Interactions between viruses and filter-feeding zooplankton can alter viral persistence in surface waters, with direct implications for water quality and public health risk. However, data on virus-zooplankton interactions and the environmental factors that influence them are still limited. This study evaluated the impact of filter feeding, in the dark and under simulated sunlight, on a bacteriophage (MS2) and a human virus (echovirus11; E11) in the presence of a ciliate (Tetrahymena pyriformis) and rotifer (Brachionus calyciflorus). Dark experiments established organism-dependent baseline removal for each virus, and rotifers showed greater removal of both viruses in comparison to ciliates. Under simulated sunlight, in contrast, experiments with ciliates resulted in greater virus removal compared to experiments with rotifers over a similar timespan (4.2 vs 2.7 log MS2 in 53-58 h; 3.5 vs 3.0 log E11 in 24-25 h). Analysis of decay rate constants reveals species-specific shifts in virus removal between dark and light that, depending on viral type and zooplankton species, either accelerate viral attenuation or protect viruses and prolong infectivity. T. pyriformis increases removal under sunlight relative to dark conditions and acts synergistically with sunlight inactivation, whereas rotifers impede sunlight inactivation. These data address important knowledge gaps on how dark biotic processes can modulate sunlight-mediated inactivation. The reported decay constants can help inform predictions of virus environmental fate with implications for natural and engineered treatment systems that rely on sunlight disinfection to inactivate viruses.

ABSTRACT Enteroviruses (EVs) are ubiquitous contaminants of surface waters, where they can remain infectious for long periods of time. Most methods used for EV monitoring are unable to distinguish between infectious and non-infectious particles or between EV types. Because different types exhibit both distinct environmental persistence and health implications, there is a need for type-resolved infectivity measurements. Here we developed Integrated Cell Culture-Nanopore Sequencing (ICC-NanoporeSeq), a method combining short-term cell culture amplification with Nanopore sequencing of the VP1 gene. The ICC approach was adapted from a previously described ICC-RTqPCR protocol, while the NanoporeSeq workflow was derived from a clinical EV typing protocol and optimized for environmentally circulating EV types. Using samples containing known concentrations of ten EV types, the NanoporeSeq method accurately and reproducibly recovered the original proportions of all EV types after correction of biases. Furthermore, type-specific calibration curves generated with ICC-NanoporeSeq enabled quantification of the infectious concentrations of six EV types, allowing a simultaneous and type-resolved assessment of infectivity in mixed samples. Overall, ICC-NanoporeSeq provides a scalable approach for the parallel analysis of multiple EV types. Compared with the predecessor ICC-RTqPCR method, it eliminates the need for multiple type-specific PCR primers and can therefore be readily expanded to include additional EV types. IMPORTANCE Current methods used to detect EVs in environmental samples generally measure viral genome copies without determining whether viruses remain infectious, limiting their use in public health risk assessment or water quality monitoring. At the same time, available infectivity assays are often labor-intensive and cannot distinguish between different EV types. Here, we developed ICC-NanoporeSeq, a method combining cell culture and Nanopore sequencing to simultaneously quantify the infectious concentrations of multiple EV types in samples containing mixed EV populations. The method provides an efficient and scalable approach for studying EVs in complex environmental matrices. ICC-NanoporeSeq has potential applications in wastewater-based epidemiology, environmental surveillance, and disinfection studies, where understanding the persistence of different EV types simultaneously is crucial.

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.

Airborne transmission of influenza A virus (IAV) poses significant challenges to public health. However, the mechanisms governing viral inactivation in aerosols remain poorly understood. IAVs exhibit morphological variability, ranging from 100 nm spherical virions to micrometer-long filaments, depending on strain and growth conditions. Although virion morphology was shown to influence transmissibility, the mechanisms of how morphology affects airborne transmission are yet to be delineated. Here, we investigated the impact of virion shape on IAV stability in bulk solutions and an aerosol system with a focus on particles in the submicrometer range to examine how physicochemical aerosol properties, such as elevated solute concentration and low pH, affect infectivity. We show that filamentous viruses exhibit enhanced stability in aerosol particles at 80% relative humidity (RH) and in bulk solution mimicking increased salinity at this RH. Similarly, filamentous viruses exhibited slower decay under acidic conditions, both in bulk solutions and in acidified aerosol particles. Using primary human airway cultures, we further found that filamentous IAVs possess an infectivity advantage under mucosal immune pressures, including neutralizing antibodies and mucus. These results reveal that filamentous shape provides IAV with enhanced stability under diverse environmental conditions in the aerosol phase and increased infectivity in the respiratory epithelium.

Climate-driven disruptions in aquatic ecosystems are amplifying cyanotoxin production, threatening drinking and recreational water safety. Monitoring of these toxins is challenged by requirements of the low μg/L detection limits and structural diversity. Here, we employ aerolysin nanopores to distinguish seven of the most prevalent microcystin congeners, both individually and in mixtures, at environmentally relevant concentrations. Importantly, we showed that aerolysin enables the detection of microcystins in spiked and real contaminated lake water samples at concentrations below the World Health Organization's intervention thresholds, reaching picomolar sensitivity. Moreover, combining experiments and molecular dynamics simulations, we further investigated the microcystin sensing mechanism, suggesting that the ionic current blockage is primarily governed by K238 in aerolysin, while dwell time is regulated by the R220 constriction site. Our results support the use of nanopore sensing technology for real-time monitoring of microcystins in drinking water sources and surface waters.

The COVID-19 pandemic has accelerated the development and adoption of wastewater-based epidemiology. Wastewater samples can provide genomic information for detecting and assessing the spread of SARS-CoV-2 variants in communities and for estimating important epidemiological parameters such as the selection advantage of a viral variant. However, despite demonstrated successes, epidemiological data derived from wastewater suffers from potential biases. Of particular concern are shedding profiles, which can affect the relationship between true viral incidence and viral loads in wastewater. Changes in shedding between variants may decouple the established relationship between wastewater loads and clinical test data. Using mathematical modeling, simulations, and Swiss surveillance data, we demonstrate that estimates of the selection advantage of a variant are not biased by shedding profiles. We show that they are robust to differences in shedding between variants under a wide range of assumptions, and identify specific conditions under which this robustness may break down. Additionally, we demonstrate that differences in shedding only briefly affect estimates of the effective reproduction number. Thus, estimates of selective advantage and reproduction numbers derived from wastewater maintain their advantages over traditional clinical data, even when there are differences in shedding among variants.

ABSTRACT Human enteric viruses can remain infective in surface waters for extended periods of time, posing a public health risk. Microbial activity contributes to the inactivation of waterborne enteric viruses, but while individual bacteria–virus interactions have been characterized, the importance of microbial diversity remains unknown. Here, we experimentally manipulated the diversity of bacterial communities from Lake Geneva across three seasons using a dilution-to-extinction approach and monitored the inactivation and genome decay of echovirus 11, a member of the Enterovirus genus. Long-read sequencing of the 16S rRNA gene revealed diversity gradients ranging between 373 and 2,722 bacterial species. Compared to sterile controls, echovirus 11 inactivation was enhanced by the presence of active bacteria and depended both on season and sample dilution. Throughout all seasons, the highest inactivation (between 3.0 and 7.9 log 10 fold reduction in infectivity over 96 h) was observed in the least diluted incubations (i.e., the highest bacterial richness). Genome decay exhibited a 24-h lag and was less pronounced than the corresponding infectivity loss (ranging between 2.3 and 3.8 log 10 fold over 96 h), indicating that microbial inactivation primarily targets the echovirus 11 capsid. We found a positive-saturating relationship between bacterial species richness and viral inactivation, suggesting functional redundancy and pointing toward the importance of rare species for viral inactivation. Biomarker analysis revealed several clades of bacteria, particularly members of Chitinophagaceae , to be significantly associated with echovirus 11 inactivation. Overall, these findings suggest that high microbial diversity enhances the capacity of surface waters to rid themselves of contamination by enteric viruses and hence protects public health. IMPORTANCE Human enteric viruses in natural waterbodies pose a public health risk. Microorganisms, particularly bacteria, contribute to the inactivation of enteroviruses, thereby mitigating this risk. We use experimental manipulations of lake water bacterial diversity to unravel the importance of diversity for the inactivation of echovirus 11, a model human pathogen. Our findings suggest that bacterial diversity is important for echovirus 11 inactivation and that specific, but numerically rare, bacteria present in the surface water of Lake Geneva across different seasons contribute to viral inactivation. These findings contribute to our understanding of the inactivation of human enteric viruses in natural waterbodies—a hitherto understudied ecosystem service.

Lakewater microorganisms secrete proteases which contribute to the turnover of dissolved organic matter and the degradation of peptidic contaminants. However, little is known about the identities and substrate specificities of these proteases. Herein, we sought to characterize the global proteolytic fingerprint of the extracellular proteases present in Lake Geneva, the largest freshwater body in Central Europe. Using Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS), we identified preferred enzymatic cleavage next to positively charged and certain nonpolar amino acids, while cleavage next to negatively charged residues was disfavored. Specifically, the detected dominant cleavage sites were surrounded by arginine and lysine, consistent with a trypsin-like substrate specificity. This pattern was conserved across seasons and water depths and was shared with two other Swiss lakes. In contrast, we observed variability in the numbers and types of less prevalent cleavage sites across samples, suggesting that the degree of heterogeneity in proteolytic substrate specificity varies spatially and temporally. Using class-specific inhibitors, we found that serine and metalloproteases contribute to both exo- and endoproteolytic activity in lakewater. Our findings expand our understanding of protein stability in lake ecosystems and may be used to predict the fate of peptidic contaminants in the environment.

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.

Climate-driven disruptions in aquatic ecosystems are amplifying cyanotoxin production, threatening drinking and recreational water safety. Monitoring of these toxins is challenged by requirements of the low µg/L detections limits and structural diversity. Here, we employ aerolysin nanopores to distinguish seven of the most prevalent microcystin congeners, both individually and in mixtures, at environmentally relevant concentrations. Importantly, we showed that aerolysin enables the detection of microcystins in different lake water samples at concentrations below the World Health Organization’s intervention thresholds, reaching picomolar sensitivity. Moreover, combining experiments and molecular dynamics simulations, we further investigated the microcystin sensing mechanism, suggesting that the ionic current blockage is primarily governed by K238 in aerolysin, while dwell time is regulated by R220 constriction site. Our results open the way to use the nanopore sensing technology for real-time monitoring of microcystins in drinking water sources and surface waters. ### Competing Interest Statement The authors have declared no competing interest. Swiss National Science Foundation, https://ror.org/00yjd3n13, 200021L_212128, PR00P3_193090

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.

Differentiated primary human respiratory epithelial cells grown at air-liquid interface have become a widely used cell culture model of the human conducting airways. These cultures contain secretory cells such as goblet and club cells, which produce and secrete mucus. Here, we characterize the composition of mucus harvested from airway cultures of nasal and bronchial origin. We find that despite inter-donor variability, the salt, sugar, lipid, and protein content and composition are very similar between nasal and bronchial mucus. However, subtle differences in the abundance of individual components in nasal versus bronchial mucus can influence its antimicrobial properties. The ability of mucus to neutralize influenza A virus varies with the anatomical origin of the airway cultures and correlates with the abundance of triglycerides and specific sialylated glycoproteins and glycolipids.IMPORTANCERespiratory mucus plays an important role during the transmission and infection process of microbes in the human respiratory tract. In the case of influenza A virus, the mucus stabilizes the virions in infectious respiratory particles and droplets but hampers virus particles before they reach the respiratory epithelium through its physicochemical properties and the presence of sialylated decoy receptors. However, it is thus far not well understood which components of mucus mediate protection and inhibition. Our study now provides a comprehensive analysis of bronchial and nasal mucus from primary human airway cultures that can be used as a resource for future experimental designs and interpretations.

SARS-CoV-2, the causative agent of COVID-19, is predominantly transmitted by respiratory aerosol and contaminated surfaces. Recent studies demonstrated that aerosols can become acidic, and acidification has been proposed as decontamination method. Here, we investigate how SARS-CoV-2 reacts to acidic pH and by which mechanism the virus is inactivated. We show that a pH below 3 is required to inactivate SARS-CoV-2 in a period of seconds to minutes. While we measured a 1000 to 10,000-fold drop in infectivity, virion structure remained intact under these conditions. Using super-resolution microscopy, we found that the attachment of virions to target cells is abrogated after acidic treatment, revealing spike protein (S) as the major inactivation target. Limited proteolysis of S combined with testing spike-specific antibodies for binding under low pH conditions revealed that exposure of SARS-CoV-2 to pH below 3 results in partial unfolding of S, thereby preventing binding of virions to target cells.

Adenovirus (HAdV), a double-stranded DNA virus, exhibits resistance to direct inactivation by UVC and solar UVB radiation in water, owing to its ability to repair UV-induced genome damage using host cellular machinery. In the presence of dissolved organic matter (DOM), however, previous studies have reported unexpectedly rapid sunlight-mediated inactivation, even after accounting for indirect effects mediated by photochemically produced reactive intermediates (PPRIs). Here, we hypothesized that a synergistic interaction between direct and indirect inactivation pathways compromises HAdV2's genome repair capacity, thereby enhancing its overall susceptibility to inactivation. First, we demonstrated that pre-exposure to singlet oxygen (1O2)─a key PPRI─significantly increased HAdV2's susceptibility to subsequent direct UV inactivation. Using host cells with differing genome repair capacities, we further showed that simultaneous exposure to direct and indirect inactivation pathways reduced HAdV2's genome repair efficiency compared to direct UV alone. Finally, although indirect inactivation caused minimal DNA damage, it impaired the DNA replication efficiency of host cells, likely due to oxidative damage to viral proteins involved in transcription-coupled repair. These findings highlight a critical interplay between direct and indirect inactivation pathways and offer new insights that can aid in optimizing light-based disinfection strategies to enhance the efficacy of water treatment processes targeting adenovirus-contaminated sources.

Toxic cyanobacteria are likely to be favored by global warming and other human impacts, posing significant threats to aquatic ecosystems. While cyanobacterial blooms in eutrophic lakes are widely investigated, the dynamics of cyanobacteria and the effects of their toxins and bioactive metabolites on the plankton communities in mesotrophic and oligotrophic lakes are less well understood. Here we investigated seasonal dynamics of cyanobacteria, eukaryotic algae and cyanotoxins in oligo-mesotrophic Lake Geneva—the largest and deepest lake in western Europe. High-throughput sequencing of the 16S rRNA genes in 143 samples along a water column revealed that Lake Geneva hosts diverse, co-dominant cyanobacterial genera, including Planktothrix, Cyanobium, Pseudanabaena, and Aphanizomenon. The abundance of the mcyA gene marker for microcystin production was highly correlated with total cyanobacteria abundance, obtained from qPCR of the 16S rRNA genes. Targeted LC-HRMS/MS analysis demonstrated peak concentrations of cyanotoxins in September and December 2021 at the deep chlorophyll-a maximum layer, reaching up to 1474 ng/l for anabaenopeptins and 144 ng/l for microcystins. The toxin peaks did not correlate with the abundance or variations in the cyanobacteria or eukaryote community, but they were correlated in time with seasonal lows in the abundances of ciliates (18S rRNA analysis). Laboratory exposure tests demonstrated that growth of the model ciliate Tetrahymena pyriformis was inhibited by Microcystin-RR and Anabaenopeptin A at environmentally relevant concentrations in the ng/l-range, in natural lake water, synthetic freshwater, and growth media spiked with the cyanotoxins. Our findings suggest that even low concentrations (in the ng/l-range) of microcystins and anabaenopeptins, reduce growth of ciliates such as T. pyriformis and can be expected to have wider impacts on the eukaryote communities.