Abstract
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.
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Acknowledgements
This work was supported by grants from the Service Public Fédéral ‘Santé Publique, Sécurité de la Chaîne alimentaire et Environnement’ (RT15/8 IQUINOR2) (R.M.R, L. F. L.-B., E. T.), the Fund Leon Fredericq for biomedical research at the University Hospital of Liège (L. F. L.-B.) and the German Academic Exchange Service (L. F. L.-B.). The authors would like to thank Dr Marco Grodzki for his expert assistance and advice on bivalve mollusc manipulation. Thanks are also due to Mrs Marie Bournonville and to Mr Jérôme Biondolillo from Liège University Aquarium-Museum for kindly providing natural sea water.
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Razafimahefa, R.M., Ludwig-Begall, L.F., Le Guyader, F.S. et al. Optimisation of a PMAxx™-RT-qPCR Assay and the Preceding Extraction Method to Selectively Detect Infectious Murine Norovirus Particles in Mussels. Food Environ Virol 13, 93–106 (2021). https://doi.org/10.1007/s12560-020-09454-w
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DOI: https://doi.org/10.1007/s12560-020-09454-w