Systematic Surveillance of Hepatitis E Virus in Pig Livers and Risk Assessment Related to Consumption in Selected Provinces of China

Hepatitis E infection is a major global health concern associated with viral hepatitis. HEV genotypes 3 and 4 viruses (HEV3, HEV4) are zoonotic pathogens and transmission to humans is primarily through the consumption of contaminated and undercooked food. HEV has emerged as a significant pathogen in China, with 28,000 cases reported annually. In Chapter 1 we present a review of the epidemiology HEV in China and examine policies for prevention and control of infection. HEV4 is considered the most common genotype identified in human infection and is also endemic in indigenous pig herds, with seropositivity rates reaching 90%. Surveys of pork-products have identified HEV4 RNA, confirming its presence in the food chain. The identification of HEV4 in humans, pigs and pork-products suggest a food-associated route of transmission. No systematic surveillance study in transmission dynamics has been conducted in China to date and the existing data is limited. Firstly, to understand HEV transmission from the food chain in China, more effective methods for detection of HEV RNA from high-risk foodstuffs were required. Due to the low concentration of HEV and the complex food matrix, this has, to-date, presented significant challenges. In Chapter 2, we established a HEV RNA extraction method from pig liver. The method improved extraction efficiencies and amplification inhibition indices. In Chapter 3, a systematic survey of HEV in pig livers in Yunnan, Guizhou, and Hebei provinces was carried out (562 pig liver samples collected from 19 cities}. Significant rates of HEV RNA positivity were observed in Hebei, 14.29% (23/161), Guizhou (10.45%, 21/201) and Yunnan (9.00%, 18/200). Supermarkets. wholesale markets and butcher shops all contained HEV positive products. ORF2 nucleotide sequence (300bp) were amplified, and genotypes 4a, 4d, and 3a were identified. In Chapter 4, an amplicon based whole genome sequencing method was established to assess genetic relatedness of viral strains found in humans and food. Six whole genome sequences (99.08 – 99.34%) were obtained from pig faeces and five partial sequences (37%-89%) were obtained from pig liver (3153bp to 6443bp). The majority were identified as HEV 4d. Phylogenetic analysis of HEV4 whole genome sequences revealed a high homology between human derived viruses isolated from cases in China and those identified in this study from pigs and pork products. The close genetic relatedness suggests indigenous transmission routes from pigs to humans via consumption of contaminated pig liver products in China. HEV3 viruses were also identified in pig liver sourced in China which clustered closely which viruses found in clinical cases in Singapore suggesting alternative routes of transmission involving sources external to China. In Chapter 5 a risk assessment from the consumption of pig liver in Yunnan and Guizhou provinces was conducted. Results revealed that the risk to consumers was significant (199 to 3,96/100,000 population) and underscores the importance of cooking pig liver to reduce the risk of HEV infection. This was the first systematic survey of HEV in pig liver in China and revealed the extent of contaminated products available at the point of retail in the country. However. there are limitations. First, the imbalance in sample numbers may have introduced data bias. Second, there are still challenges with the detection methods in pork liver samples. Third, using HEV RNA genomic copies to estimate risk may have contributed to an overestimation due to the assumption that all detected genomic copies are infectious. Nevertheless, specific public health messaging is needed to advise consumers of the risk and to recommend risk-mitigating steps such as thoroughly cooking pork products, avoiding the consumption of raw or undercooked pork liver, and practicing good hygiene when handling raw pork to prevent cross-contamination.