Tamar Kohn

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–reverse transcriptase quantitative PCR (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 10 EV types, the NanoporeSeq method accurately and reproducibly recovered the original proportions of all EV types after correction of biases. When applied to wastewater extracts, the method detected 45 EV types across five species. Furthermore, type-specific calibration curves generated with ICC-NanoporeSeq enabled quantification of the infectious concentrations of six EV types of the Enterovirus B species, 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 enteroviruses (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 integrated cell culture–nanopore sequencing (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.

Transmission of respiratory pathogens depends on their ability to remain viable in drying respiratory droplets, yet the physicochemical drivers of bacterial inactivation during droplet evaporation remain poorly quantified. This study combines controlled droplet experiments with physicochemical modeling to investigate how osmotic pressure dynamics influence bacterial survival. Using Escherichia coli and Staphylococcus epidermidis as gram-negative and gram-positive surrogates, respectively, we measured viability loss in artificial saliva droplets dried at multiple relative humidities, and reconstructed the time-resolved osmotic pressure using the respiratory aerosol model. Both organisms remained stable while droplets were liquid and osmotic pressure was low but lost viability as decreasing water activity during drying produced rapid increases in osmotic pressure. For droplets that effloresced, these changes were most pronounced around efflorescence. The extent of inactivation scaled log-linearly with the rate of osmotic pressure change: E. coli decayed faster than S. epidermidis , and relationships derived in artificial saliva predicted survival in independent phosphate-buffered saline experiments. A lower relative humidity produced a more rapid increase in osmotic pressure and greater inactivation. These results identify the rate of osmotic pressure change during respiratory droplet drying as a quantitative predictor of bacterial survival across different solution matrices and drying conditions. IMPORTANCE: The risk of infection by respiratory pathogens depends on how long microorganisms remain viable in respiratory particles after exhalation, yet the physical mechanisms controlling bacterial survival during droplet drying are not well defined. Evaporation of respiratory droplets concentrates salts and can impose sudden and extreme osmotic stress on microbes, but this process has been difficult to quantify because osmotic pressure cannot be measured directly inside microscopic droplets. Integration of droplet experiments with a physicochemical aerosol model shows that bacterial inactivation is governed primarily by the rate of osmotic pressure increase during droplet drying rather than by static values of humidity or solute concentration alone. This mechanism explains why rapid drying produces strong inactivation.

In freshwater environments, bacteria are known to enhance the removal and inactivation of human viruses. Rates of inactivation differ across freshwater ecosystems, though the influence of community diversity and composition on this process remain poorly understood. Here, we used dilution-to-extinction to generate 64 distinct freshwater communities from three lakes and three aquifers. After establishing freshwater communities in chemostat cultures, each community was challenged with two viruses, coxsackievirus B5 and human adenovirus 2. Both viruses were inactivated more rapidly in lake-derived than in groundwater-derived communities, and adenovirus was inactivated faster than coxsackievirus in both freshwater types. Community-level parameters (e.g., cell numbers, richness) were not significant predictors of viral inactivation. Instead, differential abundance analyses identified bacterial OTUs associated with inactivation: Acidovorax sp. were associated across sources and Brevundimonas sp., Sphingopyxis sp., and Hydrogenophaga sp. showed source-specific associations. Together, these findings provide new insights into the ecological drivers of viral stability and suggest that viral inactivation may depend more strongly on specific microbial taxa and their environmental context than on overall community diversity. Identifying these associations improves our understanding of the factors governing viral stability in freshwater environments. GRAPHICAL

Enteric viruses are important contaminants of surface waters and a significant burden on public health. In aquatic ecosystems, the stability of these pathogens is differentially impacted by abiotic and biotic stressors, which can exert inactivating effects. Here, we investigated the fate of two enteroviruses, echovirus 11 (E11) and coxsackievirus B5 (CVB5), and one adenovirus, human adenovirus 2 (HAdV2), in lakewater and explored the mechanisms underlying their microbial inactivation. By combining infectivity assays, genome quantification, and capsid integrity analysis, we characterized virus-specific inactivation kinetics and mechanisms and examined the relationship between infectivity loss and capsid structural integrity. We observed rapid, intermediate, and negligible decay for HAdV2, E11, and CVB5, respectively, and confirmed that microbial proteases contribute to their inactivation. In addition, we revealed that loss of capsid structural integrity drives E11 inactivation, but not HAdV2 inactivation. Genome decay did not consistently correlate with loss of infectivity, highlighting the limitations of genome-based detection for assessing the presence of infectious viruses. These findings provide new insights into the mechanisms governing virus inactivation in aquatic environments and emphasize the importance of understanding viral fate when interpreting molecular detection data to assess virus-associated microbial risks. Importance: Enteric viruses are common contaminants of surface waters and pose important risks to human health. However, the stability of these viruses in water varies widely and is influenced by many environmental factors. In this study, we investigated how microbial activity causes the inactivation of three enteric viruses in lakewater. We found substantial differences in virus stability and showed that microbial enzymes that break down proteins contribute to virus inactivation. We additionally found that virus structural damage is associated with the inactivation of one virus, but not of another, and that quantification of virus genetic material does not reliably indicate levels of infectious virus particles. These findings highlight the importance of understanding how viruses behave in surface waters and can help inform approaches for monitoring viral contamination and assessing potential risks to human health.

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 reemerging 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 viral capsid residue as one of the drivers of 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.

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.

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.

Airborne transmission of respiratory pathogens depends on their ability to remain viable in drying respiratory droplets, yet the physicochemical drivers of bacterial inactivation during droplet evaporation remain poorly quantified. This study combines controlled droplet experiments with physicochemical modeling to investigate how osmotic pressure dynamics influence bacterial survival. Using Escherichia coli and Staphylococcus epidermidis as Gram-negative and Gram-positive surrogates, respectively, we measured viability loss in artificial saliva droplets dried at multiple relative humidities and reconstructed the time-resolved osmotic pressure using the Respiratory Aerosol Model (ResAM). Both organisms remained stable while droplets were liquid but lost viability following efflorescence, when rapid solute concentration changes produced sharp osmotic pressure increases. The extent of inactivation scales log-linearly with the rate of osmotic pressure change around efflorescence: E. coli decays faster than S. epidermidis , and relationships derived in artificial saliva predict survival in independent phosphate-buffered saline experiments. A more rapid drop in humidity led to more severe osmotic shocks and greater inactivation. These results identify the rate of osmotic pressure change during efflorescence as a quantitative, medium-independent predictor of bacterial survival in drying respiratory droplets.

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.

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.