Temperature and particles interact to affect human norovirus and MS2 persistence in surface water.

Modeling the fate and transport of viruses and their genetic material in surface water is necessary to assess risks associated with contaminated surface waters and to inform environmental surveillance efforts. Temperature has been identified as a key variable affecting virus persistence in surface waters, but the effects of the presence of biological and inert particles and of their interaction with temperature have not been well characterized. We assessed these effects on the persistence of human norovirus (HuNoV) genotype II.4 purified from stool and MS2 in surface water. Raw or filter-sterilized creek water microcosms were inoculated and incubated in the dark at 10 °C, 15 °C, and 20 °C. HuNoV (i.e., genome segments and intact capsids) and MS2 (i.e., infectious MS2, genome segments, and intact capsids) concentrations were followed over 36 days. The range in positive, significant first-order decay rate constants for HuNoV in this study was 0.14 to 0.69 day-1 compared with 0.026 to 0.71 day-1 for that of MS2. Decay rate constants for HuNoV genome segments and infectious MS2 were largest in creek water that included biological and inert particles and incubated at higher temperatures. In addition, for HuNoV and MS2 incubated in raw or filter-sterilized creek water at 15 °C, capsid damage was not identified as a dominant inactivation mechanism. Environmental processes and events that affect surface water biological and inert particles, temperature, or both could lead to variable virus decay rate constants. Incorporating the effects of particles, temperature, and their interaction could enhance models of virus fate and transport in surface water.

Persistence of Human Norovirus (GII) in Surface Water: Decay Rate Constants and Inactivation Mechanisms.

Human norovirus (HuNoV) is an important cause of acute gastroenteritis and can be transmitted by water exposures, but its persistence in water is not well understood. Loss of HuNoV infectivity in surface water was compared with persistence of intact HuNoV capsids and genome segments. Surface water from a freshwater creek was filter-sterilized, inoculated with HuNoV (GII.4) purified from stool, and incubated at 15 or 20 °C. We measured HuNoV infectivity via the human intestinal enteroid system and HuNoV persistence via reverse transcription-quantitative polymerase chain reaction assays without (genome segment persistence) or with (intact viral capsid persistence) enzymatic pretreatment to digest naked RNA. For infectious HuNoV, results ranged from no significant decay to a decay rate constant ("k") of 2.2 day-1. In one creek water sample, genome damage was likely a dominant inactivation mechanism. In other samples from the same creek, loss of HuNoV infectivity could not be attributed to genome damage or capsid cleavage. The range in k and the difference in the inactivation mechanism observed in water from the same site could not be explained, but variable constituents in the environmental matrix could have contributed. Thus, a single k may be insufficient for modeling virus inactivation in surface waters.

Microbial Water Quality through a Full-Scale Advanced Wastewater Treatment Demonstration Facility.

The fates of viruses, bacteria, and antibiotic resistance genes during advanced wastewater treatment are important to assess for implementation of potable reuse systems. Here, a full-scale advanced wastewater treatment demonstration facility (ozone, biological activated carbon filtration, micro/ultrafiltration, reverse osmosis, and advanced oxidation) was sampled over three months. Atypically, no disinfectant residual was applied before the microfiltration step. Microbial cell concentrations and viability were assessed via flow cytometry and adenosine triphosphate (ATP). Concentrations of bacteria (16S rRNA gene), viruses (human adenovirus and JC polyomavirus), and antibiotic resistance genes (sul1 and bla TEM ) were assessed via quantitative PCR following the concentration of large sample volumes by dead-end ultrafiltration. In all membrane filtration permeates, microbial concentrations were higher than previously reported for chloraminated membranes, and log10 reduction values were lower than expected. Concentrations of 16S rRNA and sul1 genes were reduced by treatment but remained quantifiable in reverse osmosis permeate. It is unclear whether sul1 in the RO permeate was from the passage of resistance genes or new growth of microorganisms, but the concentrations were on the low end of those reported for conventional drinking water distribution systems. Adenovirus, JC polyomavirus, and bla TEM genes were reduced below the limit of detection (∼10-2 gene copies per mL) by microfiltration. The results provide insights into how treatment train design and operation choices affect microbial water quality as well as the use of flow cytometry and ATP for online monitoring and process control.

SARS-CoV-2 RNA is enriched by orders of magnitude in primary settled solids relative to liquid wastewater at publicly owned treatment works.

Wastewater-based epidemiology has gained attention throughout the world for detection of SARS-CoV-2 RNA in wastewater to supplement clinical testing. Raw wastewater consists of small particles, or solids, suspended in liquid. Methods have been developed to measure SARS-CoV-2 RNA in the liquid and the solid fraction of wastewater, with some studies reporting higher concentrations in the solid fraction. To investigate this relationship further, six laboratories collaborated to conduct a study across five publicly owned treatment works (POTWs) where both primary settled solids obtained from primary clarifiers and raw wastewater influent samples were collected and quantified for SARS-CoV-2 RNA. Settled solids and influent samples were processed by participating laboratories using their respective methods and retrospectively paired based on date of collection. SARS-CoV-2 RNA concentrations, on a mass equivalent basis, were higher in settled solids than in influent by approximately three orders of magnitude. Concentrations in matched settled solids and influent were positively and significantly correlated at all five POTWs. RNA concentrations in both settled solids and influent were correlated to COVID-19 incidence rates in the sewersheds and thus representative of disease occurrence; the settled solids methods appeared to produce a comparable relationship between SARS-CoV-2 RNA concentration measurements and incidence rates across all POTWs. Settled solids and influent methods showed comparable sensitivity, N gene detection frequency, and calculated empirical incidence rate lower limits. Analysis of settled solids for SARS-CoV-2 RNA has the advantage of using less sample volume to achieve similar sensitivity to influent methods.

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.

Tools for interpretation of wastewater SARS-CoV-2 temporal and spatial trends demonstrated with data collected in the San Francisco Bay Area.

Wastewater surveillance for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be integrated with COVID-19 case data to inform timely pandemic response. However, more research is needed to apply and develop systematic methods to interpret the true SARS-CoV-2 signal from noise introduced in wastewater samples (e.g., from sewer conditions, sampling and extraction methods, etc.). In this study, raw wastewater was collected weekly from five sewersheds and one residential facility. The concentrations of SARS-CoV-2 in wastewater samples were compared to geocoded COVID-19 clinical testing data. SARS-CoV-2 was reliably detected (95% positivity) in frozen wastewater samples when reported daily new COVID-19 cases were 2.4 or more per 100,000 people. To adjust for variation in sample fecal content, four normalization biomarkers were evaluated: crAssphage, pepper mild mottle virus, Bacteroides ribosomal RNA (rRNA), and human 18S rRNA. Of these, crAssphage displayed the least spatial and temporal variability. Both unnormalized SARS-CoV-2 RNA signal and signal normalized to crAssphage had positive and significant correlation with clinical testing data (Kendall's Tau-b (τ)=0.43 and 0.38, respectively), but no normalization biomarker strengthened the correlation with clinical testing data. Locational dependencies and the date associated with testing data impacted the lead time of wastewater for clinical trends, and no lead time was observed when the sample collection date (versus the result date) was used for both wastewater and clinical testing data. This study supports that trends in wastewater surveillance data reflect trends in COVID-19 disease occurrence and presents tools that could be applied to make wastewater signal more interpretable and comparable across studies.

Sewage, Salt, Silica, and SARS-CoV-2 (4S): An Economical Kit-Free Method for Direct Capture of SARS-CoV-2 RNA from Wastewater.

Wastewater-based epidemiology is an emerging tool to monitor COVID-19 infection levels by measuring the concentration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. There remains a need to improve wastewater RNA extraction methods' sensitivity, speed, and reduce reliance on often expensive commercial reagents to make wastewater-based epidemiology more accessible. We present a kit-free wastewater RNA extraction method, titled "Sewage, Salt, Silica and SARS-CoV-2" (4S), that employs the abundant and affordable reagents sodium chloride (NaCl), ethanol, and silica RNA capture matrices to recover sixfold more SARS-CoV-2 RNA from wastewater than an existing ultrafiltration-based method. The 4S method concurrently recovered pepper mild mottle virus (PMMoV) and human 18S ribosomal subunit rRNA, which have been proposed as fecal concentration controls. The SARS-CoV-2 RNA concentrations measured in three sewersheds corresponded to the relative prevalence of COVID-19 infection determined via clinical testing. Lastly, controlled experiments indicate that the 4S method prevented RNA degradation during storage of wastewater samples, was compatible with heat pasteurization, and in our experience, 20 samples can be processed by one lab technician in approximately 2 h. Overall, the 4S method is promising for effective, economical, and accessible wastewater-based epidemiology for SARS-CoV-2, providing another tool to fight the global pandemic.

Challenges in Measuring the Recovery of SARS-CoV-2 from Wastewater.

Wastewater-based epidemiology is an emerging tool for tracking the spread of SARS-CoV-2 through populations. However, many factors influence recovery and quantification of SARS-CoV-2 from wastewater, complicating data interpretation. Specifically, these factors may differentially affect the measured virus concentration, depending on the laboratory methods used to perform the test. Many laboratories add a proxy virus to wastewater samples to determine losses associated with concentration and extraction of viral RNA. While measuring recovery of a proxy virus is an important process control, in this piece, we describe the caveats and limitations to the interpretation of this control, including that it typically does not account for losses during RNA extraction. We recommend reporting the directly measured concentration data alongside the measured recovery efficiency, rather than attempting to correct the concentration for recovery efficiency. Even though the ability to directly compare SARS-CoV-2 concentrations from different sampling locations determined using different methods is limited, concentration data (uncorrected for recovery) can be useful for public health response.

Genome Sequencing of Sewage Detects Regionally Prevalent SARS-CoV-2 Variants.

Viral genome sequencing has guided our understanding of the spread and extent of genetic diversity of SARS-CoV-2 during the COVID-19 pandemic. SARS-CoV-2 viral genomes are usually sequenced from nasopharyngeal swabs of individual patients to track viral spread. Recently, RT-qPCR of municipal wastewater has been used to quantify the abundance of SARS-CoV-2 in several regions globally. However, metatranscriptomic sequencing of wastewater can be used to profile the viral genetic diversity across infected communities. Here, we sequenced RNA directly from sewage collected by municipal utility districts in the San Francisco Bay Area to generate complete and nearly complete SARS-CoV-2 genomes. The major consensus SARS-CoV-2 genotypes detected in the sewage were identical to clinical genomes from the region. Using a pipeline for single nucleotide variant calling in a metagenomic context, we characterized minor SARS-CoV-2 alleles in the wastewater and detected viral genotypes which were also found within clinical genomes throughout California. Observed wastewater variants were more similar to local California patient-derived genotypes than they were to those from other regions within the United States or globally. Additional variants detected in wastewater have only been identified in genomes from patients sampled outside California, indicating that wastewater sequencing can provide evidence for recent introductions of viral lineages before they are detected by local clinical sequencing. These results demonstrate that epidemiological surveillance through wastewater sequencing can aid in tracking exact viral strains in an epidemic context.

Sewage, Salt, Silica and SARS-CoV-2 (4S): An economical kit-free method for direct capture of SARS-CoV-2 RNA from wastewater.

Wastewater-based epidemiology is an emerging tool to monitor COVID-19 infection levels by measuring the concentration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. There remains a need to improve wastewater RNA extraction methods' sensitivity, speed, and reduce reliance on often expensive commercial reagents to make wastewater-based epidemiology more accessible. We present a kit-free wastewater RNA extraction method, titled "Sewage, Salt, Silica and SARS-CoV-2" (4S), that employs the abundant and affordable reagents sodium chloride (NaCl), ethanol and silica RNA capture matrices to recover 6-fold more SARS-CoV-2 RNA from wastewater than an existing ultrafiltration-based method. The 4S method concurrently recovered pepper mild mottle virus (PMMoV) and human 18S ribosomal subunit rRNA, both suitable as fecal concentration controls. The SARS-CoV-2 RNA concentrations measured in three sewersheds corresponded to the relative prevalence of COVID-19 infection determined via clinical testing. Lastly, controlled experiments indicate that the 4S method prevented RNA degradation during storage of wastewater samples, was compatible with heat pasteurization, and could be performed in approximately 3 hours. Overall, the 4S method is promising for effective, economical, and accessible wastewater-based epidemiology for SARS-CoV-2, providing another tool to fight the global pandemic.