Optimization of a capsid integrity RT-qPCR method to quantify thermally inactivated hepatitis E virus in suspensions...

Hepatitis E virus (HEV) belongs to the Paslahepevirus balayani species (Hepeviridae family) and harbours a positive-sense, single-stranded RNA genome. HEV genotypes 3 and 4 (HEV-3 and-4) are mainly transmitted from animal reservoirs (primarily domestic pig), either through direct contact or by consuming meat from infected animals. Heat treatment is the main food technological process used to inactivate HEV in pork products. It is crucial to implement robust methods for assessing HEV infectivity to ensure the effectiveness of such inactivation processing. Even if several cell lines are permissive to HEV, cell culture-based methods still present several drawbacks (high technical expertise, time-consuming, low throughput). The aims of this study were to optimize a capsid-integrity-RT-qPCR assay to study the thermal resistance of HEV in simple matrix (PBS, cell culture medium), to validate its application to deli meat products (cooked ham and emulsified sausage), and to compare its performance to a cell culture assay using HepaRG cells. Among several candidates, PtCl₄ was identified as the most effective capsid-integrity marker to bind viral RNA, and a concentration of 2.5 mM PtCl₄ provided the highest reduction in genomic titre for both heat-inactivated strains of HEV-3 tested (HEV-3c and HEV-3f) without affecting the viral genomes from intact viral particles. The capsid-integrity-RT-qPCR was successfully validated in food matrices (cooked ham and emulsified sausage), with reductions >4 log₁₀ achieved for heat-inactivated HEV whatever the strain tested. Sensitivity analysis showed reliable detection of infectious HEV in both suspensions and food matrices down to 2 log₁₀ genome equivalents/mL. When applied to assess the effectiveness of thermal inactivation of ≥63°C for ≥5 min or ≥75°C for 5 min, our results showed effective inactivation of HEV, with both molecular and cell culture detection methods showing parallel trends. Capsid-integrity-RT-qPCR showed a time- and temperature-dependent reduction in HEV genomic titres that correlated with the loss of infectivity observed in cell culture assays. These findings support the use of capsid-integrity-RT-qPCR as a useful first-line screening tool for evaluating HEV thermal inactivation, to be complemented by cell culture assays when feasible, particularly in complex food matrices. This complementary use of both methods is intended to be further assessed for validating viral inactivation processes in the food industry.