Molecular detection and characterization of Hepatitis E virus in wild boars (Sus scrofa) from northern Italy

Hepatitis E virus (HEV) is an emerging zoonotic pathogen with wild boars (Sus scrofa) recognized as important reservoirs in Europe. This study investigated HEV circulation in wild boar populations inhabiting the Ticino River Natural Park (Lombardy, northern Italy), an area characterized by high wild boar density and frequent human-animal interaction. Between 2016 and 2018 and 2020-2022, a total of 3040 liver samples and 582 bile samples were collected from hunted or found-dead wild boars as part of regional wildlife surveillance. HEV RNA was detected by Real-Time RT-PCR targeting the ORF3 region, and positive samples were further characterized by nested RT-PCR and Sanger sequencing of the ORF2 region. Overall, HEV RNA was detected in 8.0% of samples, with a higher prevalence observed in males (9.0%) than females (7.4%), and increased prevalence in colder months (winter 10.8%, autumn 8.1%) compared to summer 3.7%. Phylogenetic analysis of ORF2 sequences revealed all successfully sequenced strains belonged to subtype HEV-3f1, showing high similarity to human HEV strains reported in Italy. These findings confirm active circulation of zoonotic HEV-3f1 in wild boars in the region and underscore the potential risk of environmental contamination and transmission to humans, especially given area's popularity for outdoor activities and proximity to domestic swine populations. Continued surveillance and molecular characterization of HEV in wildlife and domestic animals are essential for understanding HEV dynamics and reducing zoonotic transmission risks, with important public health implications, underscoring the need to strengthen surveillance systems and implement targeted prevention measures, particularly among populations at risk of exposure.