Viromic analysis of wastewater input to a river catchment reveals a diverse assemblage of RNA viruses

Abstract

13Detection of viruses in the environment is heavily dependent on PCR-based 14 approaches that require reference sequences for primer design. While this strategy 15 can accurately detect known viruses, it will not find novel genotypes, nor emerging 16 and invasive viral species. In this study, we investigated the use of viromics, i.e. 17 high-throughput sequencing of the biosphere viral fraction, to detect human/animal 18 pathogenic RNA viruses in the Conwy river catchment area in Wales, UK. Using a 19 combination of filtering and nuclease treatment, we extracted the viral fraction from 20 wastewater, estuarine river water and sediment, followed by RNASeq analysis on 21 the Illumina HiSeq platform for the discovery of RNA virus genomes. We found a 22 the range of potentially pathogenic viruses that are present in the environment and 37 identify prevalent genotypes. The ultimate goal is to trace the fate of these 38 pathogenic viruses from origin to the point where they are a threat to human health, 39 informing reference-based detection methods and water quality management. 40Recent reviews have proposed the use of viral metagenomics or viromic approaches 58 as an alternative method to test for the presence of pathogenic viruses in the 59 environment (2, 9, 10). Provided the entire viral community is sampled and 60 4 sequenced, novel genotypes or even entirely novel viruses can be detected. 61Potential new viral markers for faecal contamination have already been revealed, 62 such as pepper mild mottle virus and crAssphage (11, 12), among the huge diversity 63 of human viruses found in sludge samples (13). 64In this pilot study, we have used viromics to investigate the presence of human 65 pathogenic RNA viruses in wastewater, estuarine surface water and sediment in a 66 single catchment. The water and sediment samples were collected at, and 67 downstream of, the wastewater treatment plant (Lanrwst, Wales, UK), at the estuary 68 of the river Conwy (Wales, UK) near Morfa beach (Figure 1). To our knowledge, this 69 is the first study to use unamplified environmental viral RNA for sequencing library 70 construction, sequence dataset production and subsequent analysis. Because we 71 used a directional library sequencing protocol on RNA, rather than amplifying to 72 cDNA, we were able to distinguish single-stranded from double-stranded RNA 73 genome fragments. 74

Tracing the fate of wastewater viruses reveals catchment-scale virome diversity and connectivity

“…5b). The RVA genome segments recovered here are markedly different to those recovered from wastewater influent from Llanrwst (LE-LI) in our pilot study 10 months previously 22 , for which the dominant genotypes of RVA were G8/G10-P[1]/P [14]/P [41], and a diverse set of rotavirus C segments were also present. We can conclude that rotavirus shedding into wastewater within the population varied both spatially and temporally, but more data are required to investigate any possible seasonal patterns.…”

“…Interestingly, we were unable to unambiguously identify any potentially pathogenic dsDNA UViGs. The ability to reconstruct a complete papillomavirus genome in our pilot study from a subset of these sites sampled at an earlier date 22 suggests that there were in fact no predicted-pathogenic dsDNA viruses circulating (above the limit of detection) in the Conwy catchment at the time of sampling (June 2017). As an additional check, 199 we did a search for similarity of the UViG dataset with members of the Coronaviridae family, but no 200 coronavirus signatures were identified in our dataset.…”

“…3a). For the family Caliciviridae, we were not able to identify any UViGs representing noroviruses, the leading cause of viral gastro-intestinal illness in the UK and indeed worldwide [24][25][26] in contrast to our pilot study performed in autumn, where we assembled a norovirus GI.2 genome 22 . We did, however, find two near-complete sapovirus UViGs and six shorter contigs grouped with the near-complete genomes (Fig.…”

Making waves: Wastewater-based epidemiology for COVID-19 – approaches and challenges for surveillance and prediction

“…The complexity of the matrix and the high abundance of genetic material in the samples often lead to more conservative results of viral detection as well as to poorer detection limits for a specific virus compared to qPCR ( Fernandez-Cassi et al., 2018 ; Martínez-Puchol et al., 2020 ). Conversely, they can also detect new variants that are missed by conventional qPCR primer sets as de virus progressively mutates ( Adriaenssens et al., 2018 ).…”

Metagenomic Insights into the Sewage RNA Virosphere of a Large City

“…Sewage has been historically used to monitor known human viral pathogens, such as noroviruses, hepatitis viruses, enteroviruses, rotaviruses and adenoviruses [ 6 , 7 ]. The presence of viral pathogens in wastewater reflects ongoing infections being transmitted in the human population served by the given WWTP [ 2 , 3 , 4 ].…”