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.