The selection of appropriate viral indicators for evaluating wastewater treatment performance remains challenging because candidate markers have rarely been compared systematically within a unified analytical framework. Here, we collected influent and effluent samples monthly for one year from two wastewater treatment plants in Japan and conducted, to our knowledge, the first comprehensive comparison of 19 viral targets and one protozoan target using high-throughput quantitative PCR. Pepper mild mottle virus (PMMoV) was consistently detected at high concentrations, showed limited seasonal variability, and exhibited an approximately 1.0 log10 reduction, comparable to those observed for pathogenic viruses. In contrast, Carjivirus, formerly known as crAssphage, was present at the highest concentrations but showed significantly greater reduction than pathogenic viruses. Tomato brown rugose fruit virus (ToBRFV), despite its high abundance and emerging recognition as a potential marker, exhibited pronounced seasonal fluctuations. Other Tobamovirus species, such as cucumber green mottle mosaic virus and tobacco mild green mosaic virus, exhibited similar removal but lower prevalence compared with PMMoV. Overall, PMMoV demonstrated the most balanced performance in terms of abundance, stability, and removal behavior, supporting its use as a robust indicator for monitoring virus removal in wastewater treatment.

Waterborne outbreaks caused by protozoan, bacterial, and viral pathogens continue to pose serious public health threats. However, comprehensive monitoring of all three major pathogen types remains uncommon, primarily due to the technical difficulty of simultaneously concentrating and characterizing these diverse microorganisms from a single water sample. Here, we evaluated a membrane adsorption followed by bead-beating-based direct nucleic acid extraction for its sensitivity in detecting waterborne pathogens. We further applied the optimized protocol to river water samples. Our results demonstrated that the use of magnesium chloride in combination with a mixed cellulose ester membrane filter (pore size: 0.8 μm) achieved the highest concentration efficiency for Cryptosporidium parvum, Legionella pneumophila, and murine norovirus (MNV) in spiked water samples, outperforming other methods utilizing membranes with alternative pore sizes or membrane with another material (i.e., nylon). Furthermore, under controlled spiking conditions, targeting rRNA of C. parvum instead of rDNA, or incorporating a preamplification step for the L. pneumophila mip gene and MNV, significantly improved the sensitivity. Notably, under the spiking conditions, the 50% limit of detection for C. parvum using our method was comparable to that of traditional immunofluorescent antibody testing. Application of this method to river water demonstrated its environmental applicability, revealing a higher abundance of Bacteroides rRNA markers compared to their corresponding rDNA, underscoring the potential of rRNA-based targets for more sensitive bacterial detection. Importantly, when the target is genomic DNA or RNA, preamplification enables the use of a larger nucleic acids input volume, thereby enhancing sensitivity. Overall, the combination of membrane adsorption and bead-beating-based direct nucleic acid extraction offers a sensitive approach for simultaneous detection of protozoa, bacteria, and viruses in environmental water. This method serves as an effective ‘screening’ tool for the presence of the three major types of waterborne microorganisms.

Enteroviruses are environmentally transmissible human pathogens whose stability in natural waters varies widely, yet the molecular determinants underlying this variability remain largely unknown. Echovirus 11 (E11), a re-emerging cause of severe neonatal infections, is efficiently transmitted via contaminated water, making its environmental stability a critical factor in infection risk. Here we identify a single viral capsid residue that governs E11 susceptibility to inactivation by extracellular microbial proteases in freshwater. By combining virus decay measurements in lakewater with proteolytic-cleavage profiling, viral capsid structural analyses, and reverse genetics, we show that the presence of VP2.Y97 renders E11 highly sensitive to microbially-mediated proteolytic decay. Strikingly, this residue is absent from multiple enteroviruses with greater environmental stability, indicating that substitution at a single capsid position is sufficient to shift virus fate in natural waters. These findings reveal that fine-scale capsid architecture controls virus-microbe interactions in aquatic environments and establish a molecular mechanism linking capsid variation to environmental transmission potential among enteroviruses.

Free chlorine reacts with viral proteins, but the protein structural determinants of viral resistance to chlorine treatment remain poorly understood. Here, we curated a dataset of 498 icosahedral virus structures, including intact virions and virus-like particles (VLPs), from the Protein Data Bank. Surprisingly, only 6.6% of these structures are associated with published viral chlorine inactivation rate constants (k obs). In these matched cases representing 12 virus families, total and maximum solvent accessible surface areas (SASA) of methionine residues within viral attachment and entry proteins correlated significantly with median k obs (Pearson's r = 0.83 and 0.45, respectively; p < 0.05), suggesting a critical role of methionine exposure in viral resistance phenotypes. Across the full curated dataset, fuzzy c-means clustering upon total and maximum SASA profiles of chlorine-reactive residues demonstrates that the common surrogate panel (MS2, PhiX174, Phi6, PRD1, and PR772) fails to represent the SASA diversity of human viruses. Instead, VLPs and novel phages may serve as better surrogates for chlorine treatment due to SASA profile similarities to human viruses. Our findings highlight that residue SASA features provide a quantitative baseline for screening viral resistance to chlorine and offer a data-driven strategy to select structurally representative virus surrogates for future disinfection studies.

Ionic liquid crystals have various potential applications enabling selective and effective transportation. Herein, we report the development of separation membranes prepared by fixing liquid-crystalline (LC) columnar (Col) nanostructures through the in situ polymerization of the film states formed by imidazolium-sulfobetaine ionic liquid crystals. We compared the properties of the nanostructured betaine liquid crystals with those of analogous Col structures containing mono-ionic groups such as imidazolium and trialkyl ammonium moieties. The betaine LC compounds formed more thermally stable LC phases than the mono-ionic LC compounds having analogous structures. For salt permeation in water treatment, the betaine LC membrane exhibited ion selectivity, which was different from those of the mono-ionic LC membranes. During virus filtration, the water flux of the betaine membrane was the highest among other analogous nanostructured Col membranes. For gas separation, these Col LC membranes showed selective CO2 permeation properties and exhibited an alpha CO2/N2 selectivity of about 30 under highly humidified conditions.

Disinfection is key to controlling the infection risk caused by viral contamination. Current disinfection guidelines often refer to a single virus resistant to the disinfectant of interest, despite the large variation in sensitivity to disinfectants among viruses or even among strains within the same species. Here, we demonstrate a statistical framework that integrates multiple experimental data sets and model the variation in sensitivity to disinfectants across different virus species using a parametric distribution termed the disinfectant sensitivity distribution. To illustrate this framework, we used 37, 9, and 28 species-dependent inactivation rate constants for ultraviolet (UV), ozone, and free chlorine, respectively, from systematic reviews. We estimated the sensitivity distributions of these disinfectants by incorporating the uncertainty in the individual inactivation rate constants using a Bayesian framework. The estimated sensitivity distributions suggested that it should be possible to achieve 4-log inactivation of 93.0% (95% credible interval (CrI): 84.2%-97.5%), 99.4% (95% CrI: 86.7%-100%), and 95.0% (95% CrI: 85.5%-98.8%) of the examined virus species using UV, ozone, and free chlorine, respectively, if the disinfectant dose complies with the values recommended by the US EPA. The proposed approach provides a reasonable extrapolation of observed inactivation kinetics to untested viruses and tools for more transparent risk assessments.

AIMS:The wider implementation of wastewater-based surveillance (WBS) has increased the necessity for nucleic acid detection and quantification from complex samples. Here, we compared the performance and the characteristics of three digital polymerase chain reaction (dPCR) platforms. METHODS AND RESULTS:The dPCR platforms selected in this study included the QX200 AutoDG Droplet Digital PCR System from Bio-Rad, the QIAcuity One, 2plex Device from Qiagen, and the 3-color Naica System manufactured by Stilla Technologies. Platforms were compared and described based on their handling in the laboratory and performance quantifying two viral targets-SARS-CoV-2 and Norovirus GII-in wastewater samples. Our findings showed that no single platform consistently outperformed the others in terms of target quantification. Moreover, we observed similarities amongst the systems with respect to resilience to inhibition, but differences in laboratory handling including sample throughput and method of quantification. CONCLUSIONS:This study suggests that all three selected dPCR platforms are similarly suitable for WBS, as quantitative performance of the systems for viral RNA targets extracted from wastewater is comparable. Decisions on platform selection can be driven by researcher preference on handling, throughput, and other differentiating characteristics.

As more data on virus concentrations in influent water from wastewater treatment plants (WWTPs) becomes available, establishing best practices for virus measurements, monitoring, and statistical modelling can improve the understanding of virus concentration distributions in wastewater. To support this, we assessed the temporal variability of norovirus, adenovirus, enterovirus, and rotavirus concentrations in influent water across multiple WWTPs in Switzerland, the USA, and Japan. Our findings demonstrate that the lognormal distribution accurately describes temporal variations in concentrations for all viruses at all sites, outperforming the gamma and Weibull distributions, which fail to capture high variability. However, notable differences in variability and uncertainty were observed across systems, underscoring the need for site-specific assessments. Using lognormal parameters, we identified optimal monitoring frequencies that balance cost-effectiveness and precision. For most sites, weekly monitoring was sufficient to estimate the annual average concentration of enteric viruses within a 95% confidence interval of 0.5 log10. We further examined the mechanistic basis of the lognormal distribution, highlighting processes that drive its prevalence and shape the behavior of its upper tail. By integrating these insights, this study provides a statistical foundation for optimizing virus monitoring frameworks and informing public health interventions targeting wastewater systems.

Noroviruses and enteroviruses are major causes of endemic gastrointestinal disease associated with substantial disease burden. However, viral gastroenteritis is often diagnosed based on symptoms, with etiology infrequently tested or reported, so little information exists on community-level transmission dynamics. In this study, we demonstrate that norovirus (NoV) genogroup II and enterovirus (EV) viral loads in wastewater reveal transmission dynamics of these viruses. We report NoV and EV concentrations in wastewater from 363 samples between December 5 2020 and October 10 2022 (sampled every second day). Virus concentrations in wastewater were low during 2021, and increased in 2022. Wastewater recapitulated periods of increased clinical cases, and also identified silent waves of transmission. We used the measured wastewater loads to estimate the effective reproductive number (Re). The Re for both NoV and EV peaked between 1.1 and 1.2. However, the usual seasonality of NoV transmission was upended by non-pharmaceutical interventions implemented to mitigate the COVID-19 pandemic, leading to correlated transmission dynamics of NoV GII and EV during 2021-2022. This highlights the use of wastewater to understand transmission dynamics of endemic enteric viruses and estimate relevant epidemiological parameters, including Re.

The sensitivity of enteroviruses to disinfectants varies among genetically similar variants and coincides with amino acid changes in capsid proteins, although the effect of individual substitutions remains unknown. Here, we employed reverse genetics to investigate how amino acid substitutions in coxsackievirus B5 (CVB5) capsid proteins affect the virus' sensitivity to free chlorine and heat treatment. Of ten amino acid changes observed in CVB5 variants with free chlorine resistance, none significantly reduced the chlorine sensitivity, indicating a minor role of the capsid composition in chlorine sensitivity of CVB5. Conversely, a subset of these amino acid changes located at the C-terminal region of viral protein 1 led to reduced heat sensitivity. Cryo-electron microscopy revealed that these changes affect the assembly of intermediate viral states (altered and empty particles), suggesting that the mechanism for reduced heat sensitivity could be related to improved molecular packing of CVB5, resulting in greater stability or altered dynamics of virus uncoating during infection.

Serine proteases are important environmental contributors of enterovirus biocontrol. However, the structural features of molecular interaction accounting for the susceptibility of enteroviruses to proteases remains unexplained. Here, we describe the molecular mechanisms involved in the recruitment of serine proteases to viral capsids. Among the virus types used, coxsackievirus A9 (CVA9), but not CVB5 and echovirus 11 (E11), was inactivated by Subtilisin A in a host-independent manner, while Bovine Pancreatic Trypsin (BPT) only reduced CVA9 infectivity in a host-dependent manner. Predictive interaction models of each protease with capsid protomers indicate the main targets as internal disordered protein (IDP) segments exposed either on the 5-fold vertex (DE loop VP1) or at the 5/2-fold intersection (C-terminal end VP1) of viral capsids. We further show that a functional binding protease/capsid depends on both the strength and the evolution over time of protease-VP1 complexes, and lastly on the local adaptation of proteases on surrounding viral regions. Finally, we predicted three residues on CVA9 capsid that trigger cleavage by Subtilisin A, one of which may act as a sensor residue contributing to enzyme recognition on the DE loop. Overall, this study describes an important biological mechanism involved in enteroviruses biocontrol. A predictive molecular model sheds light on serine proteases and Enterovirus capsids interaction leading to viral inactivation.

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.

The hollow fiber ultrafiltration (HFUF)-based microbial concentration method is widely applied for monitoring pathogenic viruses and microbial indicators in environmental water samples. However, the HFUF-based method can co-concentrate substances that interfere with downstream molecular processes—nucleic acid extraction, reverse transcription (RT), and PCR. These inhibitory substances are assumed to be hydrophobic and, therefore, expected to be excluded by a simple surfactant treatment before the silica membrane-based RNA extraction process. In this study, the efficacy and limitations of the sodium deoxycholate (SD) treatment were assessed by quantifying a process control and indigenous viruses using 42 surface water samples concentrated with HFUF. With some exceptions, which tended to be seen in samples with high turbidity (> 4.0 NTU), virus recovery by the ultrafiltration method was sufficiently high (> 10%). RNA extraction-RT-quantitative PCR (RT-qPCR) efficiency of the process control was insufficient (10%) for 30 of the 42 HFUF concentrates without any pretreatments, but it was markedly improved for 21 of the 30 inhibitory concentrates by the SD treatment. Detection rates of indigenous viruses were also improved and no substantial loss of viral RNA was observed. The SD treatment was particularly effective in mitigating RT-qPCR inhibition, although it was not effective in improving RNA extraction efficiency. The methodology is simple and easily applied. These findings indicate that SD treatment can be a good alternative to sample dilution, which is widely applied to mitigate the effect of RT-qPCR inhibition, and can be compatible with other countermeasures.

Summary Enteroviruses are human pathogens known to challenge water safety 1,2 . Among the microbial stressors found in water, bacterial serine proteases contribute to the control of enterovirus persistence 3 . However, the structural interactions accounting for the susceptibility of enteroviruses to proteases remains unexplained. Here, we describe the molecular mechanisms involved in the recruitment of serine proteases to viral capsids. Among the virus types used, coxsackievirus A9 (CVA9), but not CVB5 and echovirus 11 (E11), was inactivated by Subtilisin A in a host-independent manner, while Bovine Pancreatic Trypsin (BPT) only reduced CVA9 infectivity in a host-dependent manner. Predictive interaction models of each protease with capsid protomers indicate the main targets as internal disordered protein (IDP) segments exposed either on the 5-fold vertex (DE loop VP1) or at the 5/2-fold intersection (C-terminal end VP1) of viral capsids. We further show that a functional binding protease/capsid depends on both the strength and the evolution over time of protease-VP1 complexes, and lastly on the local adaptation of proteases on surrounding viral regions. Finally, we identified three residues on CVA9 capsid that trigger cleavage by Subtilisin A, one of which acts as a sensor residue contributing to enzyme recognition on the DE loop. Overall, this study describes an important biological mechanism involved in enteroviruses biocontrol.

Raw data underlying the journal article "Selective elimination of enterovirus genotypes by activated sludge and chlorination" by Larivé et al., Environmental Science: Water Research and Technology, 2023 (doi: 10.1039/d3ew00050h) One CSV file for each of Figures 2-6 of the main manuscript $ One CSV file for each of Figures S5, S6 and S7 of the Supplementary information. The data for Figures S2, S3 and S4 are summarized in a single CSV file.

UV-LED treatment at 265 nm was more efficient than UV-LED treatment at 280 nm in inactivating both enveloped and non-enveloped viruses. Capsid damage is not important in virus inactivation by UV-LEDs for both enveloped and non-enveloped viruses.

Chlorine disinfection is commonly applied to inactivate pathogenic viruses in drinking water treatment plants. However, the role of water quality in chlorine disinfection of viruses has not been investigated thoughtfully. In this study, we investigated the inactivation efficiency of coxsackievirus B5 (CVB5) by free chlorine using actual water samples collected from four full-scale drinking water treatment plants in Japan under strict turbidity management (less than 0.14 NTU) over a 12-month period. It was found that chlorine disinfection of CVB5 might not be affected by water quality. Japanese turbidity management might play an indirect role in controlling the efficiency of chlorine disinfection.