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Previous studies on horseshoe bats (Rhinolophus spp.) have described many coronaviruses related to SARS-CoV (SARSCoVr) in China and only a few coronaviruses related to SARS-CoV-2 (SARSCoV2r) in Yunnan (southern China), Cambodia, Laos and Thailand. Here, we report the results of several field missions carried out in 2017, 2021 and 2022 across Vietnam during which 1218 horseshoe bats were sampled from 19 locations. Sarbecoviruses were detected in 11% of faecal RNA extracts, with much more positives among Rhinolophus thomasi (46%). We assembled 38 Sarbecovirus genomes, including 32 SARSCoVr, four SARSCoV2r, and two recombinants of SARSCoVr and SARSCoV2r (RecSar), one showing a Spike protein very similar to SARS-CoV-2. We detected a bat co-infected with four coronaviruses, including two sarbecoviruses. Our analyses revealed that Sarbecovirus genomes evolve in Vietnam under strong geographical and host constraints. First, we found evidence for a deep separation between viruses from northern Vietnam and those from central and southern Vietnam. Second, we detected only SARSCoVr in Rhinolophus thomasi, both SARSCoVr and SARSCoV2r in Rhinolophus affinis, and only RecSar in Rhinolophus pusillus captured close to the border with China. Third, the bias in favour of Uracil in synonymous third codon positions of SARSCoVr extracted from R. thomasi showed a negative correlation with latitudes. Our results also provided support for an emergence of SARS-CoV in horseshoe bats from northern Yunnan and emergence of SARS-CoV-2 in horseshoe bats from northern Indochina subtropical forests (southern Yunnan, northern Laos and north-western Vietnam).
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Swine acute diarrhoea syndrome coronavirus (SADS-CoV; Coronaviridae, Rhinacovirus) was detected in 2017 in Guangdong Province (China), where it caused high mortality rates in piglets. According to previous studies, SADS-CoV evolved from horseshoe bat reservoirs. Here, we report the first five Rhinacovirus genomes sequenced in horseshoe bats from Vietnam and their comparisons with data published in China. Our phylogenetic analyses provided evidence for four groups: rhinacoviruses from Rhinolphus pusillus bats, including one from Vietnam; bat rhinacoviruses from Hainan; bat rhinacoviruses from Yunnan showing a divergent synonymous nucleotide composition; and SADS-CoV and related bat viruses, including four rhinacoviruses from Vietnam sampled in Rhinolophus affinis and Rhinolophus thomasi. Our phylogeographic analyses showed that bat rhinacoviruses from Dien Bien (Vietnam) share more affinities with those from Yunnan (China) and that the ancestor of SADS-CoVs arose in Rhinolophus affinis circulating in Guangdong. We detected sequencing errors and artificial chimeric genomes in published data. The two SADS-CoV genomes previously identified as recombinant could also be problematic. The reliable data currently available, therefore, suggests that all SADS-CoV strains originate from a single bat source and that the virus has been spreading in pig farms in several provinces of China for at least seven years since the first outbreak in August 2016.
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Alphacoronavirus , Quirópteros , Infecções por Coronavirus , Genoma Viral , Filogenia , Doenças dos Suínos , Animais , Quirópteros/virologia , Vietnã/epidemiologia , China/epidemiologia , Suínos , Doenças dos Suínos/virologia , Doenças dos Suínos/epidemiologia , Alphacoronavirus/genética , Alphacoronavirus/classificação , Alphacoronavirus/isolamento & purificação , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Infecções por Coronavirus/epidemiologia , Evolução Molecular , FilogeografiaRESUMO
BackgroundWastewater surveillance is an effective approach to monitor population health, as exemplified by its role throughout the COVID-19 pandemic.AimThis study explores the possibility of extending wastewater surveillance to the Paris 2024 Olympic and Paralympic Games, focusing on identifying priority pathogen targets that are relevant and feasible to monitor in wastewater for these events.MethodsA list of 60 pathogens of interest for general public health surveillance for the Games was compiled. Each pathogen was evaluated against three inclusion criteria: (A) analytical feasibility; (B) relevance, i.e. with regards to the specificities of the event and the characteristics of the pathogen; and (C) added value to inform public health decision-making. Analytical feasibility was assessed through evidence from peer-reviewed publications demonstrating the detectability of pathogens in sewage, refining the initial list to 25 pathogens. Criteria B and C were evaluated via expert opinion using the Delphi method. The panel consisting of some 30 experts proposed five additional pathogens meeting criterion A, totalling 30 pathogens assessed throughout the three-round iterative questionnaire. Pathogens failing to reach 70% group consensus threshold underwent further deliberation by a subgroup of experts.ResultsSix priority targets suitable for wastewater surveillance during the Games were successfully identified: poliovirus, influenza A virus, influenza B virus, mpox virus, SARS-CoV-2 and measles virus.ConclusionThis study introduced a model framework for identifying context-specific wastewater surveillance targets for a mass gathering. Successful implementation of a wastewater surveillance plan for Paris 2024 could incentivise similar monitoring efforts for other mass gatherings globally.
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COVID-19 , SARS-CoV-2 , Águas Residuárias , Humanos , Águas Residuárias/virologia , Águas Residuárias/microbiologia , COVID-19/epidemiologia , COVID-19/prevenção & controle , COVID-19/transmissão , SARS-CoV-2/isolamento & purificação , França/epidemiologia , Esportes , Saúde Pública , Pandemias , Esgotos/virologia , Paris/epidemiologia , Aniversários e Eventos Especiais , Vigilância em Saúde Pública/métodosRESUMO
The emergence of SARS-CoV-2 has heightened the need to evaluate the detection of enveloped viruses in the environment, particularly in wastewater, within the context of wastewater-based epidemiology. The studies published over the past 80 years focused primarily on non-enveloped viruses due to their ability to survive longer in environmental matrices such as wastewater or sludge compared to enveloped viruses. However, different enveloped viruses survive in the environment for different lengths of time. Therefore, it is crucial to be prepared to assess the potential infectious risk that may arise from future emerging enveloped viruses. This will require appropriate tools, notably suitable viral concentration methods that do not compromise virus infectivity. This review has a dual purpose: first, to gather all the available literature on the survival of infectious enveloped viruses, specifically at different pH and temperature conditions, and in contact with detergents; second, to select suitable concentration methods for evaluating the infectivity of these viruses in wastewater and sludge. The methodology used in this data collection review followed the systematic approach outlined in the PRISMA (Preferred Reporting Items for Systematic Review and Meta-Analysis) guidelines. Concentration methods cited in the data gathered are more tailored towards detecting the enveloped viruses' genome. There is a lack of suitable methods for detecting infectious enveloped viruses in wastewater and sludge. Ultrafiltration, ultracentrifugation, and polyethylene glycol precipitation methods, under specific/defined conditions, appear to be relevant approaches. Further studies are necessary to validate reliable concentration methods for detecting infectious enveloped viruses. The choice of culture system is also crucial for detection sensitivity. The data also show that the survival of infectious enveloped viruses, though lower than that of non-enveloped ones, may enable environmental transmission. Experimental data on a wide range of enveloped viruses is required due to the variability in virus persistence in the environment.
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Esgotos , Águas Residuárias , Esgotos/virologia , Águas Residuárias/virologia , SARS-CoV-2 , Vírus/isolamento & purificação , COVID-19/transmissãoRESUMO
Numerous SARS-CoV-2 variants are emerging as the epidemic continues, inducing new waves of contamination. In July 2023, a new variant named BA.2.86 was identified, raising concerns about its potential for viral escape, even in an immune population. The reduction in patient-centered testing and the identification of variants by sequencing means that we are now blind to the spread of this new variant. The aim of this study was to track the signature of this variant in wastewater in Paris, France. This variant showed a very rapid spread, highly correlated with national flash studies involving sequencing of clinical samples, but with a moderate impact on virus circulation. This easy-to-implement approach enabled us to monitor the emergence and spread of this new variant in real time at low cost.
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Epidemias , Águas Residuárias , Humanos , Paris , França , Contaminação de MedicamentosRESUMO
Human enteric viruses are important etiological agents of waterborne diseases. Environmental waters are usually contaminated with low virus concentration requiring large concentration factors for effective detection by (RT)-qPCR. Low-pressure reverse osmosis is often used to remove water contaminants, but very few studies focused on the effective virus removal of reverse osmosis treatment with feed concentrations as close as possible to environmental concentrations and principally relied on theoretical virus removal. The very low viral concentrations usually reported in the permeates (i.e. at least 5 log of removal rate) mean that very large volumes of water need to be analysed to have sufficient sensitivity and assess the process efficiency. This study evaluates two methods for the concentration of adenoviruses, enteroviruses and MS2 bacteriophages at different viral concentrations in large (< 200 L) and very large (> 200 L) volumes. The first method is composed of two ultrafiltration membranes with low-molecular weight cut-offs while the second method primarily relies on adsorption and elution phases using electropositive-charged filters. The recovery rates were assessed for both methods. For the ultrafiltration-based protocol, recovery rates were similar for each virus studied: 80% on average at high virus concentrations (106-107 viruses L-1) and 50% at low virus concentrations (103-104 viruses L-1). For the electropositive-charged filter-based method, the average recoveries obtained were about 36% for ADV 41, 57% for CV-B5 and 1.6% for MS2. The ultrafiltration-based method was then used to evaluate the performance of a low-pressure reverse osmosis lab-scale pilot plant. The retentions by reverse osmosis were similar for all studied viruses and the validated recovery rates applied to the system confirmed the reliability of the concentration method. This method was effective in concentrating all three viruses over a wide range of viral concentrations. Moreover, the second concentration method using electropositive-charged filters was studied, allowing the filtration of larger volumes of permeate from a semi-industrial low-pressure reverse osmosis pilot plant. This reference method was used because of the inability of the UF method to filter volumes on the order of one cubic metre.
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Enterovirus , Vírus , Purificação da Água , Humanos , Reprodutibilidade dos Testes , Filtração/métodos , Ultrafiltração/métodos , Purificação da Água/métodos , Água , OsmoseRESUMO
OBJECTIVE: There is extensive evidence that SARS-CoV-2 replicates in the gastrointestinal tract. However, the infectivity of virions in feces is poorly documented. Although the primary mode of transmission is airborne, the risk of transmission from contaminated feces remains to be assessed. DESIGN: The persistence of SARS-CoV-2 (infectivity and RNA) in human and animal feces was evaluated by virus isolation on cell culture and RT-qPCR, respectively. The exposure of golden Syrian hamsters to experimentally contaminated feces through intranasal inoculation has also been tested to assess the fecal-oral transmission route. RESULTS: For periods that are compatible with average intestinal transit, the SARS-CoV-2 genome was noticeably stable in human and animal feces, contrary to the virus infectivity that was reduced in a time- and temperature-dependent manner. In human stools, this reduction was variable depending on the donors. Viral RNA was excreted in the feces of infected hamsters, but exposure of naïve hamsters to feces of infected animals did not lead to any productive infection. Conversely, hamsters could be experimentally infected following exposure to spiked fresh feces. CONCLUSION: Infection following exposure to naturally contaminated feces has been suspected but has not been established so far. The present work demonstrates that SARS-CoV-2 rapidly lost infectivity in spiked or naturally infected feces. Although the possibility of persistent viral particles in human or animal feces cannot be fully ruled out, SARS-CoV-2 transmission after exposure to contaminated feces is unlikely.
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COVID-19 , SARS-CoV-2 , Animais , Cricetinae , Fezes , Humanos , Mesocricetus , RNA ViralRESUMO
Throughout the COVID-19 pandemic, new variants have continuously emerged and spread in populations. Among these, variants of concern (VOC) have been the main culprits of successive epidemic waves, due to their transmissibility, pathogenicity or ability to escape the immune response. Quantification of the SARS-CoV-2 genomes in raw wastewater is a reliable approach well-described and widely deployed worldwide to monitor the spread of SARS-CoV-2 in human populations connected to sewage systems. Discrimination of VOCs in wastewater is also a major issue and can be achieved by genome sequencing or by detection of specific mutations suggesting the presence of VOCs. This study aimed to date the emergence of these VOCs (from Alpha to Omicron BA.2) by monitoring wastewater from the greater Paris area, France, but also to model the propagation dynamics of these VOCs and to characterize the replacement kinetics of the prevalent populations. These dynamics were compared to various individual-centered public health data, such as regional incidence and the proportions of VOCs identified by sequencing of strains isolated from patient. The viral dynamics in wastewater highlighted the impact of the vaccination strategy on the viral circulation within human populations but also suggested its potential effect on the selection of variants most likely to be propagated in immunized populations. Normalization of concentrations to capture population movements appeared statistically more reliable using variations in local drinking water consumption rather than using PMMoV concentrations because PMMoV fecal shedding was subject to variability and was not sufficiently relevant in this study. The dynamics of viral spread was observed earlier (about 13 days on the wave related to Omicron VOC) in raw wastewater than the regional incidence alerting to a possible risk of decorrelation between incidence and actual virus circulation probably resulting from a lower severity of infection in vaccinated populations.
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COVID-19 , SARS-CoV-2 , Teste para COVID-19 , Humanos , Pandemias , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , Esgotos , Águas ResiduáriasRESUMO
Since the beginning of the Coronavirus Disease-19 (COVID-19) pandemic, multiple Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) mutations have been reported and led to the emergence of variants of concern (VOC) with increased transmissibility, virulence or immune escape. In parallel, the observation of viral fecal shedding led to the quantification of SARS-CoV-2 genomes in wastewater, providing information about the dynamics of SARS-CoV-2 infections within a population including symptomatic and asymptomatic individuals. Here, we aimed to adapt a sequencing technique initially designed for clinical samples to apply it to the challenging and mixed wastewater matrix, and hence identify the circulation of VOC at the community level. Composite raw sewage sampled over 24 h in two wastewater-treatment plants (WWTPs) from a city in western France were collected weekly and SARS-CoV-2 quantified by RT-PCR. Samples collected between October 2020 and May 2021 were submitted to whole-genome sequencing (WGS) using the primers and protocol published by the ARTIC Network and a MinION Mk1C sequencer (Oxford Nanopore Technologies, Oxford, United Kingdom). The protocol was adapted to allow near-full genome coverage from sewage samples, starting from â¼5% to reach â¼90% at depth 30. This enabled us to detect multiple single-nucleotide variant (SNV) and assess the circulation of the SARS-CoV-2 VOC Alpha, Beta, Gamma, and Delta. Retrospective analysis of sewage samples shed light on the emergence of the Alpha VOC with detection of first co-occurring signature mutations in mid-November 2020 to reach predominance of this variant in early February 2021. In parallel, a mutation-specific qRT-PCR assay confirmed the spread of the Alpha VOC but detected it later than WGS. Altogether, these data show that SARS-CoV-2 sequencing in sewage can be used for early detection of an emerging VOC in a population and confirm its ability to track shifts in variant predominance.
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Leptospirosis is a neglected zoonotic disease with a worldwide distribution caused by bacterial pathogenic Leptospira. Rodents are considered as the main reservoir of Leptospira and transmission usually occurs through exposure to urine-contaminated environment. However, interactions between environment, rodent reservoir and human leptospirosis remain poorly studied. Here, we evaluated the concentration of Leptospira in surface water and captured rats in the city of Paris (France) from 2018 to 2020 using an integrity qPCR (Quantitative Polymerase Chain Reaction). All environmental samples (n = 1031) were positive for saprophytic Leptospira but pathogenic Leptospira P1 group were only found in 40% (n = 363; 2018) to 0% (n = 264; 2020) of samples. In the same time, analysis of 200 brown rat corpses trapped in the city, showed about 15% of positivity for Leptospira but the different method used for rats conservation (based on presence or absence of conservative agent) showed important variations in the Leptospira prevalence. Metagenomic analysis, based on rrs gene sequencing, was also carried out to evaluate the distribution of Leptospira in samples. Results could indicate that some species of Leptospira are found in surface waters as well as rats, but further study is needed to accurately describe the nature of the link between these two reservoirs. Quantification of Leptospira and pathogenic species description circulating inside animal reservoir living in the vicinity of freshwater in urban areas, will be helpful to understand the eco-epidemiology of leptospirosis and to establish prevention and intervention strategies, especially in the context of organization of recreative activity events in these urban areas.
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Leptospira , Leptospirose , Animais , Água Doce , Leptospira/genética , Leptospirose/epidemiologia , Leptospirose/veterinária , Ratos , Reação em Cadeia da Polimerase em Tempo Real , RoedoresRESUMO
Since many infected people experience no or few symptoms, the SARS-CoV-2 epidemic is frequently monitored through massive virus testing of the population, an approach that may be biased and may be difficult to sustain in low-income countries. Since SARS-CoV-2 RNA can be detected in stool samples, quantifying SARS-CoV-2 genome by RT-qPCR in wastewater treatment plants (WWTPs) has been carried out as a complementary tool to monitor virus circulation among human populations. However, measuring SARS-CoV-2 viral load in WWTPs can be affected by many experimental and environmental factors. To circumvent these limits, we propose here a novel indicator, the wastewater indicator (WWI), that partly reduces and corrects the noise associated with the SARS-CoV-2 genome quantification in wastewater (average noise reduction of 19%). All data processing results in an average correlation gain of 18% with the incidence rate. The WWI can take into account the censorship linked to the limit of quantification (LOQ), allows the automatic detection of outliers to be integrated into the smoothing algorithm, estimates the average measurement error committed on the samples and proposes a solution for inter-laboratory normalization in the absence of inter-laboratory assays (ILA). This method has been successfully applied in the context of Obépine, a French national network that has been quantifying SARS-CoV-2 genome in a representative sample of French WWTPs since March 5th 2020. By August 26th, 2021, 168 WWTPs were monitored in the French metropolitan and overseas territories of France. We detail the process of elaboration of this indicator, show that it is strongly correlated to the incidence rate and that the optimal time lag between these two signals is only a few days, making our indicator an efficient complement to the incidence rate. This alternative approach may be especially important to evaluate SARS-CoV-2 dynamics in human populations when the testing rate is low.
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COVID-19 , Epidemias , Humanos , RNA Viral , SARS-CoV-2 , Águas ResiduáriasRESUMO
The uses of bivalve molluscs in environmental biomonitoring have recently gained momentum due to their ability to indicate and concentrate human pathogenic microorganisms. In the context of the health crisis caused by the COVID-19 epidemic, the objective of this study was to determine if the SARS-CoV-2 ribonucleic acid genome can be detected in zebra mussels (Dreissena polymorpha) exposed to raw and treated urban wastewaters from two separate plants to support its interest as bioindicator of the SARS-CoV-2 genome contamination in water. The zebra mussels were exposed to treated wastewater through caging at the outlet of two plants located in France, as well as to raw wastewater in controlled conditions. Within their digestive tissues, our results showed that SARS-CoV-2 genome was detected in zebra mussels, whether in raw and treated wastewaters. Moreover, the detection of the SARS-CoV-2 genome in such bivalve molluscans appeared even with low concentrations in raw wastewaters. This is the first detection of the SARS-CoV-2 genome in the tissues of a sentinel species exposed to raw and treated urban wastewaters. Despite the need for development for quantitative approaches, these results support the importance of such invertebrate organisms, especially zebra mussel, for the active surveillance of pathogenic microorganisms and their indicators in environmental waters.
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Bivalves , COVID-19 , Dreissena , Animais , Dreissena/genética , Humanos , SARS-CoV-2 , Águas ResiduáriasRESUMO
Leptospirosis is an emerging worldwide zoonotic disease, but the general biology of the causative agents is still poorly understood. Humans are an occasional host. The main risk factors are water-associated exposure during professional or recreational activities or during outbreaks in endemic areas. Detecting the presence of pathogenic bacteria in aquatic environments and their capacity to resist various inactivation processes are research fields that need to be further developed. In addition, the methods used for detecting and enumerating Leptospira still need to be improved. We aimed to describe a new quantitative polymerase chain reaction coupled to propidium monoazide treatment (PMAqPCR) that targets not only total Leptospira but also discriminates pathogenic from non-pathogenic Leptospira while also addressing PCR inhibitors, a frequently encountered problem when studying environmental water. In a second step, the killing efficiency of Leptospira to different treatments was tested and PMAqPCR compared to culture-based enumeration. This provided information about the effects of temperature, as well as ultraviolet and chlorine disinfection, that are both related to water treatment processes, in particular for the production of drinking water, on the persistence of both saprophytic and pathogenic Leptospira. Finally, PMAqPCR was used for the detection of Leptospira in freshwater samples for a proof-of-concept. In conclusion, our method could be used for routine freshwater monitoring and allows better evaluation of the presence of Leptospira, allowing evaluation of the bacterial dynamics in a designated area or assessment of the efficacy of water disinfection processes.
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DNA Bacteriano/isolamento & purificação , Desinfecção , Leptospira/isolamento & purificação , Leptospirose/microbiologia , Animais , Monitoramento Ambiental , Água Doce/microbiologia , Humanos , Leptospira/patogenicidade , Leptospirose/diagnóstico , Leptospirose/genética , Reação em Cadeia da Polimerase em Tempo Real , Água , Microbiologia da Água , Zoonoses/microbiologiaRESUMO
The crucial role of microbes in the evolution, development, health, and ecological interactions of multicellular organisms is now widely recognized in the holobiont concept. However, the structure and stability of microbiota are highly dependent on abiotic and biotic factors, especially in the gut, which can be colonized by transient bacteria depending on the host's diet. We studied these impacts by manipulating the digestive microbiota of the detritivore Armadillidium vulgare and analyzing the consequences on its structure and function. Hosts were exposed to initial starvation and then were fed diets that varied the different components of lignocellulose. A total of 72 digestive microbiota were analyzed according to the type of the diet (standard or enriched in cellulose, lignin, or hemicellulose) and the period following dysbiosis. The results showed that microbiota from the hepatopancreas were very stable and resilient, while the most diverse and labile over time were found in the hindgut. Dysbiosis and selective diets may have affected the host fitness by altering the structure of the microbiota and its predicted functions. Overall, these modifications can therefore have effects not only on the holobiont, but also on the "eco-holobiont" conceptualization of macroorganisms.
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Opportunistic premise plumbing pathogens present in drinking water are linked to a significant number of infections for health compromised patients. However, their monitoring is not required in current water potability standards and they have been poorly studied in a full-scale network. In this study, we quantified, by qPCR, three opportunistic pathogens, Mycobacterium spp., Legionella pneumophila, Pseudomonas aeruginosa throughout the Paris drinking water network over a one-year sampling campaign. While Mycobacteria spp. seemed ubiquitous whatever the distribution system and the time of the year, the occurrence of L. pneumophila and P. aeruginosa showed seasonal variations. Unlike L. pneumophila and P. aeruginosa, the concentration (copies number/L) of Mycobacterium spp. varied between sampling sites. The variation in microbial numbers did not demonstrate any correlations with temperature, pH, chlorine, conductivity, orthophosphate or nitrate levels. In conclusion, Mycobacterium spp. are common inhabitants of the Paris network while L. pneumophila and P. aeruginosa presence fluctuate over space and time. Such qPCR approach would help to better understand the behaviour of opportunistic premise plumbing pathogens.