Comparison of Three Viral Nucleic Acid Preamplification Pipelines for Sewage Viral Metagenomics.

Viral metagenomics is a useful tool for detecting multiple human viruses in urban sewage. However, more refined protocols are required for its effective use in disease surveillance. In this study, we investigated the performance of three different preamplification pipelines (specific to RNA viruses, DNA viruses or both) for viral genome sequencing using spiked-in Phosphate Buffered Saline and sewage samples containing known concentrations of viruses. We found that compared to the pipeline targeting all genome types, the RNA pipeline performed better in detecting RNA viruses in both spiked and unspiked sewage samples, allowing the detection of various mammalian viruses including members from the Reoviridae, Picornaviridae, Astroviridae and Caliciviridae. However, the DNA-specific pipeline did not improve the detection of mammalian DNA viruses. We also measured viral recovery by quantitative reverse transcription polymerase chain reaction and assessed the impact of genetic background (non-viral genetic material) on viral coverage. Our results indicate that viral recoveries were generally lower in sewage (average of 11.0%) and higher in Phosphate Buffered Saline (average of 23.4%) for most viruses. Additionally, spiked-in viruses showed lower genome coverage in sewage, demonstrating the negative effect of genetic background on sequencing. Finally, correlation analysis revealed a relationship between virus concentration and genome normalized reads per million, indicating that viral metagenomic sequencing can be semiquantitative.

Influence of Amino Acid Substitutions in Capsid Proteins of Coxsackievirus B5 on Free Chlorine and Thermal Inactivation.

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.

The neuraminidase activity of influenza A virus determines the strain-specific sensitivity to neutralization by respiratory mucus.

IMPORTANCE: The respiratory tract of humans is constantly exposed to potentially harmful agents, such as small particles or pathogens, and thus requires protective measures. Respiratory mucus that lines the airway epithelia plays a major role in the prevention of viral infections by limiting the mobility of viruses, allowing subsequent mucociliary clearance. Understanding the interplay between respiratory mucus and viruses can help elucidate host and virus characteristics that enable the initiation of infection. Here, we tested a panel of primary influenza A viruses of avian or human origin for their sensitivity to mucus derived from primary human airway cultures and found that differences between virus strains can be mapped to viral neuraminidase activity. We also show that binding of influenza A viruses to decoy receptors on highly glycosylated mucus components constitutes the major inhibitory function of mucus against influenza A viruses.

Inactivation mechanisms of influenza A virus under pH conditions encountered in aerosol particles as revealed by whole-virus HDX-MS.

Multiple respiratory viruses, including influenza A virus (IAV), can be transmitted via expiratory aerosol particles, and aerosol pH was recently identified as a major factor influencing airborne virus infectivity. Indoors, small exhaled aerosols undergo rapid acidification to pH ~4. IAV is known to be sensitive to mildly acidic conditions encountered within host endosomes; however, it is unknown whether the same mechanisms could mediate viral inactivation within the more acidic aerosol micro-environment. Here, we identified that transient exposure to pH 4 caused IAV inactivation by a two-stage process, with an initial sharp decline in infectious titers mainly attributed to premature attainment of the post-fusion conformation of viral protein haemagglutinin (HA). Protein changes were observed by hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) as early as 10 s post-exposure to acidic conditions. Our HDX-MS data are in agreement with other more labor-intensive structural analysis techniques, such as X-ray crystallography, highlighting the ease and usefulness of whole-virus HDX-MS for multiplexed protein analyses, even within enveloped viruses such as IAV. Additionally, virion integrity was partially but irreversibly affected by acidic conditions, with a progressive unfolding of the internal matrix protein 1 (M1) that aligned with a more gradual decline in viral infectivity with time. In contrast, no acid-mediated changes to the genome or lipid envelope were detected. Improved understanding of respiratory virus fate within exhaled aerosols constitutes a global public health priority, and information gained here could aid the development of novel strategies to control the airborne persistence of seasonal and/or pandemic influenza in the future. IMPORTANCE It is well established that COVID-19, influenza, and many other respiratory diseases can be transmitted by the inhalation of aerosolized viruses. Many studies have shown that the survival time of these airborne viruses is limited, but it remains an open question as to what drives their infectivity loss. Here, we address this question for influenza A virus by investigating structural protein changes incurred by the virus under conditions relevant to respiratory aerosol particles. From prior work, we know that expelled aerosols can become highly acidic due to equilibration with indoor room air, and our results indicate that two viral proteins are affected by these acidic conditions at multiple sites, leading to virus inactivation. Our findings suggest that the development of air treatments to quicken the speed of aerosol acidification would be a major strategy to control infectious bioburdens in the air.

Selective elimination of enterovirus genotypes by activated sludge and chlorination.

Enteroviruses, which are commonly circulating viruses shed in the stool, are released into the sewage system and only partially removed or inactivated, resulting in the discharge of infectious enteroviruses into the environment. Activated sludge and chlorination remove or inactivate enterovirus genotypes to different extents, and thus have the potential to shape the population that will be discharged. The goal of this study was to evaluate how activated sludge and chlorination treatment shape an enterovirus population at the genotype level, using a population of eight genotypes commonly found in sewage: CVA9, CVB1, CVB2, CVB3, CVB4, CVB5, E25, E30. Our results show that the extent of inactivation varied among genotypes, but also across sludge samples. We find that the effluent of activated sludge systems will be depleted in CVA9, CVB1 and CVB2 while E25 together with CVB3, CVB4 and CVB5 will be prevalent. Furthermore, we found that microbial inactivation was the main mechanism of infectivity loss in the activated sludge, while adsorption to the sludge flocs was not significant. During effluent chlorination, we also observed that CVB5, CVB3 and to a lesser extent E25 were less susceptible to chlorination while E30 was readily inactivated, and activated sludge-derived EPS provided further protection against chlorination. This study contributes to a better understanding of the variability of sewage treatment efficacy against different enteroviruses.

Uptake without inactivation of human adenovirus type 2 by Tetrahymena pyriformis ciliates.

Human adenoviruses are ubiquitous contaminants of surface water. Indigenous protists may interact with adenoviruses and contribute to their removal from the water column, though the associated kinetics and mechanisms differ between protist species. In this work, we investigated the interaction of human adenovirus type 2 (HAdV2) with the ciliate Tetrahymena pyriformis. In co-incubation experiments in a freshwater matrix, T. pyriformis was found to efficiently remove HAdV2 from the aqueous phase, with ≥4 log10 removal over 72 hours. Neither sorption onto the ciliate nor secreted compounds contributed to the observed loss of infectious HAdV2. Instead, internalization was shown to be the dominant removal mechanism, resulting in the presence of viral particles inside food vacuoles of T. pyriformis, as visualized by transmission electron microscopy. The fate of HAdV2 once ingested was scrutinized and no evidence of virus digestion was found over the course of 48 hours. This work shows that T. pyriformis can exert a dual role in microbial water quality: while they remove infectious adenovirus from the water column, they can also accumulate infectious viruses.

Observed Kinetics of Enterovirus Inactivation by Free Chlorine Are Host Cell-Dependent.

Virucidal efficacies of disinfectants are typically assessed by infectivity assay utilizing a single type of host cell. Enteroviruses infect multiple host cells via various entry routes, and each entry route may be impaired differently by a given disinfectant. Yet, it is unknown how the choice of host cells affects the observed inactivation kinetics. Here, we evaluated the inactivation kinetics of echovirus 11 (E11) by free chlorine, ultraviolet (UV) irradiation, and heat, using three different host cells (BGMK, RD, and A549). Inactivation rates were independent of the host cell for treatment of E11 by UV or heat. Conversely, E11 inactivation by free chlorine occurred 2-fold faster when enumerated on BGMK cells compared with RD and A549 cells. Host cell-dependent inactivation kinetics by free chlorine were also observed for echovirus 7, 9, and 13, and coxsackievirus A9. E11 inactivation by free chlorine was partly caused by a loss in host cell attachment, which was most pronounced for BGMK cells. BGMK cells lack the attachment receptor CD55 and a key subunit of the uncoating receptor ß2M, which may contribute to the differential inactivation kinetics for this cell type. Consequently, inactivation kinetics of enteroviruses should be assessed using host cells with different receptor profiles.

Expiratory Aerosol pH: The Overlooked Driver of Airborne Virus Inactivation.

Respiratory viruses, including influenza virus and SARS-CoV-2, are transmitted by the airborne route. Air filtration and ventilation mechanically reduce the concentration of airborne viruses and are necessary tools for disease mitigation. However, they ignore the potential impact of the chemical environment surrounding aerosolized viruses, which determines the aerosol pH. Atmospheric aerosol gravitates toward acidic pH, and enveloped viruses are prone to inactivation at strong acidity levels. Yet, the acidity of expiratory aerosol particles and its effect on airborne virus persistence have not been examined. Here, we combine pH-dependent inactivation rates of influenza A virus (IAV) and SARS-CoV-2 with microphysical properties of respiratory fluids using a biophysical aerosol model. We find that particles exhaled into indoor air (with relative humidity ≥ 50%) become mildly acidic (pH ∼ 4), rapidly inactivating IAV within minutes, whereas SARS-CoV-2 requires days. If indoor air is enriched with nonhazardous levels of nitric acid, aerosol pH drops by up to 2 units, decreasing 99%-inactivation times for both viruses in small aerosol particles to below 30 s. Conversely, unintentional removal of volatile acids from indoor air may elevate pH and prolong airborne virus persistence. The overlooked role of aerosol acidity has profound implications for virus transmission and mitigation strategies.

Waterborne virus transport and the associated risks in a large lake.

Waterborne enteric viruses in lakes, especially at recreational water sites, may have a negative impact on human health. However, their fate and transport in lakes are poorly understood. In this study, we propose a coupled water quality and quantitative microbial risk assessment (QMRA) model to study the transport, fate and infection risk of four common waterborne viruses (adenovirus, enterovirus, norovirus and rotavirus), using Lake Geneva as a study site. The measured virus load in raw sewage entering the lake was used as the source term in the water quality simulations for a hypothetical scenario of discharging raw wastewater at the lake surface. After discharge into the lake, virus inactivation was modeled as a function of water temperature and solar irradiance that varied both spatially and temporally during transport throughout the lake. Finally, the probability of infection, while swimming at a popular beach, was quantified and compared among the four viruses. Norovirus was found to be the most abundant virus that causes an infection probability that is at least 10 times greater than the other viruses studied. Furthermore, environmental inactivation was found to be an essential determinant in the infection risks posed by viruses to recreational water users. We determined that infection risks by enterovirus and rotavirus could be up to 1000 times lower when virus inactivation by environmental stressors was accounted for compared with the scenarios considering hydrodynamic transport only. Finally, the model highlighted the role of the wind field in conveying the contamination plume and hence in determining infection probability. Our simulations revealed that for beaches located west of the sewage discharge, the infection probability under eastward wind was 43% lower than that under westward wind conditions. This study highlights the potential of combining water quality simulation and virus-specific risk assessment for a safe water resources usage and management.

Vertical distribution and seasonal dynamics of planktonic cyanobacteria communities in a water column of deep mesotrophic Lake Geneva.

Background: Temperate subalpine lakes recovering from eutrophication in central Europe are experiencing harmful blooms due to the proliferation of Planktothrix rubescens, a potentially toxic cyanobacteria. To optimize the management of cyanobacteria blooms there is the need to better comprehend the combination of factors influencing the diversity and dominance of cyanobacteria and their impact on the lake's ecology. The goal of this study was to characterize the diversity and seasonal dynamics of cyanobacteria communities found in a water column of Lake Geneva, as well as the associated changes on bacterioplankton abundance and composition. Methods: We used 16S rRNA amplicon high throughput sequencing on more than 200 water samples collected from surface to 100 meters deep monthly over 18 months. Bacterioplankton abundance was determined by quantitative PCR and PICRUSt predictions were used to explore the functional pathways present in the community and to calculate functional diversity indices. Results: The obtained results confirmed that the most dominant cyanobacteria in Lake Geneva during autumn and winter was Planktothrix (corresponding to P. rubescens). Our data also showed an unexpectedly high relative abundance of picocyanobacterial genus Cyanobium, particularly during summertime. Multidimensional scaling of Bray Curtis dissimilarity revealed that the dominance of P. rubescens was coincident with a shift in the bacterioplankton community composition and a significant decline in bacterioplankton abundance, as well as a temporary reduction in the taxonomic and PICRUSt2 predicted functional diversity. Conclusion: Overall, this study expands our fundamental understanding of the seasonal dynamics of cyanobacteria communities along a vertical column in Lake Geneva and the ecology of P. rubescens, ultimately contributing to improve our preparedness against the potential occurrence of toxic blooms in the largest lake of western Europe.

Wastewater Reveals the Spatiotemporal Spread of SARS-CoV-2 in the Canton of Ticino (Switzerland) during the Onset of the COVID-19 Pandemic.

Wastewater-based epidemiology (WBE) has emerged as an effective tool for monitoring SARS-CoV-2 dynamics during the COVID-19 pandemic. Here, we add a spatial component to WBE and use it to investigate SARS-CoV-2 spread in the canton of Ticino during the onset of the pandemic in Switzerland (end of February 2020 to beginning of March 2020). Ticino is located at the border to Northern Italy, where a large COVID-19 outbreak occurred in February 2020. Not surprisingly, Ticino was the site of the first clinically confirmed COVID-19 case in Switzerland. We retrospectively analyzed daily influent samples from nine wastewater treatment plants in Ticino that jointly cover an area of 20 km × 60 km and 351,000 people (>99% of the population). Our result is a fine-grained view of the spatiotemporal evolution of the COVID-19 pandemic in this canton. The wastewater analysis revealed that by February 29, 2020, SARS-CoV-2 had already spread to all catchments. At the same time, only four individual cases had been clinically confirmed across the region served by the treatment plants investigated. Our results demonstrate that WBE could serve as a versatile tool to monitor the introduction and spread of an infectious agent on a regional scale. To fully exploit its utility, WBE should be implemented in real time and become an integral part of future disease surveillance efforts.

Early detection and surveillance of SARS-CoV-2 genomic variants in wastewater using COJAC.

The continuing emergence of SARS-CoV-2 variants of concern and variants of interest emphasizes the need for early detection and epidemiological surveillance of novel variants. We used genomic sequencing of 122 wastewater samples from three locations in Switzerland to monitor the local spread of B.1.1.7 (Alpha), B.1.351 (Beta) and P.1 (Gamma) variants of SARS-CoV-2 at a population level. We devised a bioinformatics method named COJAC (Co-Occurrence adJusted Analysis and Calling) that uses read pairs carrying multiple variant-specific signature mutations as a robust indicator of low-frequency variants. Application of COJAC revealed that a local outbreak of the Alpha variant in two Swiss cities was observable in wastewater up to 13 d before being first reported in clinical samples. We further confirmed the ability of COJAC to detect emerging variants early for the Delta variant by analysing an additional 1,339 wastewater samples. While sequencing data of single wastewater samples provide limited precision for the quantification of relative prevalence of a variant, we show that replicate and close-meshed longitudinal sequencing allow for robust estimation not only of the local prevalence but also of the transmission fitness advantage of any variant. We conclude that genomic sequencing and our computational analysis can provide population-level estimates of prevalence and fitness of emerging variants from wastewater samples earlier and on the basis of substantially fewer samples than from clinical samples. Our framework is being routinely used in large national projects in Switzerland and the UK.

Genotype-dependent kinetics of enterovirus inactivation by free chlorine and ultraviolet (UV) irradiation.

Inactivation kinetics of enterovirus by disinfection is often studied using a single laboratory strain of a given genotype. Environmental variants of enterovirus are genetically distinct from the corresponding laboratory strain, yet it is poorly understood how these genetic differences affect inactivation. Here we evaluated the inactivation kinetics of nine coxsackievirus B3 (CVB3), ten coxsackievirus B4 (CVB4), and two echovirus 11 (E11) variants by free chlorine and ultraviolet irradiation (UV). The inactivation kinetics by free chlorine were genotype- (i.e., susceptibility: CVB5 < CVB3 ≈ CVB4 < E11) and genogroup-dependent and exhibited up to 15-fold difference among the tested viruses. In contrast, only minor (up to 1.3-fold) differences were observed in the UV inactivation kinetics. The differences in variability between the two disinfectants could be rationalized by their respective inactivation mechanisms: inactivation by UV mainly depends on the genomic size and composition, which was similar for all viruses tested, whereas free chlorine targets the viral capsid protein, which exhibited critical differences between genogroups and genotypes. Finally, we integrated the observed variability in inactivation rate constants into an expanded Chick-Watson model to estimate the overall inactivation of an enterovirus consortium. The results highlight that the distribution of inactivation rate constants and the abundance of each genotype are essential parameters to accurately predict the overall inactivation of an enterovirus population by free chlorine. We conclude that predictions based on inactivation data of a single variant or reference pathogen alone likely overestimate the true disinfection efficiency of free chlorine.

Bacterial matrix metalloproteases and serine proteases contribute to the extra-host inactivation of enteroviruses in lake water.

Enteroviruses are ubiquitous contaminants of surface waters, yet their fate in presence of microbial congeners is poorly understood. In this work, we investigated the inactivation of Echovirus-11 (E11) and Coxsackievirus-A9 (CVA9) by bacteria isolated from Lake Geneva. Incubation of E11 or CVA9 in biologically active lake water caused inactivation of 2- and 4-log10, respectively, within 48 h. To evaluate the antiviral action of individual bacterial species, we isolated 136 bacterial strains belonging to 31 genera from Lake Geneva. The majority of isolates (92) induced decay of at least 1.5-log10 of CVA9, whereas only 13 isolates induced a comparable inactivation on E11. The most extensive viral decay was induced by bacterial isolates producing matrix metalloproteases (MMPs). Correspondingly, the addition of a specific MMP inhibitor to lake water reduced the extent of inactivation for both viruses. A lesser, though significant protective effect was also observed with inhibitors of chymotrypsin-like or trypsin-like proteases, suggesting involvement of serine proteases in enterovirus inactivation in natural systems. Overall, we demonstrate the direct effect of bacterial proteases on the inactivation of enteroviruses and identify MMPs as effective controls on enteroviruses' environmental persistence.

Wastewater-Based Estimation of the Effective Reproductive Number of SARS-CoV-2.

BACKGROUND: The effective reproductive number, Re, is a critical indicator to monitor disease dynamics, inform regional and national policies, and estimate the effectiveness of interventions. It describes the average number of new infections caused by a single infectious person through time. To date, Re estimates are based on clinical data such as observed cases, hospitalizations, and/or deaths. These estimates are temporarily biased when clinical testing or reporting strategies change. OBJECTIVES: We show that the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater can be used to estimate Re in near real time, independent of clinical data and without the associated biases. METHODS: We collected longitudinal measurements of SARS-CoV-2 RNA in wastewater in Zurich, Switzerland, and San Jose, California, USA. We combined this data with information on the temporal dynamics of shedding (the shedding load distribution) to estimate a time series proportional to the daily COVID-19 infection incidence. We estimated a wastewater-based Re from this incidence. RESULTS: The method to estimate Re from wastewater worked robustly on data from two different countries and two wastewater matrices. The resulting estimates were as similar to the Re estimates from case report data as Re estimates based on observed cases, hospitalizations, and deaths are among each other. We further provide details on the effect of sampling frequency and the shedding load distribution on the ability to infer Re. DISCUSSION: To our knowledge, this is the first time Re has been estimated from wastewater. This method provides a low-cost, rapid, and independent way to inform SARS-CoV-2 monitoring during the ongoing pandemic and is applicable to future wastewater-based epidemiology targeting other pathogens. https://doi.org/10.1289/EHP10050.

Inferring transmission fitness advantage of SARS-CoV-2 variants of concern from wastewater samples using digital PCR, Switzerland, December 2020 through March 2021.

BackgroundThroughout the COVID-19 pandemic, SARS-CoV-2 genetic variants of concern (VOCs) have repeatedly and independently arisen. VOCs are characterised by increased transmissibility, increased virulence or reduced neutralisation by antibodies obtained from prior infection or vaccination. Tracking the introduction and transmission of VOCs relies on sequencing, typically whole genome sequencing of clinical samples. Wastewater surveillance is increasingly used to track the introduction and spread of SARS-CoV-2 variants through sequencing approaches.AimHere, we adapt and apply a rapid, high-throughput method for detection and quantification of the relative frequency of two deletions characteristic of the Alpha, Beta, and Gamma VOCs in wastewater.MethodsWe developed drop-off RT-dPCR assays and an associated statistical approach implemented in the R package WWdPCR to analyse temporal dynamics of SARS-CoV-2 signature mutations (spike Δ69-70 and ORF1a Δ3675-3677) in wastewater and quantify transmission fitness advantage of the Alpha VOC.ResultsBased on analysis of Zurich wastewater samples, the estimated transmission fitness advantage of SARS-CoV-2 Alpha based on the spike Δ69-70 was 0.34 (95% confidence interval (CI): 0.30-0.39) and based on ORF1a Δ3675-3677 was 0.53 (95% CI: 0.49-0.57), aligning with the transmission fitness advantage of Alpha estimated by clinical sample sequencing in the surrounding canton of 0.49 (95% CI: 0.38-0.61).ConclusionDigital PCR assays targeting signature mutations in wastewater offer near real-time monitoring of SARS-CoV-2 VOCs and potentially earlier detection and inference on transmission fitness advantage than clinical sequencing.

Removal of Waterborne Viruses by Tetrahymena pyriformis Is Virus-Specific and Coincides with Changes in Protist Swimming Speed.

Biological treatment of waterborne viruses, specifically grazing of viruses by protists, can enhance microbial water quality while avoiding the production of toxic byproducts and high energy costs. However, tangible applications are limited by the lack of understanding of the underlying mechanisms. Here, we examined the feeding behavior of Tetrahymena pyriformis ciliates on 13 viruses, including bacteriophages, enteric viruses, and respiratory viruses. Significant differences in virus removal by T. pyriformis were observed, ranging from no removal (Qbeta, coxsackievirus B5) to ≥2.7 log10 (JC polyomavirus) after 48 h of co-incubation of the protist with the virus. Removal rates were conserved even when protists were co-incubated with multiple viruses simultaneously. Video analysis revealed that the extent of virus removal was correlated with an increase in the protists' swimming speed, a behavioral trait consistent with the protists' response to the availability of food. Protistan feeding may be driven by a virus' hydrophobicity but was independent of virus size or the presence of a lipid envelope.

An integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method to simultaneously quantify the infectious concentrations of eight environmentally relevant enterovirus serotypes.

Enterovirus (EV) infectivity is typically measured as a bulk parameter, yet EV serotypes vary in their susceptibility to natural and engineered stressors. Here we developed an integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method to simultaneously and specifically quantify the infectious concentrations of eight EV serotypes commonly encountered in sewage (coxsackieviruses A9, B1, B2, B3, B4 and B5, and echoviruses 25 and 30). The method uses two cell lines for virus replication and serotype-specific qPCR primers for quantification. Primers were designed to target multiple environmental strains of a given serotype and displayed high specificity. The ICC-RTqPCR method exhibited a linear calibration range between 50 and 1000 (echoviruses) or 5000 (coxsackieviruses) infectious units per mL. Over this range, measurements were not influenced by the presence of non-target serotypes, and calibration slopes were reproducible for different virus batches and cell ages. The ICC-RTqPCR method was able to accurately quantify the infectious concentration of a virus after inactivation by heat, and the concentration of a virus within a wastewater matrix. This method will be valuable to assess the differing fates of EV serotypes in natural or engineered systems, and to portray the associated changes in EV population composition.