivizumab neutralization and viral spread.
- To create tools for studying RSV pathogenesis and evaluating new interventions.
Main Methods:
- Generated reverse genetics systems for RSV-A and RSV-B clinical isolates using bacterial artificial chromosome (BAC) technology.
- Rescued recombinant (r) RSV expressing reporter genes (EGFP, mScarlet, NanoLuc).
- Introduced mutations in antigenic site II of the F protein and assessed their effects using cell-fusion assays and rRSVs.
Main Results:
- Successfully generated functional reverse genetics systems for clinical RSV-A and RSV-B strains.
- Demonstrated that specific F protein mutations (K272E, K272Q, S275L) confer resistance to palivizumab neutralization.
- Observed differential effects of these mutations on cell-to-cell fusion, with S275L significantly impairing syncytium formation.
Conclusions:
- The developed reverse genetics systems are valuable tools for studying RSV.
- These systems facilitate research into viral pathogenesis and the development of novel therapeutic strategies.
- Understanding mutation impacts is crucial for designing effective RSV interventions and vaccines.
Abstract:
Human respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory infection in children under 5 y of age. In the absence of a safe and effective vaccine and with limited options for therapeutic interventions, uncontrolled epidemics of RSV occur annually worldwide. Existing RSV reverse genetics systems have been predominantly based on older laboratory-adapted strains such as A2 or Long. These strains are not representative of currently circulating genotypes and have a convoluted passage history, complicating their use in studies on molecular determinants of viral pathogenesis and intervention strategies. In this study, we have generated reverse genetics systems for clinical isolates of RSV-A (ON1, 0594 strain) and RSV-B (BA9, 9671 strain) in which the full-length complementary DNA (cDNA) copy of the viral antigenome is cloned into a bacterial artificial chromosome (BAC). Additional recombinant (r) RSVs were rescued expressing enhanced green fluorescent protein (EGFP), mScarlet, or NanoLuc luciferase from an additional transcription unit inserted between the P and M genes. Mutations in antigenic site II of the F protein conferring escape from palivizumab neutralization (K272E, K272Q, S275L) were investigated using quantitative cell-fusion assays and rRSVs via the use of BAC recombineering protocols. These mutations enabled RSV-A and -B to escape palivizumab neutralization but had differential impacts on cell-to-cell fusion, as the S275L mutation resulted in an almost-complete ablation of syncytium formation. These reverse genetics systems will facilitate future cross-validation efficacy studies of novel RSV therapeutic intervention strategies and investigations into viral and host factors necessary for virus entry and cell-to-cell spread.
