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To date, sources of hepatitis E virus (HEV) in the Netherlands, including swine and wild boar, have been identified, but no direct attribution to Dutch hepatitis E cases have been demonstrated. Other animal sources may exist. To identify these species, HEV RNA detection by RT-PCR is required, but complicated. A preselection based on serology may be useful. Therefore, wildlife species were studied by serology and molecular methods. Using a species-independent double-antigen sandwich ELISA, HEV-specific antibodies were detected in sera from 12% of 1029 wild boar (Sus scrofa scrofa), in 5% of 38 red deer (Cervus elaphus) and in none of 8 studied roe deer (Capreolus capreolus). Differences in background signals were observed between species and accounted for by fitting finite mixture distributions. HEV RNA was detected in 8% of 106 wild boars, in 15% of 39 red deer and in none of 8 roe deer. In conclusion, HEV was shown to be present in European red deer for the first time. This preselection based on species-independent serological assays may be beneficial to identify new potential animal reservoirs of HEV. The consumption of Dutch undercooked wild boar and red deer meat may lead to human exposure to HEV., (Copyright 2010 Elsevier B.V. All rights reserved.)

Non-travel-related hepatitis E virus (HEV) genotype 3 infections in persons in the Netherlands may have a zoonotic, foodborne, or water-borne origin. Possible reservoirs for HEV transmission by water, food, and animals were studied. HEV genotype 3/open reading frame 2 sequences were detected in 53% of pig farms, 4% of wild boar feces, and 17% of surface water samples. HEV sequences grouped within 4 genotype 3 clusters, of which 1 is so far unique to the Netherlands. The 2 largest clusters contained 35% and 43% of the animal and environmental sequences and 75% and 6%, respectively, of human HEV sequences obtained from a study on Dutch hepatitis E patients. This finding suggests that infection risk may be also dependent on transmission routes other than the ones currently studied. Besides the route of exposure, virus characteristics may be an important determinant for HEV disease in humans.

Human hepatitis E virus (HEV) infections by genotype 3 strains in industrialized countries are hypothesized to be caused by pigs. To examine this hypothesis, the potential health risks of transmission routes should be examined. Possible foodborne transmission was studied by quantifying the presence and infectivity of HEV in commercial porcine livers in The Netherlands. A comparison of four tissue disruption and seven RNA extraction methods revealed that mechanical disruption followed by silica-based RNA extraction gave the highest RNA yields and was therefore employed on commercial porcine livers. Four (6.5%) of 62 porcine livers were HEV RNA positive by reverse transcriptase PCR and Southern blot hybridization. Each positive liver was estimated to contain approximately 65 PCR-detectable units per g. Sequences were obtained for three of four positive livers and classified as HEV genotype 3. Ninety-three percent similarity to Dutch human HEV sequences and 97% similarity to Dutch swine HEV sequences were observed. To determine whether positive livers contained infectious HEV particles, extracts from livers with known HEV RNA sequences were inoculated intravenously in pigs. Two control pigs were included: one was inoculated with a high dose known to result in infection (10(4) PCR-detectable units of HEV RNA), and the other was inoculated with a lower concentration of virus that equaled the concentration of PCR-detectable units in commercial livers ( approximately 20 PCR-detectable units). Infection was observed in the high-dose control, but not in other pigs, suggesting a dose-dependent response in pigs. Hence, the implications of HEV RNA in commercial porcine livers in The Netherlands are unknown. However, HEV RNA is present in commercial porcine livers, and sufficient heating of porcine livers before consumption as precautionary measure is recommended.