Exploring viral contamination in urban groundwater and runoff.

The reliance of the global population on urban aquifers is steadily increasing, and urban aquifers are susceptible to pathogenic contamination through sources such as sewer leakage or urban runoff. However, there is insufficient monitoring of groundwater quality in urban areas. In this study, quantitative polymerase chain reaction (qPCR) was employed to evaluate the presence of human fecal viral indicators and viral pathogens in urban wastewater (n = 13) and groundwater (n = 12) samples from four locations in Barcelona with different degrees of urbanization, as well as in runoff samples (n = 2). Additionally, a target enrichment sequencing (TES) approach was utilized to explore the viral diversity within groundwater and runoff samples, offering insights into viral contamination and potential virus transmission routes in urban areas. Human adenovirus (HAdV) was identified in all wastewater samples, 67 % (8/12) of groundwater samples, and one runoff sample by qPCR indicating human viral fecal contamination. The viral pathogen Norovirus genogroup GI (NoV GI) was detected in wastewater and two winter groundwater samples from highly and medium urbanized areas. NoV genogroup GII (NoV GII), Enterovirus (EV) and SARS-CoV-2 were exclusively detected in wastewater. Human and other vertebrate viruses were detected in groundwater and runoff samples using TES. This study gives insights about the virome present in urban water sources, emphasizing the need for thorough monitoring and deeper understanding to address emerging public health concerns.

Assessing environmental exposure to viruses in wastewater treatment plant and swine farm scenarios with next-generation sequencing and occupational risk approaches.

Occupational exposure to pathogens can pose health risks. This study investigates the viral exposure of workers in a wastewater treatment plant (WWTP) and a swine farm by analyzing aerosol and surfaces samples. Viral contamination was evaluated using quantitative polymerase chain reaction (qPCR) assays, and target enrichment sequencing (TES) was performed to identify the vertebrate viruses to which workers might be exposed. Additionally, Quantitative Microbial Risk Assessment (QMRA) was conducted to estimate the occupational risk associated with viral exposure for WWTP workers, choosing Human Adenovirus (HAdV) as the reference pathogen. In the swine farm, QMRA was performed as an extrapolation, considering a hypothetical zoonotic virus with characteristics similar to Porcine Adenovirus (PAdV). The modelled exposure routes included aerosol inhalation and oral ingestion through contaminated surfaces and hand-to-mouth contact. HAdV and PAdV were widespread viruses in the WWTP and the swine farm, respectively, by qPCR assays. TES identified human and other vertebrate viruses WWTP samples, including viruses from families such as Adenoviridae, Circoviridae, Orthoherpesviridae, Papillomaviridae, and Parvoviridae. In the swine farm, most of the identified vertebrate viruses were porcine viruses belonging to Adenoviridae, Astroviridae, Circoviridae, Herpesviridae, Papillomaviridae, Parvoviridae, Picornaviridae, and Retroviridae. QMRA analysis revealed noteworthy risks of viral infections for WWTP workers if safety measures are not taken. The probability of illness due to HAdV inhalation was higher in summer compared to winter, while the greatest risk from oral ingestion was observed in workspaces during winter. Swine farm QMRA simulation suggested a potential occupational risk in the case of exposure to a hypothetical zoonotic virus. This study provides valuable insights into WWTP and swine farm worker's occupational exposure to human and other vertebrate viruses. QMRA and NGS analyses conducted in this study will assist managers in making evidence-based decisions, facilitating the implementation of protection measures, and risk mitigation practices for workers.

Wastewater-based epidemiology applied at the building-level reveals distinct virome profiles based on the age of the contributing individuals.

BACKGROUND: Human viruses released into the environment can be detected and characterized in wastewater. The study of wastewater virome offers a consolidated perspective on the circulation of viruses within a population. Because the occurrence and severity of viral infections can vary across a person's lifetime, studying the virome in wastewater samples contributed by various demographic segments can provide valuable insights into the prevalence of viral infections within these segments. In our study, targeted enrichment sequencing was employed to characterize the human virome in wastewater at a building-level scale. This was accomplished through passive sampling of wastewater in schools, university settings, and nursing homes in two cities in Catalonia. Additionally, sewage from a large urban wastewater treatment plant was analysed to serve as a reference for examining the collective excreted human virome. RESULTS: The virome obtained from influent wastewater treatment plant samples showcased the combined viral presence from individuals of varying ages, with astroviruses and human bocaviruses being the most prevalent, followed by human adenoviruses, polyomaviruses, and papillomaviruses. Significant variations in the viral profiles were observed among the different types of buildings studied. Mamastrovirus 1 was predominant in school samples, salivirus and human polyomaviruses JC and BK in the university settings while nursing homes showed a more balanced distribution of viral families presenting papillomavirus and picornaviruses and, interestingly, some viruses linked to immunosuppression. CONCLUSIONS: This study shows the utility of building-level wastewater-based epidemiology as an effective tool for monitoring the presence of viruses circulating within specific age groups. It provides valuable insights for public health monitoring and epidemiological studies.

Evaluation of a virus concentration method based on ultrafiltration and wet foam elution for studying viruses from large-volume water samples.

Assessing the presence of viruses in large-volume samples involves cumbersome methods that require specialized training and laboratory equipment. In this study, a large volume concentration (LVC) method, based on dead-end ultrafiltration (DEUF) and Wet Foam Elution™ technology, was evaluated in different type of waters and different microorganisms. Its recovery efficiency was evaluated through different techniques (infectivity assays and molecular detection) by spiking different viral surrogates (bacteriophages PhiX174 and MS2 and Coxsackie virus B5 (CVB5) and Escherichia coli (E. coli). Furthermore, the application of a secondary concentration step was evaluated and compared with skimmed milk flocculation. Viruses present in river water, seawater and groundwater samples were concentrated by applying LVC method and a centrifugal ultrafiltration device (CeUF), as a secondary concentration step and quantified with specific qPCR Human adenoviruses (HAdV) and noroviruses (NoVs). MS2 was used as process control, obtaining a mean viral recovery of 22.0 ± 12.47%. The presence of other viruses was also characterized by applying two different next-generation sequencing approaches. LVC coupled to a secondary concentration step based on CeUF allowed to detect naturally occurring viruses such as HAdV and NoVs in different water matrices. Using HAdV as a human fecal indicator, the highest viral pollution was found in river water samples (100% of positive samples), followed by seawater (83.33%) and groundwater samples (66.67%). The LVC method has also proven to be useful as a virus concentration method in the filed since HAdV and NoVs were detected in the river water and groundwater samples concentrated in the field. All in all, LVC method presents high concentration factor and a low limit of detection and provides viral concentrates useful for subsequent molecular analysis such as PCR and massive sequencing.

NGS Techniques Reveal a High Diversity of RNA Viral Pathogens and Papillomaviruses in Fresh Produce and Irrigation Water.

Fresh fruits and vegetables are susceptible to microbial contamination at every stage of the food production chain, and as a potential source of pathogens, irrigation water quality is a critical factor. Next-generation sequencing (NGS) techniques have been flourishing and expanding to a wide variety of fields. However, their application in food safety remains insufficiently explored, and their sensitivity requires improvement. In this study, quantitative polymerase chain reaction (qPCR) assays showed low but frequent contamination of common circulating viral pathogens, which were found in 46.9% of samples of fresh produce: 6/12 lettuce samples, 4/12 strawberries samples, and 5/8 parsley samples. Furthermore, the application of two different NGS approaches, target enrichment sequencing (TES) for detecting viruses that infect vertebrates and amplicon deep sequencing (ADS), revealed a high diversity of viral pathogens, especially Norovirus (NoV) and Human Papillomavirus (HPV), in fresh produce and irrigation water. All NoV and HPV types found in fresh fruit and vegetable samples were also detected in irrigation water sources, indicating that these viruses are common circulating pathogens in the population and that irrigation water may be the most probable source of viral pathogens in food samples.

Unveiling Viruses Associated with Gastroenteritis Using a Metagenomics Approach.

Acute infectious gastroenteritis is an important illness worldwide, especially on children, with viruses accounting for approximately 70% of the acute cases. A high number of these cases have an unknown etiological agent and the rise of next generation sequencing technologies has opened new opportunities for viral pathogen detection and discovery. Viral metagenomics in routine clinical settings has the potential to identify unexpected or novel variants of viral pathogens that cause gastroenteritis. In this study, 124 samples from acute gastroenteritis patients from 2012-2014 previously tested negative for common gastroenteritis pathogens were pooled by age and analyzed by next generation sequencing (NGS) to elucidate unidentified viral infections. The most abundant sequences detected potentially associated to acute gastroenteritis were from Astroviridae and Caliciviridae families, with the detection of norovirus GIV and sapoviruses. Lower number of contigs associated to rotaviruses were detected. As expected, other viruses that may be associated to gastroenteritis but also produce persistent infections in the gut were identified including several Picornaviridae members (EV, parechoviruses, cardioviruses) and adenoviruses. According to the sequencing data, astroviruses, sapoviruses and NoV GIV should be added to the list of viral pathogens screened in routine clinical analysis.

Microbiological contamination of conventional and reclaimed irrigation water: Evaluation and management measures.

The wide diversity of irrigation water sources (i.e., drinking water, groundwater, reservoir water, river water) includes reclaimed water as a requested measure for increasing water availability, but it is also a challenge as pathogen exposure may increase. This study evaluates the level of microbial contamination in different irrigation waters to improve the knowledge and analyses management measures for safety irrigation. Over a one-year period, the occurrence of a set of viruses, bacteria and protozoa, was quantified and the performance of a wetland system, producing reclaimed water intended for irrigation, was characterized. Human fecal pollution (HAdV) was found in most of the irrigation water types analysed. Hepatitis E virus (HEV), an emerging zoonotic pathogen, was present in groundwater where porcine contamination was identified (PAdV). The skin-carcinoma associated Merkel cell polyomavirus (MCPyV), was found occasionally in river water. Noroviruses were detected, as expected, in winter, in river water and reclaimed water. Groundwater, river water and reservoir water also harboured potential bacterial pathogens, like Helicobacter pylori, Legionella spp. and Aeromonas spp. that could be internalized and viable inside amoebas like Acanthamoeba castellanii, which was also detected. Neither Giardia cysts, nor any Cryptosporidium oocysts were detected. The wetland system removed 3 Log10 of viruses and 5 Log10 of bacteria, which resembled the river water quality. Irrigation waters were prone to variable contamination levels and according to the European guidance documents, the E. coli (EC) levels were not always acceptable. Sporadic detection of viral pathogens as NoV GII and HAdV was identified in water samples presenting lower EC than the established limit (100MNP/100 mL). When dealing with reclaimed water as a source of irrigation the analysis of some viral parameters, like HAdV during the peak irrigation period (summer and spring) or NoV during the coldest months, could complement existing water management tools based on bacterial indicators.

Characterisation of the sewage virome: comparison of NGS tools and occurrence of significant pathogens.

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).

Occurrence of pathogens in the river-groundwater interface in a losing river stretch (Besòs River Delta, Spain).

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.

Phylodynamics of Merkel-cell polyomavirus and human polyomavirus 6: A long-term history with humans.

New human polyomaviruses have been described in the last years, including the Merkel-cell polyomavirus (MCPyV; Human polyomavirus 5) and the Human polyomavirus 6 (HPyV6). Although their infection is usually asymptomatic, in immunocompromised host can cause life-threatening pathologies, such as the Merkel cell carcinoma, an aggressive skin neoplasia associated to the MCPyV. Despite being prevalent viruses in population, epidemiological data from South America are scarce, as well as the characterization of the viral types circulating and their origin. The aims of this work were to describe MCPyV and HPyV6 from environmental samples with different geographical origin and to analyze their phylogenetic and evolutionary histories, particularly for MCPyV. Partial and complete genome sequences were obtained from sewage samples from Argentina, Uruguay and Spain. A total number of 87 sequences were obtained for MCPyV and 33 for HPyV6. Phylogenetic analysis showed that MCPyV sequences distributed according to their geographic origin in Europe/North America, Africa, Asia, South America and Oceania groups, suggesting that viral diversification might have followed human migrations across the globe. In particular, viruses from Argentina associated with Europe/North America and South America genotypes, whereas those from Uruguay and Spain also grouped with Africa genotype, reflecting the origin of the current population in each country, which could arrive not only during ancient human migration but also during recent migratory events. In addition, the South American group presented a high level of clusterization, showing internal clusters that could be related to specific locations, such as French Guiana and Brazil or the Southern region into South America, such as Argentina and Uruguay, suggesting a long term evolutionary process in the region. Additionally, in this work, we carried out the first analysis about the evolutionary history of MCPyV trough the integration of phylogenetic, epidemiological and historical data. Since a strong association is observed between the phylogenetic relationships and the origin of the sampled population, this analysis was based on the hypothesis of co-divergence between the virus and human populations. This analysis resulted in a substitution rate of 5.1 × 10-8 s/s/y (∼5.1% of divergence per million years) for the complete genome of MCPyV, which is in the range of those estimated for other double-stranded DNA viruses. Regarding HPyV6, a South American group with clusterization was observed (sequences from Uruguay). Meanwhile, sequences from Argentina grouped with European ones (France and Spain) and remained separated from those isolated in China, USA or Australia. The analysis of viruses from the environment allowed us to deep characterize prevalent infections in different geographic regions, reveling that viruses circulating in each population reflected its origin and that there are specific lineages associated with South America.

Evaluation of Methods for the Concentration and Extraction of Viruses from Sewage in the Context of Metagenomic Sequencing.

Viral sewage metagenomics is a novel field of study used for surveillance, epidemiological studies, and evaluation of waste water treatment efficiency. In raw sewage human waste is mixed with household, industrial and drainage water, and virus particles are, therefore, only found in low concentrations. This necessitates a step of sample concentration to allow for sensitive virus detection. Additionally, viruses harbor a large diversity of both surface and genome structures, which makes universal viral genomic extraction difficult. Current studies have tackled these challenges in many different ways employing a wide range of viral concentration and extraction procedures. However, there is limited knowledge of the efficacy and inherent biases associated with these methods in respect to viral sewage metagenomics, hampering the development of this field. By the use of next generation sequencing this study aimed to evaluate the efficiency of four commonly applied viral concentrations techniques (precipitation with polyethylene glycol, organic flocculation with skim milk, monolithic adsorption filtration and glass wool filtration) and extraction methods (Nucleospin RNA XS, QIAamp Viral RNA Mini Kit, NucliSENS® miniMAG®, or PowerViral® Environmental RNA/DNA Isolation Kit) to determine the viriome in a sewage sample. We found a significant influence of concentration and extraction protocols on the detected viriome. The viral richness was largest in samples extracted with QIAamp Viral RNA Mini Kit or PowerViral® Environmental RNA/DNA Isolation Kit. Highest viral specificity were found in samples concentrated by precipitation with polyethylene glycol or extracted with Nucleospin RNA XS. Detection of viral pathogens depended on the method used. These results contribute to the understanding of method associated biases, within the field of viral sewage metagenomics, making evaluation of the current literature easier and helping with the design of future studies.

Genome Sequence of a Cynomolgus Macaque Adenovirus (CynAdV-1) Isolate from a Primate Colony in the United Kingdom.

The genome sequence of a simian adenovirus from a cynomolgus macaque, denoted CynAdV-1, is presented here. Phylogenetic analysis supports CynAdV-1 in an independent clade, comprising a new simian adenovirus (SAdV) species. These genome data are critical for understanding the evolution and relationships of primate adenoviruses, including zoonosis and emergent human pathogens.

Human-, Ovine-, and Bovine-Specific Viral Source Tracking Tools to Discriminate Between the Major Fecal Sources in Agricultural Waters.

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.

Evidence of viral dissemination and seasonality in a Mediterranean river catchment: Implications for water pollution management.

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 log10 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.

Treating progressive multifocal leukoencephalopathy with interleukin 7 and vaccination with JC virus capsid protein VP1.

Progressive multifocal leukoencephalopathy is a currently untreatable infection of the brain. Here, we demonstrate in 2 patients that treatment with interleukin 7, JC polyomavirus (JCV) capsid protein VP1, and a Toll-like receptor 7 agonist used as adjuvant, was well tolerated, and showed a very favorable safety profile and unexpected efficacy that warrant further investigation.

Application of human and animal viral microbial source tracking tools in fresh and marine waters from five different geographical areas.

Integrated river basin management planning to mitigate the impacts of economic, demographic and climate change is an important issue for the future protection of water resources. Identifying sources of microbial contamination via the emerging science of Microbial Source Tracking (MST) plays a key role in risk assessment and the design of remediation strategies. Following an 18-month surveillance program within the EU-FP7-funded VIROCLIME project, specific MST tools were used to assess human markers such as adenoviruses (HAdV) and JC polyomaviruses (JCPyV) and porcine and bovine markers such as porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) via quantification with real-time PCR to analyze surface water collected from five sites within different climatic zones: the Negro River (Brazil), Glafkos River (Greece), Tisza River (Hungary), Llobregat River (Spain) and Umeälven River (Sweden). The utility of the viral MST tools and the prevalence and abundance of specific human and animal viruses in the five river catchments and adjacent seawater, which is impacted by riverine contributions from the upstream catchments, were examined. In areas where no sanitation systems have been implemented, sewage can directly enter surface waters, and river water exhibited high viral loads; HAdV and JCPyV could be detected at mean concentrations of 10(5) and 10(4) Genome Copies/Liter (GC/L), respectively. In general, river water samples upstream of urban discharges presented lower human viral loads than downstream sampling sites, and those differences appeared to increase with urban populations but decrease in response to high river flow, as the elevated river water volume dilutes microbial loads. During dry seasons, river water flow decreases dramatically, and secondary effluents can represent the bulk of the riverine discharge. We also observed that ice cover that formed over the river during the winter in the studied areas in North Europe could preserve viral stability due to the low temperatures and/or the lack of solar inactivation. Porcine and bovine markers were detected where intensive livestock and agricultural activities were present; mean concentration values of 10(3) GC/L indicated that farms were sometimes unexpected and important sources of fecal contamination in water. During spring and summer, when livestock is outdoors and river flows are low, animal pollution increases due to diffuse contamination and direct voiding of feces onto the catchment surface. The field studies described here demonstrate the dynamics of fecal contamination in all catchments studied, and the data obtained is currently being used to develop dissemination models of fecal contamination in water with respect to future climate change scenarios. The results concerning human and animal targets presented in this study demonstrate the specificity and applicability of the viral quantitative parameters developed to widely divergent geographical areas and their high interest as new indicators of human and animal fecal contamination in water and as MST tools.

Adenovirus and Norovirus Contaminants in Commercially Distributed Shellfish.

Shellfish complying with European Regulations based on quantification of fecal bacterial indicators (FIB) are introduced into markets; however, information on viruses, more stable than FIB, is not available in the literature. To assess the presence of noroviruses (NoVs) GI and GII and human adenoviruses (HAdV) in domestic and imported mussels and clams (n = 151) their presence was analyzed during winter seasons (2004-2008) in north-west Spanish markets through a routine surveillance system. All samples tested negative for NoV GI and 13 % were positive for NoV GII. The role of HAdV as viral indicator was evaluated in 20 negative and 10 positive NoV GII samples showing an estimated sensitivity and specificity of HAdV to predict the presence of NoV GII of 100 and 74 % (cut-off 0.5). The levels of HAdV and NoVs and the efficiency of decontamination in shellfish depuration plants (SDP) were evaluated analyzing pre- and post-depurated mussels collected in May-June 2010 from three different SDP. There were no statistically significant differences in the prevalence and quantification of HAdV between pre- and post-depurated shellfish and between seawater entering and leaving the depuration systems. Moreover, infectious HAdV were detected in depurated mussels. These results confirm previous studies showing that current controls and depuration treatments limiting the number of FIB do not guarantee the absence of viruses in shellfish.

Quantification of human and animal viruses to differentiate the origin of the fecal contamination present in environmental samples.

Many different viruses are excreted by humans and animals and are frequently detected in fecal contaminated waters causing public health concerns. Classical bacterial indicator such as E. coli and enterococci could fail to predict the risk for waterborne pathogens such as viruses. Moreover, the presence and levels of bacterial indicators do not always correlate with the presence and concentration of viruses, especially when these indicators are present in low concentrations. Our research group has proposed new viral indicators and methodologies for determining the presence of fecal pollution in environmental samples as well as for tracing the origin of this fecal contamination (microbial source tracking). In this paper, we examine to what extent have these indicators been applied by the scientific community. Recently, quantitative assays for quantification of poultry and ovine viruses have also been described. Overall, quantification by qPCR of human adenoviruses and human polyomavirus JC, porcine adenoviruses, bovine polyomaviruses, chicken/turkey parvoviruses, and ovine polyomaviruses is suggested as a toolbox for the identification of human, porcine, bovine, poultry, and ovine fecal pollution in environmental samples.

Description of a novel viral tool to identify and quantify ovine faecal pollution in the environment.

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.

Detection and quantification of classic and emerging viruses by skimmed-milk flocculation and PCR in river water from two geographical areas.

Molecular techniques and virus concentration methods have shown that previously unknown viruses are shed by humans and animals, and may be transmitted by sewage-contaminated water. In the present study, 10-L river-water samples from urban areas in Barcelona, Spain and Rio Janeiro, Brazil, have been analyzed to evaluate the viral dissemination of human viruses, validating also a low-cost concentration method for virus quantification in fresh water. Three viral groups were analyzed: (i) recently reported viruses, klassevirus (KV), asfarvirus-like virus (ASFLV), and the polyomaviruses Merkel cell (MCPyV), KI (KIPyV) and WU (WUPyV); (ii) the gastroenteritis agents noroviruses (NoV) and rotaviruses (RV); and (iii) the human fecal viral indicators in water, human adenoviruses (HAdV) and JC polyomaviruses (JCPyV). Virus detection was based on nested and quantitative PCR assays. For KV and ASFLV, nested PCR assays were developed for the present study. The method applied for virus concentration in fresh water samples is a one-step procedure based on a skimmed-milk flocculation procedure described previously for seawater. Using spiked river water samples, inter- and intra-laboratory assays showed a viral recovery rate of about 50% (20-95%) for HAdV, JCPyV, NoV and RV with a coefficient of variation ≤ 50%. HAdV and JCPyV were detected in 100% (12/12) of the river samples from Barcelona and Rio de Janeiro. Moreover, NoV GGII was detected in 83% (5/6) and MCPyV in 50% (3/6) of the samples from Barcelona, whereas none of the other viruses tested were detected. NoV GGII was detected in 33% (2/6), KV in 33% (2/6), ASFLV in 17% (1/6) and MCPyV in 50% (3/6) of the samples from Rio de Janeiro, whereas KIPyV and WUPyV were not detected. RV were only analyzed in Rio de Janeiro and resulted positive in 67% (4/6) of the samples. The procedure applied here to river water represents a useful, straightforward and cost-effective method that could be applied in routine water quality testing. The results of the assays expand our understanding of the global distribution of the viral pathogens studied here and their persistence in the environment.