Optimisation of Respiratory Syncytial Virus (RSV) whole genome sequencing of subtypes A and B

Abstract

Respiratory Syncytial Virus (RSV) is a major cause of lower respiratory tract infections, causing significant morbidity and mortality rates worldwide. RSV mainly affects infants, children under five years old, and older or immunocompromised adults. Establishing a cohesive method for the detection and identification of RSV could significantly improve monitoring and surveillance of RSV cases globally. The present study focused on optimising previously developed 400 base pair tiled-amplicon schemes for the whole genome sequencing of RSV subtypes A and B. We have developed novel ~1200 base pair primer schemes to increase sensitivity and produce a cost-efficient method for RSV genome sequencing. Samples were chosen between 2014 to 2024 and sequenced using Oxford Nanopore Technology’s PromethION device to acquire high- throughput genome data of both RSV subtypes. Our results revealed high potential for the optimised RSV A scheme, with all but two amplicons achieving genomic coverage depth of > 1000 reads per amplicon. However, the developed RSV B schemes generated insufficient results for most of RSV B samples. Moreover, the samples tested for RSV A displayed greater strain variability after phylogenetic analysis. In contrast, RSV B samples were of closely related strains and showed less genomic diversity. Acquiring high-throughput genomic data for RSV can hugely influence our understanding of the diversity of RSV, in addition to its evolutionary and seasonality patterns, which subsequently assists in the management of this virus on a global range. Furthermore, constituting a unified method for RSV sequencing largely impacts the implementation of successful surveillance systems for RSV infections.