{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T19:02:22Z","timestamp":1785438142923,"version":"3.56.0"},"reference-count":36,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,7,11]],"date-time":"2026-07-11T00:00:00Z","timestamp":1783728000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002749","name":"Belgian Federal Public Service","doi-asserted-by":"publisher","award":["RT 18\/6 DECHAHEV"],"award-info":[{"award-number":["RT 18\/6 DECHAHEV"]}],"id":[{"id":"10.13039\/501100002749","id-type":"DOI","asserted-by":"publisher"}]},{"award":["RT 18\/6 DECHAHEV"],"award-info":[{"award-number":["RT 18\/6 DECHAHEV"]}],"id":[{"id":"https:\/\/ror.org\/05fk5tr28","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003130","name":"research foundation Flanders","doi-asserted-by":"publisher","award":["18B2821N"],"award-info":[{"award-number":["18B2821N"]}],"id":[{"id":"10.13039\/501100003130","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003130","name":"research foundation Flanders","doi-asserted-by":"publisher","award":["18B2826N"],"award-info":[{"award-number":["18B2826N"]}],"id":[{"id":"10.13039\/501100003130","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>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 p\u00e2t\u00e9 bought in a Belgian supermarket, confirming the presence of infectious HEV in a ready-to-eat p\u00e2t\u00e9 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.<\/jats:p>","DOI":"10.3390\/v18070760","type":"journal-article","created":{"date-parts":[[2026,7,13]],"date-time":"2026-07-13T09:51:09Z","timestamp":1783936269000},"page":"760","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Detection of Infectious Hepatitis E Virus in Retail Pork P\u00e2t\u00e9 After Optimization of Extraction Protocols"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1357-8090","authenticated-orcid":false,"given":"Tatjana","family":"Locus","sequence":"first","affiliation":[{"name":"Fisheries and Food, Technology and Food Unit, Flemish Research Institute for Agriculture (ILVO),  Brusselsesteenweg 370, 9090 Melle, Belgium"},{"name":"Sciensano, Infectious Diseases in Humans, Viral Diseases, Engelandstraat 642, 1180 Ukkel, Belgium"},{"name":"Viral Hepatitis Research Group, Laboratory of Experimental Medicine and Pediatrics, University of Antwerp, Drie Eikenstraat 655, 2650 Edegem, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0775-7888","authenticated-orcid":false,"given":"Michael","family":"Peeters","sequence":"additional","affiliation":[{"name":"Sciensano, Infectious Diseases in Humans, Viral Diseases, Engelandstraat 642, 1180 Ukkel, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steven Van","family":"Gucht","sequence":"additional","affiliation":[{"name":"Sciensano, Infectious Diseases in Humans, Viral Diseases, Engelandstraat 642, 1180 Ukkel, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0572-8741","authenticated-orcid":false,"given":"Thomas","family":"Vanwolleghem","sequence":"additional","affiliation":[{"name":"Viral Hepatitis Research Group, Laboratory of Experimental Medicine and Pediatrics, University of Antwerp, Drie Eikenstraat 655, 2650 Edegem, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8068-4809","authenticated-orcid":false,"given":"Ellen","family":"Lambrecht","sequence":"additional","affiliation":[{"name":"Fisheries and Food, Technology and Food Unit, Flemish Research Institute for Agriculture (ILVO),  Brusselsesteenweg 370, 9090 Melle, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,11]]},"reference":[{"key":"ref_1","unstructured":"ECDC (2019). Options for National Testing and Surveillance for Hepatitis E Virus in the EU\/EEA\u2014Operational Guidance, European Centre for Disease Prevention and Control."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1038\/nrgastro.2017.150","article-title":"Hepatitis E virus: Advances and challenges","volume":"15","author":"Nimgaonkar","year":"2018","journal-title":"Nat. Rev. Gastroenterol. Hepatol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/j.coviro.2018.11.006","article-title":"Hepatitis E virus: Reasons for emergence in humans","volume":"34","author":"Sooryanarain","year":"2019","journal-title":"Curr. Opin. Virol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1016\/j.jhep.2022.08.033","article-title":"Viral clade is associated with severity of symptomatic genotype 3 hepatitis E virus infections in Belgium, 2010\u20132018","volume":"78","author":"Peeters","year":"2023","journal-title":"J. Hepatol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1007\/s12560-021-09461-5","article-title":"The foodborne transmission of hepatitis E virus to humans","volume":"13","author":"Treagus","year":"2021","journal-title":"Food Environ. Virol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Cook, N., D\u2019Agostino, M., Wood, A., and Scobie, L. (2022). Real-time PCR-based methods for detection of hepatitis E virus in pork products: A critical review. Microorganisms, 10.","DOI":"10.3390\/microorganisms10020428"},{"key":"ref_7","first-page":"1","article-title":"Assessment of hepatitis E virus RNA detection in meat samples: Optimization of pre-analytical conditions","volume":"17","author":"Fantilli","year":"2025","journal-title":"Food Environ. Virol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s12560-018-9360-6","article-title":"Interlaboratory validation of a method for hepatitis E virus RNA detection in meat and meat products","volume":"11","author":"Althof","year":"2019","journal-title":"Food Environ. Virol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1016\/j.ijfoodmicro.2015.09.013","article-title":"Detection of hepatitis E virus RNA in raw sausages and liver sausages from retail in Germany using an optimized method","volume":"215","author":"Szabo","year":"2015","journal-title":"Int. J. Food Microbiol."},{"key":"ref_10","first-page":"767","article-title":"Organic solvent-free method for HEV extraction","volume":"18","year":"2025","journal-title":"Food Anal. Methods"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"111018","DOI":"10.1016\/j.ijfoodmicro.2024.111018","article-title":"Determination of hepatitis E virus inactivation during manufacturing of spreadable pork liver sausage and salami-like raw pork sausage","volume":"429","author":"Meyer","year":"2025","journal-title":"Int. J. Food Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Locus, T., Lambrecht, E., Lamoral, S., Willems, S., Van Gucht, S., Vanwolleghem, T., and Peeters, M. (2023). A multifaceted approach for evaluating hepatitis E virus infectivity in vitro: Cell culture and innovative molecular methods for integrity assessment. Vet. Sci., 10.","DOI":"10.3390\/vetsci10120676"},{"key":"ref_13","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_14","doi-asserted-by":"crossref","unstructured":"Hakze-van der Honing, R.W., van Oort, S., Dirks, R.A.M., and van der Poel, W.H.M. (2023). Development of an Ex Vivo Assay for Identification of Infectious Hepatitis E Virus in Different Kinds of Food Samples. Pathogens, 12.","DOI":"10.3390\/pathogens12101231"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Stunnenberg, M., van Huizen, S.C., Swart, A., Lodder, W.J., Boxman, I.L.A., and Rutjes, S.A. (2023). Thermal inactivation of hepatitis E virus in pork products estimated with a semiquantitative infectivity assay. Microorganisms, 11.","DOI":"10.3390\/microorganisms11102451"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1007\/s00705-011-1153-2","article-title":"A549 and PLC\/PRF\/5 cells can support the efficient propagation of swine and wild boar hepatitis E virus (HEV) strains: Demonstration of HEV infectivity of porcine liver sold as food","volume":"157","author":"Takahashi","year":"2012","journal-title":"Arch. Virol."},{"key":"ref_17","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","volume":"88","author":"Feagins","year":"2007","journal-title":"J. Gen. Virol."},{"key":"ref_18","first-page":"3","article-title":"Hepatitis E Virus in Pork Liver Sausage, France","volume":"19","author":"Berto","year":"2013","journal-title":"Emerg. Infect. Dis."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1007\/s12560-024-09586-3","article-title":"Evaluation of food homogenates on cell survival in vitro","volume":"16","author":"Semaan","year":"2024","journal-title":"Food Environ. Virol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"110198","DOI":"10.1016\/j.ijfoodmicro.2023.110198","article-title":"Hepatitis E virus in pork meat products and exposure assessment in Belgium","volume":"397","author":"Locus","year":"2023","journal-title":"Int. J. Food Microbiol."},{"key":"ref_21","first-page":"103248","article-title":"Evaluation of methods for elution of HEV particles in food matrices","volume":"84","author":"Fraisse","year":"2019","journal-title":"Food Microbiol."},{"key":"ref_22","unstructured":"(2017). Microbiology of the Food Chain\u2014Horizontal Method for Determination of Hepatitis A Virus and Norovirus Using Real-Time RT-PCR\u2014Part 1: Method for Quantification (Standard No. ISO 15216-1:2017)."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"447","DOI":"10.1111\/jvh.12157","article-title":"An ORF1-rearranged hepatitis E virus derived from a chronically infected patient efficiently replicates in cell culture","volume":"21","author":"Johne","year":"2014","journal-title":"J. Viral Hepat."},{"key":"ref_24","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_25","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1038\/nmeth.2089","article-title":"NIH Image to ImageJ: 25 years of image analysis","volume":"9","author":"Schneider","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"869","DOI":"10.3201\/eid1805.111712","article-title":"Hepatitis E virus infection among solid organ transplant recipients, the Netherlands","volume":"18","author":"Pas","year":"2012","journal-title":"Emerg. Infect. Dis."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1016\/j.ijfoodmicro.2012.12.007","article-title":"Development of a rapid total nucleic acid extraction method for the isolation of hepatitis A virus from fresh produce","volume":"161","author":"Hida","year":"2013","journal-title":"Int. J. Food Microbiol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1016\/j.ijfoodmicro.2018.04.008","article-title":"Hepatitis E virus in lettuce and water samples: A method-comparison study","volume":"277","author":"Randazzo","year":"2018","journal-title":"Int. J. Food Microbiol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.ijfoodmicro.2011.10.014","article-title":"Extraction of food-borne viruses from food samples: A review","volume":"153","author":"Stals","year":"2012","journal-title":"Int. J. Food Microbiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1128\/AEM.01248-06","article-title":"Procedure for rapid concentration and detection of enteric viruses from berries and vegetables","volume":"73","author":"Butot","year":"2007","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1177\/1535676017713739","article-title":"Evaluation of TRIzol LS inactivation of viruses","volume":"22","author":"Kochel","year":"2017","journal-title":"Appl. Biosaf."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"134","DOI":"10.1016\/j.jceh.2013.05.003","article-title":"Chronic hepatitis E virus infection and treatment","volume":"3","author":"Kamar","year":"2013","journal-title":"J. Clin. Exp. Hepatol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"940","DOI":"10.1111\/j.1539-6924.2010.01546.x","article-title":"Estimation of the likelihood of fecal\u2013oral HEV transmission among pigs","volume":"31","author":"Bouwknegt","year":"2011","journal-title":"Risk Anal."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s12560-011-9056-7","article-title":"Minimum infective dose of major human viruses","volume":"3","author":"Yezli","year":"2011","journal-title":"Food Environ. Virol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"e1926","DOI":"10.1002\/rmv.1926","article-title":"Norovirus infection in primary immune deficiency","volume":"27","author":"Brown","year":"2017","journal-title":"Rev. Med. Virol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"111342","DOI":"10.1016\/j.ijfoodmicro.2025.111342","article-title":"Unraveling hepatitis E virus susceptibility to heat treatment, pH reduction and drying in pork meat","volume":"441","author":"Locus","year":"2025","journal-title":"Int. J. Food Microbiol."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/7\/760\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,13]],"date-time":"2026-07-13T09:52:43Z","timestamp":1783936363000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/7\/760"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,11]]},"references-count":36,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["v18070760"],"URL":"https:\/\/doi.org\/10.3390\/v18070760","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,11]]}}}