Environmental Science & Technology Letters

Sequencing-based wastewater surveillance is emerging as an important tool in pathogen-agnostic threat detection, potentially enabling early identification before capture through clinical surveillance systems. However, virus sequences of human pathogens are typically low in abundance in wastewater while much of the data is unclassifiable at the read level. This presents a challenge because genomes may not assemble well for novel pathogens of interest, but read-based methods cannot currently separate novel from previously seen unclassified sequences. Using ultra-deep untargeted sequencing of the wastewater RNA virome performed by the CASPER consortium (321 samples), we constructed a wastewater virus genome database (WVDB) with the goal of expanding the set of available high-quality non-redundant reference genomes. The first version of this database contains 21,015 near-complete viral genomes, of which the majority are ssRNA bacteriophage (79%). We additionally recovered genomes for putative plant and vertebrate-infecting viruses, human enteric viruses, and viruses whose host could not be predicted. Fewer than 4000 genomes had matches in previously published virus genome databases, and WVDB captured around one fifth of the reads that could not be classified by Kraken2. Further expansion of WVDB will provide a comprehensive resource of RNA virus genomes for characterization of viral diversity and dynamics in wastewater across space and time.

Medical demands during the COVID-19 pandemic have triggered a grave shortage of medical-grade personal protective equipment (PPE), especially, N95 respirators. N95 respirators are critical for the personal protection of medical providers and others when being exposed to individuals with infections caused by the SARS-CoV-2 coronavirus. To address the shortage of N95 respirators, innovative methods are needed to decontaminate coronaviruses from N95 respirators, allowing them to be safely reused by healthcare workers. For this research, we use a commercial ozone disinfecting cabinet to examine the efficacy of ozone-based disinfection of a conservative surrogate virus for SARS-CoV-2, the MS2 bacteriophage. Treatment of mask materials with enhanced ozone treatment resulted in 2.38-log 10 (>99%) reduction of phage from household dust masks and a range of 1.43-log 10 (96.2%) to 4-log 10 (99.99%) reductions of phage from common N95 mask materials.

Wastewater surveillance of respiratory pathogens can provide timely estimates of viral activity and disease trends in a population. Indoor air surveillance could be used similarly with some advantages but remains largely unvalidated at the community-scale. Here, an indoor air surveillance program was employed as part of public health environmental surveillance in Chicago, Illinois, USA. Ten air samplers were placed in healthcare and congregate living settings across the city. Weekly air samples were evaluated for influenza A, influenza B, respiratory syncytial virus, and SARS-CoV-2 over two respiratory virus seasons. Citywide, aggregated air sample positivity and viral load were closely correlated with local clinical case and wastewater surveillance data across all respiratory viruses. Virus trends in air data often preceded clinical and wastewater, although this varied across pathogens and respiratory virus seasons. Further, whole-genome sequencing of SARS-CoV-2 showed close correlation of variant proportions across all datasets. At the building-scale, air samples obtained from a single sampling device provided efficient respiratory virus surveillance, with well-correlated estimates of respiratory pathogens. These data demonstrate that air surveillance can provide accurate estimates of respiratory virus infections and variants at a building or community-scale, serving as an alternative or complementary tool for public health environmental surveillance.

Wastewater surveillance is a powerful tool for monitoring the prevalence of infectious disease. Systems for wastewater monitoring were put in place throughout the world during the COVID-19 pandemic. These systems use viral RNA copies as the basis of estimates of COVID-19 cases in the sewershed area, thereby providing data critical for public health responses. However, the potential to measure other biomarkers in wastewater during outbreaks has not been fully explored. Here we report a novel approach for detecting specific human antibodies from wastewater. We measured the abundance of anti-SARS-CoV-2 spike IgG and IgA from fresh samples of community wastewater and from archived frozen samples dating from 2020-22. The assay described can be performed with readily available reagents, at a moderate per-sample cost. Our findings demonstrate the feasibility of noninvasive serological surveillance via wastewater, enabling a new approach to immunity-based monitoring of populations.

MotivationSARS-CoV-2 is the causative agent of the COVID-19 pandemic. Variants of Concern (VOCs) and Variants of Interest (VOIs) are lineages that represent a greater risk to public health, and can be differentiated from the wildtype virus based on unique profiles of signature mutations. Metagenomic sequence analysis of wastewater represents an emerging form of surveillance that can capture early signal for these variants in a community prior to detection through public health testing or genomic surveillance activities. However, because multiple viral genomes are likely to be present in a metagenomic sample, additional analytical scrutiny of the sequencing reads beyond variant calling is needed to increase confidence in diagnostic determinations. ResultsWhere multiple signature mutations are present on a given read, they can be used as enhanced biomarkers to confirm the presence of a VOC/VOI in the sample. We have developed MMMVI, a tool to aggregate and report on the likely presence of a VOC/VOI in a set of metagenomic reads based on the detection of reads bearing multiple signature mutations. AvailabilityMMMVI is implemented in Python, and is available under the MIT licence from https://github.com/dorbarker/voc-identify/ Contactdillon.barker@canada.ca

Early detection of outbreaks and emerging pathogens is critical for public health and global biosecurity. Airports, as major international travel hubs with dense, enclosed populations, are high-risk settings for disease transmission and potential pathogen introduction. The U.S. Centers for Disease Control and Prevention, in collaboration with Ginkgo Biosecurity and the University of Wisconsin-Madison, implemented air monitoring for pathogen surveillance in congregate areas at four U.S. international airports. From October 2023 to August 2024, SARS-CoV-2 was detected by PCR in 98.3% of air samples and influenza A in 17.2%. These results correlated with positivity trends from other sample modalities, including aviation wastewater, traveler nasal swabs, and national clinical surveillance data. Targeted amplicon sequencing of SARS-CoV-2 from air samples correlated with contemporaneous lineages in wastewater collected and sequenced from the same airports. Metagenomic enrichment sequencing detected 30 viral species and recovered high-quality genomes for SARS-CoV-2, influenza, bocavirus, and seasonal coronaviruses. Together, these findings demonstrate that air sampling is a complementary surveillance modality to aviation wastewater for early pathogen detection at ports of entry.

Metagenomic sequencing is increasingly applied to wastewater to characterize the diversity, dynamics, and relative abundance of human and animal viruses. Among these sequencing approaches are those that enrich viral nucleic acids from the wastewater matrix, aiming to increase the viral read fraction for analysis. However, the feasibility of scaling targeted viral sequencing to diverse sewersheds across large geographic scales is currently unknown. In this study, we apply hybrid capture metagenomic sequencing to nearly 450 weekly wastewater samples collected during the respiratory virus season in the United States and evaluate sequencing performance for generating public health-relevant data. Analysis of data from 15 wastewater treatment plants demonstrates that our approach enabled efficient capture of pathogens of interest, achieving a median viral read fraction over 19%. Importantly, relative abundance estimates of common pathogens correlated with direct quantification of viral targets using RT-ddPCR. Together, our results demonstrate that hybrid capture sequencing of wastewater is a viable tool to monitor both common and rare pathogens across geographically diverse sewersheds.

The COVID-19 pandemic has given rise to diverse approaches to track infections. The causative agent, SARS-CoV-2, is a fecally-shed RNA virus, and many groups have assayed wastewater for viral RNA fragments by quantitative reverse transcription polymerase chain reaction (qRT-PCR) as a proxy for COVID-19 prevalence in the community. Most groups report low levels of viral RNA that often skirt the methods theoretical limits of detection and quantitation. Here, we demonstrate the presence of SARS-CoV-2 structural proteins in wastewater using traditional immunoblotting and quantitate them from wastewater solids using an immuno-linked PCR method called Multiplex Paired-antibody Amplified Detection (MPAD). MPAD demonstrated facile detection of SARS-CoV-2 proteins compared with SARS-CoV-2 RNA via qRT-PCR in wastewater. In this longitudinal study, we corrected for stochastic variability inherent to wastewater-based epidemiology using multiple fecal content protein biomarkers. These normalized SARS-CoV-2 protein data correlated well with public health metrics. Our method of assaying SARS-CoV-2 protein from wastewater represents a promising and sensitive epidemiological tool to assess prevalence of fecally-shed pathogens in the community.

Avian influenza (serotype H5N1) is a highly pathogenic virus that emerged in domestic waterfowl in 1996. Over the past decade, zoonotic transmission to mammals, including humans, has been reported. Although human to human transmission is rare, infection has been fatal in nearly half of patients who have contracted the virus in past outbreaks. The increasing presence of the virus in domesticated animals raises substantial concerns that viral adaptation to immunologically naive humans may result in the next flu pandemic. Wastewater-based epidemiology (WBE) to track viruses was historically used to track polio and has recently been implemented for SARS-CoV2 monitoring during the COVID-19 pandemic. Here, using an agnostic, hybrid-capture sequencing approach, we report the detection of H5N1 in wastewater in nine Texas cities, with a total catchment area population in the millions, over a two-month period from March 4th to April 25th, 2024. Sequencing reads uniquely aligning to H5N1 covered all eight genome segments, with best alignments to clade 2.3.4.4b. Notably, 19 of 23 monitored sites had at least one detection event, and the H5N1 serotype became dominant over seasonal influenza over time. A variant analysis suggests avian or bovine origin but other potential sources, especially humans, could not be excluded. We report the value of wastewater sequencing to track avian influenza.

Conventional air filtration relies on passive mechanical capture without pathogen inactivation, where viral reduction must be balanced with airflow and energy performance. We developed an Ablative Polymer Coated (APC) filtration system that converts passive filters into active pathogen-reducing surfaces while maintaining low airflow resistance. Unlike conventional approaches requiring denser, higher-resistance media, this strategy enhances biological performance at the filter surface without equivalent aerodynamic penalties. The coating incorporates benzalkonium chloride within a polyvinyl acetate/acrylate matrix for controlled ablative exposure. Performance was evaluated using transmission electron microscopy (TEM), aerosol challenge testing, and HVAC-scale filtration. Ablative exposure caused progressive structural disruption of MS2 bacteriophage, the SARS-CoV-2 simulant. In aerosol challenge testing, coated media achieved up to 99.997% viral filtration efficiency under respiratory airflow conditions. In HVAC (Heating, Ventilation, and Air Conditioning)-scale testing, coated filters achieved >85% viral filtration efficiency with minimal pressure-drop increase. Computational fluid dynamics modeling confirmed uniform airflow distribution without significant turbulence generation. Energy analysis suggested coated filters may reduce energy demand relative to conventional higher-resistance configurations while improving biological performance. These findings support ablative polymer-coated media as a strategy for reducing airborne viral burden without aerodynamic penalties of higher-efficiency passive filtration, suggesting an approach that complements rather than depends solely on tighter filter design.

Wastewater represents a composite biological sample from the entire contributing population. People infected with monkeypox excrete monkeypox virus DNA via skin lesions, saliva, feces and urine and these can enter the wastewater via toilets, sinks, and shower drains. To test whether monkeypox can be detected and monitored in wastewater during a period when publicly reported monkey cases in the region were increasing, we deployed digital PCR assays that target genomic DNA from the monkeypox virus in our routine, ongoing wastewater surveillance program in the Greater Bay Area of California, USA. We measured monkeypox virus DNA daily in settled solids samples from nine wastewater plants over the period of approximately 4 weeks. During that period, we detected monkeypox virus DNA in wastewater solids at nearly all the wastewater plants we routinely sample. Frequency of occurrence and concentrations were highest at plants serving San Francisco County. To confirm the presence of monkeypox DNA, we used two assays that target distinct sequences on the monkeypox genome on a subset of samples and results from both assays were in close agreement strongly suggesting true positives in the wastewater. Additionally, we show that concentrations of monkeypox DNA is 103 times higher in the solid fraction compared to the liquid fraction of wastewater on a mass-equivalent basis.

Methods to concentrate wastewater samples are essential for sensitive environmental surveillance of infectious diseases. We defined six main principles used to concentrate viral pathogens in wastewater and performed a systematic review and meta-analysis of their performance. PubMed and Web of Science were searched on 31 January 2025 using terms wastewater, sewage, concentration methods and wastewater surveillance. We included all studies comparing [≥]2 concentration methods for virus detection. Our search identified 49 eligible studies published since 2013 across seven continents. We ranked the performance of evaluated methods in each study and generated an overall performance metric for each method principle by virus group (enveloped vs. non-enveloped) using Plackett-Luce analysis. Precipitation and filtration methods were the most studied, while magnetic bead-based and centrifugation were least studied. Magnetic bead-based methods were more effective for concentrating enveloped viruses (63% of pairwise comparisons), whereas flocculation performed better for non-enveloped viruses (60%). However, no single method strongly dominated and method rankings were variable between studies. This study provides evidence-based guidance for selecting wastewater concentration methods to support environmental surveillance of viral pathogens.

Measles outbreaks continue to pose significant public health challenges globally despite the availability of effective vaccines. In this study, we evaluated wastewater-based surveillance for detection of measles virus during an ongoing outbreak in Texas. Weekly wastewater samples collected from two Texas cities between January 2 and March 17, 2025 were analyzed using multiple RT-PCR assays targeting the nucleoprotein and matrix genes of the measles virus. Viral RNA was detected in multiple days from both cities, with City A showing positives from January 13 and City B from January 6, both predating the first confirmed case in the state on January 23. Sequencing of PCR amplicons confirmed the specificity of detection and phylogenetic analysis using global and U.S. measles genome databases further validated that the viral RNA belonged to the currently circulating genotype D8. Our findings demonstrate that wastewater surveillance can provide early evidence of measles virus circulation in communities before clinical cases are recognized and can support public health responses to these re-emerging infectious diseases.

BackgroundRespiratory disease is a major cause of morbidity and mortality; however, current surveillance for circulating respiratory viruses is passive and biased. Seasonal circulation of respiratory viruses changed dramatically during the COVID-19 pandemic. More active methods for understanding respiratory disease dynamics are needed to better inform public health response and to guide clinical decision making. Wastewater-based epidemiology has been used to understand COVID-19, influenza A, and RSV infection rates at a community level, but has not been used to investigate other respiratory viruses. MethodsWe measured concentrations of influenza A and B, RSV A and B, human parainfluenza (1-4), rhinovirus, seasonal human coronaviruses, and human metapneumovirus RNA in wastewater solids three times per week for 17 months spanning the COVID-19 pandemic at a wastewater treatment plant in California, USA. Novel probe-based assays were developed and validated for non-influenza viral targets. We compared viral concentrations to positivity rates for viral infections from clinical specimens submitted to sentinel laboratories. FindingsWe detected RNA from all target viruses in wastewater solids. Human rhinovirus and seasonal coronaviruses were found at highest concentrations. Concentrations of viruses correlated significantly and positively with positivity rates of associated viral diseases from sentinel laboratories. Measurements from wastewater indicated limited circulation of RSV A and influenza B, and human coronavirus OC43 dominated the seasonal human coronavirus infections while human parainfluenza 1 and 4A dominated among parainfluenza infections. InterpretationWastewater-based epidemiology can be used to obtain information on circulation of respiratory viruses at a community level without the need to test many individuals because a single sample of wastewater represents the entire contributing community. Results from wastewater can be available within 24 hours of sample collection, allowing real time information to inform public health response, clinical decision making, and individual behavior modifications.

Despite wide scale assessments, it remains unclear how large-scale SARS-CoV-2 vaccination affected the wastewater concentration of the virus or the overall disease burden as measured by hospitalization rates. We used weekly SARS-CoV-2 wastewater concentration with a stratified random sampling of seroprevalence, and linked vaccination and hospitalization data, from April 2021-August 2021 in Jefferson County, Kentucky (USA). Our susceptible (S), vaccinated (V), variant-specific infected (I1 and I2), recovered (R), and seropositive (T) model (SVI2 RT) tracked prevalence longitudinally. This was related to wastewater concentration. The 64% county vaccination rate translated into about 61% decrease in SARS-CoV-2 incidence. The estimated effect of SARS-CoV-2 Delta variant emergence was a 24-fold increase of infection counts, which corresponded to an over 9-fold increase in wastewater concentration. Hospitalization burden and wastewater concentration had the strongest correlation (r = 0.95) at 1 week lag. Our study underscores the importance of continued environmental surveillance post-vaccine and provides a proof-of-concept for environmental epidemiology monitoring of infectious disease for future pandemic preparedness.

Wastewater monitoring can provide insights into respiratory disease occurrence in communities that contribute to the wastewater system. Using daily measurements of RNA of influenza A (IAV), respiratory syncytial virus (RSV), and human metapneumovirus (HMPV), as well as SARS-CoV-2 in wastewater solids from eight publicly owned treatment works in the Greater San Francisco Bay Area of California between July 2022 until early May 2023, we identify a "tripledemic" when concentrations of IAV, RSV, and SARS-CoV-2 peaked at approximately the same time. HMPV was also widely circulating. We designed novel hydrolysis probe RT-PCR assays for different IAV subtype makers to discern that the dominant circulating IAV subtype was H3N2. We show that wastewater data can be used to identify onset and offset of wastewater disease occurrence events that can provide insight into disease epidemiology and timely, localized information to inform hospital staffing and clinical decision making to respond to circulating viruses. Whereas RSV and IAV wastewater events were mostly regionally coherent, HMPV events displayed localized occurrence patterns.

BackgroundContinuous, non-invasive viral surveillance is essential to monitor emerging pathogens and guide public health responses. Most environmental surveillance studies use targeted qPCR approaches, and comparisons between wastewater and indoor air surveillance remain limited. We aimed to compare the utility of emergency department indoor air and urban wastewater for tracking circulating viruses and resolving genomic information. MethodsWe conducted a matched-pair study comparing 19 weekly indoor air samples from the central ventilation exhaust shaft of an emergency department and 19 24-hour composite municipal wastewater samples in Leuven, Belgium, from December 2024 to April 2025. Both sample sets were processed using probe-based hybrid-capture viral metagenomics targeting over 3000 viral species, using influenza A as a clinically relevant test case. FindingsWastewater captured higher overall viral diversity (233 versus 106 species) and more complete genomes compared to indoor air, showing a relatively stable composition, mainly of enteric and animal-associated viruses. Indoor air demonstrated lower overall diversity but was enriched for respiratory viruses, including influenza A, coronaviruses, metapneumovirus, and respiratory syncytial virus, and more frequently achieved high genome coverage for these pathogens. Although both sample types permitted influenza A subtype characterization, influenza A genomes from wastewater were often less well covered. When coverage thresholds were met, indoor air supported targeted antiviral resistance-site screening for influenza A and RSV-A. InterpretationWastewater and indoor air generate distinct but complementary viromes. Wastewater acts as a diverse, population-level monitor for One-Health applications, whereas indoor air serves as a targeted, human-centric sentinel system facilitating further genomic characterization for respiratory viruses. FundingMustafa Karatas is supported by a Research Foundation Flanders (FWO) fundamental research scholarship (number: 11P7I24N). C.G., L.C., E.H., S.G. and E.A. acknowledge support from the DURABLE project. The DURABLE project has been co-funded by the European Union, under the EU4Health Programme (EU4H), project no. 101102733. Research in context Evidence before this studyWe searched PubMed for studies published between Jan 2000 and March 2024 using the terms "wastewater surveillance", "metagenomics", "indoor air", and "viral metagenomics". Previous studies have shown that wastewater surveillance can detect population-level viral circulation, and more recent work has explored indoor air sampling as a method for monitoring respiratory virus transmission. However, environmental metagenomic studies have largely examined these two sample types separately. Furthermore, most studies relied on untargeted sequencing approaches, which often yield fragmented genomes in these environments. To date, no study has systematically compared indoor air and wastewater using a comprehensive hybrid-capture viral metagenomics approach for virus surveillance. Added value of this studyWe conducted a matched comparison of indoor air from a hospital emergency department and municipal wastewater collected during the same weeks in Leuven, Belgium. We analyzed both sample types using an identical hybrid-capture viral metagenomics workflow targeting more than 3000 viral species. This design enabled a direct evaluation of how the two environmental surveillance lenses differ in viral diversity, genomic recovery, and epidemiological relevance. Wastewater captured broader viral diversity and a stable background dominated by enteric and animal-associated viruses, whereas indoor air captured more respiratory viruses and more frequently yielded high genome completeness for these pathogens. When genome coverage thresholds were met, indoor air data enabled influenza subtype identification and screening for antiviral resistance markers. Implications of all the available evidenceOur findings support a layered environmental surveillance strategy in which different environmental samples provide complementary information. Wastewater offers a stable, population-level view of viral circulation and captures broad viral diversity, including human and animal-associated viruses. Indoor air sampling in human-dominated settings provides a more direct signal of respiratory virus circulation and can yield genomes suitable for subtype and mutation-level characterization. Combining these approaches could strengthen metagenomic surveillance frameworks by improving the interpretation of environmental viral signals, supporting early detection of emerging pathogens, and helping distinguish human virus circulation from environmental or animal-derived detections.

Rapid, on-site, airborne virus detection is a requirement for timely action against the spread of air-transmissible infectious diseases. This applies both to future threats and to common viral diseases, such as influenza and COVID-19, which hit vulnerable populations yearly with severe consequences. The ultra-low concentrations of virus in the air make airborne virus detection difficult, yet readily infect individuals when breathed. Here, we propose a fieldable process that includes an enrichment step to concentrate collected genetic material in a small volume. The enrichment approach uses capillary electrophoresis and an RT-qPCR-compatible buffer, which allow enrichment of the RNA by about 5-fold within only 10 minutes of operation. Our detection process consists of air sampling through electrostatic precipitation, RNA extraction via heating, RNA enrichment, and RT-qPCR for detection. We optimized each step of the process and estimated a detection sensitivity of 3106 {+/-} 2457 genome copies (gc) per m3 of air. We then performed an integration experiment and confirmed a sensitivity of 5654 gc/m3 with a detection rate of 100% and a sensitivity of 4221 gc/m3 with a detection rate of 78.6%. When using fast RT-qPCR, the latency of the whole process is down to 61 minutes. Given that our sensitivity falls in the low range of influenza and SARS-CoV-2 concentrations reported in indoor spaces, our study shows that, with enrichment, airborne pathogen detection can be made sufficiently sensitive for practical use.

Measles incidence has increased in recent years as vaccination rates have dropped globally. However, there are challenges in surveillance of measles; measles presents similarly to other diseases and can be misdiagnosed. The lag between infectivity and symptom onset also poses a challenge for surveillance, as measles is highly infectious and significant transmission can occur before case identification. Wastewater monitoring of measles RNA could help to fill gaps in clinical surveillance. In this study, we developed a novel assay to detect wild-type measles virus in wastewater; through both in silico and in vivo tests, we demonstrated assay specificity and sensitivity. We conducted both retrospective and prospective monitoring in a sewershed adjacent to ongoing outbreak areas in the region in the United States from December 2024 - May 2025. In total, 11 of 105 (10.5%) of samples were positive for measles with a median concentration of 6,900 gene copies per dry gram of wastewater solids. Overall, we demonstrate that measles is detectable in wastewater during an ongoing outbreak and that wastewater monitoring of measles can result in early warning over clinical surveillance.

Far-UVC (222 nm UV-C light) is a promising tool to mitigate aerosol transmission of pathogens indoors. However, recent studies have raised concerns related to ozone (O3) production and secondary chemistry. In this study, we measured indoor O3 and ultra-fine particle (UFP, 17.5-289 nm) concentrations with and without 222 nm far-UVC (average fluence rate 1.7-1.8 {micro}W/cm2) in a hotel quarantine facility in Baltimore (MD, USA). We obtained nearby outdoor O3 concentrations from the Environmental Protection Agency (EPA) website. In a sealed empty guest room, the average O3 concentrations were 3 ppb (UV off, 0.1-0.5 ACH), 16 ppb (UV on, 0.1 ACH) and 9 ppb (UV on, 0.5 ACH). In a standard guest room, the average O3 concentrations were 12 ppb (UV off, 1.4 ACH) and 14 ppb (UV on, 1.4 ACH), and correlated with outdoor concentrations ({rho} = 0.65 - 0.74, p = 2*10-12 - 2*10-29). A linear regression model, adjusted for outdoor O3, estimated that use of far-UVC lamps increased the O3 concentration by 5.7 ppb (95% confidence interval (CI) 4.9 - 6.5 ppb) in the standard hotel room. Indoor O3 concentrations increased with far-UVC usage, however, the concentrations remained 6-12 ppb lower, on average, than outdoors and well below EPA ambient limits. We did not find a clear relationship between indoor UFP concentrations and UV usage. Although our study was limited by absence of direct outdoor measurements of local O3 and UFPs, our findings do not support a major impact of far-UVC on UFP concentrations in the real-world environment that we studied.