Food and Environmental Virology

, Volume 11, Issue 4, pp 410–419 | Cite as

Screening and Molecular Characterization of Hepatitis E Virus in Slaughter Pigs in Serbia

  • Lazar Milojević
  • Branko VelebitEmail author
  • Vlado Teodorović
  • Andrej Kirbiš
  • Tamaš Petrović
  • Neđeljko Karabasil
  • Mirjana Dimitrijević
Original Paper


Hepatitis E virus (HEV) is a zoonotic virus that can cause acute hepatitis in humans. Besides the fecal–oral route, transmission can occur by consumption of undercooked pig liver. Genotype 3 is the most frequent genotype found in Europe. Studies on HEV in slaughter-age pigs have not been conducted in Serbia so far. Pork meat production and consumption in Serbia is on average, higher than in the rest of Europe. With the aim to identify the circulating HEV genotypes, pig livers and swab samples from three pig slaughterhouses located in three different sub-regions of Serbia were collected. A nested RT-PCR was used to amplify the hypervariable HEV ORF-1 region (334 bp). The amplicons yielded in this study were sequenced, and a molecular phylogeny analysis based on the maximum likelihood method, including HEV sequences reported in several other countries, was performed. The average prevalence of HEV genotype 3 in 3-month-old pigs was 34%. Phylogenetic analysis revealed the majority of HEV amplification fragments from Serbia were grouped in four clades within sub-genotype 3a and were also genetically related to German, Italian, Slovenian, and American HEV sequences. Sub-genotypes 3b and 3j were also found in a single pig each. This study provides the first analysis of the genetic diversity and circulation dynamics of HEV in pigs at slaughterhouses in Serbia.


Hepatitis E virus Slaughterhouse Pigs Serbia 



This research was supported by the Serbian Ministry of Education, Science and Technological Development, Grant No. III 46009.


  1. Aggarwal, R., & Jameel, S. (2011). Hepatitis E. Hepatology,54(6), 2218–2226. Scholar
  2. Aprea, G., Amoroso, M. G., Di Bartolo, I., D’Alessio, N., Di Sabatino, D., Boni, A., et al. (2018). Molecular detection and phylogenetic analysis of hepatitis E virus strains circulating in wild boars in south-central Italy. Transboundary and Emerging Disease,65(1), e25–e31. Scholar
  3. Berto, A., Martelli, F., Grierson, S., & Banks, M. (2012). Hepatitis E virus in pork food chain, United Kingdom, 2009-2010. Emerging Infectious Diseases,18(8), 1358–1360. Scholar
  4. 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
  5. Caruso, C., Peletto, S., Rosamilia, A., Modesto, P., Chiavacci, L., Sona, B., et al. (2017). Hepatitis E virus: a cross-sectional serological and virological study in pigs and humans at zoonotic risk within a high-density pig farming area. Transboundary Emerging Disease,64(5), 1443–1453. Scholar
  6. Casas, M., Cortes, R., Pina, S., Peralta, B., Allepuz, A., Cortey, M., et al. (2011). Longitudinal study of hepatitis E virus infection in Spanish farrow-to-finish swine herds. Veterinary Microbiology,148(1), 27–34. Scholar
  7. Clemente-Casares, P., Ramos-Romero, C., Ramirez-Gonzalez, E., & Mas, A. (2016). Hepatitis E virus in industrialized countries: the silent threat. BioMed Research International,2016, 9838041. Scholar
  8. Dalton, H. R., Webb, G. W., Norton, B. C., & Woolson, K. L. (2016). Hepatitis E virus: time to change the textbooks. Digestive Diseases,34(4), 308–316. Scholar
  9. De Aceituno, A. M. F., Rocks, J. J., Jaykus, L. A., & Leon, J. S. (2013). Foodborne viruses. In R. G. Labbé & S. García (Eds.), Guide to foodborne pathogens (2nd ed., pp. 352–376). Oxford: Wiley.CrossRefGoogle Scholar
  10. Di Bartolo, I., Angeloni, G., Ponterio, E., Ostanello, F., & Ruggeri, F. M. (2015). Detection of hepatitis E virus in pork liver sausages. International Journal of Food Microbiology,193, 29–33. Scholar
  11. 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
  12. Di Bartolo, I., Ponterio, E., Castellini, L., Ostanello, F., & Ruggeri, F. M. (2011). Viral and antibody HEV prevalence in swine at slaughterhouse in Italy. Veterinary Microbiology,149(3–4), 330–338. Scholar
  13. Doceul, V., Bagdassarian, E., Demange, A., & Pavio, N. (2016). Zoonotic hepatitis E virus: classification, animal reservoirs and transmission routes. Viruses,8(10), 270. Scholar
  14. European Commission (2013). Market data on national and European agriculture, provided by the European Commission's agricultural and rural development department. Retrieved March 20, 2019 from
  15. Feurer, C., Le Roux, A., Rossel, R., Barnaud, E., Dumarest, M., Garry, P., et al. (2018). High load of hepatitis E viral RNA in pork livers but absence in pork muscle at French slaughterhouses. International Journal of Food Microbiology,264, 25–30. Scholar
  16. Forgách, P., Nowotny, N., Erdelyi, K., Boncz, A., Zentai, J., Szucs, G., et al. (2010). Detection of hepatitis E virus in samples of animal origin collected in Hungary. Veterinary Microbiology,143(2–4), 106–116. Scholar
  17. Gardinali, N. R., Barry, A. F., da Silva, P. F., de Souza, C., Alfieri, A. F., & Alfieri, A. A. (2012). Molecular detection and characterization of hepatitis E virus in naturally infected pigs from Brazilian herds. Research in Veterinary Science,93(3), 1515–1519. Scholar
  18. Huang, X., Huang, Y., Wagner, A. L., Chen, X., & Lu, Y. (2019). Hepatitis E virus infection in swine workers: a meta-analysis. Zoonoses Public Health,66(1), 155–163. Scholar
  19. Johne, R., Plenge-Bonig, A., Hess, M., Ulrich, R. G., Reetz, J., & Schielke, A. (2010). Detection of a novel hepatitis E-like virus in faeces of wild rats using a nested broad-spectrum RT-PCR. Journal of General Virology,91(Pt 3), 750–758. Scholar
  20. Johne, R., Trojnar, E., Filter, M., & Hofmann, J. (2016). Thermal stability of hepatitis E virus as estimated by a cell culture method. Applied and Environment Microbiology,82(14), 4225–4231. Scholar
  21. 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 of Virological Methods,131(1), 65–71. Scholar
  22. Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution,35(6), 1547–1549. Scholar
  23. Meng, X. J. (2010). Hepatitis E virus: animal reservoirs and zoonotic risk. Veterinary Microbiology,140(3–4), 256–265. Scholar
  24. Meng, X. J., Halbur, P. G., Shapiro, M. S., Govindarajan, S., Bruna, J. D., Mushahwar, I. K., et al. (1998). Genetic and experimental evidence for cross-species infection by swine hepatitis E virus. Journal of Virology,72(12), 9714–9721.PubMedPubMedCentralGoogle Scholar
  25. Monini, M., Di Bartolo, I., Ianiro, G., Angeloni, G., Magistrali, C. F., Ostanello, F., et al. (2015). Detection and molecular characterization of zoonotic viruses in swine fecal samples in Italian pig herds. Archives of Virology,160(10), 2547–2556. Scholar
  26. Moor, D., Liniger, M., Baumgartner, A., & Felleisen, R. (2018). Screening of ready-to-eat meat products for hepatitis E virus in Switzerland. Food Environ Virol,10(3), 263–271. Scholar
  27. Mughini-Gras, L., Angeloni, G., Salata, C., Vonesch, N., D’Amico, W., Campagna, G., et al. (2017). Hepatitis E virus infection in North Italy: high seroprevalence in swine herds and increased risk for swine workers. Epidemiology and Infection,145(16), 3375–3384. Scholar
  28. Mulder, A. C., Kroneman, A., Franz, E., Vennema, H., Tulen, A. D., Takkinen, J., et al. (2019). HEVnet: A one health, collaborative, interdisciplinary network and sequence data repository for enhanced hepatitis E virus molecular typing, characterisation and epidemiological investigations. Eurosurveillance,24(10), 1800407. Scholar
  29. Nei, M., & Kumar, S. (2000). Molecular evolution and phylogenetics. Oxford: Oxford University Press.Google Scholar
  30. Park, W. J., Park, B. J., Ahn, H. S., Lee, J. B., Park, S. Y., Song, C. S., et al. (2016). Hepatitis E virus as an emerging zoonotic pathogen. Journal of Veterinary Science,17(1), 1–11. Scholar
  31. Pavio, N., Merbah, T., & Thebault, A. (2014). Frequent hepatitis E virus contamination in food containing raw pork liver, France. Emerging Infectious Diseases,20(11), 1925–1927. Scholar
  32. Pei, Y., & Yoo, D. (2002). Genetic characterization and sequence heterogeneity of a canadian isolate of Swine hepatitis E virus. Journal of Clinical Microbiology,40(11), 4021–4029. Scholar
  33. Purcell, R. H., & Emerson, S. U. (2008). Hepatitis E: an emerging awareness of an old disease. Journal of Hepatology,48(3), 494–503. Scholar
  34. Raspor Lainšček, P., Toplak, I., & Kirbiš, A. (2017). A comprehensive study of hepatitis E virus infection in pigs entering a slaughterhouse in Slovenia. Veterinary Microbiology,212, 52–58. Scholar
  35. Ricci, A., Allende, A., Bolton, D., Chemaly, M., Davies, R., et al. (2017). Public health risks associated with hepatitis E virus (HEV) as a food-borne pathogen. EFSA Journal,15(7), e04886. Scholar
  36. Rivero-Juarez, A., Frias, M., Martinez-Peinado, A., Risalde, M. A., Rodriguez-Cano, D., Camacho, A., et al. (2017). Familial hepatitis E outbreak linked to wild boar meat consumption. Zoonoses Public Health,64(7), 561–565. Scholar
  37. Ruggeri, F. M., Di Bartolo, I., Ostanello, F., & Trevisani, M. (2013). Epidemiology of the human HEV infection. In Hepatitis E virus: An emerging zoonotic and foodborne pathogen (pp. 11–21): New York: Springer. Scholar
  38. Salines, M., Andraud, M., & Rose, N. (2017). From the epidemiology of hepatitis E virus (HEV) within the swine reservoir to public health risk mitigation strategies: a comprehensive review. Veterinary Research,48(1), 31. Scholar
  39. Shirazi, R., Pozzi, P., Wax, M., Bar-Or, I., Asulin, E., Lustig, Y., et al. (2018). Hepatitis E in pigs in Israel: seroprevalence, molecular characterisation and potential impact on humans. Eurosurveillance,23(49), 1800067. Scholar
  40. Smith, D. B., Simmonds, P., Izopet, J., Oliveira-Filho, E. F., Ulrich, R. G., Johne, R., et al. (2016). Proposed reference sequences for hepatitis E virus subtypes. Journal of General Virology,97(3), 537–542. Scholar
  41. Smith, D. B., Simmonds, P., Jameel, S., Emerson, S. U., Harrison, T. J., Meng, X. J., et al. (2014). Consensus proposals for classification of the family Hepeviridae. Journal of General Virology,95(Pt 10), 2223–2232. Scholar
  42. Sridhar, S., Lau, S. K., & Woo, P. C. (2015). Hepatitis E: A disease of reemerging importance. Journal of the Formosan Medical Association,114(8), 681–690. Scholar
  43. 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
  44. Vina-Rodriguez, A., Schlosser, J., Becher, D., Kaden, V., Groschup, M. H., & Eiden, M. (2015). Hepatitis E virus genotype 3 diversity: phylogenetic analysis and presence of subtype 3b in wild boar in Europe. Viruses,7(5), 2704–2726. Scholar
  45. Weigand, K., Weigand, K., Schemmerer, M., Muller, M., & Wenzel, J. J. (2018). Hepatitis E seroprevalence and genotyping in a cohort of wild boars in Southern Germany and Eastern Alsace. Food Environ Virol,10(2), 167–175. Scholar
  46. 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
  47. Widen, F. (2016). Hepatitis E as a zoonosis. Advances in Experimental Medicine and Biology,948, 61–71. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Meat Hygiene and TechnologyBelgradeSerbia
  2. 2.Faculty of Veterinary MedicineUniversity of BelgradeBelgradeSerbia
  3. 3.Veterinary FacultyUniversity of LjubljanaLjubljanaSlovenia
  4. 4.Scientific Veterinary Institute “Novi Sad”Novi SadSerbia

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