Apport de la spectroscopie IRTF dans la caractérisation polyphasique de microorganismes impliqués dans les phénomènes de corrosion marine

CAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF

The impact of chlorine and heat on the infectivity and physicochemical properties of bacteriophage MS2

International audienceEnteric viruses and bacteriophages are exposed to various inactivating factors outside their host, and among them chlorine and heat are the most commonly used sanitizer in water industry and treatment in the food industry, respectively. Using MS2 phages as models for enteric viruses, we investigated the impact of free chlorine and heat on their physicochemical properties. Free chlorine was first evaluated alone. No increase in either capsid permeability or hydrophobicity was observed. The negative surface charge slightly increased suggesting molecular changes in the capsid. However, a weakening of the capsid by chlorine was suggested by differential scanning fluorimetry. This phenomenon was confirmed when chlorination was followed by a heat treatment. Indeed, an increase in the inactivation of MS2 phages and the permeability of their capsids to RNases was observed. More interestingly, an increase in the expression of hydrophobic domains at the phage surface was observed, but only for phages remaining infectious. The chlorine-caused weakening of the capsid suggested that, for an optimal use, the oxidant should be followed by heat. The increased permeability to RNases and the expression of hydrophobic domains may contribute to the development or improvement of molecular methods specific for infectious enteric viruses

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 on the Physicochemical Properties of Bacteriophage MS2

International audienceThe differences in physicochemical characteristics between infectious and non-infectious viral particles are poorly known. Even for heat, which is known as one of the most efficient treatments to inactivate enteric viruses, the global inactivation mechanisms have not been described yet. Such knowledge would help distinguish between both types of particles and therefore clarify the interpretation of the presence of viral genomes in food after heat treatment. In this study, we examined in particular the differences in electrostatic charge and hydrophobicity between the two particle types. MS2 phage, a common surrogate for enteric viruses, was used as a model virus. The heat-induced inactivation process of the infectious phages caused hydrophobic domains to be transiently exposed and their charge to become less negative. The particles also became progressively permeable to small molecules such as SYPRO Orange dye. The presence of non-infectious phage particles in which the genome was not accessible to RNases has been clearly demonstrated. These observations were done for MS2 phages exposed to a temperature of 60 °C. When exposed to a temperature higher than their critical temperature (72 °C), the particles were disrupted and the genome became available for RNases. At lower temperatures, 60 °C in this study, the transient expression of hydrophobic domains of remaining infectious phages appeared as an interesting parameter for improving their specific detection

Impact of reducing and oxidizing agents on the infectivity of Q beta phage and the overall structure of its capsid

Q beta phages infect Escherichia coli in the human gut by recognizing F-pili as receptors. Infection therefore occurs under reducing conditions induced by physiological agents (e.g. glutathione) or the intestinal bacterial flora. After excretion in the environment, phage particles are exposed to oxidizing conditions and sometimes disinfection. If inactivation does not occur, the phage may infect new hosts in the human gut through the oral route. During such a life cycle, we demonstrated that, outside the human gut, cysteines of the major protein capsid of Q beta phage form disulfide bonds. Disinfection with NaClO does not allow overoxidation to occur. Such oxidation induces inactivation rather by irreversible damage to the minor proteins. In the presence of glutathione, most disulfide bonds are reduced, which slightly increases the capacity of the phage to infect E. coli in vitro. Such reduction is reversible and barely alters infectivity of the phage. Reduction of all disulfide bonds by dithiothreitol leads to complete capsid destabilization. These data provide new insights into how the phages are impacted by oxidizing-reducing conditions outside their host cell and raises the possibility of the intervention of the redox during life cycle of the phage.Physiological redox conditions modify disulfide bonds of the capsid of Q beta phage without decreasing infectivity, whereas total reduction disrupts the capsid and chlorine oxidation disrupts especially minor proteins.Physiological redox conditions modify disulfide bonds of the capsid of Q beta phage without decreasing infectivity, whereas total reduction disrupts the capsid and chlorine oxidation disrupts especially minor proteins

Rapid and sensitive method to assess human viral pollution in shellfish using infectious F-specific RNA bacteriophages: Application to marketed products

International audienceF-specific RNA bacteriophages (FRNAPH) have been used as indicators of environmental fecal pollution for many years. While FRNAPH subgroup I (FRNAPH-I) are not host specific, some FRNAPH-II and -III strains appear specific to human pollution. Because a close relationship has been observed between FRNAPH-II genome and human norovirus (NoV) in shellfish, and because FRNAPH infectivity can easily be investigated unlike that of NoV, the detection of human infectious FRNAPH could therefore provide a valuable tool for assessing viral risk. In this study, an integrated cell culture real-time RT-PCR method has been developed to investigate infectious FRNAPH subgroup prevalence in oysters. This rapid screening method appears more sensitive than E. coli or NoV genome detection, and allows an FRNAPH subgroup present in low concentrations (0.05 PFU/g of oyster) to be detected in the presence of another 1000 times more concentrated, without any dissection step. Its application to marketed oysters (n = 135) over a 1-year period has allowed to identify the winter peak classically described for NoV or FRNAPH accumulation. Infectious FRNAPH were detected in 34% of batches, and 7% were suspected of having a human origin. This approach may be helpful to evaluate oyster’s depuration processes, based on an infectious viral parameter

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

Inactivation of murine norovirus and hepatitis A virus on fresh raspberries by gaseous ozone treatment

International audienceRaspberries are vulnerable products for which industrial treatment solutions ensuring both food safety and sensory quality are not easily applicable. Raspberries have been associated with numerous foodborne outbreaks in recent decades. Ozone has been proven effective as a drinking water treatment against pathogenic microorganisms. Nevertheless, to date, little information is available regarding the effect of gaseous ozone on viruses in food matrices. A comparison of the effect of gaseous ozone on murine norovirus (MNV-1) and hepatitis A virus (HAV) adsorbed on fresh raspberries was performed. Infectious MNV-1 was highly inactivated (>3.3 log10) by ozone (3 ppm, 1 min). The raspberry matrix seems to enhance inactivation by ozone compared to water. The same treatment was observed to have little effect on HAV even for the highest dose under the tested conditions (5 ppm, 3 min). Ozone treatment (5 ppm, 3 min) did not affect the appearance of raspberries even after three days post-treatment. No ozone effect was observed on the genomes detected by RT-PCR on both tested viruses, irrespective of the matrix or tested doses used. Gaseous ozone could therefore be a good candidate for human norovirus inactivation on raspberries but new conditions are needed for it to have significant effects on HAV inactivation

The Utility of Dreissena polymorpha for Assessing the Viral Contamination of Rivers by Measuring the Accumulation of F-Specific RNA Bacteriophages

International audienceRiver water that receives treated wastewater can be contaminated by pathogens including enteric viruses due to fecal pollution, which may represent an important public health hazard. There is a great diversity of enteric viruses and fecal bacteriophages, especially F-specific RNA bacteriophages (FRNAPHs), are commonly proposed as indicators of viral pollution due to a variety of characteristics such as their structural similarities to the main enteric viruses, their high concentrations in raw wastewater and their environmental survival rate, which is better than other cultivable enteric viruses. However, evaluating the viral contamination of water on the basis of FRNAPH concentration levels continues to present a challenge. This is because the quality of detection is strongly dependent on the quantity of viral particles, high spatio-temporal variabilities and the physico-chemical conditions of the water during sampling. To overcome these limitations, the present study aims to evaluate whether the bivalve mollusk Dreissena polymorpha (zebra mussel) could be considered a suitable experimental model for assessing the viral contamination of rivers. In order to determine this, the capacity of D. polymorpha to accumulate FRNAPHs and assimilate them into their soft tissue was studied. This provided a proof of concept for the use of D. polymorpha to evaluate the viral contamination of surface water. Two experiments were conducted: (1) an in situ experiment to confirm that zebra mussels naturally accumulated FRNAPHs and (2) a laboratory experiment to determine the accumulation and depuration kinetics of FRNAPHs in D. polymorpha tissue. The study highlights the capacity of the mussels to accumulate infectious FRNAPHs both on a laboratory scale under controlled conditions as well as in situ at different sites that are representative of different bodies of water. An analysis of the mussels’ soft tissue showed that they were capable of reflecting the water’s contamination level very quickly (within less than 24 h). Moreover, the soft tissue retained the viral load much longer than the water due to a low depuration rate. The analysis of FRNAPH concentrations in mussels exposed in situ suggested that there were differences in contamination levels between sites. These preliminary results underline the potential utility of zebra mussels in assessing viral contamination by measuring the accumulation of FRNAPHs in their tissue. This may ultimately enable stakeholders to use zebra mussels as a means of monitoring viral pollution in surface water

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