Linear Mixed Model of Virus Disinfection by Free Chlorine to Harmonize Data Collected across Broad Environmental Conditions.

Despite the critical importance of virus disinfection by chlorine, our fundamental understanding of the relative susceptibility of different viruses to chlorine and robust quantitative relationships between virus disinfection rate constants and environmental parameters remains limited. We conducted a systematic review of virus inactivation by free chlorine and used the resulting data set to develop a linear mixed model that estimates chlorine inactivation rate constants for viruses based on experimental conditions. 570 data points were collected in our systematic review, representing 82 viruses over a broad range of environmental conditions. The harmonized inactivation rate constants under reference conditions (pH = 7.53, T = 20 °C, [Cl-] < 50 mM) spanned 5 orders of magnitude, ranging from 0.0196 to 1150 L mg-1 min-1, and uncovered important trends between viruses. Whereas common surrogate bacteriophage MS2 does not serve as a conservative chlorine disinfection surrogate for many human viruses, CVB5 was one of the most resistant viruses in the data set. The model quantifies the role of pH, temperature, and chloride levels across viruses, and an online tool allows users to estimate rate constants for viruses and conditions of interest. Results from the model identified potential shortcomings in current U.S. EPA drinking water disinfection requirements.

The Protective Effect of Virus Capsids on RNA and DNA Virus Genomes in Wastewater.

Virus concentrations measured in municipal wastewater help inform both the water treatment necessary to protect human health and wastewater-based epidemiology. Wastewater measurements are typically PCR-based, and interpreting gene copy concentrations requires an understanding of the form and stability of the nucleic acids. Here, we study the persistence of model virus genomes in wastewater, the protective effects provided by the virus capsids, and the relative decay rates of the genome and infectious viruses. In benchtop batch experiments in wastewater influent at 25 °C, extraviral (+)ssRNA and dsDNA amplicons degraded by 90% within 15-19 min and 1.6-1.9 h, respectively. When encapsidated, the T90 for MS2 (+)ssRNA increased by 424× and the T90 for T4 dsDNA increased by 52×. The (+)ssRNA decay rates were similar for a range of amplicon sizes. For our model phages MS2 and T4, the nucleic acid signal in untreated wastewater disappeared shortly after the viruses lost infectivity. Combined, these results suggest that most viral genome copies measured in wastewater are encapsidated, that measured concentrations are independent of assay amplicon sizes, and that the virus genome decay rates of nonenveloped (i.e., naked) viruses are similar to inactivation rates. These findings are valuable for the interpretation of wastewater virus measurements.

Estimating Relative Abundance of 2 SARS-CoV-2 Variants through Wastewater Surveillance at 2 Large Metropolitan Sites, United States.

Monitoring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) is critical for public health management of coronavirus disease. Sequencing is resource-intensive and incompletely representative, and not all isolates can be sequenced. Because wastewater SARS-CoV-2 RNA concentrations correlate with coronavirus disease incidence in sewersheds, tracking VOCs through wastewater is appealing. We developed digital reverse transcription PCRs to monitor abundance of select mutations in Alpha and Delta VOCs in wastewater settled solids, applied these to July 2020-August 2021 samples from 2 large US metropolitan sewersheds, and compared results to estimates of VOC abundance from case isolate sequencing. Wastewater measurements tracked closely with case isolate estimates (Alpha, rp 0.82-0.88; Delta, rp 0.97). Mutations were detected in wastewater even at levels <5% of total SARS-CoV-2 RNA and in samples available 1-3 weeks before case isolate results. Wastewater variant monitoring should be strategically deployed to complement case isolate sequencing.

Detection of SARS-CoV-2 Variants Mu, Beta, Gamma, Lambda, Delta, Alpha, and Omicron in Wastewater Settled Solids Using Mutation-Specific Assays Is Associated with Regional Detection of Variants in Clinical Samples.

Changes in the circulation of SARS-CoV-2 variants of concern (VOCs) may require changes in the public health response to the COVID-19 pandemic, as they have the potential to evade vaccines and pharmaceutical interventions and may be more transmissive than other SARS-CoV-2 variants. As such, it is essential to track and prevent their spread in susceptible communities. We developed digital reverse transcription (RT)-PCR assays for mutations characteristic of VOCs and used them to quantify those mutations in samples of wastewater settled solids collected from a publicly owned treatment works (POTW) during different phases of the COVID-19 pandemic. Wastewater concentrations of single mutations characteristic of each VOC, normalized by the concentration of a conserved SARS-CoV-2 N gene, correlate with regional estimates of the proportion of clinical infections caused by each VOC. These results suggest that targeted RT-PCR assays can be used to detect variants circulating in communities and inform the public health response to the pandemic. IMPORTANCE Wastewater represents a pooled biological sample of the contributing community and thus a resource for assessing community health. Here, we show that emergence, spread, and disappearance of SARS-CoV-2 infections caused by variants of concern are reflected in the presence of variant genomic RNA in wastewater settled solids. This work highlights an important public health use case for wastewater.

Reactivity of Viral Nucleic Acids with Chlorine and the Impact of Virus Encapsidation.

Free chlorine disinfection is widely applied to inactivate viruses by reacting with their biomolecules, which include nucleic acids, proteins, and lipids. Knowing the reactivities of viral genomes with free chlorine and the protection that encapsidation provides would ultimately help predict virus susceptibility to the disinfectant. The relative reactivities of different viral genome types and the impact of viral higher order structure with free chlorine are poorly characterized. Here, we studied the reactivity of viral genomes representing four genome types from virus particles with diverse structures, namely, (+)ssRNA (MS2), dsRNA (φ6), ssDNA (φX174), and dsDNA (T3) with free chlorine. We compared the reactivities of these viral nucleic acids when they were suspended in phosphate buffer solutions (naked forms) and when they were in the native virus particles (encapsidated forms). The reactivities of nucleic acids were tracked by polymerase chain reaction (PCR)-based assays. The naked dsDNA of T3 was the least reactive with free chlorine, with an average second order rate constant normalized by the number of bases in the measured regions (in M-1 s-1 b-1) that was 34×, 65×, and 189× lower than those of the dsRNA of φ6, ssRNA of MS2, and ssDNA of φX174, respectively. Moreover, different regions in the ssRNA genome of MS2 and the dsRNA genome of φ6 exhibited statistically different reaction kinetics. The genomes within virus particles reacted slower than the naked genomes overall, but the extent of these differences varied among the four viruses. The results on viral nucleic acid reactivity help explain different susceptibilities of viruses to inactivation by free chlorine and also provide a valuable comparison of the susceptibilities of different nucleic acids to oxidants.

Chloride Enhances DNA Reactivity with Chlorine under Conditions Relevant to Water Treatment.

Free available chlorine (FAC) is widely used to inactivate viruses by oxidizing viral components, including genomes. It is commonly assumed that hypochlorous acid (HOCl) is the chlorinating agent responsible for virus inactivation; however, recent studies have underscored that minor constituents of FAC existing in equilibrium with HOCl, such as molecular chlorine (Cl2), can influence FAC reactivity toward select organic compounds. This study measures the FAC reaction kinetics with dsDNA and ssDNA extracted from representative bacteriophages (T3 and ϕX174) in samples augmented with chloride. Herein, chloride enhances FAC reactivity toward dsDNA and, to a lesser extent, toward ssDNA, especially at pH < 7.5. The enhanced reactivity can be attributed to the formation of Cl2. Second-order rate constants were determined for reactions of ssDNA and dsDNA with HOCl and Cl2. DNA chlorination kinetics followed the reactivity-selectivity principle, where the more-reactive nucleophilic species (ssDNA, ∼100× more reactive than dsDNA) reacted less selectively with electrophilic FAC species. The addition of chloride was also shown to enhance the inactivation of bacteriophage T3 (dsDNA genome) by FAC but did not enhance the inactivation of bacteriophage ϕX174 (ssDNA genome). Overall, the results suggest that Cl2 is an important chlorinating agent of nucleic acids and viruses.

Predictive Modeling of Virus Inactivation by UV.

UV254 disinfection strategies are commonly applied to inactivate pathogenic viruses in water, food, air, and on surfaces. There is a need for methods that rapidly predict the kinetics of virus inactivation by UV254, particularly for emerging and difficult-to-culture viruses. We conducted a systematic literature review of inactivation rate constants for a wide range of viruses. Using these data and virus characteristics, we developed and evaluated linear and nonlinear models for predicting inactivation rate constants. Multiple linear regressions performed best for predicting the inactivation kinetics of (+) ssRNA and dsDNA viruses, with cross-validated root mean squared relative prediction errors similar to those associated with experimental rate constants. We tested the models by predicting and measuring inactivation rate constants of a (+) ssRNA mouse coronavirus and a dsDNA marine bacteriophage; the predicted rate constants were within 7% and 71% of the experimental rate constants, respectively, indicating that the prediction was more accurate for the (+) ssRNA virus than the dsDNA virus. Finally, we applied our models to predict the UV254 rate constants of several viruses for which high-quality UV254 inactivation data are not available. Our models will be valuable for predicting inactivation kinetics of emerging or difficult-to-culture viruses.

The Environmental Microbiology Minimum Information (EMMI) Guidelines: qPCR and dPCR Quality and Reporting for Environmental Microbiology.

Real-time quantitative polymerase chain reaction (qPCR) and digital PCR (dPCR) methods have revolutionized environmental microbiology, yielding quantitative organism-specific data of nucleic acid targets in the environment. Such data are essential for characterizing interactions and processes of microbial communities, assessing microbial contaminants in the environment (water, air, fomites), and developing interventions (water treatment, surface disinfection, air purification) to curb infectious disease transmission. However, our review of recent qPCR and dPCR literature in our field of health-related environmental microbiology showed that many researchers are not reporting necessary and sufficient controls and methods, which would serve to strengthen their study results and conclusions. Here, we describe the application, utility, and interpretation of the suite of controls needed to make high quality qPCR and dPCR measurements of microorganisms in the environment. Our presentation is organized by the discrete steps and operations typical of this measurement process. We propose systematic terminology to minimize ambiguity and aid comparisons among studies. Example schemes for batching and combining controls for efficient work flow are demonstrated. We describe critical reporting elements for enhancing data credibility, and we provide an element checklist in the Supporting Information. Additionally, we present several key principles in metrology as context for laboratories to devise their own quality assurance and quality control reporting framework. Following the EMMI guidelines will improve comparability and reproducibility among qPCR and dPCR studies in environmental microbiology, better inform engineering and public health actions for preventing disease transmission through environmental pathways, and for the most pressing issues in the discipline, focus the weight of evidence in the direction toward solutions.

SARS-CoV-2 RNA in Wastewater Settled Solids Is Associated with COVID-19 Cases in a Large Urban Sewershed.

Wastewater-based epidemiology may be useful for informing public health response to viral diseases like COVID-19 caused by SARS-CoV-2. We quantified SARS-CoV-2 RNA in wastewater influent and primary settled solids in two wastewater treatment plants to inform the preanalytical and analytical approaches and to assess whether influent or solids harbored more viral targets. The primary settled solids samples resulted in higher SARS-CoV-2 detection frequencies than the corresponding influent samples. Likewise, SARS-CoV-2 RNA was more readily detected in solids using one-step digital droplet (dd)RT-PCR than with two-step RT-QPCR and two-step ddRT-PCR, likely owing to reduced inhibition with the one-step ddRT-PCR assay. We subsequently analyzed a longitudinal time series of 89 settled solids samples from a single plant for SARS-CoV-2 RNA as well as coronavirus recovery (bovine coronavirus) and fecal strength (pepper mild mottle virus) controls. SARS-CoV-2 RNA targets N1 and N2 concentrations correlated positively and significantly with COVID-19 clinically confirmed case counts in the sewershed. Together, the results demonstrate that measuring SARS-CoV-2 RNA concentrations in settled solids may be a more sensitive approach than measuring SARS-CoV-2 in influent.

Fate of Extracellular DNA in the Production of Fertilizers from Source-Separated Urine.

The practice of urine source-separation for fertilizer production necessitates an understanding of the presence and impact of extracellular DNA in the urine. This study examines the fate of plasmid DNA carrying ampicillin and tetracycline resistance genes in aged urine, including its ability to be taken up and expressed by competent bacteria. Plasmid DNA incubated in aged urine resulted in a >2 log loss of bacterial transformation efficiency in Acinetobacter baylyi within 24 h. The concentration of ampicillin and tetracycline resistance genes, as measured with quantitative polymerase chain reaction, did not correspond with the observed transformation loss. When the plasmid DNA was incubated in aged urine that had been filtered (0.22 µm) or heated (75 °C), the transformation efficiencies were more stable than when the plasmids were incubated in unfiltered and unheated aged urine. Gel electrophoresis results indicated that plasmid linearization by materials larger than 100 kDa in the aged urine caused the observed transformation efficiency decreases. The results of this study suggest that extracellular DNA released into aged urine poses a low potential for the spread of antibiotic resistance genes to bacteria once it is released to the environment.

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.

Fate of the Urinary Tract Virus BK Human Polyomavirus in Source-Separated Urine.

Human polyomaviruses are emerging pathogens that infect a large percentage of the human population and are excreted in urine. Consequently, urine that is collected for fertilizer production often has high concentrations of polyomavirus genes. We studied the fate of infectious double-stranded DNA (dsDNA) BK human polyomavirus (BKPyV) in hydrolyzed source-separated urine with infectivity assays and quantitative PCR (qPCR). Although BKPyV genomes persisted in the hydrolyzed urine for long periods of time (T90 [time required for 90% reduction in infectivity or gene copies] of >3 weeks), the viruses were rapidly inactivated (T90 of 1.1 to 11 h) in most of the tested urine samples. Interestingly, the infectivity of dsDNA bacteriophage surrogate T3 (T90 of 24 to 46 days) was much more persistent than that of BKPyV, highlighting a major shortcoming of using bacteriophages as human virus surrogates. Pasteurization and filtration experiments suggest that BKPyV virus inactivation was due to microorganism activity in the source-separated urine, and SDS-PAGE Western blots showed that BKPyV protein capsid disassembly is concurrent with inactivation. Our results imply that stored urine does not pose a substantial risk of BKPyV transmission, that qPCR and infectivity of the dsDNA surrogate do not accurately depict BKPyV fate, and that microbial inactivation is driven by structural elements of the BKPyV capsid.IMPORTANCE We demonstrate that a common urinary tract virus has a high susceptibility to the conditions in hydrolyzed urine and consequently would not be a substantial exposure route to humans using urine-derived fertilizers. The results have significant implications for understanding virus fate. First, by demonstrating that the dsDNA (double-stranded DNA) genome of the polyomavirus lasts for weeks despite infectivity lasting for hours to days, our work highlights the shortcomings of using qPCR to estimate risks from unculturable viruses. Second, commonly used dsDNA surrogate viruses survived for weeks under the same conditions that BK polyomavirus survived for only hours, highlighting issues with using virus surrogates to predict how human viruses will behave in the environment. Finally, our mechanistic inactivation analysis provides strong evidence that microbial activity drives rapid virus inactivation, likely through capsid disassembly. Overall, our work underlines how subtle structural differences between viruses can greatly impact their environmental fate.

Reactivity of Enveloped Virus Genome, Proteins, and Lipids with Free Chlorine and UV254.

The survivability of viruses in natural and engineered systems impacts public health. Inactivation mechanisms in the environment have been described for nonenveloped viruses, but it remains unclear how the membrane layer of enveloped viruses influences inactivation. We applied molecular tools and high-resolution mass spectrometry to measure reactions in the genome, proteins, and lipids of enveloped Pseudomonas phage Phi6 during inactivation by free chlorine and UV254. Free chlorine readily penetrated the lipid membrane to react with proteins in the nucleocapsid and polymerase complex. The most reactive Phi6 peptides were approximately 150 times more reactive with free chlorine than the most reactive peptides reported in nonenveloped coliphage MS2. The inactivation kinetics of Phi6 by UV254 was comparable with those of nonenveloped adenovirus and coliphage MS2 and were driven by UV254 reactions with viral genomes. Our research identifies molecular features of an enveloped virus that are susceptible to chemical oxidants or UV radiation. Finally, the framework developed in the manuscript for studying molecular reactivities of Phi6 can be adopted to investigate enveloped virus survivability under various environmental conditions.

Nucleic Acid Photolysis by UV254 and the Impact of Virus Encapsidation.

Determining the influence of higher order structure on UVC photolysis will help inform predictions of nucleic acid fate and microorganism inactivation. We measured the direct UV254 photolysis kinetics of four model viral genomes composed of single-stranded and double-stranded RNA (ssRNA and dsRNA, respectively), as well as single-stranded and double-stranded DNA (ssDNA and dsDNA, respectively), in ultrapure water, in phosphate buffered saline (PBS), and encapsidated in their native virus particles. The photolysis rate constants of naked nucleic acids measured by qPCR (RT-qPCR for RNA) and normalized by the number of bases measured in a particular sequence exhibited the following trend: ssDNA > ssRNA ≈ dsDNA > dsRNA. In PBS, naked ssRNA bases reacted, on average, 24× faster than the dsRNA bases, whereas naked ssDNA bases reacted 4.3× faster than dsDNA bases. Endogenous indirect photolysis involving 1O2 and ·OH was ruled out as a major contributing factor in the reactions. A comparison of our measured rate constants with rate constants reported in the literature shows a general agreement among the nucleic acid UV254 direct photolysis kinetics. Our results underscore the high resistance of dsRNA to UVC photolysis and demonstrate the role that nucleic acid structure and solution chemistry play in photoreactivity.

Degradation of Extracellular Antibiotic Resistance Genes with UV254 Treatment.

Disinfected wastewater effluent contains a complex mixture of biomolecules including DNA. If intact genes conveying antibiotic resistance survive the disinfection process, environmental bacteria may take them up. We treated plasmid pWH1266, which contains ampicillin resistance gene blaTEM-1 and tetracycline resistance gene tetA, with UV254 doses up to 430 mJ/cm2 and studied the ability of those genes to be acquired by Acinetobacter baylyi. The plasmids required approximately 20-25 mJ/cm2 per log10 loss of transformation efficiency. We monitored plasmid DNA degradation using gel electrophoresis and qPCR with both short amplicons (∼200 bps, representative of ARG amplicon lengths commonly used for environmental monitoring) and long amplicons (800-1200 bps, designed to cover the entire resistance genes). The rate of transformability loss due to UV254 treatment was approximately 20× and 2× larger than the rate of gene degradation measured with the short and long amplicons qPCR, respectively. When extrapolated to account for the length of the entire pWH1266 plasmid, the qPCR rate constants were 2-7× larger than the rate constants measured with transformation assays. Gel electrophoresis results confirmed that DNA cleavage was not a major inactivating mechanism. Overall, our results demonstrate that qPCR conservatively measures the potential for a gene to be transformed by environmental bacteria following UV254 treatment.

Direct and Indirect Photochemical Reactions in Viral RNA Measured with RT-qPCR and Mass Spectrometry.

RNA carries the genetic instructions for many viruses to replicate in their host cells. The photochemical reactions that take place in RNA and affect viral infectivity in natural and engineered environments, however, remain poorly understood. We exposed RNA oligomer segments from the genome of bacteriophage MS2 to UV254, simulated sunlight, and singlet oxygen (1O2) and analyzed the oligomer reaction kinetics with reverse transcription quantitative PCR (RT-qPCR) and quantitative matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Following UV254 exposure, quantitative MALDI-TOF-MS detected significantly more RNA modifications than did RT-qPCR, suggesting that certain chemical modifications in the RNA were not detected by the reverse transcriptase enzyme. In contrast, MALDI-TOF-MS tracked as much 1O2-induced RNA damage as RT-qPCR. After 5 h of simulated sunlight exposure (5100 J/m2 UVB and 1.2 × 105 J/m2 UVA), neither MALDI-TOF-MS nor RT-qPCR detected significant decreases in the oligomer concentrations. High-resolution electrospray ionization (ESI)-Orbitrap MS analyses identified pyrimidine photohydrates as the major UV254 products, which likely contributed to the discrepancy between the MS- and RT-qPCR-based results. Reactions between RNA oligomers and 1O2 resulted in an unidentified major product with a mass change of +6 Da. These results shed light on the photochemical reactions that take place in RNA and suggest that the analytical techniques used to detect RNA reactivity could bias the observed reaction kinetics.