Institute of Environmental Science and Research Ltd (ESR), Kenepuru Science Centre, PO Box 50348, Porirua, 5240, New Zealand.
The New Zealand Institute for Plant and Food Research Ltd (PFR), Private Bag 92169, Auckland, 1142, New Zealand.
Food Environ Virol. 2024 Jun;16(2):171-179. doi: 10.1007/s12560-024-09585-4. Epub 2024 Mar 8.
Norovirus is the leading cause of viral gastroenteritis globally. While person-to-person transmission is most commonly reported route of infection, human norovirus is frequently associated with foodborne transmission, including through consumption of contaminated bivalve molluscan shellfish. Reverse transcription (RT)-qPCR is most commonly used method for detecting human norovirus detection in foods, but does not inform on its infectivity, posing challenges for assessing intervention strategies aimed at risk elimination. In this study, RT-qPCR was used in conjunction with a derivative of the photoreactive DNA binding dye propidium monoazide (PMAxx™) (PMAxx-RT-qPCR) to evaluate the viral capsid integrity of norovirus genogroup I and II (GI and GII) in shellfish following high pressure processing (HPP). Norovirus GI.3 and GII.4 bioaccumulated oysters were subjected to HPP at pressures of 300 and 450 MPa at 15 °C, and 300, 450 and 600 MPa at 20 °C. Samples were analysed using both RT-qPCR and PMAxx-RT-qPCR. For each sample, norovirus concentration (genome copies/g digestive tissue) determined by RT-qPCR was divided by the PMAxx-RT-qPCR concentration, giving the relative non-intact (RNI) ratio. The RNI ratio values relate to the amount of non-intact (non-infectious) viruses compared to fully intact (possible infectious) viruses. Our findings revealed an increasing RNI ratio value, indicating decreasing virus integrity, with increasing pressure and decreasing pressure. At 300 MPa, for norovirus GI, the median [95% confidence interval, CI] RNI ratio values were 2.6 [1.9, 3.0] at 15 °C compared to 1.1 [0.9, 1.8] at 20 °C. At 450 MPa, the RNI ratio values were 5.5 [2.9, 7.0] at 15 °C compared to 1.3 [1.0, 1.6] at 20 °C. At 600 MPa, the RNI ratio value was 5.1 [2.9, 13.4] at 20 °C. For norovirus GII, RT-qPCR and PMAxx-RT-qPCR detections were significantly reduced at 450 and 600 MPa at both 15 °C and 20 °C, with the median [95% CI] RNI ratio value at 300 MPa being 1.1 [0.8, 1.6]. Following HPP treatment, the use of PMAxx-RT-qPCR enables the selective detection of intact and potential infectious norovirus, enhancing our understanding of the inactivation profiles and supporting the development of more effective risk assessment strategies.
诺如病毒是全球病毒性肠胃炎的主要病因。虽然人际传播是最常见的感染途径，但人类诺如病毒经常与食源性传播有关，包括食用受污染的双壳贝类软体动物。逆转录（RT）-qPCR 是最常用于检测食品中人类诺如病毒的方法，但它不能说明其感染力，这给评估旨在消除风险的干预策略带来了挑战。在这项研究中，RT-qPCR 与光反应性 DNA 结合染料吖啶单甲醚（PMAxx）的衍生物（PMAxx-RT-qPCR）一起用于评估高压处理（HPP）后贝类中诺如病毒基因组 I 和 II（GI 和 GII）的病毒衣壳完整性。GI.3 和 GII.4 型诺如病毒生物累积的牡蛎在 15°C 时分别在 300 和 450 MPa、20°C 时在 300、450 和 600 MPa 的压力下进行 HPP。使用 RT-qPCR 和 PMAxx-RT-qPCR 对样品进行分析。对于每个样品，通过 RT-qPCR 确定的诺如病毒浓度（消化组织中的基因组拷贝/g）除以 PMAxx-RT-qPCR 浓度，得出相对非完整（RNI）比。RNI 比值与完整（可能感染）病毒相比，与非完整（无传染性）病毒的数量有关。我们的研究结果表明，随着压力的增加和降低，RNI 比值值（表示病毒完整性降低的指标）呈上升趋势。在 300 MPa 时，对于 GI 型诺如病毒，在 15°C 时的中位数[95%置信区间，CI]RNI 比值值为 2.6[1.9，3.0]，而在 20°C 时为 1.1[0.9，1.8]。在 450 MPa 时，在 15°C 时的 RNI 比值值为 5.5[2.9，7.0]，而在 20°C 时为 1.3[1.0，1.6]。在 600 MPa 时，在 20°C 时的 RNI 比值值为 5.1[2.9，13.4]。对于 GII 型诺如病毒，在 15°C 和 20°C 时，450 和 600 MPa 下的 RT-qPCR 和 PMAxx-RT-qPCR 检测均显著降低，在 300 MPa 时的中位数[95%CI]RNI 比值值为 1.1[0.8，1.6]。经过 HPP 处理后，PMAxx-RT-qPCR 的使用能够选择性地检测完整和潜在传染性的诺如病毒，这增强了我们对失活特征的理解，并支持开发更有效的风险评估策略。
