Hepatitis E Virus: Identification and Evaluation of the Potential for Zoonotic Transmission in the Pork Food Chain.

Hepatitis E is an acute hepatitis in humans, first recognised in 1980 and caused by hepatitis E virus (HEV). The principal mode of spread of HEV is faecal-oral from contaminated water supplies, almost exclusively in developing regions. Accumulating evidence indicates that HEV transmission may be zoonotic in developed regions from swine and perhaps other animal species serving as reservoirs for the virus. The exact transmission routes are unclear, largely because HEV is extremely difficult to propagate in vitro, but retail pig products have been shown to contain HEV RNA. This PhD project was part of the EU FP7 project VITAL (Integrated Monitoring and Control of Food borne Viruses in European Food Supply Chains). The main aim of this PhD project was to investigate the presence and residual infectivity of HEV in the pork food chain. This helped to assess the potential importance of the pig and its products in zoonotic transmission of HEV. A cell culture system for HEV was further optimised for HEV detection in food samples. A productive HEV infection was established in 3D cell culture (Alexander hepatoma PLC/PRF/S) that was permissive for HEV replication. Furthermore, a trial to compare the efficiency of 3D, 2D and 3D transferred to 2D cells culture systems was performed indicating that replication in the 3D cell culture system was the most efficient. In addition, these studies showed that cells grown in 3D and then transferred to 2D for infection were able to support HEV replication. Further refinements such as heat, UV light and sodium hypochlorite inactivation studies were performed. These approaches should enable an assessment of the significance 1 of the pork food chain in transmission of HEV and facilitate the development of control measures. Within the VITAL project standard methods were developed to have common viral detection and extraction methods between all laboratories, and ring trials were organized between 15 EU laboratories to assess the efficacy of the Standard Operating Procedures (SOPs) developed. Since all the laboratories involved were able to detect the viruses with the common SOPs the ring trial was considered successful and the second step of the project began, involving the screening by real time RT-PCR for HEV throughout the pork food chain. One of 40 pig livers and 6 of 63 pork sausages were found to be HEV positive. Virus viability was tested using the 3D cell culture system but no evidence of viral replication was detected. A mathematical model' suggested that the Circulation of HEV in six European countries is endemic. In addition, HEV prevalence in pig's faeces was investigated showing that pigs close to the slaughter age can still be HEV positive. In conclusion, the work carried out in this PhD projected contributed to our understanding of HEV replication in-vitro and provided useful information on the prevalence of HEV in the pork food chain in the UK. In addition, progress was made with possible inactivation methods and control strategies