Detection of hepatitis E virus (HEV) from porcine livers in Southeastern Germany and high sequence homology to human HEV isolates☆

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Abstract

Background

Hepatitis E virus (HEV) has been identified as an emerging cause of infectious hepatitis over the last years in developed countries. In contrast to travel associated hepatitis E, zoonotic sources of infection are suspected for autochthonous cases in Europe.

Objective

Since pigs are known reservoirs of HEV, we tested porcine livers sold as food in Southeastern Germany for the presence of hepatitis E virus RNA.

Study design

We purchased 200 porcine liver samples in 81 butcher shops and grocery stores in Regensburg, Germany. Nucleic acid preparations were tested for the presence of HEV RNA by quantitative real-time PCR (RT-qPCR). HEV isolates from positive samples were characterized by partial sequencing of ORF1 and ORF2 regions in the HEV genome and by phylogenetic analysis.

Results

Specimens from eight (4%) of 200 purchased pig livers had detectable HEV RNA amounts. Sequence determination and phylogenetic analysis allowed two novel isolates to be classified as HEV genotype 3, subgenotype 3a (swR437) and 3c (swR269), respectively. Both novel swine HEV isolates showed high sequence homology to isolates obtained from patients with acute HEV infection from the same geographic region.

Conclusions

These results support the suggested role of undercooked pig products in food as a source of zoonotic HEV infection for humans. It remains to be clarified if this mechanism of transmission is responsible for the surprisingly high anti-HEV IgG prevalence recently observed in some European countries and the USA.

Section snippets

Background

Hepatitis E virus (HEV) is a small, non-enveloped virus with an icosahedral capsid of ∼33 nm in diameter. It contains a positive-sense, single-stranded RNA genome of approximately 7.2 kb in size. The genome is composed of a short 5′ untranslated region (UTR), three open reading frames (ORF1–3) and a short 3′ UTR terminated by a poly(A) tail.1 Sequencing and analysis of several full-length viral genomes led to the identification of four major mammalian HEV genotypes (1–4) with unique geographic

Objectives

In the present study raw porcine livers sold as food in Southeastern Germany were tested for the presence of hepatitis E virus RNA in order to determine whether autochthonous hepatitis E in this region is likely to be food-borne.

Study design

Between April and August 2010, a total of 200 raw porcine liver samples were purchased in 81 butcher shops and grocery stores in Regensburg and its close surroundings (Southeastern Germany). According to the information available at the stores, the domestic pigs were raised, slaughtered and processed in Germany. Liver blocks weighed 100–500 g per package. Pieces of liver tissue (∼200 mg each) were dissected from each block and stored in RNAlater preservative reagent (Ambion) at −80 °C. Specimens

Results

To test the hypothesis whether porcine liver sold as food is contaminated with HEV, raw porcine livers, purchased between April and August 2010 from butcher shops and grocery stores in the Regensburg area, were tested for the presence of HEV RNA by RT-qPCR. Specimens from eight (4%) of 200 purchased porcine livers had detectable HEV RNA amounts. All positive samples were reanalyzed by using a nested PCR protocol targeting the HEV ORF1 and ORF2 genomic regions (Table 1). Two of seven samples

Conclusions

Hepatitis E has been generally perceived as a strictly travel associated disease in industrialized countries. Thus it was often eliminated from differential diagnosis if a patient with clinical signs of acute hepatitis had no travel history to known HEV endemic regions. However, during the last years a growing body of evidence has accumulated supporting the notion that autochthonous HEV infections are also widespread in many industrialized countries.9, 10, 11 The majority of autochthonous

Funding

None.

Conflict of interest statement

None declared.

Ethical approval

Not required.

Acknowledgements

The authors wish to thank J. Klein, B. Kreuzpaintner, E. Kreuzpaintner, A. Rohrhofer and S. Schreder-Meindl for expert technical assistance.

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    ☆

    The nucleotide sequences of two swine and six human HEV isolates reported herein have been assigned DDBJ/EMBL/GenBank accession nos. FR846453, FR846454, FR846455, FN995000, FN995001, FR846450, FN985025, FR846451, FR846452, FR728255, FR728256, GU479457, GU479458.

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