Development of a Sensitive and Specific Quantitative RT-qPCR Method for the Detection of Hepatitis E Virus Genotype 3 in Porcine Liver and Foodstuff.

As an international and zoonotic cause of hepatitis, hepatitis E virus (HEV) poses a significant risk to public health. However, the frequency of occurrence and the degree of contamination of food of animal origin require further research. The aim of this study was to develop and validate a highly sensitive quantitative RT-qPCR assay for the detection and quantification of HEV contamination in porcine liver and food. The focus was on genotype 3, which is most common as a food contaminant in developed countries and Europe. The selected assay has its target sequence in the open reading frame 1 (ORF1) of the HEV genome and showed good results in inclusivity testing, especially for HEV genotype 3. The developed assay seems to show high efficiency and a low intercept when compared to other assays, while having a comparable limit of detection (LOD). In addition, a standard curve was generated using artificially spiked liver to provide more accurate quantitative results for contamination assessment and tracking in this matrix. Application of the assay to test 67 pig livers from different origins resulted in a positivity rate of 7.5%, which is consistent with the results of numerous other prevalence studies. Quantitative detection of the viral genome in the food chain, particularly in pig livers, is essential for understanding the presence and evolution of HEV contamination and thus ensures consumer safety.

Hepatitis E virus neutralization by porcine serum antibodies.

The consumption of raw or undercooked meat products poses a serious risk for human hepatitis E virus (HEV) infections. In many high-income countries, domestic pigs and wild boars represent the main animal reservoirs for HEV and are usually identified by reverse transcription-PCR and antibody enzyme-linked immunosorbent assay (ELISA). In order to characterize the humoral immune response in more detail, a cell culture-based serum neutralization assay using a culture-adapted HEV strain was established here. Measurement of neutralizing antibodies was only possible after removing the viral quasi-envelope by detergent treatment. Serum samples of 343 wild boars from Northern Germany were first analyzed for anti-HEV IgG using an in-house ELISA, resulting in 19% positive samples. Subsequently, a subset of 41 representative samples was tested with the neutralization assay, and the results correlated well with those obtained by ELISA. Not only the human HEV strain 47832c but also two porcine HEV strains were shown to be neutralized by porcine serum antibodies. Neutralizing activity was also found in samples containing both HEV-specific antibodies and HEV RNA. Testing of serum samples derived from two experimentally infected domestic pigs showed a steep increase in neutralizing activity at 24 or 51 days post infection, dependent on the used infectious dose. The developed assay can be useful for characterization of the humoral immune response after HEV infection and for assessing the efficiency of HEV vaccine candidates.

Isolation of 15 hepatitis E virus strains lacking ORF1 rearrangements from wild boar and pig organ samples and efficient replication in cell culture.

As a zoonotic pathogen, the hepatitis E virus (HEV) leads to numerous infections in humans with different clinical manifestations. Especially genotype 3, as causative agent of a foodborne zoonosis, is transmitted to humans by ingestion of undercooked or raw meat containing liver from HEV-infected animals. Although the virus' prevalence and dissemination in hosts like wild boar and pig have been well characterized, HEV is greatly understudied on a molecular level and reliable cell culture models are lacking. For this reason, the present study concentrated on the isolation and subsequent characterization of porcine HEV from tissue samples derived from wild boar and domestic pigs: 222 wild boars hunted in Northern Germany were investigated for the presence of HEV RNA with a detection rate of 5.9%. Three additional HEV-positive wild boar liver samples as well as an HEV-positive spleen and a positive kidney from domestic pigs were included. After inoculation of positive samples onto the human hepatoma cell line PLC/PRF/5, cells were grown for several weeks. Successful isolation was confirmed by RT-qPCR, virus passage, immunofluorescence staining and titration. Overall, 15 strains from a total of 18 RNA-positive organ samples could be obtained and viral loads >109  RNA copies/ml were measured in cell culture supernatants. Accordingly, 83.3% of the HEV RNA-positive samples contained infectious hepatitis E viral particles and therefore must be considered as a potential source for human infections. Phylogenetic analyses revealed that all isolated strains belong to genotype 3. Further genetic characterization showed a high degree of sequence variability, but no sequence insertions, in the hypervariable region within the open reading frame 1.