Polyomaviruses are a family of DNA tumor viruses that are known to infect mammals and birds. To investigate the deeper evolutionary history of the family, we used a combination of viral metagenomics, bioinformatics, and structural modeling approaches to identify and characterize polyomavirus sequences associated with fish and arthropods. Analyses drawing upon the divergent new sequences indicate that polyomaviruses have been gradually co-evolving with their animal hosts for at least half a billion years. Phylogenetic analyses of individual polyomavirus genes suggest that some modern polyomavirus species arose after ancient recombination events involving distantly related polyomavirus lineages. The improved evolutionary model provides a useful platform for developing a more accurate taxonomic classification system for the viral family Polyomaviridae. Polyomaviruses are a family of DNA-based viruses that are known to infect various terrestrial vertebrates, including humans. In this report, we describe our discovery of highly divergent polyomaviruses associated with various marine fish. Searches of public deep sequencing databases unexpectedly revealed the existence of polyomavirus-like sequences in scorpion and spider datasets. Our analysis of these new sequences suggests that polyomaviruses have slowly co-evolved with individual host animal lineages through an established mechanism known as intrahost divergence. The proposed model is similar to the mechanisms through with other DNA viruses, such as papillomaviruses, are thought to have evolved. Our analysis also suggests that distantly related polyomaviruses sometimes recombine to produce new chimeric lineages. We propose a possible taxonomic scheme that can account for these inferred ancient recombination events.

Enteric viruses represent a global public health threat and are implicated in numerous foodborne and waterborne disease outbreaks. Nonetheless, relatively little is known of their fate and stability in the environment. In this study we used carefully validated methods to monitor enteric viruses, namely adenovirus (AdV), JC polyomavirus (JCV), noroviruses (NoVs), sapovirus (SaV) and hepatitis A and E viruses (HAV and HEV) from wastewater source to beaches and shellfish beds. Wastewater influent and effluent, surface water, sediment and shellfish samples were collected in the Conwy catchment (North Wales, UK) once a month for one year. High concentrations of AdV and JCV were found in the majority of samples, and no seasonal patterns were observed. No HAV and HEV were detected and no related illnesses were reported in the area during the period of sampling. Noroviruses and SaV were also detected at high concentrations in wastewater and surface water, and their presence correlated with local gastroenteritis outbreaks during the spring and autumn seasons. Noroviruses were also found in estuarine sediment and in shellfish harvested for human consumption. As PCR-based methods were used for quantification, viral infectivity and degradation was estimated using a NoV capsid integrity assay. The assay revealed low-levels of viral decay in wastewater effluent compared to influent, and more significant decay in environmental waters and sediment. Results suggest that AdV and JCV may be suitable markers for the assessment of the spatial distribution of wastewater contamination in the environment; and pathogenic viruses can be directly monitored during and after reported outbreaks to prevent further environment-derived illnesses.

Waterborne enteric viruses are an emerging cause of disease outbreaks and represent a major threat to global public health. Enteric viruses may originate from human wastewater and can undergo rapid transport through aquatic environments with minimal decay. Surveillance and source apportionment of enteric viruses in environmental waters is therefore essential for accurate risk management. However, individual monitoring of the >100 enteric viral strains that have been identified as aquatic contaminants is unfeasible. Instead, viral indicators are often used for quantitative assessments of wastewater contamination, viral decay and transport in water. An ideal indicator for tracking wastewater contamination should be (i) easy to detect and quantify, (ii) source-specific, (iii) resistant to wastewater treatment processes, and (iv) persistent in the aquatic environment, with similar behaviour to viral pathogens. Here, we conducted a comprehensive review of 127 peer-reviewed publications, to critically evaluate the effectiveness of several viral indicators of wastewater pollution, including common enteric viruses (mastadenoviruses, polyomaviruses, and Aichi viruses), the pepper mild mottle virus (PMMoV), and gut-associated bacteriophages (Type II/III FRNA phages and phages infecting human Bacteroides species, including crAssphage). Our analysis suggests that overall, human mastadenoviruses have the greatest potential to indicate contamination by domestic wastewater due to their easy detection, culturability, and high prevalence in wastewater and in the polluted environment. Aichi virus, crAssphage and PMMoV are also widely detected in wastewater and in the environment, and may be used as molecular markers for human-derived contamination. We conclude that viral indicators are suitable for the long-term monitoring of viral contamination in freshwater and marine environments and that these should be implemented within monitoring programmes to provide a holistic assessment of microbiological water quality and wastewater-based epidemiology, improve current risk management strategies and protect global human health.

The discharge of human-derived wastewater represents a major threat to water quality with the potential for waterborne disease outbreaks mainly associated with enteric viruses. To prevent illnesses, indicators associated with fecal contamination are monitored in polluted areas, however, their prevalence often does not correlate well with viral pathogens. In this study, we used crAssphage, a recently discovered human-specific gut-associated bacteriophage, for the surveillance of wastewater-derived viral contamination. Untreated and treated wastewater, surface water, sediment and mussel samples were collected monthly over 1 year from the Conwy River and estuary (UK) and were analyzed for crAssphage marker by quantitative PCR. This is the first long-term catchment-to-coast scale study of environmental crAssphage concentrations. CrAssphage was detected in all sample types and showed no distinct seasonal pattern. CrAssphage concentrations were 2 × 10⁵–10⁹ genome copies (gc)/L in all untreated wastewater influent and 10⁷–10⁸ gc/L in secondary treated effluent samples, 3 × 10³ gc/L–3 × 10⁷ gc/L in surface water samples (94% positive) and 2 × 10²–10⁴ gc/g sediment (68% positive) and mussel digestive tissue (79% positive). CrAssphage concentrations were 1–5 log₁₀ higher than human enteric virus titers (norovirus, sapovirus, adenovirus, polyomavirus). Our results indicate that crAssphage is well suited to tracking human wastewater contamination and pollution risk assessment in aquatic environments.

Wastewater-based epidemiology has become an important tool for the surveillance of SARS-CoV-2 outbreaks. However, the detection of viruses in sewage is challenging and to date there is no standard method available which has been validated for the sensitive detection of SARS-CoV-2. In this paper, we describe a simple concentration method based on polyethylene glycol (PEG) precipitation, followed by RNA extraction and a one-step quantitative reverse transcription PCR (qRT-PCR) for viral detection in wastewater. PEG-based concentration of viruses is a simple procedure which is not limited by the availability of expensive equipment and has reduced risk of disruption to consumable supply chains. The concentration and RNA extraction steps enable 900–1500× concentration of wastewater samples and sufficiently eliminates the majority of organic matter, which could inhibit the subsequent qRT-PCR assay. Due to the high variation in the physico-chemical properties of wastewater samples, we recommend the use of process control viruses to determine the efficiency of each step. This procedure enables the concentration and the extraction the DNA/RNA of different viruses and hence can be used for the surveillance of different viral targets for the comprehensive assessment of viral diseases in a community.