Hepatitis E virus is a prominent cause of viral hepatitis worldwide. In Western countries, most infections are asymptomatic. However, acute self-limiting hepatitis and chronic cases in immunocompromised individuals can occur. Studying HEV is challenging due to its difficulty to grow in cell culture. Consequently, the detection of the virus mainly relies on RT-qPCR, which cannot differentiate between infectious and non-infectious particles. To overcome this problem, methods assessing viral integrity offer a possible solution to differentiate between intact and damaged viruses. This study aims at optimizing existing HEV cell culture models and RT-qPCR-based assays for selectively detecting intact virions to establish a reliable model for assessing HEV infectivity. In conclusion, these newly developed methods hold promise for enhancing food safety by identifying approaches for inactivating HEV in food processing, thereby increasing food safety measures.

Hepatitis E virus (HEV) is an emerging foodborne pathogen in Europe, primarily associated with the consumption of pork products. While conventional destructive TRIzol extraction methods followed by RT-qPCR can detect HEV RNA in processed meat, this approach fails to distinguish between infectious and inactivated virus, complicating risk assessment. Currently, no standardized method exists for extracting intact, infectious HEV from pork products, limiting our ability to evaluate viral infectivity post-processing. In this study, we developed and evaluated three extraction methods to recover intact, infectious HEV from pork meat products. Our optimized soft extraction method using TGBE buffer and stomacher-based homogenization allowed us to successfully isolate infectious HEV from pork liver pâté bought in a Belgian supermarket, confirming the presence of infectious HEV in a ready-to-eat pâté intended for direct consumer consumption. Furthermore, viral RNA was detected following extraction with this method at concentrations (copies/g) similar to those obtained with the TRIzol method, while preserving viral integrity, as confirmed by capsid integrity assays and cell culture infectivity models. As a critical tool for assessing the impact of food processing on viral infectivity, the method holds significant potential for advancing food safety research and improving risk evaluations related to HEV transmission through pork products.

Hepatitis E Virus (HEV) poses a significant public health risk in Europe, particularly through the consumption of contaminated pork products. With increasing concern about foodborne transmission, understanding the persistence of HEV in meat products is crucial for improving food safety and preventing infections. This thesis investigates HEV in a Belgian context, with the primary aim of enhancing knowledge of HEV survival in pork meat products. The first part of the study involved conducting risk analyses to identify which pork products present the highest risk of HEV infection for consumers. The findings revealed that ready-to-eat pork products carry the greatest theoretical risk, leading to the sampling and testing of these products from Belgian supermarkets for the presence of HEV RNA. HEV RNA was detected in 65% of the tested samples, particularly in pork liver pâtés, confirming the presence of HEV in pork products available for sale. The second part of the project focused on developing and refining methods to detect HEV in meat products. Existing detection methods are labor-intensive, not standardized, and often fail to extract intact viral particles. The goal was to 1) optimize a cell culture model for directly assessing infectivity and 2) develop proxy methods for evaluating HEV infectivity indirectly by assessing the intactness of viral particles. Afterwards we optimized an extraction method that successfully retrieved intact and infectious HEV from pork products, enabling differentiation between infectious and inactivated HEV—a capability lacking in current RT-qPCR methods. In the final part of this project, the optimized methods were used to assess the impact of common meat processing techniques on HEV infectivity. Pork liver pâtés were made to evaluate the effects of heat treatment, showing that HEV is remarkably heat-stable, requiring temperatures up to 99C for complete inactivation (>3.5 log reduction). Similarly, dried pork sausages were used to study the effects of drying and pH reduction, revealing that HEV remains stable for up to 14 days, with pH reduction having very limited effect on inactivation. These findings indicate that current meat processing techniques may be inadequate for fully inactivating HEV, leaving residual infectious virus in pork products available for sale. This underscores the potential risk of foodborne HEV infection from consuming these products. In summary, this project provided the first comprehensive demonstration of HEV presence in Belgian meat products, alongside the development and optimization of methods for detecting infectious HEV. Additionally, the study assessed the effects of thermal processing, pH reduction, and drying on HEV survival, offering valuable insights into the persistence of the virus during meat processing and emphasizing the need for improved food safety practices.