{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,13]],"date-time":"2026-07-13T21:56:32Z","timestamp":1783979792231,"version":"3.55.0"},"reference-count":30,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2023,10,11]],"date-time":"2023-10-11T00:00:00Z","timestamp":1696982400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"European Union\u2019s Horizon 2020 Research and Innovation programme","award":["773830"],"award-info":[{"award-number":["773830"]}]},{"name":"European Union\u2019s Horizon 2020 Research and Innovation programme","award":["WOT-01-003-069"],"award-info":[{"award-number":["WOT-01-003-069"]}]},{"name":"European Union\u2019s Horizon 2020 Research and Innovation programme","award":["KB-37-003-003"],"award-info":[{"award-number":["KB-37-003-003"]}]},{"name":"Dutch ministry of Agriculture, Nature and Food quality","award":["773830"],"award-info":[{"award-number":["773830"]}]},{"name":"Dutch ministry of Agriculture, Nature and Food quality","award":["WOT-01-003-069"],"award-info":[{"award-number":["WOT-01-003-069"]}]},{"name":"Dutch ministry of Agriculture, Nature and Food quality","award":["KB-37-003-003"],"award-info":[{"award-number":["KB-37-003-003"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Hepatitis E virus (HEV) is a positive-sense single-stranded RNA virus and a major cause of acute viral hepatitis. HEV is responsible for 20 million infections worldwide in humans every year. HEV-3 and HEV-4 are zoonotic and are responsible for most of the HEV cases in developed countries. Consumption of contaminated pig meat or pig products is considered to be the main transmission route of HEV HEV-3 in Europe. Prevalence studies for HEV generally use PCR methods to detect the presence or absence of genomic RNA. However, these methods do not discriminate infectious virus particles from non-infectious material. Previously developed HEV cell culture systems only worked with high efficiency after cell line adaptation of the subjected virus strains. In this manuscript, the development of a culture system for the detection of infectious HEV strains is described. For this purpose, we optimized the isolation and the growth of primary hepatocytes from young piglets. Subsequently, the isolated hepatocytes were used to culture HEV of different origins, such as liver tissue samples and sausage samples. This method can be applied to better assess the risk of infection through consumption of food products associated with HEV RNA contamination.<\/jats:p>","DOI":"10.3390\/pathogens12101231","type":"journal-article","created":{"date-parts":[[2023,10,11]],"date-time":"2023-10-11T08:07:12Z","timestamp":1697011632000},"page":"1231","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Development of an Ex Vivo Assay for Identification of Infectious Hepatitis E Virus in Different Kinds of Food Samples"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7621-2729","authenticated-orcid":false,"given":"Renate W.","family":"Hakze-van der Honing","sequence":"first","affiliation":[{"name":"Wageningen Bioveterinary Research (WBVR), Wageningen University and Research, Houtribweg 39, 8221 Lelystad, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sophie","family":"van Oort","sequence":"additional","affiliation":[{"name":"Wageningen Bioveterinary Research (WBVR), Wageningen University and Research, Houtribweg 39, 8221 Lelystad, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3093-5662","authenticated-orcid":false,"given":"Ren\u00e9 A. M.","family":"Dirks","sequence":"additional","affiliation":[{"name":"Wageningen Food Safety Research (WFSR), Wageningen University and Research, Akkermaalsbos 2, 6708 Wageningen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7498-8002","authenticated-orcid":false,"given":"Wim H. M.","family":"van der Poel","sequence":"additional","affiliation":[{"name":"Wageningen Bioveterinary Research (WBVR), Wageningen University and Research, Houtribweg 39, 8221 Lelystad, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,10,11]]},"reference":[{"key":"ref_1","unstructured":"World Health Organization (2021). Hepatitis E."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"692","DOI":"10.1099\/jgv.0.001435","article-title":"Update: Proposed reference sequences for subtypes of hepatitis E virus (species Orthohepevirus A)","volume":"101","author":"Smith","year":"2020","journal-title":"J. Gen. Virol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"17086","DOI":"10.1038\/nrdp.2017.86","article-title":"Hepatitis E virus infection","volume":"3","author":"Kamar","year":"2017","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Meng, X.-J. (2016). Expanding Host Range and Cross-Species Infection of Hepatitis E Virus. PLoS Pathog., 12.","DOI":"10.1371\/journal.ppat.1005695"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1053\/j.gastro.2015.10.048","article-title":"Chronic Infection with Camelid Hepatitis E Virus in a Liver Transplant Recipient Who Regularly Consumes Camel Meat and Milk","volume":"150","author":"Lee","year":"2016","journal-title":"Gastroenterology"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Lhomme, S., Marion, O., Abravanel, F., Izopet, J., and Kamar, N. (2020). Clinical Manifestations, Pathogenesis and Treatment of Hepatitis E Virus Infections. J. Clin. Med., 9.","DOI":"10.3390\/jcm9020331"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"811","DOI":"10.1056\/NEJMoa0706992","article-title":"Faculty Opinions recommendation of Hepatitis E virus and chronic hepatitis in organ-transplant recipients","volume":"358","author":"Kamar","year":"2008","journal-title":"N. Engl. J. Med."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"EFSA Panel on Biological Hazards (BIOHAZ), Ricci, A., Allende, A., Bolton, D., Chemaly, M., Davies, R., Fernandez Escamez, P.S., Herman, L., Koutsoumanis, K., and Lindqvist, R. (2017). Scientific opinion on the public health risks associated with hepatitis E virus (HEV) as a food-borne pathogen. EFSA J., 15, e04886.","DOI":"10.2903\/j.efsa.2017.4886"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"825","DOI":"10.1086\/655898","article-title":"Pig Liver Sausage as a Source of Hepatitis E Virus Transmission to Humans","volume":"202","author":"Colson","year":"2010","journal-title":"J. Infect. Dis."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"264","DOI":"10.3201\/eid1902.121255","article-title":"Hepatitis E Virus in Pork Liver Sausage, France","volume":"19","author":"Berto","year":"2012","journal-title":"Emerg. Infect. Dis."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"912","DOI":"10.1099\/vir.0.82613-0","article-title":"Detection and characterization of infectious Hepatitis E virus from commercial pig livers sold in local grocery stores in the USA","volume":"88","author":"Feagins","year":"2007","journal-title":"J. Gen. Virol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1007\/s12560-015-9196-2","article-title":"Survival and Elimination of Hepatitis E Virus: A Review","volume":"7","author":"Cook","year":"2015","journal-title":"Food Environ. Virol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/j.coviro.2014.01.006","article-title":"Food and environmental routes of Hepatitis E virus transmission","volume":"4","year":"2014","journal-title":"Curr. Opin. Virol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"381","DOI":"10.3201\/eid1503.071472","article-title":"Sources of Hepatitis E Virus Genotype 3 in The Netherlands","volume":"15","author":"Rutjes","year":"2009","journal-title":"Emerg. Infect. Dis."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1186\/s13567-022-01068-3","article-title":"Repeated cross-sectional sampling of pigs at slaughter indicates varying age of hepatitis E virus infection within and between pig farms","volume":"53","author":"Meester","year":"2022","journal-title":"Veter. Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1016\/j.jviromet.2012.10.017","article-title":"Replication of hepatitis E virus in three-dimensional cell culture","volume":"187","author":"Berto","year":"2013","journal-title":"J. Virol. Methods"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1016\/j.antiviral.2019.01.007","article-title":"Cell culture systems for the study of hepatitis E virus","volume":"163","author":"Meister","year":"2019","journal-title":"Antivir. Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"4225","DOI":"10.1128\/AEM.00951-16","article-title":"Thermal Stability of Hepatitis E Virus as Estimated by a Cell Culture Method","volume":"82","author":"Johne","year":"2016","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Schemmerer, M., Apelt, S., Trojnar, E., Ulrich, R.G., Wenzel, J.J., and Johne, R. (2016). Enhanced Replication of Hepatitis E Virus Strain 47832c in an A549-Derived Subclonal Cell Line. Viruses, 8.","DOI":"10.3390\/v8100267"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Scholz, J., B\u00e4chlein, C., Gadicherla, A.K., Falkenhagen, A., Tausch, S.H., and Johne, R. (2020). Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832c. Pathogens, 9.","DOI":"10.3390\/pathogens9030157"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1016\/j.jviromet.2005.07.004","article-title":"A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus","volume":"131","author":"Jothikumar","year":"2006","journal-title":"J. Virol. Methods"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2889","DOI":"10.4315\/0362-028X-70.12.2889","article-title":"Hepatitis E virus RNA in commercially available porcine livers in The Netherlands","volume":"70","author":"Bouwknegt","year":"2007","journal-title":"J. Food Prot."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Hakze-Van Der Honing, R.W., Van Coillie, E., Antonis, A.F., and van der Poel, W.H. (2011). First isolation of hepatitis E virus genotype 4 in Europe through swine surveillance in the Netherlands and Belgium. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0022673"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Boxman, I.L., Verhoef, L., Dop, P.Y., Vennema, H., Dirks, R.A., and Opsteegh, M. (2022). High prevalence of acute hepatitis E virus infection in pigs in Dutch slaughterhouses. Int. J. Food Microbiol., 379.","DOI":"10.1016\/j.ijfoodmicro.2022.109830"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"487","DOI":"10.1186\/1743-422X-8-487","article-title":"Thermal stability of hepatitis E virus assessed by a molecular biological approach","volume":"8","author":"Schielke","year":"2011","journal-title":"Virol. J."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Capelli, N., Dubois, M., Pucelle, M., Da Silva, I., Lhomme, S., Abravanel, F., Chapuy-Regaud, S., and Izopet, J. (2020). Optimized Hepatitis E Virus (HEV) Culture and Its Application to Measurements of HEV Infectivity. Viruses, 12.","DOI":"10.3390\/v12020139"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Chew, N., Situ, J., Wu, S., Yao, W., and Sridhar, S. (2022). Independent Evaluation of Cell Culture Systems for Hepatitis E Virus. Viruses, 14.","DOI":"10.3390\/v14061254"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1016\/j.trsl.2009.05.007","article-title":"In vitro hepatic differentiation of human bone marrow mesenchymal stem cells under differential exposure to liver-specific factors","volume":"154","author":"Chivu","year":"2009","journal-title":"Transl. Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"940","DOI":"10.1111\/j.1539-6924.2010.01546.x","article-title":"Estimation of the Likelihood of Fecal-Oral HEV Transmission Among Pigs","volume":"31","author":"Bouwknegt","year":"2010","journal-title":"Risk Anal."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1186\/1297-9716-44-102","article-title":"Direct contact and environmental contaminations are responsible for HEV transmission in pigs","volume":"44","author":"Andraud","year":"2013","journal-title":"Veter. Res."}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/12\/10\/1231\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T21:04:51Z","timestamp":1760130291000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/12\/10\/1231"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,10,11]]},"references-count":30,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2023,10]]}},"alternative-id":["pathogens12101231"],"URL":"https:\/\/doi.org\/10.3390\/pathogens12101231","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,10,11]]}}}