Molecular characteristics and transmission dynamics of human respiratory syncytial virus

Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in young children and an increasingly recognised cause of illness in older adults. To date, the role of viral genetic diversity and infection dynamics in RSV have not been fully understood, and viral genetic studies of clinical samples are critical for developing and deploying preventative measures (e.g., vaccines, monoclonal antibodies) against RSV infection. In this thesis my aim is to identify viral and microbial factors associated with severe RSV disease, to explore RSV diversity during seasonal outbreaks both at the population level and within an individual infection, and to reconstruct the global epidemic history of the recently dominant RSV genotype ON1. RSV genomes and transcriptomes were reconstructed from target-enriched quantitative deep sequencing of >800 RSV-positive nasopharyngeal swabs collected from infants <1 year of age and adults ≥60 years of age in the United Kingdom, Spain, and the Netherland during 2017–2020. In addition to RSV, the sequencing targets included a panel of >100 common bacterial and viral pathogens, enabling simultaneous genotyping of RSV with that of any co-infecting organisms. Two subgroups of RSV, A and B, co-circulated in each RSV season, with RSV-B (genotype BA) dominating the 2017/18 and 2018/19 seasons and RSV-A (genotype ON1) dominating the 2019/20 season. Differential gene expression analysis showed that RSV-A had a lower expression of the P gene (encoding the phosphoprotein, a cofactor of the viral RNA-dependent RNA polymerase) and a higher expression of the open reading frame M2-1 (encoding the M2-1 protein, facilitating transcription of full-length mRNA) than RSV-B. Analysis of the deep sequencing data identified two RSV-B consensus strains and one minor variant carrying amino acid alterations potentially associated with resistance to RSV monoclonal antibodies. RSV-B had greater within-host diversity than RSV-A, while RSV-A exhibited greater population diversity than RSV-B. Further analysis combining metagenomic sequencing data with clinical data showed that RSV-A and RSV-B caused disease of similar severity and exhibited similar viral load dynamics in the infant cohort. Young age and co-infection with Haemophilus spp. were significant risk factors for severe RSV disease in infants. Delayed viral clearance was found in infants requiring mechanical ventilation. Transmission dynamics of RSV were examined by reconstructing the epidemic history of genotype ON1 from sequences generated in this thesis along with publicly available sequences downloaded from GenBank. ON1 was estimated to emerge in the Oceania region between October 2008 and May 2009. After its emergence, it rapidly spread to the Americas and was first detected in Mexico in late 2009. It reached every region of the world within 2 years and gained dominance within 3 years. This thesis provides new insights into the molecular characteristics and transmission dynamics of RSV and emphasises the importance of continued genomic surveillance of RSV to promptly detect emerging variants that may confer resistance to forthcoming RSV preventative measures.

Keywords: Within-host diversity
Respiratory syncytial virus
Molecular epidemiology
Genotype ON1
Viral transcriptomics
Phylogeographic analysis
Transmission dynamics
Evolutionary dynamics
Respiratory microbiome
Whole-genome sequencing
Advisor: Drysdale, S
Pollard, A
Golubchik, T