Of masks and methylene blue-The use of methylene blue photochemical treatment to decontaminate surgical masks contaminated with a tenacious small nonenveloped norovirus.

BACKGROUND: In the context of the SARS-CoV-2 pandemic, reuse of personal protective equipment, specifically that of medical face coverings, has been recommended. The reuse of these typically single-use only items necessitates procedures to inactivate contaminating human respiratory and gastrointestinal pathogens. We previously demonstrated decontamination of surgical masks and respirators contaminated with infectious SARS-CoV-2 and various animal coronaviruses via low concentration- and short exposure methylene blue photochemical treatment (10 µM methylene blue, 30 minutes of 12,500-lux red light or 50,000 lux white light exposure). METHODS: Here, we describe the adaptation of this protocol to the decontamination of a more resistant, non-enveloped gastrointestinal virus and demonstrate efficient photodynamic inactivation of murine norovirus, a human norovirus surrogate. RESULTS: Methylene blue photochemical treatment (100 µM methylene blue, 30 minutes of 12,500-lux red light exposure) of murine norovirus-contaminated masks reduced infectious viral titers by over four orders of magnitude on surgical mask surfaces. DISCUSSION AND CONCLUSIONS: Inactivation of a norovirus, the most difficult to inactivate of the respiratory and gastrointestinal human viruses, can predict the inactivation of any less resistant viral mask contaminant. The protocol developed here thus solidifies the position of methylene blue photochemical decontamination as an important tool in the package of practical pandemic preparedness.

Development of a Specific Anti-capsid Antibody- and Magnetic Bead-Based Immunoassay to Detect Human Norovirus Particles in Stool Samples and Spiked Mussels via Flow Cytometry.

Human noroviruses impose a considerable health burden globally. Here, a flow cytometry approach designed for their detection in biological waste and food samples was developed using antibody-coated magnetic beads. Antipeptide antibodies against murine norovirus and various human norovirus genotypes were generated for capture and coated onto magnetic beads. A flow cytometry assay was then implemented to detect bead-bound human norovirus GI.3 in patient stool samples and in norovirus-spiked mussel digestive tissues. The detection limit for stool samples was 105 gc/mL, thus bettering detection limits of commercially available norovirus diagnosis quick kits of 100-fold; the detection limit in spiked mussels however was ten-fold higher than in stool samples. Further assays showed a decrease in fluorescence intensity for heat- or UV-inactivated virus particles. Overall, we demonstrate the application of a flow cytometry approach for direct detection of small non-enveloped virus particles such as noroviruses. An adaptation of the technology to routine diagnostics has the potential to contribute a rapid and sensitive tool to norovirus outbreak investigations. Further improvements to the method, notably decreasing the detection limit of the approach, may allow the analysis of naturally contaminated food and environmental samples.

The use of germicidal ultraviolet light, vaporised hydrogen peroxide and dry heat to decontaminate face masks and filtering respirators contaminated with an infectious norovirus.

In the context of the SARS-CoV-2 pandemic, reuse of surgical masks and filtering facepiece respirators has been recommended. Their reuse necessitates procedures to inactivate contaminating human respiratory and oral pathogens. We previously demonstrated decontamination of masks and respirators contaminated with an infectious SARS-CoV-2 surrogate via ultraviolet germicidal irradiation, vaporised hydrogen peroxide, and use of dry heat. Here, we show that these same methods efficiently inactivate a more resistant, non-enveloped oral virus; decontamination of infectious murine norovirus-contaminated masks and respirators reduced viral titres by over four orders of magnitude on mask or respirator coupons.

"Don, doff, discard" to "don, doff, decontaminate"-FFR and mask integrity and inactivation of a SARS-CoV-2 surrogate and a norovirus following multiple vaporised hydrogen peroxide-, ultraviolet germicidal irradiation-, and dry heat decontaminations.

BACKGROUND: As the SARS-CoV-2 pandemic accelerates, the supply of personal protective equipment remains under strain. To combat shortages, re-use of surgical masks and filtering facepiece respirators has been recommended. Prior decontamination is paramount to the re-use of these typically single-use only items and, without compromising their integrity, must guarantee inactivation of SARS-CoV-2 and other contaminating pathogens. AIM: We provide information on the effect of time-dependent passive decontamination (infectivity loss over time during room temperature storage in a breathable bag) and evaluate inactivation of a SARS-CoV-2 surrogate and a non-enveloped model virus as well as mask and respirator integrity following active multiple-cycle vaporised hydrogen peroxide (VHP), ultraviolet germicidal irradiation (UVGI), and dry heat (DH) decontamination. METHODS: Masks and respirators, inoculated with infectious porcine respiratory coronavirus or murine norovirus, were submitted to passive decontamination or single or multiple active decontamination cycles; viruses were recovered from sample materials and viral titres were measured via TCID50 assay. In parallel, filtration efficiency tests and breathability tests were performed according to EN standard 14683 and NIOSH regulations. RESULTS AND DISCUSSION: Infectious porcine respiratory coronavirus and murine norovirus remained detectable on masks and respirators up to five and seven days of passive decontamination. Single and multiple cycles of VHP-, UVGI-, and DH were shown to not adversely affect bacterial filtration efficiency of masks. Single- and multiple UVGI did not adversely affect respirator filtration efficiency, while VHP and DH induced a decrease in filtration efficiency after one or three decontamination cycles. Multiple cycles of VHP-, UVGI-, and DH slightly decreased airflow resistance of masks but did not adversely affect respirator breathability. VHP and UVGI efficiently inactivated both viruses after five, DH after three, decontamination cycles, permitting demonstration of a loss of infectivity by more than three orders of magnitude. This multi-disciplinal approach provides important information on how often a given PPE item may be safely reused.

Optimisation of a PMAxx™-RT-qPCR Assay and the Preceding Extraction Method to Selectively Detect Infectious Murine Norovirus Particles in Mussels.

Human noroviruses are a major cause for gastroenteritis outbreaks. Filter-feeding bivalve molluscs, which accumulate noroviruses in their digestive tissues, are a typical vector for human infection. RT-qPCR, the established method for human norovirus detection in food, does not allow discrimination between infectious and non-infectious viruses and can overestimate potentially infectious viral loads. To develop a more accurate method of infectious norovirus load estimation, we combined intercalating agent propidium monoazide (PMAxx™)-pre-treatment with RT-qPCR assay using in vitro-cultivable murine norovirus. Three primer sets targeting different genome regions and diverse amplicon sizes were used to compare one-step amplification of a short genome fragment to three two-step long-range RT-qPCRs (7 kbp, 3.6 kbp and 2.3 kbp amplicons). Following initial assays performed on untreated infectious, heat-, or ultraviolet-inactivated murine noroviruses in PBS suspension, PMAxx™ RT-qPCRs were implemented to detect murine noroviruses subsequent to their extraction from mussel digestive tissues; virus extraction via anionic polymer-coated magnetic beads was compared with the proteinase K-dependent ISO norm. The long-range RT-qPCR process detecting fragments of more than 2.3 kbp allowed accurate estimation of the infectivity of UV-damaged murine noroviruses. While proteinase K extraction limited later estimation of PMAxx™ pre-treatment effects and was found to be unsuited to the assay, magnetic bead-captured murine noroviruses retained their infectivity. Genome copies of heat-inactivated murine noroviruses differed by 2.3 log10 between RT-qPCR and PMAxx™-RT-qPCR analysis in bivalve molluscs, the PMAxx™ pre-treatment allowing a closer approximation of infectious titres. The combination of bead-based virus extraction and PMAxx™ RT-qPCR thus provides a more accurate model for the estimation of noroviral bivalve mollusc contamination than the conjunction of proteinase K extraction and RT-qPCR and has the potential (once validated utilising infectious human norovirus) to provide an added measure of security to food safety authorities in the hazard assessment of potential bivalve mollusc contamination.

Human Exposure to Hantaviruses Associated with Rodents of the Murinae Subfamily, Madagascar.

We conducted a national human serologic study of a hantavirus detected in Madagascar rodents using a commercial kit and a new ELISA targeting the virus. Our results suggest a conservative estimate of 2.7% (46/1,680) IgG seroprevalence. A second single-district study using the new ELISA revealed a higher prevalence (7.2%; 10/139).

Cockles and mussels, alive, alive, oh-The role of bivalve molluscs as transmission vehicles for human norovirus infections.

Human noroviruses are recognized as the leading worldwide cause of sporadic and epidemic viral gastroenteritis, causing morbidity and mortality in impoverished developing countries and engendering enormous economic losses in developed countries. Transmitted faecal-orally, either via person-to-person contact, or by consumption of contaminated foods or water, norovirus outbreaks are often reported in institutional settings or in the context of communal dining. Bivalve molluscs, which accumulate noroviruses via filter feeding and are often eaten raw or insufficiently cooked, are a common food vehicle implicated in gastroenteritis outbreaks. The involvement of bivalve molluscs in norovirus outbreaks and epidemiology over the past two decades are reviewed. The authors describe how their physiology of filter feeding can render them concentrated vehicles of norovirus contamination in polluted environments and how high viral loads persist in molluscs even after application of depuration practices and typical food preparation steps. The global prevalence of noroviruses in bivalve molluscs as detected by different monitoring efforts is determined and the various methods currently utilized for norovirus extraction and detection from bivalve matrices described. An overview of gastroenteritis outbreaks affirmatively associated with norovirus-contaminated bivalve molluscs as reported in the past 18 years is also provided. Strategies for risk reduction in shellfish contamination and subsequent human infection are discussed.

The Effect of Heat and Free Chlorine Treatments on the Surface Properties of Murine Norovirus.

Heat and free chlorine are among the most efficient and commonly used treatments to inactivate enteric viruses, but their global inactivation mechanisms have not been elucidated yet. These treatments have been shown to affect at least the capsid proteins of viruses and thus may affect the surface properties (i.e. electrostatic charge and hydrophobicity) of such particles. Our aim was to study the effects of heat and free chlorine on surface properties for a murine norovirus chosen as surrogate for human norovirus. No changes in the surface properties were observed with our methods for murine norovirus exposed to free chlorine. Only the heat treatment led to major changes in the surface properties of the virus with the expression of hydrophobic domains at the surface of the particles after exposure to a temperature of 55 °C. No modification of the expression of hydrophobic domains occurred after exposure to 60 °C, and the low hydrophobic state exhibited by infectious and inactivated particles after exposure to 60 °C appeared to be irreversible for inactivated particles only, which may provide a means to discriminate infectious from inactivated murine noroviruses. When exposed to a temperature of 72 °C or to free chlorine at a concentration of 50 mg/L, the genome became available for RNases.