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Enteroviruses (EVs), which belong to the Picornaviridae family, infect individuals asymptomatically or cause mild symptoms (fever, runny nose, cough, skin rash, sneezing, mouth blister). Severe cases can cause various diseases, such as acute hemorrhagic conjunctivitis, aseptic meningitis, or myocarditis, especially in infants. These viruses can be transmitted via the fecal-oral route via contaminated water. In this study, we established a polymerase chain reaction (PCR) method for detecting EVs in water sample using Coxsackievirus B5 (CV-B5) and Echovirus 30 (E-30), which belong to species B of the four species of EVs (EV-A to D). Several methods have been investigated and compared for the detection of EVs, including real-time reverse transcription (RT) polymerase chain reaction and conventional RT-PCR. The most sensitive primer sets were selected, and the PCR conditions were modified to increase sensitivity. We also quantified the detection limits of real-time and conventional RT-PCR. The detection limits of conventional RT-PCR were detected in 105-106 copy/mL for CV-B5 and 106-107 copy/mL for E-30, respectively. This optimized method for detecting EVs is expected to contribute substantially to the investigation of EV outbreaks in water samples.

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Although wastewater-based surveillance (WBS) is an efficient community-wide surveillance tool, its implementation for pathogen surveillance remains limited by ineffective sample treatment procedures, as the complex composition of wastewater often interferes with biomarker recovery. Moreover, current sampling protocols based on grab samples are susceptible to fluctuant biomarker concentrations and may increase operative costs, often rendering such systems inaccessible to communities in low-to-middle-income countries (LMICs). As a response, passive samplers have emerged as a way to make wastewater sampling more efficient and obtain more reliable, consistent data. Therefore, this study aims to review recent developments in passive sampling technologies to provide researchers with the tools to develop novel passive sampling strategies. Although promising advances in the development of nanostructured passive samplers have been reported, optimization remains a significant area of opportunity for researchers in the area, as methods for flexible, robust adsorption and recovery of viral genetic materials would greatly improve the efficacy of WBS systems while making them more accessible for communities worldwide.

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Waterborne viruses that can be harmful to human health pose significant challenges globally,affecting health care systems and the economy.Identifying these waterborne pathogens is essential for preventing diseases and protecting public health.However,handling complex samples such as human and waste-water can be challenging due to their dynamic and complex composition and the ultralow concentration of target analytes.This review presents a comprehensive overview of the latest breakthroughs in waterborne virus biosensors.It begins by highlighting several promising strategies that enhance the sensing performance of optical and electrochemical biosensors in human samples.These strategies include optimizing bioreceptor selection,transduction elements,signal amplification,and integrated sensing systems.Furthermore,the insights gained from biosensing waterborne viruses in human sam-ples are applied to improve biosensing in wastewater,with a particular focus on sampling and sample pretreatment due to the dispersion characteristics of waterborne viruses in wastewater.This review suggests that implementing a comprehensive system that integrates the entire waterborne virus detection process with high-accuracy analysis could enhance virus monitoring.These findings provide valuable insights for improving the effectiveness of waterborne virus detection,which could have sig-nificant implications for public health and environmental management.

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Quantifying viruses in wastewater via RT-qPCR provides total genomic data but does not indicate the virus capsid integrity or the potential risk for human infection. Assessing virus capsid integrity in sewage is important for wastewater-based surveillance, since discharged effluent may pose a public health hazard. While integrity assays using cell cultures can provide this information, they require specialised laboratories and expertise. One solution to overcome this limitation is the use of photo-reactive monoazide dyes (e.g., propidium monoazide [PMAxx]) in a capsid integrity-RT-qPCR assay (ci-RT-qPCR). In this study, we tested the efficiency of PMAxx dye at 50 µM and 100 µM concentrations on live and heat-inactivated model viruses commonly detected in wastewater, including adenovirus (AdV), hepatitis A (HAV), influenza A virus (IAV), and norovirus GI (NoV GI). The 100 µM PMAxx dye concentration effectively differentiated live from heat-inactivated viruses for all targets in buffer solution. This method was then applied to wastewater samples (n = 19) for the detection of encapsulated AdV, enterovirus (EV), HAV, IAV, influenza B virus (IBV), NoV GI, NoV GII, and SARS-CoV-2. Samples were negative for AdV, HAV, IAV, and IBV but positive for EV, NoV GI, NoV GII, and SARS-CoV-2. In the PMAxx-treated samples, EV, NoV GI, and NoV GII showed -0.52-1.15, 0.9-1.51, and 0.31-1.69 log reductions in capsid integrity, indicating a high degree of potentially infectious virus in wastewater. In contrast, SARS-CoV-2 was only detected using RT-qPCR but not after PMAxx treatment, indicating the absence of encapsulated and potentially infectious virus. In conclusion, this study demonstrates the utility of PMAxx dyes to evaluate capsid integrity across a diverse range of viruses commonly monitored in wastewater.

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Waterborne viruses that can be harmful to human health pose significant challenges globally, affecting health care systems and the economy. Identifying these waterborne pathogens is essential for preventing diseases and protecting public health. However, handling complex samples such as human and wastewater can be challenging due to their dynamic and complex composition and the ultralow concentration of target analytes. This review presents a comprehensive overview of the latest breakthroughs in waterborne virus biosensors. It begins by highlighting several promising strategies that enhance the sensing performance of optical and electrochemical biosensors in human samples. These strategies include optimizing bioreceptor selection, transduction elements, signal amplification, and integrated sensing systems. Furthermore, the insights gained from biosensing waterborne viruses in human samples are applied to improve biosensing in wastewater, with a particular focus on sampling and sample pretreatment due to the dispersion characteristics of waterborne viruses in wastewater. This review suggests that implementing a comprehensive system that integrates the entire waterborne virus detection process with high-accuracy analysis could enhance virus monitoring. These findings provide valuable insights for improving the effectiveness of waterborne virus detection, which could have significant implications for public health and environmental management.

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Water contamination is a global health problem, and the need for safe water is ever-growing due to the public health implications of unsafe water. Contaminated water could contain pathogenic bacteria, protozoa, and viruses that are implicated in several debilitating human diseases. The prevalence and survival of waterborne viruses differ from bacteria and other waterborne microorganisms. In addition, viruses are responsible for more severe waterborne diseases such as gastroenteritis, myocarditis, and encephalitis among others, hence the need for dedicated attention to viral inactivation. Disinfection is vital to water treatment because it removes pathogens, including viruses. The commonly used methods and techniques of disinfection for viral inactivation in water comprise physical disinfection such as membrane filtration, ultraviolet (UV) irradiation, and conventional chemical processes such as chlorine, monochloramine, chlorine dioxide, and ozone among others. However, the production of disinfection by-products (DBPs) that accompanies chemical methods of disinfection is an issue of great concern due to the increase in the risks of harm to humans, for example, the development of cancer of the bladder and adverse reproductive outcomes. Therefore, this review examines the conventional disinfection approaches alongside emerging disinfection technologies, such as photocatalytic disinfection, cavitation, and electrochemical disinfection. Moreover, the merits, limitations, and log reduction values (LRVs) of the different disinfection methods discussed were compared concerning virus removal efficiency. Future research needs to merge single disinfection techniques into one to achieve improved viral disinfection, and the development of medicinal plant-based materials as disinfectants due to their antimicrobial and safety benefits to avoid toxicity is also highlighted.

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Las precipitaciones extremas representan uno de los eventos naturales climáticos más importantes y pueden originar inundaciones devastadoras. De junio a agosto del 2014 se registró una de las más graves inundaciones en la historia de la ciudad de Asunción. Ocasionó un incremento considerable del nivel del río Paraguay y el desplazamiento de 300.000 personas a campamentos provisionales. Debido a que el contacto directo con el agua de inundación, el consumo de agua contaminada y la congregación de los afectados en refugios provisorios son factores de riesgo para enfermedades infecciosas, el objetivo de este estudio fue la implementación de una metodología estandarizada para la concentración y detección de virus entéricos y micobacterias no tuberculosas, por PCR en tiempo real y PCR-asociada al análisis de restricción enzimática (PRA), en muestras de agua de inundaciones y el reporte de los patógenos detectados en las zonas afectadas de Asunción y en la Bahía del Río Paraguay. La metodología propuesta demostró poseer buena sensibilidad y se registró la presencia de rotavirus, norovirus (genogrupos I y II), astrovirus, adenovirus entéricos y micobacterias no tuberculosas en 50% (N=4/8) de las muestras de los barrios Sajonia, San Jerónimo y Ricardo Brugada, Chacarita. Además, reportamos datos secundarios de casos de enfermedades infecciosas, registrados en los servicios de salud de los barrios afectados durante el periodo de inundación.

Extreme rainfall represents one of the most important natural climatic events and can cause devastating floods. From June to August 2014, one of the most serious floods in the history of the city of Asunción was recorded. It caused a considerable increase in the level of the Paraguay River and the displacement of 300,000 people to temporary camps. Since direct contact with flood water, consumption of contaminated water and the congregation of those affected in temporary shelters are risk factors for infectious diseases, the objective of this study was the implementation of a standardized methodology for the concentration and detection of enteric viruses and non-tuberculous mycobacteria, by real-time PCR and PCR-associated enzyme restriction analysis (PRA), in samples of flood water and the report of the pathogens detected in the affected areas of Asunción and in the Bay of the Paraguay River. The proposed methodology proved to have good sensitivity and the presence of rotavirus, norovirus (genogroups I and II), astrovirus, enteric adenovirus and non-tuberculous mycobacteria was recorded in 50% (N=4/8) of the samples from the Sajonia, San Jeronimo and Ricardo Brugada, Chacarita neighborhoods. In addition, we report secondary data on cases of infectious diseases, registered in the health services of the affected neighborhoods during the flood period.

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Water is one of the primary vectors for African swine fever virus (ASFV) transmission among swine herds. However, the low concentrations of ASFV in water represent a challenge for the detection of the virus by conventional PCR methods, and enrichment of the virus would increase the test sensitivity. In this study, aiming to enrich ASFV in water quickly and efficiently, a rapid and efficient water-borne virus enrichment system (MDEF, modified diatomaceous earth by ferric hydroxide colloid) was used to enrich ASFV in water. After enrichment by MDEF, conventional real-time PCR (qPCR) was used for ASFV detection. ASFV were inactivated and diluted in 10 L of water, of which 4 mL were collected after 60 min treatment using the MDEF system. Two thousand five hundred times reduction of the sample volume was achieved after enrichment. A high adsorption rate of about 99.99 (±0.01)% and a high recovery rate of 64.01 (±10.20)% to 179.65 (±25.53)% was achieved by using 1g modified diatomaceous earth for 10 L ASFV contaminated water. The limit of qPCR detection of ASFV decreased to 1 × 10-1.11 GU ml-1 (genomic units per milliliter) from 1 × 102.71 GU ml-1 after concentrating the spiked water from 10 L to 4 ml. Preliminary application of MDEF allowed successful detection of African swine fever virus (ASFV), porcine circovirus type 2 (PCV2), and pseudorabies virus (PRV) in sewage. Thus, the combination of modified diatomaceous earth and real-time PCR is a promising strategy for the detection of viruses in water.

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As the coronavirus disease 2019 continues to spread globally, its culprit, the severe acute respiratory syndrome coronavirus 2 has been brought under scrutiny. In addition to inhalation transmission, the possible fecal-oral viral transmission via water/wastewater has also been brought under the spotlight, necessitating a timely global review on the current knowledge about waterborne viruses in drinking water treatment system - the very barrier that intercepts waterborne pathogens to terminal water users. In this article we reviewed the occurrence, concentration methods, and control strategies, also, treatment performance on waterborne viruses during drinking water treatment were summarized. Additionally, we emphasized the potential of applying the quantitative microbial risk assessment to guide drinking water treatment to mitigate the viral exposure risks, especially when the unregulated novel viral pathogens are of concern. This review paves road for better control of viruses at drinking water treatment plants to protect public health.

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Wastewater reclamation and reuse have been practically applied to water-stressed regions, but waterborne pathogens remaining in insufficiently treated wastewater are of concern. Sanitation Safety Planning adopts the hazard analysis and critical control point (HACCP) approach to manage human health risks upon exposure to reclaimed wastewater. HACCP requires a predetermined reference value (critical limit: CL) at critical control points (CCPs), in which specific parameters are monitored and recorded in real time. A disinfection reactor of a wastewater treatment plant (WWTP) is regarded as a CCP, and one of the CCP parameters is the disinfection intensity (e.g., initial disinfectant concentration and contact time), which is proportional to the log reduction value (LRV) of waterborne pathogens. However, the achievable LRVs are not always stable because the disinfection intensity is affected by water quality parameters, which vary among WWTPs. In this study, we established models for projecting virus LRVs using ozone, in which water quality and operational parameters were used as explanatory variables. For the model construction, we used five machine learning algorithms and found that automatic relevance determination with interaction terms resulted in better prediction performances for norovirus and rotavirus LRVs. Poliovirus and coxsackievirus LRVs were predicted well by a Bayesian ridge with interaction terms and lasso with quadratic terms, respectively. The established models were relatively robust to predict LRV using new datasets that were out of the range of the training data used here, but it is important to collect LRV datasets further to make the models more predictable and flexible for newly obtained datasets. The modeling framework proposed here can help WWTP operators and risk assessors determine the appropriate CL to protect human health in wastewater reclamation and reuse.

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Waterborne diseases have critical public health issues and socioeconomic relevancy worldwide. Various viral pathogens are ordinarily associated with waterborne diseases. Six-year-surveillance (a total of 20 times) of norovirus, hepatitis A virus, group C rotavirus, and enterovirus was conducted at five raw water sampling sites including two lakes (Lakes Soyang and Juam), Hyundo region of Geum River in Daejeon City, and Guui region of Han River in Seoul Metropolitan City and Moolgeum region of Nakdong River in Gimhae City which are located near two water intake plants. In this study, we routinely investigated virus contamination in water samples through reverse transcription polymerase chain reaction (RT-PCR) and integrated cell culture RT-PCR with high sensitivity and specificity. A total 100 samples were tested. Most of the targeted viruses were found in 32% of the samples and at least one of the indicator bacteria was detected in 65% of these occurrences. Among all the detected viruses, enterovirus was the most prevalent with a detection frequency of 12% and 2.71 MPN/10 L on average, while hepatitis A virus was the least prevalent with a detection frequency of 4%. Nearly all of the analyzed viruses (except for group C rotavirus) were present in samples from Han River (the Guui region), Geum River (the Hyundo region), Lake Juam, and Nakdong River (the Moolgeum region), while group C rotavirus was detected in those from the Guui region. During the six-year sampling period, the targeted waterborne viruses in water samples exhibited seasonal patterns in their occurrence that were different from the indicator bacteria levels in the water samples. The fact that they were detected in the five representative Korean water environments makes it necessary to establish the chemical and biological analysis systems for waterborne viruses and sophisticated management systems.

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Human viruses are ubiquitous contaminants in surface waters, where they can persist over extended periods of time. Among the factors governing their environmental persistence, the control (removal or inactivation) by microorganisms remains poorly understood. Here, we determined the contribution of indigenous bacteria and protists to the decay of human viruses in surface waters. Incubation of echovirus 11 (E11) in freshwater from Lake Geneva and seawater from the Mediterranean Sea led to a 2.5-log10 reduction in the infectious virus concentration within 48 h at 22°C, whereas E11 was stable in sterile controls. The observed virus reduction was attributed to the action of both bacteria and protists in the biologically active matrices. The effect of microorganisms on viruses was temperature dependent, with a complete inhibition of microbial virus control in lake water at temperatures of ≤16°C. Among three protist isolates tested (Paraphysomonas sp., Uronema marinum, and Caecitellus paraparvulus), Caecitellus paraparvulus was particularly efficient at controlling E11 (2.1-log10 reduction over 4 days with an initial protist concentration of 103 cells ml-1). In addition, other viruses (human adenovirus type 2 and bacteriophage H6) exhibited different grazing kinetics than E11, indicating that the efficacy of antiviral action also depended on the type of virus. In conclusion, indigenous bacteria and protists in lake water and seawater can modulate the persistence of E11. These results pave the way for further research to understand how microorganisms control human viral pathogens in aquatic ecosystems and to exploit this process as a treatment solution to enhance microbial water safety.IMPORTANCE Waterborne human viruses can persist in the environment, causing a risk to human health over long periods of time. In this work, we demonstrate that in both freshwater and seawater environments, indigenous bacteria and protists can graze on waterborne viruses and thereby reduce their persistence. We furthermore demonstrate that the efficiency of the grazing process depends on temperature, virus type, and protist species. These findings may facilitate the design of biological methods for the disinfection of water and wastewater.

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This study aimed to assess anthropogenic impact of surrounding population in the Private Reserve of Natural Heritage at Pantanal, the world's largest freshwater wetland ecosystem located in the centre of South America. Viral aetiological agents of acute gastroenteritis as rotavirus A (RVA), noroviruses, human adenoviruses, klassevirus and of hepatitis, as hepatitis A virus, were investigated in different aquatic matrices. Annual collection campaigns were carried out from 2009 to 2012, alternating dry and rainy seasons. Viral particles present in the samples were concentrated by the adsorption-elution method, with negatively charged membranes, and detected by qualitative and quantitative PCR. From a total of 43 samples at least one virus was detected in 65% (28) of them. Viruses were detected in all matrices with concentrations ranging from 2 × 102 to 8·3 × 104 genome copies per litre. A significant higher RVA frequency was observed in the dry season. Our data revealing dissemination of human enteric viruses in water matrices both inside and outside the reserve could be useful to trace faecal contamination in the environment and to minimize the risk of infection by exposure of susceptible individuals. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is part of a collaborative project designed to investigate the environmental and health conditions of the Private Reserve of Natural Heritage at Pantanal, the largest seasonally flooded wetland in the world. The project aimed to promote health and quality of human and wildlife extending technical-scientific knowledge about pathogens present in the region. By assessing the occurrence of human enteric viruses in different water matrices we demonstrated the anthropogenic impact of surrounding population and pointed out the potential risk of infection by exposure of susceptible individuals.

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A quadruplex Real-Time RT-PCR assay for the simultaneous quantitative detection of hepatitis A virus (HAV), norovirus (NoV) GI and GII, and mengovirus (used as process control for determination of the virus/nucleic acid extraction efficiency) has been developed. This multiplex assay has been comparatively evaluated with the individual monoplex assays and showed to be slightly less sensitive, with average ΔCq values of 0.90, 0.28 and 0.44 for HAV, NoV GI and NoV GII, respectively, in standard curves of viral RNA, or 0.32, 0.37 and 0.51 for the same viruses respectively, in naturally-contaminated samples. These ΔCq values were mostly negligible since it represented, in the worst case scenario, a loss of 0.43 log in genome copy numbers. The quadruplex assay shows similar theoretical detection limits than the monoplex assay for NoV GII, and 10 times higher for HAV and NoV GI. However, when naturally-contaminated food and water samples were tested, these theoretical detection thresholds were often exceeded and very low genome copy numbers (below the limit of detection) could be quantified. The quadruplex assay fulfills the requirements of the method developed by the European Committee on Standardization (CEN) for virus detection in selected foodstuffs with significant advantages in labor and reagent costs.

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Human enteric viruses are shed in extremely high numbers in the feces of infected individuals, becoming environmental contaminants and eventually leading to contamination of a variety of foodstuffs at the pre-harvest stage. Among these foods at risk is fresh produce and irrigation water is a major vehicle for crop contamination. In the present study, a standardized molecular method for quantitative virus assay has been evaluated in different types of fresh produce and in irrigation water for human norovirus (NoV) detection. Two different virus concentration procedures, polyethylene-glycol precipitation (PEG) and organic flocculation (OF), were employed. The procedures were initially assayed in spiked samples and later validated on naturally contaminated samples from the Nile Delta in Egypt. Overall, PEG provided significantly (p<0.05) better virus recoveries than OF for both irrigation water and salad vegetable virus analysis. NoV GI and GII recoveries in spiked irrigation water ranged from 22.0% to 43.3% and from 12.6% to 16.4% with the PEG and OF methods, respectively. In experimentally contaminated salad vegetables, virus recoveries ranged from 28.0% to 48.0% and from 14.0% to 18.8% by PEG precipitation and OF, respectively. Using PEG precipitation, NoV was found in 31.9% of naturally contaminated irrigation water samples. Both NoV GI and GII were detected in these samples with genome copy numbers of around 10(2) per liter. Virus analyses performed in naturally contaminated fresh produce that included green onion, watercress, radish, leek, and lettuce show that NoV GI was present in 20.8%-34.0% of the samples with genome copy numbers of around 10(2) per gram. When OF was employed, NoV was found in 25.0% of the irrigation water samples. Both genogroups could be found in these samples with genome copy numbers of around 10 per liter. In fresh produce, GI was present in 16.0%-25.7% of the samples with genome copy numbers per gram of around 10. Surprisingly, NoV GII was not detected in any salad vegetable despite highly satisfactory virus/nucleic acid extraction and enzyme efficiencies reported in the assays. Available reliable standardized assays for virus detection in food matrices including appropriate quality assurance and quality control measures to assess the efficiency of critical steps in virus analysis open the possibility to produce consistent and accurate exposure data to be used in QMRA (quantitative microbial risk assessment) and at the same time may enable the formulation of guidelines to ensure the virological quality of selected commodities in specific scenarios to reduce the risk of foodborne virus infections.

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In order to survey the occurrence of waterborne viruses in Korean surface water, a total of 192 water samples from July 2003 to January 2006 were collected and analyzed. The presence of waterborne viruses was investigated by total culturable virus assay (TCVA) using buffalo green monkey kidney (BGMK) cells. The results showed that 63 of 192 samples (32.8%) were positive for waterborne viruses with the average concentration of 3.1+/-18 most probable numbers (MPN)/100 L. The relationship between the occurrence of the viruses and the physicochemical environmental factors revealed that there was a significant correlation between the turbidity of water and the occurrence of the viruses. It was also noted that the water temperature might have some relationship with the occurrence of the viruses, as the frequency of the viruses was higher in low temperature or winter season. Therefore, the occurrence of waterborne viruses in Korean surface water might be affected by the physicochemical environmental factors such as turbidity and water temperature.

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