No abstract

Respiratory syncytial virus (RSV) causes severe infections in infants, immunocompromised or elderly individuals resulting in annual epidemics of respiratory disease. Currently, limited clinical RSV surveillance and the lack of predictable RSV seasonal dynamics and limits the public health response. Wastewater-based epidemiology (WBE) has the capacity to determine levels of health-associated biomarkers and has recently been used globally as a key metric in determining prevalence of SARS-CoV-2 in the community. However, the application of genomic WBE for the surveillance of other respiratory viruses is limited. In this study, we present an integrated genomic WBE approach, using RT-qPCR and partial sequencing of the G gene to monitor RSV levels and variants in the community across 2 years encompassing two periods of high RSV clinical positivity in Northern Ireland. We report increasing detection of RSV in wastewater concomitant with increasing numbers of RSV positive clinical cases. Furthermore, analysis of wastewater-derived RSV sequences permitted subtyping, genotyping, and identification of distinct circulating lineages within and between seasons. Altogether, our genomic WBE platform has the potential to complement ongoing global surveillance efforts and aid the management of RSV by informing the timely deployment of pharmaceutical and non-pharmaceutical interventions.

Background: Influenza A viruses (IAV) are significant pathogens of humans and other animals. Although endemic in humans and birds, novel IAV strains can emerge, jump species, and cause epidemics, like the latest variant of H5N1. Wastewater-based epidemiology (WBE) has very recently been shown to detect human IAV but whether it can detect avian-origin IAV, and if whole genome sequencing (WGS) can be used to discriminate circulating strains of IAV in wastewater remains unknown. Methods: Using a pan-IAV RT-qPCR assay, six wastewater treatment works (WWTWs) across Northern Ireland (NI), were screened from August to December 2022. A WGS approach using Oxford Nanopore technology was employed to sequence positive samples. Phylogenetic analysis of sequences relative to currently circulating human and avian IAVs was performed. Findings: We detected a dynamic IAV signal in wastewater from September 2022 onwards across NI. Meta whole genome sequences were generated displaying homology to both human and avian IAV strains. The relative proportion of human versus avian-origin IAV reads differed across time and sample site. A diversity in subtypes and lineages was detected (e.g. H1N1, H3N2, and several avian). Avian segment 8 related to those found in recent H5N1 clade 2.3.4.4b was identified. Interpretation: WBE affords a means to monitor circulating human and avian IAV strains and provide crucial genetic information. As such WBE can provide rapid, cost-effective, 57 year-round one-health IAV surveillance to help control epidemic and pandemic threats.

Wastewater Based Epidemiology (WBE) has become an integral part of the public health effort to track the levels of SARS-CoV-2 within communities. Detection of SARS-CoV-2 in wastewater can be challenging due to relatively low levels of virus within the sample. The wastewater matrix is also comprised of commercial and domestically derived contaminants, as well as RNases, all of which can adversely affect RT-qPCR analysis. To improve SARS-CoV-2 detection within wastewater samples we investigated both the effect of template dilution (as a means to reduce RT-qPCR inhibition) and sample stabilisation via addition of DNA/RNA Shield™ and/or RNA Later™ (to prevent RNA degradation via RNases) as a means to improve viral fragment detection. Using both methodologies a significant improvement in SARS-CoV-2 detection from wastewater samples was observed. No adverse effects of stabilising agent addition on downstream Next Generation Sequencing workflows were detected.