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Relevant studies on waterborne viral occurrence published in the past 2 years have been analyzed showing that most of them were performed in surface waters and wastewater. Viruses were concentrated from surface water samples by applying, mainly, electronegative filtration or ultrafiltration combined with polyethyleneglycol precipitation, centrifugation, or centrifugal ultrafiltration, whereas for wastewater, most authors used flocculation/precipitation. Main novelties include monolithic filtration/chromatography and ultrafiltration-based new approaches as well as methods deployable in the field. Considering future climate change impacts, more studies on viral occurrence in groundwater, seawater, and reclaimed water are needed as well as standardized methods in view of future regulations comprising analysis of viral parameters in water matrices.

Wastewater based epidemiology was employed to track the spread of SARS-CoV-2 within the sewershed areas of 10 wastewater treatment plants (WWTPs) in Catalonia, Spain. A total of 185 WWTPs inflow samples were collected over the period consisting of both the first wave (mid-March to June) and the second wave (July to November). Concentrations of SARS-CoV-2 RNA (N1 and N2 assays) were quantified in these wastewaters as well as those of Human adenoviruses (HAdV) and JC polyomavirus (JCPyV), as indicators of human faecal contamination. SARS-CoV-2 N gene daily loads strongly correlated with the number of cases diagnosed one week after sampling i.e. wastewater levels were a good predictor of cases to be diagnosed in the immediate future. The conditions present at small WWTPs relative to larger WWTPs influence the ability to follow the pandemic. Small WWTPs (, This work was partially funded by the projects PCI2019-103643, RTI2018-097346-B-I00, and AGL2017-86797-C2-1-R projects, all funded by the Spanish Ministry of Science, Innovation and Universities. S. Bofill-Mas is a Serra-Hunter fellow at the University of Barcelona. This study is performed with partial support from the Water Research Institute of the University of Barcelona. IDAEA-CSIC is a Centre of Excellence Severo Ochoa (Spanish Ministry of Science and Innovation, Project CEX2018-000794-S). ICRA authors acknowledge the support from the Economy and Knowledge Department of the Catalan Government through Consolidated Research Group (ICRA-ENV 2017 SGR 1124). ICRA is part of the CERCA program. We would like to give a big thanks to our families and colleagues, for their support and help behind the scenes.

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.

Quantitative measurements of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in raw wastewater have been implemented worldwide since the beginning of the pandemic. Recent efforts are being made to evaluate different viral concentration methodologies to overcome supplier shortages during lockdowns. A set of 22-wastewater samples seeded with murine hepatitis virus (MHV), a member of the Coronaviridae family, and the bacteriophage MS2, were used to characterize and compare two ultrafiltration-based methods: a centrifugal ultrafiltration device (Centricon® Plus-70) and the automated concentrating pipette CP-Select™. Based on the recovery efficiencies, significant differences were observed for MHV, with Centricon® Plus-70 (24%) being the most efficient method. Nevertheless, concentrations of naturally occurring SARS-CoV-2, Human adenoviruses and JC polyomaviruses in these samples did not result in significant differences between methods suggesting that testing naturally occurring viruses may complement the evaluation of viral concentration methodologies. Based on the virus adsorption to solids and the necessity of a pre-centrifugation step to remove larger particles and avoid clogging when using ultrafiltration methods, we assessed the percentage of viruses not quantified after ultrafiltration. Around 23% of the detected SARS-CoV-2 would be discarded during the debris removal step. The CP-Select™ provided the highest concentration factor (up to 333×) and the lowest LoD (6.19 × 103 GC/l) for MHV and proved to be fast, automatic, highly reproducible and suitable to work under BSL-2 measures., Graphical abstract Unlabelled Image, Highlights • Centricon® and CP-Select™ performed equally for naturally occurring SARS-CoV-2 • Higher MHV recoveries were calculated using centrifugal ultrafiltration devices. • Naturally occurring viruses complement concentration methods comparison. • A 23% of detected SARS-CoV-2 adsorb to the solid fraction of wastewater. • CP-Select™ fits into a BSL-2 cabinet enabling to work under biosafety containment.

Since the novel SARS-CoV-2 was detected in faeces, environmental researchers have been using centrifugal ultrafiltration, polyethylene glycol precipitation and aluminium hydroxide flocculation to describe its presence in wastewater samples. High recoveries (up to 65%) are described with electronegative filtration when using surrogate viruses, but few literature reports recovery efficiencies using accurate quantification of enveloped viruses. Considering that every single virus will have a different behaviour during viral concentration, it is recommended to use an enveloped virus, and if possible, a betacoronaviruses as murine hepatitis virus (MHV), as a surrogate. In this review we show new data from a new available technology that provides a quick ultrafiltration protocol for SARS-CoV-2. Wastewater surveillance is an efficient system for the evaluation of the relative prevalence of SARS-CoV-2 infections in a community, and there is the need of using reliable concentration methods for an accurate and sensitive quantification of the virus in water., Graphical abstract Image 1, Highlights • There are efficient methods to concentrate enveloped virus such as coronaviruses. • More data on the recovery of the specific pathogen of interest is needed. • Viral surrogates, ideally betacoronavirus, may be used as SARS-CoV-2 process control. • Recovery calculation needs an accurate quantification of the viral stock.

[EN] Viruses (e.g., noroviruses and hepatitis A and E virus), bacteria (e.g., Salmonella spp. and pathogenic Escherichia coli) and protozoa (e.g., Cryptosporidium parvum and Giardia intestinalis) are well-known contributors to food-borne illnesses linked to contaminated fresh produce. As agricultural irrigation increases the total amount of water used annually, reclaimed water is a good alternative to reduce dependency on conventional irrigation water sources. European guidelines have established acceptable concentrations of certain pathogens and/or indicators in irrigation water, depending on the irrigation system used and the irrigated crop. However, the incidences of food-borne infections are known to be underestimated and all the different pathogens contributing to these infections are not known. Next-generation sequencing (NGS) enables the determination of the viral, bacterial and protozoan populations present in a water sample, providing an opportunity to detect emerging pathogens and develop improved tools for monitoring the quality of irrigation water. This is a descriptive study of the virome, bacteriome and parasitome present in different irrigation water sources. We applied the same concentration method for all the studied samples and specific metagenomic approaches to characterize both DNA and RNA viruses, bacteria and protozoa. In general, most of the known viral species corresponded to plant viruses and bacteriophages. Viral diversity in river water varied over the year, with higher bacteriophage prevalences during the autumn and winter. Reservoir water contained Enterobacter cloacae, an opportunistic human pathogen and an indicator of fecal contamination, as well as Naegleria australiensis and Naegleria clarki. Hepatitis E virus and Naegleria fowleri, emerging human pathogens, were detected in groundwater. Reclaimed water produced in a constructed wetland system presented a virome and bacteriome that resembled those of freshwater samples (river and reservoir water). Viral, bacterial and protozoan pathogens were occasionally detected in the different irrigation water sources included in this study, justifying the use of improved NGS techniques to get a comprehensive evaluation of microbial species and potential environmental health hazards associated to irrigation water., This work was supported through a grant funded by the Spanish Ministry of Economy and Competitiveness (MINECO) in the frame of the collaborative international consortium JPIW2013-095-C03-01, JPIW2013-095-C03-02 and JPIW2013-095-C03-03 of the Water Challenges for a Changing World Joint Programming Initiative (Water JPI) Pilot Call and partially by AGL2017-86797-C2-1-R. Silvia Bofill-Mas is a Serra-Hunter fellow at the University of Barcelona.

NGS techniques are excellent tools to monitor and identify viral pathogens circulating among the population with some limitations that need to be overcome, especially in complex matrices. Sewage contains a high amount of other microorganisms that could interfere when trying to sequence viruses for which random PCR amplifications are needed before NGS. The selection of appropriate NGS tools is important for reliable identification of viral diversity among the population. We have compared different NGS methodologies (Untargeted Viral Metagenomics, Target Enrichment Sequencing and Amplicon Deep Sequencing) for the detection and characterisation of viruses in urban sewage, focusing on three important human pathogens: papillomaviruses, adenoviruses and enteroviruses. A full picture of excreted viruses was obtained by applying Untargeted Viral Metagenomics, which detected members of four different vertebrate viral families in addition to bacteriophages, plant viruses and viruses infecting other hosts. Target Enrichment Sequencing, using specific vertebrate viral probes, allowed the detection of up to eight families containing human viruses, with high variety of types within the families and with a high genome coverage. By applying Amplicon Deep Sequencing, the diversity of enteroviruses, adenoviruses and papillomaviruses observed was higher than when applying the other two strategies and this technique allowed the subtyping of an enterovirus A71 C1 strain related to a brainstem encephalitis outbreak occurring at the same time in the sampling area. From the data obtained, we concluded that the different strategies studied provided different levels of analysis: TES is the best strategy to obtain a broad picture of human viruses present in complex samples such as sewage. Other NGS strategies are useful for studying the virome of complex samples when also targeting viruses infecting plants, bacteria, invertebrates or fungi (Untargeted Viral Metagenomics) or when observing the variety within a sole viral family is the objective of the study (Amplicon Deep Sequencing).

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent. At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes. The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

The use of lagooning as a complementary natural method of treating secondary effluents of wastewater treatment plants has been employed as an affordable and easy means of producing reclaimed water. However, using reclaimed water for some purposes, for example, for food irrigation, presents some risks if the effluents contain microbial pathogens. Classical bacterial indicators that are used to assess faecal contamination in water do not always properly indicate the presence of bacterial or viral pathogens. In the current study, the presence of faecal indicator bacteria (FIB), heterotrophic bacterial counts (HBC), pathogens and opportunistic pathogens, such as Legionella spp., Aeromonas spp., Arcobacter spp., free-living amoeba (FLA), several viral indicators (human adenovirus and polyomavirus JC) and viral pathogens (noroviruses and hepatitis E virus) were analysed for 1 year in inlet and outlet water to assess the removal efficiency of a lagooning system. We observed 2.58 (1.17-4.59) and 1.65 (0.15-3.14) log reductions in Escherichia coli (EC) and intestinal enterococci (IE), respectively, between the inlet and outlet samples. Genomic copies of the viruses were log reduced by 1.18 (0.24-2.93), 0.64 (0.12-1.97), 0.45 (0.04-2.54) and 0.72 (0.22-2.50) for human adenovirus (HAdV), JC polyomavirus (JCPyV) and human noroviruses (NoV GI and GII), respectively. No regrowth of opportunistic pathogens was observed within the system. FLA, detected in all samples, did not show a clear trend. The reduction of faecal pathogens was irregular with 6 out of 12 samples and 4 out of 12 samples exceeding the EC and IE values, specified in the Spanish legislation for reclaimed water (RD 1620/2007). This data evidences that there is a need for more studies to evaluate the removal mechanisms of lagooning systems in order to optimize pathogen reduction. Moreover, surveillance of water used to irrigate raw edible vegetables should be conducted to ensure the fulfilment of the microbial requirements for the production of safe reclaimed water.

The aim of this study is to investigate the occurrence of faecal indicator and microbial pathogens (bacteria and virus) in the shallow urban aquifer of the Besòs River Delta (NE Spain). To this end, human adenovirus (HAdV) and Norovirus of genogroups I and II (NoV GI and NoV GII) as well as the faecal indicator bacteria (FIB) Escherichia coli (EC) and faecal enterococci (FE) were monitored in groundwater and in the River Besòs in December 2013 and in July 2104. None of the targeted pathogens were detected in groundwater in December 2013 but contamination of human origin was observed in approximately 50% of the points sampled in July 2014 reaching concentrations up to 99 GC/100 mL for HAdV. Generally, microbial concentrations in river water were higher than those detected in groundwater. This observation indicates that pathogens are naturally attenuated when river water infiltrates and flows through the aquifer, however HAdV were detected at a sampling point located at 380 m from the river in the absence of FIB. The presence of human viral contamination may represent a risk for the use of groundwater as a drinking water source. Further research is needed to understand the dynamics of pathogens in river–groundwater interface over long time periods and a wide range of flow conditions (wet and dry periods) since the urban groundwater of this aquifer might be a valuable drinking water resource in Barcelona especially during drought periods. The methodology followed in this research can be applied to other urban aquifers with similar purposes since the scarcity and contamination of freshwater resources are worldwide issues. © 2019 Elsevier B.V., A. J gratefully acknowledges the financial support from the Excellence Initiative of the German Federal and State Governments through the “Open Topic Postdoc Position Fellow” at the Technische Universität Dresden. The authors would like to thank the European Commission, the Spanish Foundation for Science and Technology (FECYT) and Spanish State Research Agency (AEI) for funding in the frame of the collaborative international consortium (URBANWAT) financed under the 2018 Joint call of the WaterWorks2017 ERA-NET Cofund. This ERA-NET is an integral part of the activities developed by the Water JPI. Additionally authors would also thank the Ministry of Science, Innovation and Universities, for funding the project UNBIASED (Ref: RTI2018-097346-B-I00) under the 2018 call of the “Proyectos de I+D Retos Investigación.

Wastewater is an important resource in water-scarce regions of the world, and its use in agriculture requires the guarantee of acceptable public health risks. The use of fecal indicator bacteria to evaluate safety does not represent viruses, the main potential health hazards. Viral pathogens could complement the use of fecal indicator bacteria in the evaluation of water quality. In this study, we characterized the concentration and removal of human adenovirus (HAdV) and norovirus genogroup II (NoV GII), highly abundant and important viral pathogens found in wastewater, in two wastewater treatment plants (WWTPs) that use different tertiary treatments (constructed wetland vs conventional UV, chlorination and Actiflo® treatments) for a year in Catalonia. The main objective of this study was to develop a Quantitative Microbial Risk Assessment for viral gastroenteritis caused by norovirus GII and adenovirus, associated with the ingestion of lettuce irrigated with tertiary effluents from these WWTPs. The results show that the disease burden of NoV GII and HAdV for the consumption of lettuce irrigated with tertiary effluent from either WWTP was higher than the WHO recommendation of 10−6 DALYs for both viruses. The WWTP with constructed wetland showed a higher viral reduction on average (3.9 and 2.8 logs for NoV GII and HAdV, respectively) than conventional treatment (1.9 and 2.5 logs) but a higher variability than the conventional WWTP. Sensitivity analysis demonstrated that the input parameters used to estimate the viral reduction by treatment and viral concentrations accounted for much of the model output variability. The estimated reductions required to reach the WHO recommended levels in tertiary effluent are influenced by the characteristics of the treatments developed in the WWTPs, and additional average reductions are necessary (in WWTP with a constructed wetland: A total of 6.7 and 5.1 logs for NoV GII and HAdV, respectively; and in the more conventional treatment: 7 and 5.6 logs). This recommendation would be achieved with an average quantification of 0.5 genome copies per 100 mL in reclaimed water for both viruses. The results suggest that the analyzed reclaimed water would require additional treatments to achieve acceptable risk in the irrigation of vegetables with reclaimed water.

This study evaluated the sources of fecal contamination in different river catchments, using a combination of microbial source tracking tools, for human, ruminant, ovine and bovine livestock, in order to define appropriate water management strategies. Every source of waterway pollution was evaluated in river water samples from one urban river catchment and two important farming regions in New Zealand. Fecal pollution was initially measured by testing Escherichia coli and evaluating the presence of human- and ruminant-associated DNA markers of Bacteroidales (BiAdo, BacHum-UCD, BacH, and BacR) and human and ruminant fecal sterols/stanols ratios. Then specific fecal pollution sources were assessed with previously reported quantitative PCR assays targeting human-, bovine-, and ovine-specific viruses: human adenoviruses (HAdV), human JC polyomaviruses, bovine polyomaviruses (BPyV), and ovine polyomaviruses (OPyV). High level of ruminant fecal contamination was detected all over the farming areas, whereas no ruminant sources were identified in the urban river sampling sites. BacR was the most frequently observed ruminant marker and OPyV and BPyV allowed the identification of ovine and bovine fecal sources. The human fecal viral marker (HAdV) was the most frequently observed human marker, highly abundant in the urban sites, and also present in farming areas. This is the first study using simultaneously the ovine and the bovine viral markers to identify and quantify both bovine and ovine fecal pollution.

Conventional wastewater treatment does not completely remove and/or inactive viruses; consequently, viruses excreted by the population can be detected in the environment. This study was undertaken to investigate the distribution and seasonality of human viruses and faecal indicator bacteria (FIB) in a river catchment located in a typical Mediterranean climate region and to discuss future trends in relation to climate change. Sample matrices included river water, untreated and treated wastewater from a wastewater treatment plant within the catchment area, and seawater from potentially impacted bathing water. Five viruses were analysed in the study. Human adenovirus (HAdV) and JC polyomavirus (JCPyV) were analysed as indicators of human faecal contamination of human pathogens; both were reported in urban wastewater (mean values of 10 6 and 10 5 GC/L, respectively), river water (10 3 and 10 2 GC/L) and seawater (10 2 and 10 1 GC/L). Human Merkel Cell polyomavirus (MCPyV), which is associated with Merkel Cell carcinoma, was detected in 75% of the raw wastewater samples (31/37) and quantified by a newly developed quantitative polymerase chain reaction (qPCR) assay with mean concentrations of 10 4 GC/L. This virus is related to skin cancer in susceptible individuals and was found in 29% and 18% of river water and seawater samples, respectively. Seasonality was only observed for norovirus genogroup II (NoV GGII), which was more abundant in cold months with levels up to 10 4 GC/L in river water. Human hepatitis E virus (HEV) was detected in 13.5% of the wastewater samples when analysed by nested PCR (nPCR). Secondary biological treatment (i.e., activated sludge) and tertiary sewage disinfection including chlorination, flocculation and UV radiation removed between 2.22 and 4.52 log 10 of the viral concentrations. Climate projections for the Mediterranean climate areas and the selected river catchment estimate general warming and changes in precipitation distribution. Persistent decreases in precipitation during summer can lead to a higher presence of human viruses because river and sea water present the highest viral concentrations during warmer months. In a global context, wastewater management will be the key to preventing environmental dispersion of human faecal pathogens in future climate change scenarios.

Environmental factors are highly relevant to the global dissemination of viral pathogens. However, the specific contribution of major effectors such as temperature and sunlight on the inactivation of waterborne viruses is not well characterized. In this study, the effect of temperature (7, 20, and 37 °C), UVB and UVA radiation on viral inactivation was evaluated in phosphate buffered saline (PBS), mineral water, wastewater, 1,000-fold diluted wastewater and seawater. The stability of human adenoviruses infectivity, known as human pathogens and indicators of fecal contamination, was monitored during 24 h, both in the dark and exposed to UV radiation by immunofluorescence assays. In the dark, no Human adenovirus (HAdV) inactivation was observed in PBS and mineral water at any of the temperatures studied, whereas at 37 °C in reactors with higher microbial concentration (wastewater, diluted wastewater, and seawater), decays between 2.5 and 5 log were recorded. UVB radiation showed a dramatic effect on HAdV inactivation and 6-log were achieved in all reactors by the end of the experiments. The effect of UVA showed to be dependent on the water matrix analyzed. At 20 °C, HAdV showed a 2-log decay in all reactors radiation while at 37 °C, results in wastewater, diluted wastewater, and seawater reactors were equivalent to those observed in the dark. These results suggest UVB radiation as the major environmental factor challenging viral inactivation, followed by biotic activity indirectly associated to higher temperatures and finally, by UVA radiation.

Determining the stability, or persistence in an infectious state, of foodborne viral pathogens attached to surfaces of soft fruits and salad vegetables is essential to underpin risk assessment studies in food safety. Here, we evaluate the effect of temperature and sunlight on the stability of infectious human adenoviruses type 2 and MS2 bacteriophages on lettuce and strawberry surfaces as representative fresh products. Human adenoviruses have been selected because of their double role as viral pathogens and viral indicators of human fecal contamination. Stability assays were performed with artificially contaminated fresh samples kept in the dark or under sunlight exposure at 4 and 30°C over 24h. The results indicate that temperature is the major factor affecting HAdV stability in fresh produce surfaces, effecting decay between 3 and 4 log after 24h at 30°C. The inactivation times to achieve a reduction between 1 and 4-log are calculated for each experimental condition. This work provides useful information to be considered for improving food safety regarding the transmission of foodborne viruses through supply chains.

Farmed animals such as sheep, cattle, swine and poultry play an important role in microbial contamination of water, crops and food, and introduce large quantities of pathogens into the environment. The ability to determine the origin of faecal pollution in water resources is essential when establishing a robust and efficient water management system. Animal-specific viruses have previously been suggested as microbial source tracking tools, but specific ovine viral markers have not been reported before now. Previous studies have shown that polyomaviruses are host-specific, highly prevalent and are commonly excreted in urine. Furthermore, they have been reported to infect several vertebrate species but not sheep. That situation encouraged the study of a new putative ovine polyomavirus (OPyV) and its use to determine whether faecal pollution originates from ovine faecal/urine contamination. Putative OPyV DNA was amplified from ovine urine and faecal samples using a broad-spectrum nested PCR (nPCR). Specific nested PCR and quantitative PCR assays were developed and applied to faecal and environmental samples, including sheep slurries, slaughterhouse wastewater effluents, urban sewage and river water samples. Successful amplification by PCR was achieved in sheep urine samples, sheep slaughterhouse wastewater and downstream sewage effluents. The assay was specific and was negative in samples of human, bovine, goat, swine and chicken origin. Ovine faecal pollution was detected in river water samples by applying the designed methods. These results provide a quantitative tool for the analysis of OPyV as a suitable viral indicator of sheep faecal contamination that may be present in the environment.