Food and Environmental Virology

, Volume 11, Issue 1, pp 1–8 | Cite as

Interlaboratory Validation of a Method for Hepatitis E Virus RNA Detection in Meat and Meat Products

  • Nadine Althof
  • Eva Trojnar
  • Thomas Böhm
  • Sabine Burkhardt
  • Anja Carl
  • Matthias Contzen
  • Jochen Kilwinski
  • Steffen Mergemeier
  • Dominik Moor
  • Dietrich Mäde
  • Reimar JohneEmail author
Original Paper


Increasing numbers of hepatitis E cases are currently recognized in many European countries. The zoonotic hepatitis E virus (HEV) genotype 3 mainly circulates in domestic pigs and wild boars, and can be transmitted to humans via consumption of insufficiently heated meat or meat products produced from those animals. Here, a detailed protocol for detection of HEV RNA in meat products is provided, which is based on the method originally described by Szabo et al. (Intl J Food Microbiol 215:149–156, 2015). It consists of a TRI Reagent®/chloroform-based food matrix homogenization, a silica bead-based RNA extraction and a real-time RT-PCR-based RNA detection. The method was further validated in a ring trial with nine independent laboratories using pork liver sausage samples artificially contaminated with different amounts of HEV. The results indicate sufficient sensitivity, specificity, and accuracy of the method for its broad future use in survey studies, routine food control or outbreak investigations.


Hepatitis E virus Meat products Detection method Validation Ring trial 



The authors wish to thank Anja Schlosser and Silke Apelt for excellent technical assistance during preparation of the samples for the collaborative trial. Eva Trojnar was supported by a grant of the German Federal Ministry of Education and Research (BMBF) executed within the framework of the project ZooGloW (FKZ 13 N12697). The collaborative trial was in part financially supported by the Federal Office of Consumer Protection and Food Safety, Germany.

Supplementary material

12560_2018_9360_MOESM1_ESM.pdf (208 kb)
Supplementary material 1 (PDF 208 KB)


  1. Aspinall, E. J., Couturier, E., Faber, M., Said, B., Ijaz, S., Tavoschi, L., et al. (2017). Hepatitis E virus infection in Europe: surveillance and descriptive epidemiology of confirmed cases, 2005 to 2015. Euro Surveillannce. Scholar
  2. Bajpai, M., & Gupta, E. (2011). Transfusion-transmitted hepatitis E: Is screening warranted? Indian Journal of Medical Microbiology, 29(4), 353–358. Scholar
  3. Berto, A., Grierson, S., Hakze-van der Honing, R., Martelli, F., Johne, R., Reetz, J., et al. (2013). Hepatitis E virus in pork liver sausage, France. Emerging Infectious Diseases, 19(2), 264–266. Scholar
  4. Berto, A., Martelli, F., Grierson, S., & Banks, M. (2012). Hepatitis E virus in pork food chain, United Kingdom, 2009–2010. Emerging Infectious Disiseases, 18(8), 1358–1360. Scholar
  5. Bouwknegt, M., Lodder-Verschoor, F., van der Poel, W. H., Rutjes, S. A., & de Roda Husman, A. M. (2007). Hepatitis E virus RNA in commercial porcine livers in The Netherlands. Journal of Food Protection, 70(12), 2889–2895.CrossRefGoogle Scholar
  6. Centers for Disease Control and Prevention. (2013). Investigation of hepatitis E outbreak among refugees—Upper Nile, South Sudan, 2012–2013. Morbibity and Mortality Weekly Report, 62(29), 581–586.Google Scholar
  7. Colson, P., Borentain, P., Queyriaux, B., Kaba, M., Moal, V., Gallian, P., et al. (2010). Pig liver sausage as a source of hepatitis E virus transmission to humans. Journal of Infectious Diseases, 202(6), 825–834. Scholar
  8. Cook, N., D’Agostino, M., & Johne, R. (2017). Potential approaches to assess the infectivity of hepatitis E virus in pork products: A review. Food and Environmental Virology, 9(3), 243–255. Scholar
  9. Di Bartolo, I., Diez-Valcarce, M., Vasickova, P., Kralik, P., Hernandez, M., Angeloni, G., et al. (2012). Hepatitis E virus in pork production chain in Czech Republic, Italy, and Spain, 2010. Emerging Infectious Diseases, 18(8), 1282–1289. Scholar
  10. Dreier, J., Stormer, M., & Kleesiek, K. (2005). Use of bacteriophage MS2 as an internal control in viral reverse transcription-PCR assays. Journal of Clinical Microbiology, 43(9), 4551–4557. Scholar
  11. European Food Safety Authority. (2016). Summary Report of Joint Scientific Workshop on Foodborne Viruses. Retrieved August 22, 2018, from
  12. European Food Safety Authority. (2017). Public health risks associated with hepatitis E virus (HEV) as a food-borne pathogen. EFSA Journal, 15(7), 4886.Google Scholar
  13. Feagins, A. R., Opriessnig, T., Guenette, D. K., Halbur, P. G., & Meng, X. J. (2007). Detection and characterization of infectious Hepatitis E virus from commercial pig livers sold in local grocery stores in the USA. Journal of General Virology, 88(Pt 3), 912–917. Scholar
  14. Gerolami, R., Moal, V., & Colson, P. (2008). Chronic hepatitis E with cirrhosis in a kidney-transplant recipient. New England Journal of Medicine, 358(8), 859–860. Scholar
  15. Johne, R., Dremsek, P., Reetz, J., Heckel, G., Hess, M., & Ulrich, R. G. (2014). Hepeviridae: An expanding family of vertebrate viruses. Infection, Genetics and Evolution, 27, 212–229. Scholar
  16. Jothikumar, N., Cromeans, T. L., Robertson, B. H., Meng, X. J., & Hill, V. R. (2006). A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus. Journal Virological Methods, 131(1), 65–71. Scholar
  17. Kamar, N., Selves, J., Mansuy, J. M., Ouezzani, L., Peron, J. M., Guitard, J., et al. (2008). Hepatitis E virus and chronic hepatitis in organ-transplant recipients. New England Journal of Medicine, 358(8), 811–817. Scholar
  18. Kubacki, J., Fraefel, C., Jermini, M., Giannini, P., Martinetti, G., Ripellino, P., et al. (2017). Complete genome sequences of two Swiss hepatitis E virus isolates from human stool and raw pork sausage. Genome Announcements. Scholar
  19. Martin-Latil, S., Hennechart-Collette, C., Guillier, L., & Perelle, S. (2014). Method for HEV detection in raw pig liver products and its implementation for naturally contaminated food. International Journal of Food Microbiology, 176, 1–8. Scholar
  20. Meng, X. J. (2011). From barnyard to food table: the omnipresence of hepatitis E virus and risk for zoonotic infection and food safety. Virus Research, 161(1), 23–30. Scholar
  21. Meng, X. J., Purcell, R. H., Halbur, P. G., Lehman, J. R., Webb, D. M., Tsareva, T. S., et al. (1997). A novel virus in swine is closely related to the human hepatitis E virus. Proceedings of the National Academy of Sciences USA, 94(18), 9860–9865.Google Scholar
  22. Moor, D., Liniger, M., Baumgartner, A., & Felleisen, R. (2018). Screening of ready-to-eat meat products for hepatitis E virus in Switzerland. Food and Environmental Virology. Scholar
  23. Pavio, N., Doceul, V., Bagdassarian, E., & Johne, R. (2017). Recent knowledge on hepatitis E virus in Suidae reservoirs and transmission routes to human. Veterinary Research, 48(1), 78. Scholar
  24. Peron, J. M., Dalton, H., Izopet, J., & Kamar, N. (2011). Acute autochthonous hepatitis E in western patients with underlying chronic liver disease: A role for ribavirin? Journal of Hepatology, 54(6), 1323–1324. (author reply 1324–1325).CrossRefGoogle Scholar
  25. Purcell, R. H., & Emerson, S. U. (2008). Hepatitis E: An emerging awareness of an old disease. Journal of Hepatology, 48(3), 494–503. Scholar
  26. Renou, C., Roque-Afonso, A. M., & Pavio, N. (2014). Foodborne transmission of hepatitis E virus from raw pork liver sausage, France. Emerging Infectious Diseases, 20(11), 1945–1947. Scholar
  27. Scherer, K., Johne, R., Schrader, C., Ellerbroek, L., Schulenburg, J., & Klein, G. (2010). Comparison of two extraction methods for viruses in food and application in a norovirus gastroenteritis outbreak. Journal of Virological Methods, 169(1), 22–27. Scholar
  28. Schielke, A., Sachs, K., Lierz, M., Appel, B., Jansen, A., & Johne, R. (2009). Detection of hepatitis E virus in wild boars of rural and urban regions in Germany and whole genome characterization of an endemic strain. Virology Journal, 6, 58. Scholar
  29. Schlosser, B., Stein, A., Neuhaus, R., Pahl, S., Ramez, B., Kruger, D. H., et al. (2012). Liver transplant from a donor with occult HEV infection induced chronic hepatitis and cirrhosis in the recipient. Journal of Hepatology, 56(2), 500–502. Scholar
  30. Schmittgen, T. D., & Livak, K. J. (2008). Analyzing real-time PCR data by the comparative C(T) method. Nature Protocols, 3(6), 1101–1108. PubMed PMID: 18546601..CrossRefGoogle Scholar
  31. Szabo, K., Trojnar, E., Anheyer-Behmenburg, H., Binder, A., Schotte, U., Ellerbroek, L., et al. (2015). Detection of hepatitis E virus RNA in raw sausages and liver sausages from retail in Germany using an optimized method. International Journal of Food Microbiology, 215, 149–156. Scholar
  32. van den Berg, B., van der Eijk, A. A., Pas, S. D., Hunter, J. G., Madden, R. G., Tio-Gillen, A. P., et al. (2014). Guillain-Barre syndrome associated with preceding hepatitis E virus infection. Neurology, 82(6), 491–497. Scholar
  33. van der Poel, W. H., Verschoor, F., van der Heide, R., Herrera, M. I., Vivo, A., Kooreman, M., et al. (2001). Hepatitis E virus sequences in swine related to sequences in humans, The Netherlands. Emerging Infectious Diseases, 7(6), 970–976. Scholar
  34. Vollmer, T., Diekmann, J., Johne, R., Eberhardt, M., Knabbe, C., & Dreier, J. (2012). Novel approach for detection of hepatitis E virus infection in German blood donors. Journal of Clinical Microbiology, 50(8), 2708–2713. Scholar
  35. Wedemeyer, H., Pischke, S., & Manns, M. P. (2012). Pathogenesis and treatment of hepatitis e virus infection. Gastroenterology, 142(6), 1388–1397 e1381. Scholar
  36. Wenzel, J. J., Preiss, J., Schemmerer, M., Huber, B., Plentz, A., & Jilg, W. (2011). Detection of hepatitis E virus (HEV) from porcine livers in Southeastern Germany and high sequence homology to human HEV isolates. Journal of Clinical Virology, 52(1), 50–54. Scholar
  37. World Health Organization. (2017). Hepatitis E. Retrieved August 22, 2018, from
  38. Yazaki, Y., Mizuo, H., Takahashi, M., Nishizawa, T., Sasaki, N., Gotanda, Y., et al. (2003). Sporadic acute or fulminant hepatitis E in Hokkaido, Japan, may be food-borne, as suggested by the presence of hepatitis E virus in pig liver as food. Journal of General Virology, 84(Pt 9), 2351–2357. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Nadine Althof
    • 1
  • Eva Trojnar
    • 1
  • Thomas Böhm
    • 2
  • Sabine Burkhardt
    • 3
  • Anja Carl
    • 4
  • Matthias Contzen
    • 5
  • Jochen Kilwinski
    • 6
  • Steffen Mergemeier
    • 7
  • Dominik Moor
    • 8
  • Dietrich Mäde
    • 9
  • Reimar Johne
    • 1
    Email author
  1. 1.German Federal Institute for Risk AssessmentBerlinGermany
  2. 2.Landesbetrieb Hessisches LandeslaborKasselGermany
  3. 3.Landeslabor Berlin-BrandenburgBerlinGermany
  4. 4.Bayrisches Landesamt für Gesundheit und LebensmittelsicherheitErlangenGermany
  5. 5.Chemisches und Veterinäruntersuchungsamt StuttgartFellbachGermany
  6. 6.Chemisches und Veterinäruntersuchungsamt WestfalenArnsbergGermany
  7. 7.CONGEN – Biotechnologie GmbHBerlinGermany
  8. 8.Bundesamt für Lebensmittelsicherheit und VeterinärwesenBernSwitzerland
  9. 9.Landesamt für Verbraucherschutz Sachsen-AnhaltHalle (Saale)Germany

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