PMAxx-RT-qPCR to Determine Human Norovirus Inactivation Following High-Pressure Processing of Oysters.

Abstract

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 ofnorovirus 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 450MPa at 15°C, and 300, 450 and 600MPa 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 300MPa, 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 450MPa, 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 600MPa, 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 600MPa at both 15°C and 20°C, with the median [95% CI] RNI ratio value at 300MPa 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.