F-specific RNA Bacteriophages Research Articles

Quality of cellulose and biostimulant extracts from Oedogonium calcareum cultivated during primary wastewater treatment

A practical two-product cascading biorefinery was developed to extract a biostimulant and cellulose from the freshwater filamentous macroalga Oedogonium calcareum grown while treating primary wastewater. Biostimulant production provides a valuable extract with production of disinfected residual biomass for further product development. Both Escherichia coli and F-specific RNA bacteriophage, indicators of human pathogens contamination, were absent from the residual biomass. The chemical composition of the biostimulant was complex, consisting of growth-promoting substances, free amino acids, and minerals. The O. calcareum cellulose fractions yielded between 9.5% and 10.1% (w/w) with purities from 84% to 90% and closely resembled microcrystalline cellulose. Biostimulant extraction improved cellulose quality by increasing crystallinity from 59% to 62%. Biomass condition, drying process, and biostimulant production influenced the crystallinity index. This study demonstrates a two-step process of biostimulant and cellulose extraction from wastewater-grown Oedogonium, simultaneously disinfecting biomass and isolating high-quality cellulose as a sustainable alternative to conventional extraction methods.

Evaluation of virus removal in membrane bioreactor (MBR) and conventional activated sludge (CAS) processes based on long-term monitoring at two wastewater treatment plants

The membrane bioreactor (MBR) process always offers better wastewater treatment than conventional activated sludge (CAS) treatment. However, the difference in their efficacy of virus reduction remains unknown. To investigate this, we monitored virus concentrations before and after MBR and CAS processes over 2 years. Concentrations of norovirus genotypes I and II (NoV GI and GII), aichivirus (AiV), F-specific RNA phage genotypes I, II, and III (GI-, GII-, and GIII-FRNAPHs), and pepper mild mottle virus (PMMoV) were measured by a quantitative polymerase chain reaction (qPCR) method at two municipal wastewater treatment plants (WWTPs A and B) in Japan. Virus concentration datasets containing left-censored data were estimated by using both maximum likelihood estimation (MLE) and robust regression on order statistics (rROS) approaches. PMMoV was the most prevalent at both WWTPs, with median concentrations of 7.5 to 8.8 log10 copies/L before treatment. Log10 removal values (LRVs) of all viruses based on means and standard deviations of concentrations before and after treatment were consistently higher following MBR than following CAS. We used NoV GII as a model pathogen in a quantitative microbial risk assessment of the treated water, and we estimated the additional reductions required following MBR and CAS processes to meet the guideline of 10−6 DALYs pppy for safe wastewater reuse.

Assessing viral freshwater hazard using a toxicokinetic model and Dreissena polymorpha

The detection all pathogenic enteric viruses in water is expensive, time-consuming, and limited by numerous technical difficulties. Consequently, using reliable indicators such as F-specific RNA phages (FRNAPH) can be well adapted to assess the risk of viral contamination of fecal origin in surface waters. However, the variability of results inherent to the water matrix makes it difficult to use them routinely and to interpret viral risk. Spatial and temporal variability of surface waters can lead to underestimate this risk, in particular in the case of low loading. The use of bivalve mollusks as accumulating systems appears as a promising alternative, as recently highlighted with the freshwater mussel Dreissena polymorpha, but its capacity to accumulate and depurate FRNAPH needs to be better understood and described. The purpose of this study is to characterise the kinetics of accumulation and elimination of infectious FRNAPH by D. polymorpha in laboratory conditions, formalised by a toxico-kinetic (TK) mechanistic model. Accumulation and depuration experiments were performed at a laboratory scale to determine the relationship between the concentration of infectious FRNAPH in water and the concentration accumulated by D. polymorpha. The mussels accumulated infectious FRNAPH (3–5.4 × 104 PFU/g) in a fast and concentration-dependent way in only 48 h, as already recently demonstrated. The second exposure demonstrated that the kinetics of infectious FRNAPH depuration by D. polymorpha was independent to the exposure dose, with a T90 (time required to depurate 90 % of the accumulated concentration) of approximately 6 days. These results highlight the capacities of D. polymorpha to detect and reflect the viral pollution in an integrative way and over time, which is not possible with point water sampling. Different TK models were fitted based on the concentrations measured in the digestive tissues (DT) of D. polymorpha. The model has been developed to formalise the kinetics of phage accumulation in mussels tissues through the simultaneous estimation of accumulation and depuration rates. This model showed that accumulation depended on the exposure concentration, while depuration did not. Standardized D. polymorpha could be easily transplanted to the environment to predict viral concentrations using the TK model defined in the present study to predict the level of contamination of bodies of water on the basis of the level of phages accumulated by the organisms. It will be also provide a better understanding of the dynamics of the virus in continental waters at different time and spatial scales, and thereby contribute to the protection of freshwater resources.

Highly efficient and sensitive membrane-based concentration process allows quantification, surveillance, and sequencing of viruses in large volumes of wastewater

Wastewater-based epidemiology is experiencing exponential development. Despite undeniable advantages compared to patient-centered approaches (cost, anonymity, survey of large populations without bias, detection of asymptomatic infected peoples…), major technical limitations persist. Among them is the low sensitivity of the current methods used for quantifying and sequencing viral genomes from wastewater. In situations of low viral circulation, during initial stages of viral emergences, or in areas experiencing heavy rains, the extremely low concentrations of viruses in wastewater may fall below the limit of detection of the current methods. The availability during crisis and the cost of the commercial kits, as well as the requirement of expensive materials such as high-speed centrifuge, can also present major blocks to the development of wastewater-based epidemiological survey, specifically in low-income countries. Thereby, highly sensitive, low cost and standardized methods are still needed, to increase the predictability of the viral emergences, to survey low-circulating viruses and to make the results from different labs comparable. Here, we outline and characterize new protocols for concentrating and quantifying SARS-CoV-2 from large volumes (500 mL-1 L) of untreated wastewater. In addition, we report that the methods are applicable for monitoring and sequencing. Our nucleic acid extraction technique (the routine C: 5 mL method) does not require sophisticated equipment such as automatons and is not reliant on commercial kits, making it readily available to a broader range of laboratories for routine epidemiological survey. Furthermore, we demonstrate the efficiency, the repeatability, and the high sensitivity of a new membrane-based concentration method (MBC: 500 mL method) for enveloped (SARS-CoV-2) and non-enveloped (F-specific RNA phages of genogroup II / FRNAPH GGII) viruses. We show that the MBC method allows the quantification and the monitoring of viruses in wastewater with a significantly improved sensitivity compared to the routine C method. In contexts of low viral circulation, we report quantifications of SARS-CoV-2 in wastewater at concentrations as low as 40 genome copies per liter. In highly diluted samples collected in wastewater treatment plants of French Guiana, we confirmed the accuracy of the MBC method compared to the estimations done with the routine C method. Finally, we demonstrate that both the routine C method processing 5 mL and the MBC method processing 500 mL of untreated wastewater are both compatible with SARS-CoV-2 sequencing. We show that the quality of the sequence is correlated with the concentration of the extracted viral genome. Of note, the quality of the sequences obtained with some MBC processed wastewater was improved by dilutions or enzyme substitutions suggesting the presence of specific enzyme inhibitors in some wastewater. To the best of our knowledge, our MBC method is one of the first efficient, sensitive, and repeatable method characterized for SARS-CoV-2 quantification and sequencing from large volumes of wastewater.

Detection and Quantification of Bacteriophages in Wastewater Samples by Culture and Molecular Methods.

Bacteriophages are promising tools for the detection of fecal pollution in water bodies and particularly for viral pathogen risk assessment. Having similar morphological and biological characteristics, bacteriophages are perfect surrogates for the study of the fate and transport of enteric viruses, generally better than any other group of indicators.Different groups of bacteriophages, such as somatic coliphages, F-specific RNA bacteriophages, and bacteriophages infecting selected strains of Bacteroides, have been comprehensively tested as indicators of fecal pollution. Somatic coliphages and F-specific RNA bacteriophages can be used as indicators of general fecal contamination, whereas Bacteroides phages can be used to detect a particular fecal source, for instance, human, bovine, porcine, or poultry fecal contamination.Feasible and cost-effective protocols standardized by the International Standardization Organization and the United States Environmental Protection Agency for the detection of infectious bacteriophages belonging to these three groups are available. Molecular methods for the detection of some particular phages have also been developed. Here we introduce those methods for the detection, enumeration, and isolation of bacteriophages in wastewater samples.

Toward better monitoring of human noroviruses and F-specific RNA bacteriophages in aquatic environments using bivalve mollusks and passive samplers: A case study.

Monitoring pathogenic enteric viruses in continental and marine water bodies is essential to control the viral contamination of human populations. Human Noroviruses (NoV) are the main enteric viruses present in surface waters and foodstuff. In a context of global change, it is currently a challenge to improve the management of viral pollutions in aquatic environments and thereby limit the contamination of vulnerable water bodies or foodstuffs. The aim of this study is to evaluate the potential of specific accumulation systems for improving the detection of NoV in water bodies, compared to direct water analyses. Passive samplers (Zetapor filters) and three species of bivalve molluscan shellfish (BMS) (Dreissena polymorpha, Mytilus edulis and Crassostreas gigas) were used as accumulation systems to determine their performance in monitoring continental and marine waters for viruses. F-specific RNA bacteriophages (FRNAPH) were also analyzed since they are described as indicators of NoV hazard in many studies. During a one-year study in a specific area frequently affected by fecal pollution, twelve campaigns of exposure of passive samplers and BMS in continental and coastal waters were conducted. Using suitable methods, NoV (genome) and FRNAPH (infectious and genome) were detected in these accumulation systems and in water at the same time points to determine the frequency of detection but also to gain a better understanding of viral pollution in this area. The reliability of FRNAPH as a NoV indicator was also investigated. Our results clearly showed that BMS were significantly better than passive samplers and direct water analyses for monitoring NoV and FRNAPH contamination in water bodies. A dilution of viral pollution between the continental and the coastal area was observed and can be explained by the distance from the source of the pollution. Viral pollution is clearly greater during the winter period, and stakeholders should take this into consideration in their attempts to limit the contamination of food and water. A significant correlation was once again shown between NoV and FRNAPH genomes in BMS, confirming the reliability of FRNAPH as a NoV indicator. Moreover, a strong correlation was observed between NoV genomes and infectious FRNAPH, suggesting recent viral pollution since infectious particles had not been inactivated at sufficient levels in the environment. More generally, this study shows the value of using BMS as an active method for improving knowledge on the behavior of viral contamination in water bodies, the ranking of the contamination sources, and the vulnerability of downstream water bodies.

Activity- and gene-based quantification of enteric viruses, F- specific RNA phage genogroups, pepper mild mottle virus, and Escherichia coli in surface water

Polymerase chain reaction (PCR) is widely applied for the monitoring of pathogenic viruses in water environments. To date, several pretreatments to selectively detect genes from infectious viruses via PCR have been developed. This study was aimed to characterize and validate methods for quantifying active viruses and indicators and to evaluate the proportion of their active fractions in surface water (n = 42). Active E. coli and F-specific RNA phage (FRNAPH) genogroups were quantified using culture assays. In addition to these microbes, norovirus genogroups I (GI) and II, Aichi virus 1, and pepper mild mottle virus (PMMoV) were quantified by (reverse transcription)-quantitative PCR (RT-qPCR) with and without cis-dichlorodiammineplatinum (CDDP) treatment to exclude genes in inactive viruses. CDDP-RT-qPCR showed concentrations and detection frequencies comparable to or higher than culture assays. Consequently, although CDDP-RT-qPCR can suggest the presence of an inactive virus, it can also overestimate the activity of the virus in the environment. Differences between culture and CDDP-RT-qPCR and between CDDP-RT-qPCR and RT-qPCR varied among the viruses. CDDP-RT-qPCR showed a concentration comparable to the culture assay (within 1 log10 difference) in 93 % of positive samples for GI-FRNAPH but in <63 % of positive samples for GII- and GIII-FRNAPHs. GII-NoV was detected from 5 and 30 out of 42 samples via CDDP-RT-qPCR and RT-qPCR, respectively, and was suggested as inactivated by 2.0 log10 or higher in most of the samples. By contrast, concentrations of PMMoV determined by these two assays were not notably different. It is suggested that the operational conditions of wastewater treatment plants around the sites, rather than environmental stresses, affected the microbial inactivation. To better understand the infectivity of viruses in the environment, it is important to investigate them using sensitive detection methods at various sites, including the source of contamination.

Effects of surface hydrophobicity on the removal of F-specific RNA phages from reclaimed water by coagulation and ceramic membrane microfiltration.

Microfiltration (MF) has been widely adopted as an advanced treatment process to reduce suspended solids and turbidity in treated wastewater effluents designated for potable reuse. Although microfilter pores are much larger than viruses, the addition of a coagulant upstream of a microfilter system can achieve stable virus removal. Ceramic membranes have a narrow pore size distribution to achieve the high removal of contaminants. This study aims to evaluate virus log reduction using bench-scale coagulation and ceramic membrane MF. To investigate the effects of differences in net surface hydrophobicity, 18 sewage-derived F-specific RNA phages (FRNAPHs) were used for batch hydrophobicity and coagulation-MF tests. The capability of bench-scale coagulation and ceramic membrane MF under continuous automated long-term operation was tested to remove the lab reference strain MS2 and three selected FRNAPH isolates which varied by surface property. Median virus log reduction values (LRVs) exceeding 6.2 were obtained for all three isolates and MS2. Although coagulation and hydrophobicity were positively correlated, the virus isolate demonstrating the lowest level of hydrophobicity and coagulation (genogroup I) still exhibited a high LRV. Thus, coagulation and ceramic membrane MF systems may serve as viable options for virus removal during water reclamation and advanced treatment.

Peroxydisulfate activation by CuO pellets in a fixed-bed column, operating mode and assessments for antibiotics degradation and urban wastewater disinfection.

A fixed-bed column packed with copper oxide pellets (FBC-CuO) combined with peroxydisulfate (PDS) as a primary oxidant was assessed as an option for simultaneously wastewater decontamination (antibiotics) and disinfection (bacteria, viruses, and protozoa). Preliminary to these experiments, phenol was used as the target molecule to investigate the working mode of FBC-CuO under various operating conditions, such as varying flow rates, initial persulfate, and phenol concentrations. Then, the removal of a mix of five representative antibiotics (amoxicillin (AMX), cefalexin (CFX), ofloxacin (OFL), sulfamethoxazole (SMX), and clarithromycin (CLA)) in secondary treated urban wastewater (STWW) was evaluated. AMX, CFX, and OFL were effectively removed by simply flowing through the FBC-CuO, and the addition of PDS (500µM) systematically enhanced the degradation of all targeted antibiotics, which is also the necessary condition for the removal of SMX and CLA. Urban wastewater disinfection was evaluated by monitoring targeted pathogens originally in the STWW. A significant reduction of Escherichia coli, Enterococcus, F-specific RNA bacteriophages was observed after the treatment by FBC-CuO with 500µM PDS. X-ray diffraction measurement and scanning electron microscopy performed on CuO pellets before and after treatment confirmed that the structure of the catalyst was preserved without any phase segregation. Finally, quantification of Cu(II) at the outlet of FBC-CuO indicate a non-negligible but limited released. All these results underline the potential of the FBC-CuO combined with PDS at the field scale for the degradation of micropollutants and inactivation of pathogens in wastewater.

Copper oxide/peroxydisulfate system for urban wastewater disinfection: Performances, reactive species, and antibiotic resistance genes removal

In this study, copper oxide (CuO) catalyzed peroxydisulfate (PDS) system was investigated for the inactivation of a broad range of pathogenic microorganisms from urban wastewater. Complete inactivation of Escherichia coli, Enterococcus, F-specific RNA bacteriophages from secondary treated wastewater was achieved after a short time (15–30 min) treatment with CuO (10 g/L)/PDS (1 mM) system, but spores of sulfite-reducing bacteria took 120 min. No bacterial regrowth occurred during storage after treatment. Significant reduction of the pathogens was explained by the generation of the highly selective Cu(III) oxidant, as the predominant reactive species, which could quickly oxidize guanine through a one-electron oxidation pathway. Additionally, the potential of the CuO (10 g/L)/PDS (1 mM) system to inactivate antibiotic-resistant bacteria and antibiotic resistance genes (ARB&Gs) was explored. Sulfamethoxazole-resistant E. coli was used as the model ARB and a 3.2 log of reduction was observed after 10 min of treatment. A considerable reduction (0.7–2.3 log) of selected ARGs including blaTEM, qnrS, emrB, sul1, and genes related to the dissemination of antibiotic resistance, including the Class 1 integron-integrase (intI1), and the insertion sequence (IS613) was achieved after 60 min treatment. All these findings indicated the promising applicability of the CuO/PDS system as a disinfection technology for wastewater reuse in agriculture.

Assessment of Microbiological Quality of Fresh Vegetables and Oysters Produced in Buenos Aires Province, Argentina.

Fresh vegetables and shellfish are prone to microbial contamination through irrigation or breeding with sewage-polluted waters, as well as by infected food handlers. In this work, we studied the presence of human and bovine polyomaviruses and human norovirus in fresh lettuces, strawberries and oysters produced in Buenos Aires province, Argentina. In oysters, we also investigated F-specific RNA bacteriophages, indicator Escherichia coli (E. coli) and pathogen bacteria of concern (Salmonella spp., Vibrio spp.). Within vegetables, we found viral contamination of human origin given the presence of human-associated polyomaviruses -MCPyV, HPyV6, JCPyV, and SV40- in lettuce and strawberry samples (16 and 10%, respectively), probably coming from irrigation waters and food handling. Among oysters, human (MCPyV, 4.2%) and bovine (BPyV1, 8.4%) polyomaviruses were detected even with low counts of E. coli. Bacteriophages (n = 3) and Salmonella spp. (n = 1) were also found, while Vibrio spp. was not detected. These results may indicate that the contamination in oysters comes from human and animal excreta, probably present in breeding waters. Norovirus was not detected in any food sample. To our knowledge, this is the first description of SV40 in lettuces and MCPyV and BPyV1 in oysters. The detection of different viral contaminants encourages further studies to evaluate the need for including viral indicators in microbiological standards. The identification of possible sources and routes of contamination using viral markers during routine microbiological controls, such as the polyomaviruses used in this work, would be useful to focus attention on the most hazardous stages of the food production chain.

F-Specific RNA Bacteriophage Transport in Stream Water: Hydro-Meteorological Controls and Association with Suspended Solids

F-specific RNA bacteriophages (FRNAPHs) are commonly used as indicators of faecal and viral contamination in waters. Once they enter surface waters, the exact role of suspended solids, sediments and hydro-meteorological factors in their fluvial fate and transport is poorly understood, and long-term studies (e.g., over years) are lacking. In this study, FRNAPH concentrations and genogroup distribution were measured in the Orne River (France) during two years at weekly intervals, and during four storm runoff events. Hydro-meteorological driving factors were investigated at both time scales. FRNAPH concentrations and genogroups at different depths of a riverbank sediment core were also examined to better discriminate the origin of the faecal pollution. During low flows, the FRNAPH and the suspended solid transport were decoupled and the FRNAPH concentrations were mainly correlated with the air and water temperature. During storm runoff events, the FRNAPH concentrations only showed a significant correlation with conductivity, turbidity and water discharge. Despite the uncertainty of the predictions, multi parameter regression models using hydro-meteorological variables were suitable to predict log transformed FRNAPHs’ concentrations at low flows with a standard error of 0.46. Model performance using the storm runoff events dataset was low. This study highlights different driving factors at low flows and during storm runoff events, and the need to measure at both time scales to better understand phage transport dynamics in surface water.

Elimination of SARS-CoV-2 along wastewater and sludge treatment processes

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is shed in the feces of infected people. As a consequence, genomic RNA of the virus can be detected in wastewater. Although the presence of viral RNA does not inform on the infectivity of the virus, this presence of genetic material raised the question of the effectiveness of treatment processes in reducing the virus in wastewater and sludge. In this work, treatment lines of 16 wastewater treatment plants were monitored to evaluate the removal of SARS-CoV-2 RNA in raw, processed waters and sludge, from March to May 2020. Viral RNA copies were enumerated using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in 5 different laboratories. These laboratories participated in proficiency testing scheme and their results demonstrated the reliability and comparability of the results obtained for each one. SARS-CoV-2 RNA was found in 50.5% of the 101 influent wastewater samples characterized. Positive results were detected more frequently in those regions with a COVID-19 incidence higher than 100 cases per 100,000 inhabitants. Wastewater treatment plants (WWTPs) significantly reduced the occurrence of virus RNA along the water treatment lines. Secondary treatment effluents showed an occurrence of SARS-CoV-2 RNA in 23.3% of the samples and no positive results were found after MBR and chlorination. Non-treated sludge (from primary and secondary treatments) presented a higher occurrence of SARS-CoV-2 RNA than the corresponding water samples, demonstrating the affinity of virus particles for solids. Furthermore, SARS-CoV-2 RNA was detected in treated sludge after thickening and anaerobic digestion, whereas viral RNA was completely eliminated from sludge only when thermal hydrolysis was applied. Finally, co-analysis of SARS-CoV-2 and F-specific RNA bacteriophages was done in the same water and sludge samples in order to investigate the potential use of these bacteriophages as indicators of SARS-CoV-2 fate and reduction along the wastewater treatment.

Characterization of a novel group I F-specific RNA bacteriophage isolated from human stool.

A novel F-specific RNA bacteriophage (FRNAPH) YM1, affiliating to genogroup I (GI) of Levivirus, is isolated for the first time from human stool samples using double-layer agar plates with the Escherichia coli ATCC700891 as the host. The complete genomic sequence of YM1 is 3551 nt in length, obtained through next-generation sequencing, and contains four genes encoding for maturation protein, coat protein, lysis protein, and RNA-dependent RNA polymerase (RdRp). The genomic sequence of YM1 shares the highest similarity of 95.3% with that of a GI FRNAPH DL16 isolated from surface water of Great Bay. The YM1 possesses a non-enveloped, icosahedral virion of 23 ± 0.45nm in diameter. One-step growth curve analysis shows that the burst time of YM1 is 30min post-infection (p.i.) with the average burst size of 264 PFU/cell. The YM1 lyses only E. coli strains tested, revealing high host specificity. This newly discovered phage may serve as a candidate for viral indicator to monitor human enteric virus, especially norovirus, contamination in the environments.

Application of crAssphage, F-RNA phage and pepper mild mottle virus as indicators of human faecal and norovirus contamination in shellfish

Shellfish growing waters contaminated with inadequately treated human wastewater is a major source of norovirus in shellfish and poses a significant human health risk to consumers. Microbial source tracking (MST) markers have been widely used to identify the source (s) of faecal contamination in water but data are limited on their use for shellfish safety. This study evaluated the source specificity, sensitivity, occurrence and concentration of three viral MST markers i.e. cross-assembly phage (crAssphage), F-specific RNA bacteriophage genogroup II (F-RNA phage GII) and pepper mild mottle virus (PMMoV) using animal faeces (n = 119; 16 animal groups), influent wastewater (n = 12), effluent wastewater (n = 16) and shellfish (n = 33). CrAssphage, F-RNA phage GII and PMMoV had source specific values of 0.97, 0.99 and 0.91, respectively. The sensitivity of MST markers was confirmed by their 100% detection frequency in influent wastewaters. The frequency of detection in effluent wastewater ranged from 81.3% (F-RNA phage GII) to 100% (PMMoV). Concentration of F-RNA phage GII was one log10 (influent wastewater) and 2–3 log10 (effluent wastewater) lower than crAssphage and PMMoV, respectively. Despite lower prevalence of F-RNA phage GII in oysters and mussels compared to crAssphage and PMMoV, concentrations of the three MST markers were similar in mussels. As an indicator of norovirus contamination in shellfish, crAssphage and PMMoV had greater predictive sensitivity (100%; [95% CI; 81.5%–100%)]) and F-RNA phage GII had greater predictive specificity (93.3%; [95% CI; 68.1%–99.8%]). In contrast, crAssphage and F-RNA phage GII have similar accuracy for predicting norovirus in shellfish, however, PMMoV significantly overestimated its presence. Therefore, a combination of crAssphage and F-RNA phage GII analysis of shellfish could provide a robust estimation of the presence of human faecal and norovirus contamination.

The Utility of Dreissena polymorpha for Assessing the Viral Contamination of Rivers by Measuring the Accumulation of F-Specific RNA Bacteriophages

River water that receives treated wastewater can be contaminated by pathogens including enteric viruses due to fecal pollution, which may represent an important public health hazard. There is a great diversity of enteric viruses and fecal bacteriophages, especially F-specific RNA bacteriophages (FRNAPHs), are commonly proposed as indicators of viral pollution due to a variety of characteristics such as their structural similarities to the main enteric viruses, their high concentrations in raw wastewater and their environmental survival rate, which is better than other cultivable enteric viruses. However, evaluating the viral contamination of water on the basis of FRNAPH concentration levels continues to present a challenge. This is because the quality of detection is strongly dependent on the quantity of viral particles, high spatio-temporal variabilities and the physico-chemical conditions of the water during sampling. To overcome these limitations, the present study aims to evaluate whether the bivalve mollusk Dreissena polymorpha (zebra mussel) could be considered a suitable experimental model for assessing the viral contamination of rivers. In order to determine this, the capacity of D. polymorpha to accumulate FRNAPHs and assimilate them into their soft tissue was studied. This provided a proof of concept for the use of D. polymorpha to evaluate the viral contamination of surface water. Two experiments were conducted: (1) an in situ experiment to confirm that zebra mussels naturally accumulated FRNAPHs and (2) a laboratory experiment to determine the accumulation and depuration kinetics of FRNAPHs in D. polymorpha tissue. The study highlights the capacity of the mussels to accumulate infectious FRNAPHs both on a laboratory scale under controlled conditions as well as in situ at different sites that are representative of different bodies of water. An analysis of the mussels’ soft tissue showed that they were capable of reflecting the water’s contamination level very quickly (within less than 24 h). Moreover, the soft tissue retained the viral load much longer than the water due to a low depuration rate. The analysis of FRNAPH concentrations in mussels exposed in situ suggested that there were differences in contamination levels between sites. These preliminary results underline the potential utility of zebra mussels in assessing viral contamination by measuring the accumulation of FRNAPHs in their tissue. This may ultimately enable stakeholders to use zebra mussels as a means of monitoring viral pollution in surface water.

Spatial and temporal distributions of enteric viruses and indicators in a lake receiving municipal wastewater treatment plant discharge.

Although lake water can be used as a source of drinking water and recreational activities, there is a dearth of research on the occurrence and fate of enteric viruses. Over a period of 14months at six points in 2014-2015, we conducted monthly monitoring of the virological water quality of a Japanese lake. The lake receives effluent from three surrounding wastewater treatment plants and retains water for about two weeks. These features allowed us to investigate the occurrence and fate of viruses in the lake environment. Human enteric viruses such as noroviruses and their indicators (pepper mild mottle virus and F-specific RNA bacteriophage [FRNAPH] genogroups) were quantified by PCR-based assays. Additionally, FRNAPH genogroups were quantified by infectivity-based assays to estimate the degree of virus inactivation. Pepper mild mottle virus, genogroup II (GII) norovirus, and GI-FRNAPH were identified in relatively high frequencies (positive in >40% out of 64 samples), with concentrations ranging from 1.3×101 to 2.9×104 copies/L. Human enteric viruses and some indicators were not detected and less prevalent, respectively, after April 2015. Principal component analysis revealed that the virological water quality changed gradually over time, but its differences between the sampling points were not apparent. FRNAPH genogroups were inactivated during the warm season (averaged water temperature of >20°C) compared to the cool season (averaged water temperature of <20°C), which may have been due to the more severe environmental stresses such as sunlight and water temperature. This suggests that the infection risk associated with the use of the lake water may have been overestimated by the gene quantification assay during the warm season.

Epidemiological surveillance of SARS-CoV-2 by genome quantification in wastewater applied to a city in the northeast of France: Comparison of ultrafiltration- and protein precipitation-based methods.

The aim of the present study was to develop a simple, sensitive, and specific approach to quantifying the SARS-CoV-2 genome in wastewater and to evaluate this approach as a means of epidemiological surveillance. Twelve wastewater samples were collected from a metropolitan area in north-eastern France during April and May 2020. In addition to the quantification of the SARS-CoV-2 genome, F-specific RNA phages of genogroup II (FRNAPH GGII), naturally present in wastewater, were used as an internal process control for the viral concentration and processing of RT-PCR inhibitors. A concentration method was required to allow the quantification of the SARS-CoV-2 genome over the longest possible period. A procedure combining ultrafiltration, phenol-chloroform-isoamyl alcohol purification, and the additional purification of the RNA extracts was chosen for the quantification of the SARS-CoV-2 genome in 100-mL wastewater samples. At the same time, the COVID-19 outbreak was evaluated through patients from the neighbouring University Hospital of Nancy, France. A regular decrease in the concentration of the SARS-CoV-2 genome from ~104 gc/L to ~102 gc/L of wastewater was observed over the eight weeks of the study, during which the population was placed under lockdown. The SARS-CoV-2 genome was even undetectable during one week in the second half of May and present but non-quantifiable in the last sample (28 May). A concordant circulation in the human community was highlighted by virological diagnosis using respiratory samples, which showed a decrease in the number of COVID-19 cases from 677 to 52 per week over the same period. The environmental surveillance of COVID-19 using a reliable viral quantification procedure to test wastewater is a key approach. The real-time detection of viral genomes can allow us to predict and monitor the circulation of SARS-CoV-2 in clinical settings and survey the entire urban human population.

F-Specific RNA Bacteriophages Model the Behavior of Human Noroviruses during Purification of Oysters: the Main Mechanism Is Probably Inactivation Rather than Release.

Noroviruses (NoV) are responsible for many shellfish outbreaks. Purification processes may be applied to oysters before marketing to decrease potential fecal pollution. This step is rapidly highly effective in reducing Escherichia coli; nevertheless, the elimination of virus genomes has been described to be much slower. It is therefore important to identify (i) the purification conditions that optimize virus removal and (ii) the mechanism involved. To this end, the effects of oyster stress, nutrients, and the presence of a potential competitor to NoV adhesion during purification were investigated using naturally contaminated oysters. Concentrations of NoV (genomes) and of the viral indicator F-specific RNA bacteriophage (FRNAPH; genomes and infectious particles) were regularly monitored. No significant differences were observed under the test conditions. The decrease kinetics of both virus genomes were similar, again showing the potential of FRNAPH as an indicator of NoV behavior during purification. The T90 (time to reduce 90% of the initial titer) values were 47.8 days for the genogroup I NoV genome, 26.7 days for the genogroup II NoV genome, and 43.9 days for the FRNAPH-II genome. Conversely, monitoring of the viral genomes could not be used to determine the behavior of infectious viruses because the T90 values were more than two times lower for infectious FRNAPH (20.6 days) compared to their genomes (43.9 days). Finally, this study highlighted that viruses are primarily inactivated in oysters rather than released in the water during purification processes.IMPORTANCE This study provides new data about the behavior of viruses in oysters under purification processes and about their elimination mechanism. First, a high correlation has been observed between F-specific RNA bacteriophages of subgroup II (FRNAPH-II) and norovirus (NoV) in oysters impacted by fecal contamination when both are detected using molecular approaches. Second, when using reverse transcription-quantitative PCR and culture to detect FRNAPH-II genomes and infectious FRNAPH in oysters, respectively, it appears that genome detection provides limited information about the presence of infectious particles. The comparison of both genomes and infectious particles highlights that the main mechanism of virus elimination in oysters is inactivation. Finally, this study shows that none of the conditions tested modify virus removal.

Spatial and temporal profiles of enteric viruses in the coastal waters of Tokyo Bay during and after a series of rainfall events

Recreational activities in coastal waters that are polluted by enteric viruses can result in gastroenteritis etc. In this study, the pollution profiles of enteric viruses were examined in the coastal area of Tokyo Bay, Japan, by collecting 57 water samples from three different depths (0.5 m, 3.0 m, and 5.0 m) during and after a series of heavy rainfall events. Vertically spatial and temporal changes in the concentrations of NoV genogroup I (GI) and genogroup II (GII), pepper mild mottle virus (PMMoV), and Aichi virus (AiV) were determined using quantitative reverse transcription-polymerase chain reaction, while those of the bacterial indicator, Escherichia coli, and F-specific RNA bacteriophages (FRNA phages) were monitored using culture methods. PMMoV was highly abundant (1.4 × 104–6.8 × 106 genome copies/L), whereas the concentrations of the other enteric viruses were relatively low (AiV, 1.3 × 102–2.9 × 104; GI, 2.9 × 10–5.6 × 103; GII, 2.5 × 10–1.2 × 104 genome copies/L). All of the viruses showed lower fluctuations in concentration than E. coli, which increased up to 460-fold after the rainfall event and then decreased over the subsequent two weeks. The maximum vertical difference in E. coli concentration was observed immediately after the rainfall. The E. coli reached the surface and then gradually spread down, whereas the virus concentrations exhibited few fluctuations due to the remaining effects of the previous combined sewer overflows. These findings indicate that viruses have a relatively long retention period over fecal indicator bacteria in this coastal area.