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 log(10) 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

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.

peer reviewedHuman 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 10(5) 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

Development of tools to detect norovirus infectious particles in bivalve molluscs and to screen norovirus in biological and bivalve mollusc samples for human norovirus-associated food safety and outbreaks management

Human noroviruses (HuNoVs) are the leading cause of epidemic and sporadic acute gastroenteritis worldwide and are the primary cause of food-borne viral gastroenteritis. Human norovirus infections resulted in a societal cost estimated at 50 billion euros per year worldwide. The consumption of raw or under-cooked contaminated food, such as shellfish is the most suspected or the most common cause of foodborne norovirus outbreaks. HuNoVs are highly contagious; their main transmission route is the fecal-oral route. It is necessary to set up sensitive and efficient tools for viral detection to ensure food safety for consumers. Nonetheless, the absence of a robust and less cumbersome in vitro culture system for detection of HuNoV represents an obstacle to the establishment of a routine analysis to selectively detect infectious HuNoV, which is not the case of molecular methods used for routine detection. Currently, these molecular methods are the gold standard to detect noroviruses in biological, water and food samples. However, these methods cannot make the distinction between infectious and noninfectious virus particles and they are very sensitive. The viral load of infectious particles detected with these methods are thus non-representative of the residual potential viral infectivity. The first objective of this thesis is to propose an approach for detection of infectious human noroviruses in food samples to mitigate the public health risks and to lower norovirus foodborne outbreaks. For this, the murine norovirus (MNV) which propagates in murine macrophages cells was used as surrogate to HuNoV. A new virus extraction method based on anionic magnetic beads was developed to separate and concentrate MNV from mussel (Mytilus edilus) digestive tissues when keeping their structure (capsid) intact. This concentration step is then followed by a RT-qPCR with PMAxx, an intercalating agent able to enter viruses with compromising capsid integrity. This approach can enhance by 2 log10 the detection limit of infectious MNV genome bioaccumulated in mussels. Despite the measures for prevention and control of food safety to limit the risk of infection, the norovirus foodborne outbreaks still occur. The second objective of this thesis is to develop a rapid and sensitive tool to screen human noroviruses in biological and food samples during investigations of norovirus foodborne outbreaks. This approach also relies on detection of particles with non-damaged structure. A flow cytometry approach based on magnetic beads coated with capture antibodies that bind to a conserved region of the capsid and detection by biotinylated antibodies for fluorescence labeling, was developed. Applied on HuNoV strain, this approach is a useful tool to diagnose noroviruses in stool samples with a better detection limit than the commercialised rapid tests. The method was tested in mussels after spiking with HuNoV. The approach needs further optimisation to enhance the detection limit for food application. Yet, this approach represents an innovative proof-of-concept. The perspectives of this PhD study are to apply this prototype of flow cytometry approach to a broad range of HuNoV strains and the synergistic application of both developed tools in this thesis for a rapid, robust and efficient diagnostic of noroviruses in order to discriminate the infectious particles from noninfectious particles in the field of food safety.IQUINO

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

Human noroviruses are recognised 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 utilised 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 of shellfish contamination and subsequent human infection are discussed

The effect of heat treatment on the surface properties of murine norovirus

International audienceIntroduction: Inactivation rates of enteric viruses by heat treatments have been widely described in different matrices (Bertrand et al., 2012), but the mechanisms that lead to viral inactivation remain poorly understood (Wigginton et al., 2012). Temperature has been shown to affect capsid of poliovirus by different ways. Among them, the externalization of the amino terminus of VP1 has been directly linked to an increase in hydrophobicity (Fricks and Hogle, 1990). The inactivation by heat has also been related to the loss of binding capacity to host cells for poliovirus, hepatitis A virus, feline calicivirus (Nuanualsuwan and Cliver, 2003), and MS2 F-specific RNA phage (Wigginton et al., 2012). In that context, the study of surface properties of pathogenic enteric viruses and their surrogates during heat exposure, in relation with loss in infectivity and alterations of capsid functions, may contribute to the understanding of the mechanisms of inactivation, and then to the development of detection methods specific for infectious viral particles.Objectives: Our study aimed to evaluate the effect of heat inactivation on the surface properties of murine norovirus (MNV), a current surrogate of human norovirus. We have also investigated the effect of heat on host cells binding capacity, genome stability, and capsid permeability to RNase.Methods: MNV-1 strain S99 in PBS suspensions at a concentration of 106 TCID50/ml was treated by heat at 50°C, 55°C, 60°C, 72°C, and 80°C for 10 min in a water bath. The inactivation rate and genome degradation were determined by TCID50 and RT-qPCR methods, respectively. The specific adhesion of MNV-1 on RAW 264.7 cells was compared before and after heat treatment. Global electrostatic charge and hydrophobicity were determined by adhesion on charged and hydrophobic beads, respectively. The charge and the hydrophobicity were independently estimated for infectious and total (infectious and non-infectious) MNV particles by TCID50 and RT-qPCR methods, respectively.Results: The decrease in infectivity followed the increase in temperature between 50°C and 60°C with- 1.1 log10 at 50°C, - 2.6 log10 at 55°C, and - 5.6 log10 at 60°C. Complete inactivation of MNV was noted after 10 min heating at temperature ≥ 72°C. On the contrary, heat had a very low impact on viral genome quantified by RT-qPCR (< - 0.4 log10) even for the highest temperatures. Degradation of genome by RNase (- 2 log10) was observed only for temperatures ≥ 72°C.The modification of binding capacity to host cells, charge and hydrophobicity were then studied only for MNV exposed to temperatures below 72°C. The decrease in binding capacity to RAW 264.7 cells followed the increase in temperature. The global charge remained negative and unchanged after heat treatment. Exposure to 50°C and 55°C led to an increase in hydrophobicity for inactivated and remaining infectious MNV as well, whereas MNV exposed to 60°C exhibited a very low hydrophobicity similar to that of native particles. Thus, heat modified the capsid of MNV and led, at least, to the transient expression of hydrophobic domains at their surface and to the loss in ability to bind to the host cells.Conclusions: By focusing on surface properties, we report elements to puzzle out the mechanisms of MNV inactivation by heat. The global electrostatic charge of MNV seemed not be modified. In contrast, an exposure to 50°C and 55°C increased the hydrophobicity for both inactivated and remaining infectious MNV. Almost all MNV particles were inactivated by exposure to 60°C, but they went back to a low hydrophobicity level. These heated particles were also resistant to RNase and mainly unable to bind to their host cells

The effect of heat treatment on the surface properties of murine norovirus

International audienceIntroduction: Inactivation rates of enteric viruses by heat treatments have been widely described in different matrices (Bertrand et al., 2012), but the mechanisms that lead to viral inactivation remain poorly understood (Wigginton et al., 2012). Temperature has been shown to affect capsid of poliovirus by different ways. Among them, the externalization of the amino terminus of VP1 has been directly linked to an increase in hydrophobicity (Fricks and Hogle, 1990). The inactivation by heat has also been related to the loss of binding capacity to host cells for poliovirus, hepatitis A virus, feline calicivirus (Nuanualsuwan and Cliver, 2003), and MS2 F-specific RNA phage (Wigginton et al., 2012). In that context, the study of surface properties of pathogenic enteric viruses and their surrogates during heat exposure, in relation with loss in infectivity and alterations of capsid functions, may contribute to the understanding of the mechanisms of inactivation, and then to the development of detection methods specific for infectious viral particles.Objectives: Our study aimed to evaluate the effect of heat inactivation on the surface properties of murine norovirus (MNV), a current surrogate of human norovirus. We have also investigated the effect of heat on host cells binding capacity, genome stability, and capsid permeability to RNase.Methods: MNV-1 strain S99 in PBS suspensions at a concentration of 106 TCID50/ml was treated by heat at 50°C, 55°C, 60°C, 72°C, and 80°C for 10 min in a water bath. The inactivation rate and genome degradation were determined by TCID50 and RT-qPCR methods, respectively. The specific adhesion of MNV-1 on RAW 264.7 cells was compared before and after heat treatment. Global electrostatic charge and hydrophobicity were determined by adhesion on charged and hydrophobic beads, respectively. The charge and the hydrophobicity were independently estimated for infectious and total (infectious and non-infectious) MNV particles by TCID50 and RT-qPCR methods, respectively.Results: The decrease in infectivity followed the increase in temperature between 50°C and 60°C with- 1.1 log10 at 50°C, - 2.6 log10 at 55°C, and - 5.6 log10 at 60°C. Complete inactivation of MNV was noted after 10 min heating at temperature ≥ 72°C. On the contrary, heat had a very low impact on viral genome quantified by RT-qPCR (< - 0.4 log10) even for the highest temperatures. Degradation of genome by RNase (- 2 log10) was observed only for temperatures ≥ 72°C.The modification of binding capacity to host cells, charge and hydrophobicity were then studied only for MNV exposed to temperatures below 72°C. The decrease in binding capacity to RAW 264.7 cells followed the increase in temperature. The global charge remained negative and unchanged after heat treatment. Exposure to 50°C and 55°C led to an increase in hydrophobicity for inactivated and remaining infectious MNV as well, whereas MNV exposed to 60°C exhibited a very low hydrophobicity similar to that of native particles. Thus, heat modified the capsid of MNV and led, at least, to the transient expression of hydrophobic domains at their surface and to the loss in ability to bind to the host cells.Conclusions: By focusing on surface properties, we report elements to puzzle out the mechanisms of MNV inactivation by heat. The global electrostatic charge of MNV seemed not be modified. In contrast, an exposure to 50°C and 55°C increased the hydrophobicity for both inactivated and remaining infectious MNV. Almost all MNV particles were inactivated by exposure to 60°C, but they went back to a low hydrophobicity level. These heated particles were also resistant to RNase and mainly unable to bind to their host cells

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

International audienceHeat 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

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

"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.

BackgroundAs 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.AimWe 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.MethodsMasks 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 discussionInfectious 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