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

Abstract : Introduction: 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.
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https://hal.univ-lorraine.fr/hal-01803288
Contributor : Isabelle Bertrand <>
Submitted on : Wednesday, May 30, 2018 - 10:54:51 AM
Last modification on : Tuesday, October 9, 2018 - 3:22:02 PM

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Adrien Brié, Ravo Razafimahefa, Nicolas Boudaud, Christophe Gantzer, Isabelle Bertrand. The effect of heat treatment on the surface properties of murine norovirus. 4th Food and Environmental Virology Conference, 2016, Kusatsu, Japan. ⟨hal-01803288⟩

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