Hepatitis E Virus in Pork Liver Sausage, France - Vol. 19 No. 2 - February 2013 - Emerging Infectious Disease journal - CDC

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Hepatitis E Virus in Pork Liver Sausage, France - Vol. 19 No. 2 - February 2013 - Emerging Infectious Disease journal - CDC

Georges de La Tour (1593‒1652) La Femme à la puce (The Flea Catcher) (1638) Oil on canvas (90 cm × 120 cm) Musée Lorrain, Nancy. Photo P. Mignot

Volume 19, Number 2—February 2013

Dispatch

Hepatitis E Virus in Pork Liver Sausage, France

Article Contents

• The Study


• Conclusions


• References


• Figure 1


• Figure 2


• Suggested Citation

Alessandra Berto , Sylvia Grierson, Renate Hakze-van der Honing, Francesca Martelli, Reimar Johne, Jochen Reetz, Rainer G. Ulrich, Nicole Pavio, Wim H.M. Van der Poel, and Malcolm Banks

Author affiliations: Author affiliations: Animal Health and Veterinary Laboratories Agency, Weybridge, UK (A. Berto, S. Grierson, F. Martelli, M. Banks); Wageningen University and Research Centre Central Veterinary Institute, Lelystad, the Netherlands (A. Berto, R. Hakze-van der Honing, W.H.M. Van der Poel); Federal Institute for Risk Assessment, Berlin, Germany (R. Johne, J. Reetz); Friedrich-Loeffler-Institut Institute for Novel and Emerging Infectious Diseases, Greifswald-Insel Riems, Germany (R.G. Ulrich); French Agency for Food, Environmental and Occupational Health and Safety, Paris, France (N. Pavio); ‘; University of Liverpool National Consortium for Zoonosis Research, Liverpool, UK (W.H.M. Van der Poel)

Suggested citation for this article

Abstract

We investigated viability of hepatitis E virus (HEV) identified in contaminated pork liver sausages obtained from France. HEV replication was demonstrated in 1 of 4 samples by using a 3-dimensional cell culture system. The risk for human infection with HEV by consumption of these sausages should be considered to be high.

Foodborne transmission of hepatitis E virus (HEV) to humans from consumption of undercooked pig liver and deer meat has been reported in Japan (1–3). In addition, commercial pig livers purchased from local grocery stores in Japan, the United States, and Europe were contaminated with HEV (4–6). Epidemiologic and PCR results linked a cluster of autochthonous acute hepatitis E cases to ingestion of raw figatelli, which is a dried, cold-smoked sausage containing ≈30% pig liver (7). We investigated the viability of HEV in pork liver sausages produced in France.

The Study

Four samples of pork liver sausage (designated sausages A–D) collected at the final production stage from 4 independent manufacturers in 3 locations in southern France were found to be HEV positive by using real-time reverse transcription PCR (RT-PCR). Samples were tested in 2 institutes, the Animal Health and Veterinary Laboratories Agency, United Kingdom, and Wageningen University and Research Centre, the Netherlands. Three-dimensional (3D) cell culture propagation was performed to investigate the presence of infectious HEV particles. PLC/PRF/5 hepatocarcinoma cells (American Type Culture Collection 8024) were cultured as a monolayer (2D) at 37°C in GTSF-2 medium (8) in a 5% CO₂ environment. Cells were trypsinized at 95% confluence and resuspended in fresh medium to a density of 2 × 10⁵ cells/mL. Fifty milliliters of cell suspension was then introduced into a rotating wall vessel with 10 mg/mL of porous Cytodex-3 microcarrier beads (collagen type I–coated porous microspheres, average diameter 175 μm (Sigma, Dorset, UK and Zwijndrecht, the Netherlands) and incubated at 37°C in 5% CO₂. The cells were incubated for >28 d before inoculation to enable complete differentiation.
The inoculum was prepared by homogenizing a 2.5-mg fragment of each sample with mortar and pestle in 5 mL of culture medium. The homogenate was centrifuged at 8,000 × g for 3 min, and the supernatant was filtered sequentially through 1.2-µm, 0.45-µm, and 0.2-µm filters to reduce the risk for bacterial contamination. The medium was removed from the rotating wall vessel, and 2.5 mL of inoculum was incubated with the cells for 2 h at 35.5°C; at that point, 47.5 mL of fresh medium was added. Subsamples of medium (140 µL) were collected in duplicate, added to 560 µL of lysis buffer (QIAamp Viral RNA Mini Kit; QIAGEN, Crawley, UK, and Venlo, the Netherlands), and stored at −20°C until RNA extraction was performed.
Both institutes performed real-time RT-PCR by using primers and probe as described (9), using the Superscript III Platinum One-Step Quantitative RT-PCR System (Invitrogen, Paisley, UK, and Bleiswijk, the Netherlands). Negative (water) and positive (extract from positive fecal sample, genotype 3) controls were included.