Viromic Analysis of Wastewater Input to a River Catchment Reveals a Diverse Assemblage of RNA Viruses

Detection of viruses in the environment is heavily dependent on PCR-based approaches that require reference sequences for primer design. While this strategy can accurately detect known viruses, it will not find novel genotypes or emerging and invasive viral species. In this study, we investigated the use of viromics, i.e., high-throughput sequencing of the biosphere's viral fraction, to detect human-/animal-pathogenic RNA viruses in the Conwy river catchment area in Wales, United Kingdom. Using a combination of filtering and nuclease treatment, we extracted the viral fraction from wastewater and estuarine river water and sediment, followed by high-throughput RNA sequencing (RNA-Seq) analysis on the Illumina HiSeq platform, for the discovery of RNA virus genomes. We found a higher richness of RNA viruses in wastewater samples than in river water and sediment, and we assembled a complete norovirus genotype GI.2 genome from wastewater effluent, which was not contemporaneously detected by conventional reverse transcription-quantitative PCR (qRT-PCR). The simultaneous presence of diverse rotavirus signatures in wastewater indicated the potential for zoonotic infections in the area and suggested runoff from pig farms as a possible origin of these viruses. Our results show that viromics can be an important tool in the discovery of pathogenic viruses in the environment and can be used to inform and optimize reference-based detection methods provided appropriate and rigorous controls are included. IMPORTANCE Enteric viruses cause gastrointestinal illness and are commonly transmitted through the fecal-oral route. When wastewater is released into river systems, these viruses can contaminate the environment. Our results show that we can use viromics to find the range of potentially pathogenic viruses that are present in the environment and identify prevalent genotypes. The ultimate goal is to trace the fate of these pathogenic viruses from origin to the point where they are a threat to human health, informing reference-based detection methods and water quality management.