Environmental Stability of Enveloped Viruses Is Impacted by Initial Volume and Evaporation Kinetics of Droplets.

Efficient spread of respiratory viruses requires the virus to maintain infectivity in the environment. Environmental stability of viruses can be influenced by many factors, including temperature and humidity. Our study measured the impact of initial droplet volume (50, 5, and 1 µL) and relative humidity (RH; 40%, 65%, and 85%) on the stability of influenza A virus, bacteriophage Phi6 (a common surrogate for enveloped viruses), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) under a limited set of conditions. Our data suggest that the drying time required for the droplets to reach quasi-equilibrium (i.e., a plateau in mass) varied with RH and initial droplet volume. The macroscale physical characteristics of the droplets at quasi-equilibrium varied with RH but not with the initial droplet volume. Virus decay rates differed between the wet phase, while the droplets were still evaporating, and the dry phase. For Phi6, decay was faster in the wet phase than in the dry phase under most conditions. For H1N1pdm09, decay rates between the two phases were distinct and initial droplet volume had an effect on virus viability within 2 h. Importantly, we observed differences in virus decay characteristics by droplet size and virus. In general, influenza virus and SARS-CoV-2 decayed similarly, whereas Phi6 decayed more rapidly under certain conditions. Overall, this study suggests that virus decay in media is related to the extent of droplet evaporation, which is controlled by RH. Importantly, accurate assessment of transmission risk requires the use of physiologically relevant droplet volumes and careful consideration of the use of surrogates. IMPORTANCE During the COVID-19 pandemic, policy decisions were being driven by virus stability experiments with SARS-CoV-2 in different droplet volumes under various humidity conditions. Our study, the first of its kind, provides a model for the decay of multiple enveloped RNA viruses in cell culture medium deposited in 50-, 5-, and 1-µL droplets at 40%, 65%, and 85% RH over time. The results of our study indicate that determination of half-lives for emerging pathogens in large droplets may overestimate transmission risk for contaminated surfaces, as observed during the COVID-19 pandemic. Our study implicates the need for the use of physiologically relevant droplet sizes with use of relevant surrogates in addition to what is already known about the importance of physiologically relevant media for risk assessment of future emerging pathogens.

Environmental routes of virus transmission and the application of nanomaterial-based sensors for virus detection

Many outbreaks of emerging disease (e.g., avian influenza, SARS, MERS, Ebola, COVID-19) are caused by viruses. In addition to direct person-to-person transfer, the movement of these viruses through environmental matrices (water, air, and food) can further disease transmission. There is a pressing need for rapid and sensitive virus detection in environmental matrices. Nanomaterial-based sensors (nanosensors), which take advantage of the unique optical, electrical, or magnetic properties of nanomaterials, exhibit significant potential for environmental virus detection. Interactions between viruses and nanomaterials (or recognition agents on the nanomaterials) can induce detectable signals and provide rapid response times, high sensitivity, and high specificity. Facile and field-deployable operations can be envisioned due to the small size of the sensing elements. In this frontier review, we summarize virus transmission via environmental pathways and then comprehensively discuss recent applications of nanosensors to detect various viruses. This review provides guidelines for virus detection in the environment through the use of nanosensors as a tool to decrease environmental transmission of current and emerging diseases.

Environmental routes of virus transmission and the application of nanomaterial-based sensors for virus detection

Many outbreaks of emerging disease (e.g., avian influenza, SARS, MERS, Ebola, COVID-19) are caused by viruses. In addition to direct person-to-person transfer, the movement of these viruses through environmental matrices (water, air, and food) can further disease transmission. There is a pressing need for rapid and sensitive virus detection in environmental matrices. Nanomaterial-based sensors (nanosensors), which take advantage of the unique optical, electrical, or magnetic properties of nanomaterials, exhibit significant potential for environmental virus detection. Interactions between viruses and nanomaterials (or recognition agents on the nanomaterials) can induce detectable signals and provide rapid response times, high sensitivity, and high specificity. Facile and field-deployable operations can be envisioned due to the small size of the sensing elements. In this frontier review, we summarize virus transmission via environmental pathways and then comprehensively discuss recent applications of nanosensors to detect various viruses. This review provides guidelines for virus detection in the environment through the use of nanosensors as a tool to decrease environmental transmission of current and emerging diseases.

Subsewershed SARS-CoV-2 Wastewater Surveillance and COVID-19 Epidemiology Using Building-Specific Occupancy and Case Data

To evaluate the use of wastewater-based surveillance and epidemiology to monitor and predict SARS-CoV-2 virus trends, over the 2020–2021 academic year we collected wastewater samples twice weekly from 17 manholes across Virginia Tech’s main campus. We used data from external door swipe card readers and student isolation/quarantine status to estimate building-specific occupancy and COVID-19 case counts at a daily resolution. After analyzing 673 wastewater samples using reverse transcription quantitative polymerase chain reaction (RT-qPCR), we reanalyzed 329 samples from isolation and nonisolation dormitories and the campus sewage outflow using reverse transcription digital droplet polymerase chain reaction (RT-ddPCR). Population-adjusted viral copy means from isolation dormitory wastewater were 48% and 66% higher than unadjusted viral copy means for N and E genes (1846/100 mL to 2733/100 mL/100 people and 2312/100 mL to 3828/100 mL/100 people, respectively;n = 46). Prespecified analyses with random-effects Poisson regression and dormitory/cluster-robust standard errors showed that the detection of N and E genes were associated with increases of 85% and 99% in the likelihood of COVID-19 cases 8 days later (incident–rate ratio (IRR) = 1.845, p = 0.013 and IRR = 1.994, p = 0.007, respectively;n = 215), and one-log increases in swipe card normalized viral copies (copies/100 mL/100 people) for N and E were associated with increases of 21% and 27% in the likelihood of observing COVID-19 cases 8 days following sample collection (IRR = 1.206, p < 0.001, n = 211 for N;IRR = 1.265, p < 0.001, n = 211 for E). One-log increases in swipe normalized copies were also associated with 40% and 43% increases in the likelihood of observing COVID-19 cases 5 days after sample collection (IRR = 1.403, p = 0.002, n = 212 for N;IRR = 1.426, p < 0.001, n = 212 for E). Our findings highlight the use of building-specific occupancy data and add to the evidence for the potential of wastewater-based epidemiology to predict COVID-19 trends at subsewershed scales. Wastewater samples, population estimates, and case outcome data from individual buildings can be used to monitor and predict COVID-19 trends.

Seizing the moment: now is the time for integrated global surveillance of antimicrobial resistance in wastewater environments.

Antimicrobial resistance (AMR) is a growing global health threat that requires coordinated action across One Health sectors (humans, animals, environment) to stem its spread. Environmental surveillance of AMR is largely behind the curve in current One Health surveillance programs, but recent momentum in the establishment of infrastructure for monitoring of the SARS-CoV-2 virus in sewage provides an impetus for analogous AMR monitoring. Simultaneous advances in research have identified striking trends in various AMR measures in wastewater and other impacted environments across global transects. Methodologies for tracking AMR, including metagenomics, are rapidly advancing, but need to be standardized and made modular for access by LMICs, while also developing systems for sample archiving and data sharing. Such efforts will help optimize effective global AMR policy.

Implications of the Coffee-Ring Effect on Virus Infectivity.

The factors contributing to the survival of enveloped viruses (e.g., influenza and SARS-CoV-2) on fomite surfaces are of societal interest. The bacteriophage Phi6 is an enveloped viral surrogate commonly used to study viability. To investigate how viability changes during the evaporation of droplets on polypropylene, we conducted experiments using a fixed initial Phi6 concentration while systematically varying the culture concentration and composition (by amendment with 2% fetal bovine serum (FBS), 0.08 wt % BSA, or 0.5 wt % SDS). The results were consistent with the well-founded relative humidity (RH) effect on virus viability; however, the measured viability change was greater than that previously reported for droplets containing either inorganic salts or proteins alone, and the protein effects diverged in 1× Dulbecco's modified Eagle's medium (DMEM). We attribute this discrepancy to changes in virus distribution during droplet evaporation that arise due to the variable solute drying patterns (i.e., the "coffee-ring" effect) that are a function of the droplet biochemical composition. To test this hypothesis, we used surface-enhanced Raman spectroscopy (SERS) imaging and three types of gold nanoparticles (pH nanoprobe, positively charged (AuNPs(+)), and negatively charged (AuNPs(-))) as physical surrogates for Phi6 and determined that lower DMEM concentrations, as well as lower protein concentrations, suppressed the coffee-ring effect. This result was observed irrespective of particle surface charge. The trends in the coffee-ring effect correlate well with the measured changes in virus infectivity. The correlation suggests that conditions resulting in more concentrated coffee rings provide protective effects against inactivation when viruses and proteins aggregate.

Mechanistic theory predicts the effects of temperature and humidity on inactivation of SARS-CoV-2 and other enveloped viruses.

Ambient temperature and humidity strongly affect inactivation rates of enveloped viruses, but a mechanistic, quantitative theory of these effects has been elusive. We measure the stability of SARS-CoV-2 on an inert surface at nine temperature and humidity conditions and develop a mechanistic model to explain and predict how temperature and humidity alter virus inactivation. We find SARS-CoV-2 survives longest at low temperatures and extreme relative humidities (RH); median estimated virus half-life is >24 hr at 10°C and 40% RH, but ∼1.5 hr at 27°C and 65% RH. Our mechanistic model uses fundamental chemistry to explain why inactivation rate increases with increased temperature and shows a U-shaped dependence on RH. The model accurately predicts existing measurements of five different human coronaviruses, suggesting that shared mechanisms may affect stability for many viruses. The results indicate scenarios of high transmission risk, point to mitigation strategies, and advance the mechanistic study of virus transmission.

Standardizing data reporting in the research community to enhance the utility of open data for SARS-CoV-2 wastewater surveillance.

SARS-CoV-2 RNA detection in wastewater is being rapidly developed and adopted as a public health monitoring tool worldwide. With wastewater surveillance programs being implemented across many different scales and by many different stakeholders, it is critical that data collected and shared are accompanied by an appropriate minimal amount of metainformation to enable meaningful interpretation and use of this new information source and intercomparison across datasets. While some databases are being developed for specific surveillance programs locally, regionally, nationally, and internationally, common globally-adopted data standards have not yet been established within the research community. Establishing such standards will require national and international consensus on what metainformation should accompany SARS-CoV-2 wastewater measurements. To establish a recommendation on minimum information to accompany reporting of SARS-CoV-2 occurrence in wastewater for the research community, the United States National Science Foundation (NSF) Research Coordination Network on Wastewater Surveillance for SARS-CoV-2 hosted a workshop in February 2021 with participants from academia, government agencies, private companies, wastewater utilities, public health laboratories, and research institutes. This report presents the primary two outcomes of the workshop: (i) a recommendation on the set of minimum meta-information that is needed to confidently interpret wastewater SARS-CoV-2 data, and (ii) insights from workshop discussions on how to improve standardization of data reporting.

Nanobiotechnology enabled approaches for wastewater based epidemiology.

The impacts of the ongoing coronavirus pandemic highlight the importance of environmental monitoring to inform public health safety. Wastewater based epidemiology (WBE) has drawn interest as a tool for analysis of biomarkers in wastewater networks. Wide scale implementation of WBE requires a variety of field deployable analytical tools for real-time monitoring. Nanobiotechnology enabled sensing platforms offer potential as biosensors capable of highly efficient and sensitive detection of target analytes. This review provides an overview of the design and working principles of nanobiotechnology enabled biosensors and recent progress on the use of biosensors in detection of biomarkers. In addition, applications of biosensors for analysis of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus are highlighted as they relate to the potential expanded use of biosensors for WBE-based monitoring. Finally, we discuss the opportunities and challenges in future applications of biosensors in WBE for effective monitoring and investigation of public health threats.