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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.
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Proteínas do Capsídeo , Capsídeo , Serina Proteases , Capsídeo/metabolismo , Serina Proteases/metabolismo , Serina Proteases/química , Serina Proteases/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/química , Humanos , Enterovirus/enzimologia , Enterovirus/fisiologia , Animais , Enterovirus Humano B/fisiologia , Enterovirus Humano B/enzimologiaRESUMO
Aerosol transmission remains a major challenge for control of respiratory viruses, particularly those causing recurrent epidemics, like influenza A virus (IAV). These viruses are rarely expelled alone, but instead are embedded in a consortium of microorganisms that populate the respiratory tract. The impact of microbial communities and inter-pathogen interactions upon stability of transmitted viruses is well-characterized for enteric pathogens, but is under-studied in the respiratory niche. Here, we assessed whether the presence of five different species of commensal respiratory bacteria could influence the persistence of IAV within phosphate-buffered saline and artificial saliva droplets deposited on surfaces at typical indoor air humidity, and within airborne aerosol particles. In droplets, presence of individual species or a mixed bacterial community resulted in 10- to 100-fold more infectious IAV remaining after 1 h, due to bacterial-mediated flattening of drying droplets and early efflorescence. Even when no efflorescence occurred at high humidity or the bacteria-induced changes in droplet morphology were abolished by aerosolization instead of deposition on a well plate, the bacteria remained protective. Staphylococcus aureus and Streptococcus pneumoniae were the most stabilizing compared to other commensals at equivalent density, indicating the composition of an individual's respiratory microbiota is a previously unconsidered factor influencing expelled virus persistence.IMPORTANCEIt is known that respiratory infections such as coronavirus disease 2019 and influenza are transmitted by release of virus-containing aerosols and larger droplets by an infected host. The survival time of viruses expelled into the environment can vary depending on temperature, room air humidity, UV exposure, air composition, and suspending fluid. However, few studies consider the fact that respiratory viruses are not alone in the respiratory tract-we are constantly colonized by a plethora of bacteria in our noses, mouth, and lower respiratory system. In the gut, enteric viruses are known to be stabilized against inactivation and environmental decay by gut bacteria. Despite the presence of a similarly complex bacterial microbiota in the respiratory tract, few studies have investigated whether viral stabilization could occur in this niche. Here, we address this question by investigating influenza A virus stabilization by a range of commensal bacteria in systems representing respiratory aerosols and droplets.
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Aerossóis , Vírus da Influenza A , Vírus da Influenza A/fisiologia , Humanos , Staphylococcus aureus/fisiologia , Streptococcus pneumoniae/fisiologia , Sistema Respiratório/microbiologia , Sistema Respiratório/virologia , Animais , Influenza Humana/virologia , Influenza Humana/transmissão , Bactérias , Microbiota , Cães , Simbiose , Células Madin Darby de Rim CaninoRESUMO
Wastewater-based epidemiology offers a complementary approach to clinical case-based surveillance of emergent diseases and can help identify regions with infected people to prioritize clinical surveillance strategies. However, tracking emergent diseases in wastewater requires reliance on novel testing assays with uncertain sensitivity and specificity. Limited pathogen shedding may cause detection to be below the limit of quantification or bordering the limit of detection. Here, we investigated how the definition of limit of detection for quantitative polymerase chain reaction (qPCR) impacts epidemiological insights during an mpox outbreak in Switzerland. 365 wastewater samples from three wastewater treatment plants in Switzerland from 9 March through 31 October 2022 were analyzed for mpox DNA using qPCR. We detected mpox DNA in 22% (79 of 365) wastewater samples based on a liberal definition of qPCR detection as any exponentially increasing fluorescence above the threshold. Based on a more restrictive definition as the lowest concentration at which there is 95% likelihood of detection, detection was 1% (5 of 365). The liberal definition shows high specificity (90%) and accuracy (78%), but moderate sensitivity (64%) when benchmarked against available clinical case reporting, which contrasts with higher specificity (98%) but lower sensitivity (10%) and accuracy (56%) of the 95% likelihood definition. Wastewater-based epidemiology applied to an emergent pathogen will require optimizing public health trade-offs between reporting data with high degrees of uncertainty and delaying communication and associated action. Information sharing with relevant public health stakeholders could couple early results with clear descriptions of uncertainty.Impact Statement: When a novel pathogen threatens to enter a community, wastewater-based epidemiology offers an opportunity to track its emergence and spread. However, rapid deployment of methods for to detect a novel pathogen may rely on assays with uncertain sensitivity and specificity. Benchmarking the detection of mpox DNA in Swiss wastewaters with reported clinical cases in 2022, we demonstrate how definitions of detection of a qPCR assay influence epidemiological insights from wastewater. The results highlight the need for information sharing between public health stakeholders that couple early insights from wastewater with descriptions of methodological uncertainty to optimize public health actions.
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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.
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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.
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Proteínas do Capsídeo , Cloro , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/química , Cloro/farmacologia , Microscopia Crioeletrônica , Substituição de Aminoácidos , Enterovirus Humano B/genética , AminoácidosRESUMO
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.
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Vírus da Influenza A , Influenza Humana , Muco , Neuraminidase , Animais , Humanos , Aves , Vírus da Influenza A/metabolismo , Muco/metabolismo , Neuraminidase/metabolismo , Sistema Respiratório/metabolismoRESUMO
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.
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Vírus da Influenza A , Influenza Humana , Humanos , Vírus da Influenza A/fisiologia , Aerossóis e Gotículas Respiratórios , Concentração de Íons de HidrogênioRESUMO
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.
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Adenovírus Humanos , Tetrahymena pyriformis , Humanos , Tetrahymena pyriformis/fisiologia , Água Doce , Microscopia Eletrônica de Transmissão , AdenoviridaeRESUMO
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.
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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.
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Desinfetantes , Enterovirus , Purificação da Água , Cloro/farmacologia , Desinfecção , Desinfetantes/farmacologia , Enterovirus Humano B , CinéticaRESUMO
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.
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Enterovirus , Norovirus , Vírus , Humanos , Lagos , Esgotos , Microbiologia da Água , Monitoramento AmbientalRESUMO
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 â¼ 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.
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Poluição do Ar em Ambientes Fechados , COVID-19 , Aerossóis e Gotículas Respiratórios , Humanos , Concentração de Íons de Hidrogênio , SARS-CoV-2 , Inativação de Vírus , Transmissão de Doença InfecciosaRESUMO
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.
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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.
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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.
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COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , COVID-19/epidemiologia , Genômica , Humanos , SARS-CoV-2/genética , Águas ResiduáriasRESUMO
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.
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Desinfetantes , Enterovirus , Vírus , Purificação da Água , Cloro/farmacologia , Desinfecção/métodos , Enterovirus Humano B , Genótipo , Cinética , Raios Ultravioleta , Inativação de Vírus , Purificação da Água/métodosRESUMO
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.