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

Viruses are present at low concentrations in wastewater, and therefore an effective concentration of 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 concentrated over 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 became significantly higher when calculated based on virus titers than genome concentration, the PGM-MBs method could be an appropriate tool for assessing the risk due to wastewater contaminated with infectious enteric viruses. PCR inhibitors were not concentrated by PGM-MBs, suggesting this tool will be successful for use with environmental samples. The PGM-MBs method is cost-effective (0.43 USD/sample) and fast turnaround (3 hours from virus concentration to genome quantification), and thus this method can be implemented for high throughput facilities. Based on good performance, intrinsic characteristics of targeting the infectious virus, robustness to wastewater, and adaptability to high throughput systems, we are confident that the PGM-MBs method can be applied for successful WBE and ultimately provides valuable public health information.Graphical abstract

Inactivation mechanism and efficacy of grape seed extract for Human Norovirus surrogate

Proper disinfection of harvested food and water is critical to minimize infectious disease. Grape seed extract (GSE), a commonly used health supplement, is a mixture of plant-derived polyphenols. Polyphenols possess anti-microbial and -fungal properties, but anti-viral effects are not well-known. Here we show that GSE outperformed chemical disinfectants (e.g., free chlorine and peracetic acids) in inactivating Tulane virus, a human norovirus surrogate. GSE induced virus aggregation, an event that correlated with a decrease in virus titers. This aggregation and disinfection was not reversible. Molecular docking simulations indicate that polyphenols potentially formed hydrogen bonds and strong hydrophobic interactions with specific residues in viral capsid proteins. Together, these data suggest that polyphenols physically associate with viral capsid proteins to aggregate viruses as a means to inhibit virus entry into the host cell. Plant-based polyphenols like GSE are an attractive alternative to chemical disinfectants to remove infectious viruses from water or food.ImportanceHuman noroviruses are major food- and water-borne pathogens, causing approximately 20% of all cases of acute gastroenteritis cases in developing and developed countries. Proper sanitation or disinfection are critical strategies to minimize human norovirus-caused disease until a reliable vaccine is created. Grape seed extract (GSE) is a mixture of plant-derived polyphenols that is used as a health supplement. Polyphenols are known for antimicrobial, antifungal, and antibiofilm activities, but antiviral effects are not well-known. In studies here, plant-derived polyphenols outperformed chemical disinfectants (e.g., free chlorine and peracetic acids) in inactivating Tulane virus, a human norovirus surrogate. Based on data from additional molecular assays and molecular docking simulations, the current model is that the polyphenols in GSE bind to the Tulane virus capsid, an event that triggers virion aggregation. It is thought that this aggregation prevents Tulane virus from entering host cells.

Application of chitosan microparticles against human norovirus

Human norovirus (HuNoV) is the leading causative agent of foodborne outbreaks and is associated with the second most prevalent cause of waterborne infections in the United States. The goal of this research was to investigate the antiviral activity of chitosan microparticles (CM) against HuNoV GII.4 Sydney and its cultivable surrogate, Tulane virus (TuV), in suspensions mimicking fecally-contaminated water. CM was prepared by crosslinking chitosan molecules with sodium sulfate, and then its anti-noroviral activity was assessed using infectivity assay on TuV and RT-qPCR on TuV and HuNoV. A 3% CM suspension in PBS (pH 7.2) showed binding to TuV particles but with a negligible impact on virus infectivity (p>0.05). TuV and HuNoV suspended in fecal suspensions showed a 1.5-log10 reduction in genomic copies per ml following a 10-min contact time (p<0.05). Despite the negligible impact on viral infectivity, CM moderately binds to virus particles and helps purify environmental water by removing infectious virus particles. In this study, TuV served as a suitable surrogate for HuNoV by showing a similar log10 reduction in fecal suspension. Overall, the outcomes of thisresearch highlight the potential application of CM as a novel, natural treatment to minimize the spread of water-transmitted viral pathogens.

Microbial Inactivation and Quality Preservation of Chicken Breast Salad Using Atmospheric Dielectric Barrier Discharge Cold Plasma Treatment

Microbiological safety of ready-to-eat foods is paramount for consumer acceptability. The effects of in-package atmospheric dielectric barrier discharge cold plasma (ADCP) treatment on the microbiological safety and quality of model chicken salad (CS) were investigated in this study. CS, packaged in a commercial polyethylene terephthalate container, was treated with ADCP at 24 kV for 2 min. The inactivation of indigenous mesophilic bacteria, Salmonella, and Tulane virus in CS; growth of indigenous mesophilic bacteria and Salmonella in CS; and quality of CS during storage at 4 °C were then investigated. ADCP inactivated indigenous mesophilic bacteria, Salmonella, and Tulane virus by 1.2 ± 0.3 log CFU/g, 1.0–1.5 ± 0.2 log CFU/g, and 1.0 ± 0.1 log PFU/g, respectively. Furthermore, it effectively retarded the growth of the microorganisms, while not significantly affecting the color of chicken, romaine lettuce, and carrot, and the antioxidant capacity of all vegetables throughout storage at the tested temperatures (p > 0.05). The color, smell, and appearance of all vegetables evaluated on day 0 were not significantly different in the sensory test, regardless of the treatment (p > 0.05). Collectively, ADCP treatment effectively decontaminates packaged CS without altering its quality-related properties.

Effect of Plant-derived Proteases on Infectivity of Tulane Virus, Murine Norovirus, and Hepatitis A Virus

Plant-derived proteases, bromelain, papain, and ficin are broad-acting enzymes with generally recognized as safe (GRAS) status for foods and have current application in several food industries. These proteases have also been reported to have antimicrobial properties. This study investigated the efficacy of commercially-prepared bromelain, papain, and ficin, individually and combined (2500 ppm crude extract), for inactivation of hepatitis A virus (HAV) and HuNoV surrogates, Tulane virus (TV), and murine norovirus (MNV). Various treatment temperatures (45, 50, 55°C), times (10 or 60 min), and pH values (5.5, 7.0) in the presence of cysteine (2 mM) were evaluated. Inactivation was assessed by infectivity in plaque assay for TV and MNV and by TCID50 for HAV. No reduction in infectious TV or HAV was attributed to the plant-derived proteases at any of the conditions tested. Infectious MNV was reduced by one to 3 log10 PFU/ml; the most effective treatment was bromelain at pH 7 and 50°C for 10 minutes. A time course study with MNV in bromelain at 50°C indicated that a 2-log10 PFU/ml reduction could be achieved within 6 minutes, but extended treatment of 15 minutes was still insufficient to eliminate infectious MNV. The lack of or limited efficacy of bromelain, papain, and ficin on HAV, TV, and MNV even at elevated temperatures and exposure times suggests the plant-derived proteases are not commercially applicable for inactivation of virus on commodities or materials that could not also withstand mild heat treatment. The variable susceptibilities observed between TV and MNV illustrate limitations in utilization of surrogates for predicting pathogen behavior for a structure-specific treatment.

Evaluation of Copper Alloy Surfaces for Inactivation of Tulane Virus and Human Noroviruses

ABSTRACT This study evaluated the efficacy of copper alloy surfaces for inactivation of Tulane virus (TV), assessed by plaque assay and porcine gastric mucin–conjugated magnetic bead (PGM-MB) binding assay, followed by quantitative reverse transcription PCR (PGM-MB–RT-qPCR assay). In addition, the efficacy of a copper surface for inactivation of human norovirus (HuNoV) GII.4 Sydney and GI.3B Potsdam strains was evaluated by PGM-MB–RT-qPCR assay. Results of time-dependent inactivation of viruses on copper, bronze, and brass coupons revealed that 15 min of surface treatments of each of the copper and copper alloys achieved >4-log reduction of purified TV, as assessed by plaque assay, while up to 20 min of copper alloy surface treatments only achieved ∼2-log reduction, as assessed by PGM-MB–RT-qPCR assay. As assessed by PGM-MB–RT-qPCR assay, 10 min of copper surface treatments achieved reductions of 3 and 4 log units for HuNoVs GII.4 Sydney and GI.3B Potsdam, respectively. Results from this study suggest that even though PGM-MB–RT-qPCR assay underestimated the efficacy of copper alloy surface inactivation of TV, copper alloy surfaces were able to effectively inactivate TV and HuNoVs. Therefore, copper alloys can be used as a preventive measure to prevent HuNoV infection and are an effective surface treatment for HuNoVs. HIGHLIGHTS

Converting Microwave Ovens into Plasma-Generating Decontamination Units for N-95 Respirators

We show how a common microwave oven, a coat-hanger and a coffee cup can be used to decontaminate N-95 respirators in 30 seconds. Tulane virus in the artificial saliva was reduced by >3 log and Geobacillus stearothermophilus spores were reduced by >6 log. Respirators maintained filtration and fit even after 10 cycles. Filtration and fit tests performed by the CDC confirmed there was no damage to the respirators. Spectroscopy of the plasma reveals that OH and C-containing radicals as the most prevalent active species.