Robust and sensitive amplicon-based whole-genome sequencing assay of respiratory syncytial virus subtype A and B
Author(s)
Type
Journal Article
Abstract
Prevention of respiratory syncytial virus (RSV) infection is now a global health priority, with a long-acting monoclonal antibody and two RSV vaccines recently licenced for clinical use. Most licenced and candidate interventions target the RSV fusion (RSV-F) protein. New interventions may be associated with the spread of mutations, reducing susceptibility to antibody neutralization in RSV-F. There is a need for ongoing longitudinal global surveillance of circulating RSV strains. To achieve this large-scale genomic surveillance, a reliable, high-throughput RSV sequencing assay is required. Here we report an improved high-throughput RSV whole-genome sequencing (WGS) assay performed directly on clinical samples without additional enrichment, using a 4-primer-pool, short-amplicon PCR-tiling approach that is suitable for short-read sequencing platforms. Using upper respiratory tract (URT) RSV-positive clinical samples obtained from a sentinel network of primary care providers and from hospital patients (29.7% and 70.2%, respectively; n = 1,037), collected over the period 2019 to 2023, this assay had a threshold of approximately 4 × 103 to 8 × 103 copies/mL (RSV-B and RSV-A sub-types, respectively) as the lowest amount of virus needed in the sample to achieve >96% of whole-genome coverage at a high-quality level. Using a Ct value of 31 as an empirical cut-off, the overall assay success rate of obtaining >90% genome coverage at a read depth minimum of 20 was 96.83% for clinical specimens successfully sequenced from a total of 1,071. The RSV WGS approach described in this study has increased sensitivity compared to previous approaches and can be applied to clinical specimens without the requirement for enrichment. The updated approach produces sequences of high quality consistently and cost-effectively, suitable for implementation to underpin national programs for the surveillance of RSV genomic variation.
Date Issued
2024-04-02
Date Acceptance
2024-02-06
Citation
Microbiology Spectrum, 2024, 12 (4)
ISSN
2165-0497
Publisher
American Society for Microbiology
Journal / Book Title
Microbiology Spectrum
Volume
12
Issue
4
Copyright Statement
© 2024 Crown. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
License URL
Identifier
https://journals.asm.org/doi/10.1128/spectrum.03067-23
Subjects
DIAGNOSIS
INFECTIONS
Life Sciences & Biomedicine
Microbiology
next-generation sequencing
PROTEIN
respiratory syncytial virus
Science & Technology
whole-genome sequencing
Publication Status
Published
Article Number
e03067-23
Date Publish Online
2024-02-27