Evaluation of woodchips-amended biosand filter for nitrate and MS2 bacteriophage reduction.

In this study, two types of woodchip-amended biosand filters (Filter A sand: woodchip = 33%: 67% versus Filter B sand: woodchip = 50%: 50%, by volume) were constructed, and their abilities to remove MS2 bacteriophage and nitrate were investigated. The results indicated that Filter A and Filter B could reduce nitrate up to 40 and 36%, respectively, indicating that the nitrate reduction increased with the increase in woodchip proportion. The study underscores a positive correlation between nitrate reduction and proportional increase in woodchip content, implying the potential for fine-tuning nitrate removal by varying sand-woodchip compositions. W-BSFs could remove MS2 bacteriophage to 1.91-log10 (98.8%) by Filter A and 1.88-log10 (98.7%) by Filter B over 39 weeks. The difference in sand-woodchip proportion did not significantly impact the MS2 reduction, demonstrating that a single W-BSF can maintain its virus removal performance fairly well over a long-term period. These results indicated that the nitrate reduction could be adjusted by varying sand-woodchip contents without impacting virus removal performance. Microbial community analysis indicated that the nitrate removal by the W-BSFs could be attributed to the denitrifying bacteria, such as the family Streptomycetaceae, the genera Pseudomonas, and Bacillus, and relative abundances of the phylum Nitrospirae.

Low-Cost UVBot Using SLAM to Mitigate the Spread of Noroviruses in Occupational Spaces.

Noroviruses (NoVs) cause over 90% of non-bacterial gastroenteritis outbreaks in adults and children in developed countries. Therefore, there is a need for approaches to mitigate the transmission of noroviruses in workplaces to reduce their substantial health burden. We developed and validated a low-cost, autonomous robot called the UVBot to disinfect occupational spaces using ultraviolet (UV) lamps. The total cost of the UVBOT is less than USD 1000, which is much lower than existing commercial robots that cost as much as USD 35,000. The user-friendly desktop application allows users to control the robot remotely, check the disinfection map, and add virtual walls to the map. A 2D LiDAR and a simultaneous localization and mapping (SLAM) algorithm was used to generate a map of the space being disinfected. Tulane virus (TV), a human norovirus surrogate, was used to validate the UVBot's effectiveness. TV was deposited on a painted drywall and exposed to UV radiation at different doses. A 3-log (99.9%) reduction of TV infectivity was achieved at a UV dose of 45 mJ/cm2. We further calculated the sanitizing speed as 3.5 cm/s and the efficient sanitizing distance reached up to 40 cm from the UV bulb. The design, software, and environment test data are available to the public so that any organization with minimal engineering capabilities can reproduce the UVBot system.

Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Ultrasensitive Detection of SARS-CoV-2 in Saliva and Viral Transport Medium Clinical Samples.

The COVID-19 pandemic has underscored the shortcomings in the deployment of state-of-the-art diagnostics platforms. Although several polymerase chain reaction (PCR)-based techniques have been rapidly developed to meet the growing testing needs, such techniques often need samples collected through a swab, the use of RNA extraction kits, and expensive thermocyclers in order to successfully perform the test. Isothermal amplification-based approaches have also been recently demonstrated for rapid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection by minimizing sample preparation while also reducing the instrumentation and reaction complexity. In addition, there are limited reports of saliva as the sample source, and some of these indicate inferior sensitivity when comparing reverse transcription loop-mediated isothermal amplification (RT-LAMP) with PCR-based techniques. In this paper, we demonstrate an improved sensitivity assay from saliva using a two-step RT-LAMP assay, where a short 10 min RT step is performed with only B3 and backward inner primers before the final reaction. We show that while the one-step RT-LAMP demonstrates satisfactory results, the optimized two-step approach allows detection of only few molecules per reaction and performs significantly better than the one-step RT-LAMP and conventional two-step RT-LAMP approaches with all primers included in the RT step. We show control measurements with RT-PCR, and importantly, we demonstrate RNA extraction-free RT-LAMP-based assays for detection of SARS-CoV-2 from viral transport media and saliva clinical samples.

UV Inactivation of Rotavirus and Tulane Virus Targets Different Components of the Virions.

Enteric viruses are shed in fecal material by humans and other animals and are common contaminants in wastewater and surface water. Wastewater treatment plants often disinfect this effluent with low-pressure and medium-pressure UV lamps, which emit 254-nm and 220- to 280-nm irradiation, respectively. It is not known whether this treatment is efficacious against enteric viruses or how such treatments may inactivate these enteric viruses. This study examined UV disinfection for two enteric viruses: rotavirus (RV) (strain OSU with double-stranded RNA and a three-layer capsid) and Tulane virus (TV) (a cultivable surrogate for human norovirus with single-stranded RNA and a single-layer capsid). Viruses were treated with UV irradiation at 220 or 254 nm under conditions relevant to wastewater stabilization ponds, whose water is often used for irrigation. TV was susceptible to 220- or 254-nm UV at similar levels. It appears that UV irradiation inactivated TV by mutagenizing both its genome and capsid binding proteins. RV was more susceptible to UV at 220 nm than to UV at 254 nm. UV irradiation of RV at either 220 or 254 nm resulted in a virus that retained its ability to bind to its host cell receptor. After 220-nm treatment, the VP7 segment of the RV genome could not be amplified by PCR, suggesting that this treatment mutagenized the viral genome. However, this correlation was not observed when UV at 254 nm was used. Thus, RV and TV, with different genome and capsid contents, are targeted by UV irradiation in different ways.IMPORTANCE UV irradiation is becoming common for disinfection in water treatment plants, but little is known about the effectiveness of this treatment for enteric RNA viruses. Here, we observed that 220-nm UV irradiation was efficacious against rotavirus (RV) and Tulane virus (TV). UV irradiation at 254 nm inactivated TV to a greater extent than RV. Additional assays showed that UV irradiation compromised different portions of the RV and TV life cycles. UV irradiation decreased the binding of TV to its host receptor and mutagenized the TV genome. UV irradiation at 220 nm appeared to allow RV-host receptor interaction but halted RV genome replication. These findings provide knowledge about the disinfection of waterborne viruses, information that is important for the safe reuse or release of treated wastewater.

Free Chlorine Disinfection Mechanisms of Rotaviruses and Human Norovirus Surrogate Tulane Virus Attached to Fresh Produce Surfaces.

To fill the knowledge gap on how effective free chlorine is against viral-contaminated produce, we inoculated the surfaces of outdoor- or greenhouse-grown kale and mustard with Rotavirus (RV) or a human norovirus surrogate (Tulane virus, TV) and then disinfected the leaves with free chlorine. Disinfection efficacies for RV strain OSU and Wa were approximately 1-log10 higher when attached to mustard than to kale. Similar disinfection efficacies were observed for TV attached to mustard or kale. When examining TV and RV OSU in suspension (not attached to leaf surfaces), TV was more resistant to free chlorine than RV OSU. Inactivation efficacies were higher for these viruses in suspension versus viruses attached to produce the surface. We also found that free chlorine damaged viral capsids, allowing free chlorine access to viral RNA to damage viral genomes. Exposure to free chlorine at 1.7 ppm over 1 min caused VP8* of RV OSU to lose its ability to bind to its host receptors. TV lost its ability to bind to its receptor only after exposure to free chlorine at 29 ppm over 1 min. Thus, to reduce foodborne viral infections, it is important to consider the differences in virus' reactivity and inactivation mechanisms with free chlorine.

Inactivation Mechanisms of Human and Animal Rotaviruses by Solar UVA and Visible Light.

Two rotavirus (RV) strains (sialidase-resistant Wa and sialidase-sensitive OSU) were irradiated with simulated solar UVA and visible light in sensitizer-free phosphate buffered solution (PBS) (lacking exogenous reactive oxygen species (ROS)) or secondary effluent wastewater (producing ROS). Although light attenuated for up to 15% through the secondary effluent wastewater (SEW), the inactivation efficacies increased by 0.7 log10 for Wa and 2 log10 for OSU compared to those in sensitizer-free phosphate buffered solution (PBS) after 4 h of irradiation. A binding assay using magnetic beads coated with porcine gastric mucin containing receptors for rotaviruses (PGM-MB) was developed to determine if inactivation influenced RV binding to its receptors. The linear correlation between the reduction in infectivity and the reduction in binding after irradiation in sensitizer-free solution suggests that the main mechanism of RV inactivation in the absence of exogenous ROS was due to damage to VP8*, the RV protein that binds to host cell receptors. For a given reduction in infectivity, greater damage in VP8* was observed with sialidase-resistant Wa compared to sialidase-sensitive OSU. The lack of correlation between the reduction in infectivity and the reduction in binding, in SEW, led us to include RNase treatment before the binding step to quantify virions with intact protein capsids and exclude virions that can bind to the receptors but have their capsid permeable after irradiation. This assay showed a linear correlation between the reduction in RV infectivity and RV-receptor interactions, suggesting that RV inactivation in SEW was due to compromised capsid proteins other than the VP8* protein. Thus, rotavirus inactivation by UVA and visible light irradiation depends on both the formation of ROS and the stability of viral proteins.

Thermal Inactivation of Enteric Viruses and Bioaccumulation of Enteric Foodborne Viruses in Live Oysters (Crassostrea virginica).

Human enteric viruses are among the main causative agents of shellfish-associated outbreaks. In this study, the kinetics of viral bioaccumulation in live oysters and the heat stabilities of the predominant enteric viruses were determined both in tissue culture and in oyster tissues. A human norovirus (HuNoV) GII.4 strain, HuNoV surrogates (murine norovirus [MNV-1], Tulane virus [TV]), hepatitis A virus (HAV), and human rotavirus (RV) bioaccumulated to high titers within oyster tissues, with different patterns of bioaccumulation for the different viruses. We tested the thermal stability of each virus at 62, 72, and 80°C in culture medium. The viruses can be ranked from the most heat resistant to the least stable as follows: HAV, RV, TV, MNV-1. In addition, we found that oyster tissues provided protection to the viruses during heat treatment. To decipher the mechanism underlying viral inactivation by heat, purified TV was treated at 80°C for increasing time intervals. It was found that the integrity of the viral capsid was disrupted, whereas viral genomic RNA remained intact. Interestingly, heat treatment leading to complete loss of TV infectivity was not sufficient to completely disrupt the receptor binding activity of TV, as determined by the porcine gastric mucin-magnetic bead binding assay. Similarly, HuNoV virus-like particles (VLPs) and a HuNoV GII.4 strain retained some receptor binding ability following heat treatment. Although foodborne viruses have variable heat stability, 80°C for >6 min was sufficient to completely inactivate enteric viruses in oysters, with the exception of HAV.

High-Pressure Inactivation of Rotaviruses: Role of Treatment Temperature and Strain Diversity in Virus Inactivation.

Rotavirus (RV) is the major etiological agent of acute gastroenteritis in infants worldwide. Although high-pressure processing (HPP) is a popular method to inactivate enteric pathogens in food, the sensitivity of different virus strains within same species and serotype to HPP is variable. This study aimed to compare the barosensitivities of seven RV strains derived from four serotypes (serotype G1, strains Wa, Ku, and K8; serotype G2, strain S2; serotype G3, strains SA-11 and YO; and serotype G4, strain ST3) following high-pressure treatment. RV strains showed various responses to HPP based on the initial temperature and had different inactivation profiles. Ku, K8, S2, SA-11, YO, and ST3 showed enhanced inactivation at 4°C compared to 20°C. In contrast, strain Wa was not significantly impacted by the initial treatment temperature. Within serotype G1, strain Wa was significantly (P < 0.05) more resistant to HPP than strains Ku and K8. Overall, the resistance of the human RV strains to HPP at 4°C can be ranked as Wa > Ku = K8 > S2 > YO > ST3, and in terms of serotype the ranking is G1 > G2 > G3 > G4. In addition, pressure treatment of 400 MPa for 2 min was sufficient to eliminate the Wa strain, the most pressure-resistant RV, from oyster tissues. HPP disrupted virion structure but did not degrade viral protein or RNA, providing insight into the mechanism of viral inactivation by HPP. In conclusion, HPP is capable of inactivating RV at commercially acceptable pressures, and the efficacy of inactivation is strain dependent.

A novel approach to concentrate human and animal viruses from wastewater using receptors-conjugated magnetic beads.

Viruses are present at low concentrations in wastewater; therefore, an effective method for concentrating virus particles is necessary for accurate wastewater-based epidemiology (WBE). We designed a novel approach to concentrate human and animal viruses from wastewater using porcine gastric mucin-conjugated magnetic beads (PGM-MBs). We systematically evaluated the performances of the PGM-MBs method (sensitivity, specificity, and robustness to environmental inhibitors) with six viral species, including Tulane virus (a surrogate for human norovirus), rotavirus, adenovirus, porcine coronavirus (transmissible gastroenteritis virus or TGEV), and two human coronaviruses (NL63 and SARS-CoV-2) in influent wastewater and raw sewage samples. We determined the multiplication factor (the ratio of genome concentration of the final solution to that of the initial solution) for the PGM-MBs method, which ranged from 1.3 to 64.0 depending on the viral species. Because the recovery efficiency was significantly higher when calculated with virus titers than it was with genome concentration, the PGM-MBs method could be an appropriate tool for assessing the risk to humans who are inadvertently exposed to wastewater contaminated with infectious viruses. Furthermore, PCR inhibitors were not concentrated by PGM-MBs, suggesting that this tool will be successful for use with environmental samples. In addition, the PGM-MBs method is cost-effective (0.5 USD/sample) and has a fast turnaround time (3 h from virus concentration to genome quantification). Thus, this method can be implemented in high throughput facilities. Because of its strong performance, intrinsic characteristics of targeting the infectious virus, robustness to wastewater, and adaptability to high throughput systems, the PGM-MBs method can be successfully applied to WBE and ultimately provides valuable public health information.

Mechanism and efficacy of virus inactivation by a microplasma UV lamp generating monochromatic UV irradiation at 222 nm.

This study evaluated the potential of a microplasma UV lamp as an alternative UV source to the current mercury-based (Hg-based) UV lamp for water disinfection. We developed a set of PCR-based molecular assays (long-range qPCR, DNase, and binding assay) to quantify the adenovirus genome, capsid, and fiber damage with a wide detection range (100.5-106.5 PFU/mL). We used these molecular assays to characterize adenovirus (AdV) inactivation kinetics by microplasma UV that produced monochromatic UV at 222 nm. We found that the inactivation rate constant (0.142 cm2/mJ) due to microplasma UV was 4.4 times higher than that of low-pressure Hg UV (0.032 cm2/mJ). This high efficacy was attributed to monochromatic UV wavelength at 222 nm damaging the AdV capsid protein. The results of these molecular assays also proved that microplasma UV and medium-pressure Hg UV with a bandpass filter at 223 nm (MPUV223nm) have a similar influence on AdV (p>0.05). We then estimated the relative energy efficiency of MPUV and microplasma UV to LPUV for 4 log reduction of the viruses. We found that the microplasma UV resulted in higher inactivation rate constants for viruses than the current Hg-based UV. Consequently, microplasma UV could be more energy efficient than low-pressure Hg UV for water disinfection if the wall-plug efficiency of the microplasma UV lamp improved to 8.4% (currently 1.5%). Therefore, the microplasma UV lamp is a promising option for water disinfection.

Dry Heat as a Decontamination Method for N95 Respirator Reuse.

A pandemic such as COVID-19 can cause a sudden depletion of the worldwide supply of respirators, forcing healthcare providers to reuse them. In this study, we systematically evaluated dry heat treatment as a viable option for the safe decontamination of N95 respirators (1860, 3M) before their reuse. We found that the dry heat generated by an electric cooker (100 °C, 5% relative humidity, 50 min) effectively inactivated Tulane virus (TV, >5.2-log10 reduction), rotavirus (RV, >6.6-log10 reduction), adenovirus (AdV, >4.0-log10 reduction), and transmissible gastroenteritis virus (TGEV, >4.7-log10 reduction). The respirator integrity (determined on the basis of the particle filtration efficiency and quantitative fit testing) was not compromised after 20 cycles of a 50 min dry heat treatment. On the basis of these results, dry heat decontamination generated by an electric cooker (e.g., rice cookers, instant pots, and ovens) could be an effective and accessible decontamination method for the safe reuse of N95 respirators. We recommend users measure the temperature during decontamination to ensure the respirator temperature can be maintained at 100 °C for 50 min.

Influence of algal organic matter on MS2 bacteriophage inactivation by ultraviolet irradiation at 220 nm and 254 nm.

We determined the potential interference of extracellular algal organic matter (EAOM) and intracellular algal organic matter (IAOM) extracted from Microcystis aeruginosa on MS2 bacteriophage inactivation under UV irradiation at two wavelengths (220 and 254 nm). UV irradiation at 220 nm doubled the inactivation rate of MS2 in water containing EAOM than in organic-free phosphate buffered solution. In contrast, EAOM did not change MS2 inactivation by exposure to UV 254 nm. The presence of IAOM did not significantly influence MS2 inactivation by exposure to either UV 254 or UV 220 nm. To achieve 3 log10 inactivation of MS2, UV254 nm required more than double the dose of UV220 nm (45 mJ/cm2 vs. 20 mJ/cm2). Linear correlations between the reduction in infectivity and the reduction in genome copies detected by reverse transcription quantitative polymerase chain reaction suggested that genomic damage is the main mechanism responsible for MS2 inactivation in water containing algal organic matter (AOM) by exposure to UV irradiation. These findings suggest that the presence of AOM did not negatively influence MS2 inactivation by either 220 or 254 nm irradiation, and that a lower UV dose of 220 nm irradiation can be used to achieve the same level of inactivation in water containing AOM.

Disintegration of simulated drinking water biofilms with arrays of microchannel plasma jets.

Biofilms exist and thrive within drinking water distribution networks, and can present human health concerns. Exposure of simulated drinking water biofilms, grown from groundwater, to a 9 × 9 array of microchannel plasma jets has the effect of severely eroding the biofilm and deactivating the organisms they harbor. In-situ measurements of biofilm structure and thickness with an optical coherence tomography (OCT) system show the biofilm thickness to fall from 122 ± 17 µm to 55 ± 13 µm after 15 min. of exposure of the biofilm to the microplasma column array, when the plasmas are dissipating a power density of 58 W/cm2. All biofilms investigated vanish with 20 min. of exposure. Confocal laser scanning microscopy (CLSM) demonstrates that the number of living cells in the biofilms declines by more than 93% with 15 min. of biofilm exposure to the plasma arrays. Concentrations of several oxygen-bearing species, generated by the plasma array, were found to be 0.4-21 nM/s for the hydroxyl radical (OH), 85-396 nM/s for the 1O2 excited molecule, 98-280 µM for H2O2, and 24-42 µM for O3 when the power density delivered to the array was varied between 3.6 W/cm2 and 79 W/cm2. The data presented here demonstrate the potential of microplasma arrays as a tool for controlling, through non-thermal disruption and removal, mixed-species biofilms prevalent in commercial and residential water systems.

Prevalence of Human Noroviruses in Commercial Food Establishment Bathrooms.

Although transmission of human norovirus in food establishments is commonly attributed to consumption of contaminated food, transmission via contaminated environmental surfaces, such as those in bathrooms, may also play a role. Our aim was to determine the prevalence of human norovirus on bathroom surfaces in commercial food establishments in New Jersey, Ohio, and South Carolina under nonoutbreak conditions and to determine characteristics associated with the presence of human norovirus. Food establishments (751) were randomly selected from nine counties in each state. Four surfaces (underside of toilet seat, flush handle of toilet, inner door handle of stall or outer door, and sink faucet handle) were swabbed in male and female bathrooms using premoistened macrofoam swabs. A checklist was used to collect information about the characteristics, materials, and mechanisms of objects in bathrooms. In total, 61 (1.5%) of 4,163 swabs tested were presumptively positive for human norovirus, 9 of which were confirmed by sequencing. Some factors associated with the presence of human norovirus included being from South Carolina (odd ratio [OR], 2.4; 95% confidence interval [CI], 1.2 to 4.9; P < 0.05) or New Jersey (OR, 1.7; 95% CI, 0.9 to 3.3; 0.05 < P < 0.10), being a chain establishment (OR, 1.9; 95% CI, 1.1 to 3.3; P < 0.05), being a unisex bathroom (versus male: OR, 2.0; 95% CI, 0.9 to 4.1; 0.05 < P < 0.10; versus female: OR, 2.6; 95% CI, 1.2 to 5.7; P < 0.05), having a touchless outer door handle (OR, 3.3; 95% CI, 0.79 to 13.63; 0.05 < P < 0.10), and having an automatic flush toilet (OR, 2.5, 95% CI, 1.1 to 5.3; 0.05 < P < 0.10). Our findings confirm that the presence of human norovirus on bathroom surfaces in commercial food establishments under nonoutbreak conditions is a rare event. Therefore, routine environmental monitoring for human norovirus contamination during nonoutbreak periods is not an efficient method of monitoring norovirus infection risk.