Stability of influenza A virus in droplets and aerosols is heightened by the presence of commensal respiratory bacteria.

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.

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.

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.

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.

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.

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.

Salt Enhances the Thermostability of Enteroviruses by Stabilizing Capsid Protein Interfaces.

Enteroviruses are common agents of infectious disease that are spread by the fecal-oral route. They are readily inactivated by mild heat, which causes the viral capsid to disintegrate or undergo conformational change. While beneficial for the thermal treatment of food or water, this heat sensitivity poses challenges for the stability of enterovirus vaccines. The thermostability of an enterovirus can be modulated by the composition of the suspending matrix, though the effects of the matrix on virus stability are not understood. Here, we determined the thermostability of four enterovirus strains in solutions with various concentrations of NaCl and different pH values. The experimental findings were combined with molecular modeling of the protein interaction forces at the pentamer and the protomer interfaces of the viral capsids. While pH only had a modest effect on thermostability, increasing NaCl concentrations raised the breakpoint temperatures of all viruses tested by up to 20°C. This breakpoint shift could be explained by an enhancement of the van der Waals attraction forces at the two protein interfaces. In comparison, the (net repulsive) electrostatic interactions were less affected by NaCl. Depending on the interface considered, the breakpoint temperature shifted by 7.5 or 5.6°C per 100-kcal/(mol·Å) increase in protein interaction force.IMPORTANCE The genus Enterovirus encompasses important contaminants of water and food (e.g., coxsackieviruses), as well as viruses of acute public health concern (e.g., poliovirus). Depending on the properties of the surrounding matrix, enteroviruses exhibit different sensitivities to heat, which in turn influences their persistence in the environment, during food treatment, and during vaccine storage. Here, we determined the effect of NaCl and pH on the heat stability of different enteroviruses and related the observed effects to changes in protein interaction forces in the viral capsid. We demonstrate that NaCl renders enteroviruses thermotolerant and that this effect stems from an increase in van der Waals forces at different protein subunits in the viral capsid. This work sheds light on the mechanism by which salt enhances virus stability.

Control of Waterborne Human Viruses by Indigenous Bacteria and Protists Is Influenced by Temperature, Virus Type, and Microbial Species.

Human viruses are ubiquitous contaminants in surface waters, where they can persist over extended periods of time. Among the factors governing their environmental persistence, the control (removal or inactivation) by microorganisms remains poorly understood. Here, we determined the contribution of indigenous bacteria and protists to the decay of human viruses in surface waters. Incubation of echovirus 11 (E11) in freshwater from Lake Geneva and seawater from the Mediterranean Sea led to a 2.5-log10 reduction in the infectious virus concentration within 48 h at 22°C, whereas E11 was stable in sterile controls. The observed virus reduction was attributed to the action of both bacteria and protists in the biologically active matrices. The effect of microorganisms on viruses was temperature dependent, with a complete inhibition of microbial virus control in lake water at temperatures of ≤16°C. Among three protist isolates tested (Paraphysomonas sp., Uronema marinum, and Caecitellus paraparvulus), Caecitellus paraparvulus was particularly efficient at controlling E11 (2.1-log10 reduction over 4 days with an initial protist concentration of 103 cells ml-1). In addition, other viruses (human adenovirus type 2 and bacteriophage H6) exhibited different grazing kinetics than E11, indicating that the efficacy of antiviral action also depended on the type of virus. In conclusion, indigenous bacteria and protists in lake water and seawater can modulate the persistence of E11. These results pave the way for further research to understand how microorganisms control human viral pathogens in aquatic ecosystems and to exploit this process as a treatment solution to enhance microbial water safety.IMPORTANCE Waterborne human viruses can persist in the environment, causing a risk to human health over long periods of time. In this work, we demonstrate that in both freshwater and seawater environments, indigenous bacteria and protists can graze on waterborne viruses and thereby reduce their persistence. We furthermore demonstrate that the efficiency of the grazing process depends on temperature, virus type, and protist species. These findings may facilitate the design of biological methods for the disinfection of water and wastewater.

Adaptation of Human Enterovirus to Warm Environments Leads to Resistance against Chlorine Disinfection.

Sunlight, temperature, and microbial grazing are among the environmental factors promoting the inactivation of viral pathogens in surface waters. Globally, these factors vary across time and space. The persistence of viral pathogens, and ultimately their ecology and dispersion, hinges on their ability to withstand the environmental conditions encountered. To understand how virus populations evolve under changing environmental conditions, we experimentally adapted echovirus 11 (E11) to four climate regimes. Specifically, we incubated E11 in lake water at 10 and 30 °C and in the presence and absence of sunlight. Temperature was the main driver of adaptation, resulting in an increased thermotolerance of the 30 °C adapted populations, whereas the 10 °C adapted strains were rapidly inactivated at higher temperatures. This finding is consistent with a source-sink model in which strains emerging in warm climates can persist in temperate regions, but not vice versa. A microbial risk assessment revealed that the enhanced thermotolerance increases the length of time in which there is an elevated probability of illness associated with swimming in contaminated water. Notably, 30 °C-adapted viruses also exhibited an increased tolerance toward disinfection by free chlorine. Viruses adapting to warm environments may thus become harder to eliminate by common disinfection strategies.

Viral Transfer and Inactivation through Zooplankton Trophic Interactions.

Waterborne viruses are responsible for numerous diseases and are abundant in aquatic systems. Understanding the fate of viruses in natural systems has important implications for human health. This research quantifies the uptake of the bacteriophage T4 and the enteric virus echovirus 11 when exposed to the filter feeders Tetrahymena pyriformis and Daphnia magna, and also examines the potential of viral transfer due to trophic interactions. Experiments co-incubating each species with the viruses over 72-96 h showed up to a 4 log virus removal for T. pyriformis, while direct viral uptake by D. magna was not observed. However, viral uptake by D. magna occurred indirectly by viral transfer from prey to predator, through D. magna feeding on virus-loaded T. pyriformis. This prey-predator interaction resulted in a 1 log additional virus removal compared to removal by T. pyriformis alone. Incomplete viral inactivation by D. magna was observed through recovery of infective viruses from the daphnid tissue. This research furthers our understanding of the impacts of zooplankton filter feeding on viral inactivation and shows the potential for viral transfer through the food chain. The viral-zooplankton interactions observed in these studies indicate that zooplankton may improve water quality through viral uptake or may serve as vectors for infection by accumulating viruses.

UV Disinfection of Human Norovirus: Evaluating Infectivity Using a Genome-Wide PCR-Based Approach.

The removal and inactivation of infectious human norovirus (HuNoV) is a major focus in water purification, but the effectiveness of disinfection processes on norovirus is largely unknown owing to the lack of a readily available infectivity assay. In particular, norovirus behavior through unit processes may be over- or underestimated using current approaches for assessing HuNoV infectivity (e.g., surrogates, molecular methods). Here, we fill a critical knowledge gap by estimating inactivation data for HuNoV after exposure to UV254, a commonly used disinfection process in the water industry. Specifically, we used a PCR-based approach that accurately tracks positive-sense single-stranded RNA virus inactivation without relying on culturing methods. We first confirmed that the approach is valid with a culturable positive-sense single-stranded RNA human virus, coxsackievirus B5, by applying both qPCR- and culture-based methods to measure inactivation kinetics with UV254 treatment. We then applied the qPCR-based method to establish a UV254 inactivation curve for HuNoV (inactivation rate constant = 0.27 cm2 mJ-1). Based on a comparison with previously published data, HuNoV exhibited similar UV254 susceptibility compared with other enteric single-stranded RNA viruses (e.g., Echovirus 12, feline calicivirus) but degraded much faster than MS2 (inactivation rate constant = 0.14 cm2 mJ-1). In addition to establishing a HuNoV inactivation rate constant, we developed an approach using a single qPCR assay that can be applied to estimate HuNoV inactivation in UV254 disinfection systems.

Global Sensitivity Analysis of Environmental, Water Quality, Photoreactivity, and Engineering Design Parameters in Sunlight Inactivation of Viruses.

Sunlight-mediated inactivation of microorganisms is a low-cost approach to disinfect drinking water and wastewater. The reactions involved are affected by a wide range of factors, and a lack of knowledge about their relative importance makes it challenging to optimize treatment systems. To characterize the relative importance of environmental conditions, photoreactivity, water quality, and engineering design in the sunlight inactivation of viruses, we modeled the inactivation of three-human adenovirus and two bacteriophages-MS2 and phiX174-in surface waters and waste stabilization ponds by integrating solar irradiance and aquatic photochemistry models under uncertainty. Through global sensitivity analyses, we quantitatively apportioned the variability of predicted sunlight inactivation rate constants to different factors. Most variance was associated with the variability in and interactions among time, location, nonpurgeable organic carbon (NPOC) concentration, and pond depth. The photolysis quantum yield of the virus outweighed the seasonal solar motion in the impact on inactivation rates. Further, comparison of simulated sunlight inactivation efficacy in maturation ponds under different design decisions showed that reducing pond depth can increase the log inactivation at the cost of larger land area, but increasing hydraulic retention time by adding ponds in series yielded greater improvements in inactivation.

The Metal Catalyst Influences the Kinetics and Mechanisms of MS2 Inactivation in Fenton-like Systems.

Human enteric viruses are frequent microbial contaminants of surface water and groundwater. Waterborne viruses can be effectively inactivated by oxidants, such as those generated in Fenton-like systems. However, the mechanisms by which this inactivation occurs are not understood. Here we investigated how two Fenton-like systems, Cu/H2O2 and Fe/H2O2/light, affect the infectivity and structural integrity of MS2 coliphage, a frequently used surrogate for human enteric viruses. The extent of MS2 genome and capsid protein degradation was evaluated by quantitative PCR and protein mass spectrometry, and was related to the observed level of inactivation. Even though inactivation in both systems occurred via the same oxidant, hydroxyl radical, the contributions of genome and capsid protein degradation to inactivation differed. Inactivation in the Cu/H2O2 system was rapid and involved both genome and protein damage. In contrast, inactivation in Fe/H2O2/light proceeded at a slower rate and encompassed solely genome damage. Our findings demonstrate that not only the oxidant, but also its source, the metal catalyst, determines the inactivation kinetics and mechanism in Fenton-like systems. This work provides the first evidence of the impact of the metal catalyst on virus inactivation in Fenton-like systems.

Differences in Viral Disinfection Mechanisms as Revealed by Quantitative Transfection of Echovirus 11 Genomes.

Virus inactivation mechanisms can be elucidated by methods that measure the loss of specific virus functionality (e.g., host attachment, genome internalization, and genome replication). Genome functionality is frequently assessed by PCR-based methods, which are indirect and potentially inaccurate; genome damage that affects detection by high-fidelity PCR enzymes may not adversely affect the ability of actual cellular enzymes to produce functional virus. Therefore, we developed here a transfection-based assay to quantitatively determine viral genome functionality by inserting viral RNA into host cells directly to measure their ability to produce new functional viruses from damaged viral genomes. Echovirus 11 was treated with ozone, free chlorine (FC), UV light at 254 nm (UV254), or heat, and then the reductions in genome functionality and infectivity were compared. Ozone reduced genome functionality proportionally to infectivity, indicating that genome damage is the main mechanism of virus inactivation. In contrast, FC caused little or no loss of genome functionality compared to infectivity, indicating a larger role for protein damage. For UV254, genome functionality loss accounted for approximately 60% of virus inactivation, with the remainder presumably due to protein damage. Heat treatment resulted in no reduction in genome functionality, in agreement with the understanding that heat inactivation results from capsid damage. Our results indicate that there is a fundamental difference between genome integrity reductions measured by PCR enzymes in previous studies and actual genome functionality (whether the genome can produce virus) after disinfection. Compared to PCR, quantitative transfection assays provide a more realistic picture of actual viral genome functionality and overall inactivation mechanisms during disinfection.IMPORTANCE This study provides a new tool for assessing virus inactivation mechanisms by directly measuring a viral genome's ability to produce new viruses after disinfection. In addition, we identify a potential pitfall of PCR for determining virus genome damage, which does not reflect whether a genome is truly functional. The results presented here using quantitative transfection corroborate previously suggested virus inactivation mechanisms for some virus inactivation methods (heat) while bringing additional insights for others (ozone, FC, and UV254). The developed transfection method provides a more mechanistic approach for the assessment of actual virus inactivation by common water disinfectants.

Health Risks for Sanitation Service Workers along a Container-Based Urine Collection System and Resource Recovery Value Chain.

Container-based sanitation (CBS) within a comprehensive service system value chain offers a low-cost sanitation option with potential for revenue generation but may increase microbial health risks to sanitation service workers. This study assessed occupational exposure to rotavirus and Shigella spp. during CBS urine collection and subsequent struvite fertilizer production in eThekwini, South Africa. Primary data included high resolution sequences of hand-object contacts from annotated video and measurement of fecal contamination from urine and surfaces likely to be contacted. A stochastic model incorporated chronological surface contacts, pathogen concentrations in urine, and literature data on transfer efficiencies of pathogens to model pathogen concentrations on hands and risk of infection from hand-to-mouth contacts. The probability of infection was highest from exposure to rotavirus during urine collection (∼10-1) and struvite production (∼10-2), though risks from Shigella spp. during urine collection (∼10-3) and struvite production (∼10-4) were non-negligible. Notably, risk of infection was higher during urine collection than during struvite production due to contact with contaminated urine transport containers. In the scale-up of CBS, disinfection of urine transport containers is expected to reduce pathogen transmission. Exposure data from this study can be used to evaluate the effectiveness of measures to protect sanitation service workers.

Transfer of Enteric Viruses Adenovirus and Coxsackievirus and Bacteriophage MS2 from Liquid to Human Skin.

Indirect exposure to waterborne viruses increases the risk of infection, especially among children with frequent hand-to-mouth contacts. Here, we quantified the transfer of one bacteriophage (MS2) and two enteric viruses (adenovirus and coxsackievirus) from liquid to skin. MS2, a commonly used enteric virus surrogate, was used to compare virus transfer rates in a volunteer trial to those obtained using human cadaver skin and synthetic skin. MS2 transfer to volunteer skin was similar to transfer to cadaver skin but significantly different from transfer to synthetic skin. The transfer of MS2, adenovirus, and coxsackievirus to cadaver skin was modeled using measurements for viruses attaching to the skin (adsorbed) and viruses in liquid residual on skin (unadsorbed). We find virus transfer per surface area is a function of the concentration of virus in the liquid and the film thickness of liquid retained on the skin and is estimable using a linear model. Notably, the amount of MS2 adsorbed on the skin was on average 5 times higher than the amount of adenovirus and 4 times higher than the amount of coxsackievirus. Quantification of pathogenic virus retention to skin would thus be overestimated using MS2 adsorption data. This study provides models of virus transfer useful for risk assessments of water-related activities, demonstrates significant differences in the transfer of pathogenic virus and MS2, and suggests cadaver skin as an alternative testing system for studying interactions between viruses and skin.IMPORTANCE Enteric viruses (viruses that infect the gastrointestinal tract) are responsible for most water-transmitted diseases. They are shed in high concentrations in the feces of infected individuals, persist for an extended period of time in water, and are highly infective. Exposure to contaminated water directly (through ingestion) or indirectly (for example, through hand-water contacts followed by hand-to-mouth contacts) increases the risk of virus transmission. The work described herein provides a quantitative model for estimating human-pathogenic virus retention on skin following contact with contaminated water. The work will be important in refining the contribution of indirect transmission of virus to risks associated with water-related activities.

Kinetics of Inactivation of Waterborne Enteric Viruses by Ozone.

Ozone is an effective disinfectant against all types of waterborne pathogens. However, accurate and quantitative kinetic data regarding virus inactivation by ozone are scarce, because of the experimental challenges associated with the high reactivity of ozone toward viruses. Here, we established an experimental batch system that allows tailoring and quantifying of very low ozone exposures and simultaneously measuring virus inactivation. Second-order ozone inactivation rate constants (kO3-virus) of five enteric viruses [laboratory and two environmental strains of coxsackievirus B5 (CVF, CVEnv1, and CVEnv2), human adenovirus (HAdV), and echovirus 11 (EV)] and four bacteriophages (MS2, Qß, T4, and Φ174) were measured in buffered solutions. The kO3-virus values of all tested viruses ranged from 4.5 × 105 to 3.3 × 106 M-1 s-1. For MS2, kO3-MS2 depended only weakly on temperature (2-22 °C; Ea = 22.2 kJ mol-1) and pH (6.5-8.5), with an increase in kO3-MS2 with increasing pH. The susceptibility of the selected viruses toward ozone decreases in the following order: Qß > CVEnv2 > EV ≈ MS2 > Φ174 ≈ T4 > HAdV > CVF ≈ CVEnv1. On the basis of the measured kO3-Virus and typical ozone exposures applied in water and wastewater treatment, we conclude that ozone is a highly effective disinfectant for virus control.