Handbook of laboratory protocols for respiratory syncytial virus (RSV) characterisation

New vaccines and monoclonal antibodies against respiratory syncytial virus (RSV) represent a significant shift in the prevention of severe disease in populations at greater risk of infection, such as infants and older adults (65 years old and above). Continuous phenotypic and genomic surveillance is essential to detect mutations in pre-fusion F epitopes, monitor potential decreases in sensitivity to monoclonal antibodies and track the dynamics of RSV-A and RSV-B genotype replacement. Even for relatively stable viruses, broad immune pressure can accelerate certain evolutionary trajectories. Therefore, integrated surveillance (clinical, virological and antigenic) is critical to maintaining an optimal match between preventive strategies and circulating viruses.

This handbook has been issued by the European Centre for Disease Prevention and Control to provide laboratory professionals in the EU/EEA with a comprehensive overview of commonly used laboratory protocols for the characterisation of respiratory syncytial virus (RSV). It compiles harmonised methodologies designed to support the study of RSV biology, molecular evolution and antigenic properties across participating laboratories. The handbook serves to support the application of consistent laboratory protocols across the EU/EEA. This enables data and results generated by different laboratories to be harmonised and compared, thereby strengthening integrated RSV surveillance and the timely detection of changes in antigenic properties and susceptibility to monoclonal antibodies.

The laboratory methodologies presented in this handbook include:

• Cell culture procedures;
• Virus isolation and amplification from positive clinical specimens to generate standardised viral stocks; and
• Infectious virus quantification using RSV plaque titration assays;
• RSV-specific RT-qPCR for molecular detection and viral load measurement;
• Genome sequencing for surveillance purposes; and
• Plaque reduction neutralisation assays to evaluate resistance of viral strains to Nirsevimab, Clesrovimab or future monoclonal antibodies, or to measure anti-RSV antibody activity in human serum samples.