Veterinary Virology and Animal Viral Diseases, Department of Infectious and Parasitic Diseases, FARAH Research Centre, Faculty of Veterinary Medicine, Liège University, B43b, Quartier Vallée 2, Avenue de Cureghem, 10, 4000, Liège, Belgium.
Ifremer, Laboratoire de Microbiologie, LSEM-SG2M, BP 21105, 44311, Nantes, France.
Food Environ Virol. 2021 Mar;13(1):93-106. doi: 10.1007/s12560-020-09454-w. Epub 2021 Jan 3.
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 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.
人诺如病毒是导致肠胃炎爆发的主要原因。滤食性双壳贝类在其消化组织中积累诺如病毒，是人类感染的典型载体。RT-qPCR 是检测食品中人诺如病毒的既定方法，但不能区分感染性和非感染性病毒，并且可能高估潜在的感染性病毒载量。为了开发更准确的传染性诺如病毒载量估计方法，我们结合了嵌入剂吖啶橙（PMAxxTM）预处理和使用体外培养的鼠诺如病毒的 RT-qPCR 检测。使用三种针对不同基因组区域和不同扩增子大小的引物组，比较了一步扩增短基因组片段与三个两步长距离 RT-qPCR（7 kbp、3.6 kbp 和 2.3 kbp 扩增子）。在对未经处理的感染性、热处理或紫外线失活的鼠诺如病毒在 PBS 悬浮液中的初始测定后，进行了 PMAxxTM RT-qPCR 以检测从贻贝消化组织中提取的鼠诺如病毒；比较了阴离子聚合物包被的磁性珠法提取与蛋白酶 K 依赖性 ISO 规范。检测超过 2.3 kbp 片段的长距离 RT-qPCR 过程允许准确估计紫外线损伤的鼠诺如病毒的感染力。虽然蛋白酶 K 提取限制了 PMAxxTM 预处理效果的后期估计，并且发现不适合该测定，但磁性珠捕获的鼠诺如病毒保留了其感染力。贝类中热灭活的鼠诺如病毒的基因组拷贝数在 RT-qPCR 和 PMAxxTM-RT-qPCR 分析之间相差 2.3 个对数，PMAxxTM 预处理更接近感染滴度。基于珠的病毒提取和 PMAxxTM-RT-qPCR 的结合因此提供了比蛋白酶 K 提取和 RT-qPCR 结合更准确的贝类诺如病毒污染估计模型，并且具有潜力（一旦利用感染性人诺如病毒验证）为食品安全当局在评估潜在贝类污染时提供对贝类污染的额外安全措施。
