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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.
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Monitoramento Ambiental , Água Subterrânea , Água Subterrânea/virologia , Humanos , Águas Residuárias/virologia , Microbiologia da Água , Cidades , Norovirus/isolamento & purificação , Norovirus/genética , Fezes/virologia , Espanha , SARS-CoV-2 , Vírus/isolamento & purificaçãoRESUMO
The COVID-19 pandemic has underscored the importance of understanding the role of animals in the transmission of coronaviruses (CoVs) and their impact on human health. A One Health approach, integrating human, animal, and environmental health, is essential for effective CoVs control. Next-generation sequencing has played a pivotal role in identifying and monitoring the evolution of novel CoVs strains, like SARS-CoV-2. However, viral occurrence and diversity studies in environmental and animal samples are challenging because of the complexity of viral communities and low abundance of viruses in these samples. Target enrichment sequencing (TES) has emerged as a valuable tool for investigating viral families in challenging samples. This approach involves the specific capture and enrichment of viral genomes using sequence-specific probes, thereby enhancing the efficiency of detection and characterization. In this study, we aimed to develop and validate a TES panel to study CoVs in various complex environmental and animal derived samples. The results demonstrated the panel's effectiveness in capturing and sequencing a wide diversity of CoVs providing valuable insights into their abundance and host diversity in urban wastewater, farm animal corpses lixiviates and bat guano samples. In sewage samples, CoVs were detected solely when TES was employed while in guano samples, sequencing of CoVs species was achieved in 2 out of 4 samples showing an almost three-logarithmic increase in the number of reads obtained in comparison with the untargeted approach. For animal lixiviates, only the TES application enabled the acquisition of CoVs reads. The information obtained can significantly contribute to early detection, surveillance, and control measures for CoVs, including viral discovery and potential spillover events. Additionally, this sequencing panel shows potential for studying other significant viral families and monitoring viral diversity in different animal populations.
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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.
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Fazendas , Sequenciamento de Nucleotídeos em Larga Escala , Exposição Ocupacional , Águas Residuárias , Animais , Suínos , Águas Residuárias/virologia , Humanos , Medição de Risco , Vírus/isolamento & purificação , Vírus/genética , Monitoramento Ambiental/métodosRESUMO
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.
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Viroses , Vírus , Humanos , Águas Residuárias , Vigilância Epidemiológica Baseada em Águas Residuárias , Viroma/genética , Vírus/genéticaRESUMO
During the last three years, various restrictions have been set up to limit the transmission of the Coronavirus Disease (COVID-19). While these rules apply at a large scale (e.g., country-wide level) human-to-human transmission of the virus that causes COVID-19, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), occurs at a small scale. Different preventive policies and testing protocols were implemented in buildings where COVID-19 poses a threat (e.g., elderly residences) or constitutes a disruptive force (e.g., schools). In this study, we sampled sewage from different buildings (a school, a university campus, a university residence, and an elderly residence) that host residents of different levels of vulnerability. Our main goal was to assess the agreement between the SARS-CoV-2 concentration in wastewater and the policies applied in these buildings. All buildings were sampled using passive samplers while 24 h composite samples were also collected from the elderly residence. Results showed that passive samplers performed comparably well to composite samples while being cost-effective to keep track of COVID-19 prevalence. In the elderly residence, the comparison of sampling protocols (passive vs. active) combined with the strict clinical testing allowed us to compare the sensitivities of the two methods. Active sampling was more sensitive than passive sampling, as the former was able to detect a COVID-19 prevalence of 0.4 %, compared to a prevalence of 2.2 % for passive sampling. The number of COVID-19-positive individuals was tracked clinically in all the monitored buildings. More frequent detection of SARS-CoV-2 in wastewater was observed in residential buildings than in non-residential buildings using passive samplers. In all buildings, sewage surveillance can be used to complement COVID-19 clinical testing regimes, as the detection of SARS-CoV-2 in wastewater remained positive even when no COVID-19-positive individuals were reported. Passive sampling is useful for building managers to adapt their COVID-19 mitigation policies.
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COVID-19 , Esgotos , Idoso , Humanos , Águas Residuárias , SARS-CoV-2 , Habitação , COVID-19/epidemiologiaRESUMO
Viruses linked to animals inhabiting Antarctic latitudes remain poorly studied. Remote environments hosting large pinniped populations may be prone to exposure of immunologically naïve animals to new infectious agents due to increasing human presence or introduction of new animal species. Antarctic fur seals (Arctocephalus gazella) inhabiting the Western Antarctic Peninsula and the South Shetland Islands are challenged because of climate change and increased anthropogenic activity. In the present study, the fecal and serum virome of A. gazella was characterized by applying target enrichment next generation sequencing. The resulting viromes were dominated by CRESS-DNA sequences. Viruses known to infect vertebrate and invertebrate hosts were also observed in fecal samples. Fur seal picornavirus was present in all the fecal pools studied suggesting it is a prevalent virus in these species. Six different viruses presenting similarities with previously described A. gazella viruses or other otariids and mammal viruses were identified as potential new A. gazella viruses. Also, diet-derived viruses such as crustacean viruses were present in fecal content. Penguin viruses, but not fish viruses, were also detected. Obtained results contribute to a better understanding of the viral community present in these species, which is relevant for its conservation.
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Otárias , Vírus , Animais , Humanos , Vírus/genética , Metagenômica , Mudança Climática , Dieta , Regiões AntárticasRESUMO
Wastewater-based epidemiology has shown to be an efficient tool to track the circulation of SARS-CoV-2 in communities assisted by wastewater treatment plants (WWTPs). The challenge comes when this approach is employed to help Health authorities in their decision-making. Here, we describe the roadmap for the design and deployment of SARSAIGUA, the Catalan Surveillance Network of SARS-CoV-2 in Sewage. The network monitors, weekly or biweekly, 56 WWTPs evenly distributed across the territory and serving 6 M inhabitants (80% of the Catalan population). Each week, samples from 45 WWTPs are collected, analyzed, results reported to Health authorities, and finally published within less than 72 h in an online dashboard ( https://sarsaigua.icra.cat ). After 20 months of monitoring (July 20-March 22), the standardized viral load (gene copies/day) in all the WWTPs monitored fairly matched the cumulative number of COVID-19 cases along the successive pandemic waves, showing a good fit with the diagnosed cases in the served municipalities (Spearman Rho = 0.69). Here we describe the roadmap of the design and deployment of SARSAIGUA while providing several open-access tools for the management and visualization of the surveillance data.
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COVID-19 , SARS-CoV-2 , COVID-19/epidemiologia , Humanos , Pandemias , RNA Viral , Esgotos , Águas Residuárias , Vigilância Epidemiológica Baseada em Águas ResiduáriasRESUMO
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.
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Adenovírus Humanos , Norovirus , Escherichia coli , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Ultrafiltração , Água , Microbiologia da ÁguaRESUMO
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.
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COVID-19 , Pandemias , Humanos , Estudos Prospectivos , RNA Viral , Reprodutibilidade dos Testes , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2 , Águas Residuárias , Vigilância Epidemiológica Baseada em Águas ResiduáriasRESUMO
SARS-CoV-2 variants are emerging worldwide, and monitoring them is key in providing early warnings. Here, we summarize the different analytical approaches currently used to study the dissemination of SARS-CoV-2 variants in wastewater and discuss their advantages and disadvantages. We also provide preliminary results of two sensitive and cost-effective approaches: variant-specific reverse transcription-nested PCR assays and a nonvariant-specific amplicon deep sequencing strategy that targets three key regions of the viral spike protein. Next-generation sequencing approaches enable the simultaneous detection of signature mutations of different variants of concern in a single assay and may be the best option to explore the real picture at a particular time. Targeted PCR approaches focused on specific signature mutations will need continuous updating but are sensitive and cost-effective.
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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.
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In the wake of the COVID-19 pandemic, the use of next generation sequencing (NGS) has proved to be an important tool for the genetic characterization of SARS-CoV-2 from clinical samples. The use of different available NGS tools applied to wastewater samples could be the key for an in-depth study of the excreted virome, not only focusing on SARS-CoV-2 circulation and typing, but also to detect other potentially pandemic viruses within the same family. With this aim, 24-hours composite wastewater samples from March and July 2020 were sequenced by applying specific viral NGS as well as target enrichment NGS. The full virome of the analyzed samples was obtained, with human Coronaviridae members (CoV) present in one of those samples after applying the enrichment. One contig was identified as HCoV-OC43 and 8 contigs as SARS-CoV-2. CoVs from other animal hosts were also detected when applying this technique. These contigs were compared with those obtained from contemporary clinical specimens by applying the same target enrichment approach. The results showed that there is a co-circulation in urban areas of human and animal coronaviruses infecting domestic animals and rodents. NGS enrichment-based protocols might be crucial to describe the occurrence and genetic characteristics of SARS-CoV-2 and other Coronaviridae family members within the excreted virome present in wastewater.
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COVID-19 , Pandemias , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , SARS-CoV-2 , EsgotosRESUMO
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.
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Gastroenterite/virologia , Metagenoma , Metagenômica , Viroses/virologia , Fatores Etários , Criança , Pré-Escolar , Biologia Computacional/métodos , Fezes/virologia , Feminino , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Metagenômica/métodos , Filogenia , Carga ViralRESUMO
Much of the knowledge on viruses is focused on those that can be propagated using cell-cultures or that can cause disease in humans or in economically important animals and plants. However, this only reflects a small portion of the virosphere. Therefore, in this study, we explore by targeted next-generation sequencing, how the virome varies between Atlantic horse mackerels and gilthead seabreams from fisheries and aquaculture from the center and south regions of Portugal. Viral genomes potentially pathogenic to fish and crustaceans, as well as to humans, were identified namelyese included Astroviridae, Nodaviridae, Hepadnaviridae, Birnaviridae, Caliciviridae, and Picornaviridae families. Also bacteriophages sequences were identified corresponding to the majority of sequencese detected, with Myoviridae, Podoviridae, and Siphoviridae, the most widespread families in both fish species. However, these findings can also be due to the presence of bacteria in fish tissues, or even to contamination. Overall, seabreams harbored viruses from a smaller number of families in comparison with mackerels. Therefore, the obtained data show that fish sold for consumption can harbor a high diversity of viruses, many of which are unknown, reflecting the overall uncharacterized virome of fish. While cross-species transmission of bonafide fish viruses to humans is unlikely, the finding of human pathogenic viruses in fish suggest that fish virome can be a potential threat regarding food safety.
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As 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, as a surrogate. In this review, we show new data from a newly 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.
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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/100â¯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.
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Água , Irrigação Agrícola , Animais , Cryptosporidium , Escherichia coli , Humanos , Suínos , Microbiologia da ÁguaRESUMO
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.
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Irrigação Agrícola , Monitoramento Ambiental , Microbiologia da Água , Criptosporidiose , Cryptosporidium , Água Doce/microbiologia , Água Doce/parasitologiaRESUMO
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).
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Esgotos , Bacteriófagos , Sequenciamento de Nucleotídeos em Larga Escala , Metagenômica , VírusRESUMO
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 99â¯GC/100â¯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 380â¯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.
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Monitoramento Ambiental , Rios/microbiologia , Microbiologia da Água , Poluição da Água/análise , Poluição da Água/estatística & dados numéricosRESUMO
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.