Molecular Detection and Genetic Typing of Hepatitis E Virus in Wild Animals from Slovakia

Abstract

Hepatitis E is an emerging zoonosis caused by the hepatitis E virus (HEV) and is recognised worldwide. Wild boars are considered one of the main reservoirs of the zoonotic HEV-3 genotype. However, HEV-3 has also been detected in many other wildlife species. In this study, we investigated 284 liver and muscle tissue samples from wild boars and 107 liver and muscle tissue samples from four different wild ruminant species (red deer, roe deer, European mouflon and fallow deer) across 35 hunting areas in Slovakia. HEV RNA was detected in 14.2% (95% CI 9.8-18.6%) of the liver and 10.5% (95% CI 0.4-20.6%) of the muscle tissue samples from wild boars but in none of the samples from the wild ruminant species. Phylogenetic analysis based on partial ORF1 and ORF2 of the HEV genome revealed that the Slovak wild boar HEV sequences clustered within the zoonotic genotype HEV-3. Depending on their geographical origin, the obtained sequences clustered into three HEV-3 subtypes: HEV-3a, HEV-3i and HEV-3e. Our findings confirm the circulation of HEV in the wild boar population in the Slovak Republic but not in wild ruminant species.

Keywords: HEV-3 genotype; Hepatitis E virus; Wild boar; Wild ungulates; Zoonosis.

Fig. 1

Slovakia map showing the approximate geographical origin of the collected samples (hunting areas). Locations with positive samples are marked with a symbol of red dot, whilst negative samples are marked with a symbol of green dot

Fig. 2

Phylogenetic tree based on the 242 bp nucleotide sequences of the ORF1 fragment of the HEV genome. The tree was constructed using the Maximum Likelihood method and the General Time Reversible model (GTR+G+I) incorporated into MEGA X (Kumar et al., 2018). The sequences from Slovak wild boars are shown in bold and marked with a symbol of black circle, whilst those taken from Slovak domestic swine are marked with a symbol of black square. The reference sequences of the virus strains are marked with a symbol of white triangle and an accession number. All sequences from the GenBank database are marked with an accession number and the country of origin according to ISO 3166-1 alpha-2 codes. The HEV-4 strain was used as an outgroup and the bootstrap values > 70% were shown. The scale bar indicates nucleotide substitutions

Fig. 3

Phylogenetic tree based on the 304 bp nucleotide sequences of the ORF2 fragment of the HEV genome. The tree was constructed by applying the Maximum Likelihood method and the Tamura—Nei model (TN93+G+I) incorporated into MEGA X (Kumar et al., 2018). The HEV sequences from Slovak wild boars are shown in bold and marked with a symbol of black circle, whilst those taken from Slovak domestic swine are marked with a symbol of black square. The reference sequences of the virus strains are marked with a symbol of white triangle and an accession number. All sequences from the GenBank database are marked with an accession number and the country of origin according to ISO 3166-1 alpha-2 codes. The HEV-5 strain was used as an outgroup and the bootstrap values > 70% were shown. The scale bar indicates nucleotide substitutions