Wild Boars as a Reservoir of Zoonotic Hepatitis E Virus in Portugal with Full-Genome Evidence of Genotype 3m

The Hepatitis E virus (HEV) is a zoonotic virus of global concern that circulates in both domestic and wild pig populations. Understanding its presence and dynamics in wildlife reservoirs is crucial for assessing spillover risks and designing One Health surveillance strategies. HEV is classified into eight distinct genotypes, two of which (genotypes 3 and 4) infect both humans and swine, representing a major public health concern in Europe. This study explored the occurrence, genetic diversity, and evolutionary relationships of HEV in wild boars (Sus scrofa scrofa) from mainland Portugal. Organ samples from a total of 120 wild boar were collected from seven different districts in Portugal. HEV RNA was detected in four animals (3.3%), all of which were collected from municipalities in the Évora district, near the land border with Spain. Of the four wild boars that tested positive for HEV in the diagnostic context, it was possible to genotype and characterize one through full sequencing. Phylogenetic analysis based on the complete genome, revealed that this strain clustered with predominantly human-derived HEV-3m sequences from Spain and France, highlighting its zoonotic potential. The inclusion of 14 sequences from domestic swine in a second phylogenetic analysis performed with a small fragment allowed to conclude that sub-genotypes 3e, 3f and 3m are circulating in this population with no spatial or temporal segregation. Phylogeographic analysis suggests that there have been several transmission events between Spain and France and estimated that this HEV strain is most likely to have been introduced from Spain. The fact that these four positive samples from wild boar in south-eastern Portugal all have a common spatial origin, together with the absence of detections in the other six districts tested and the presence of known HEV hotspots in south-western Spain, supports the hypothesis that this strain in Portugal is most likely from HEV circulation in Spanish wild boar, facilitated by cross-border movement of wild boar along the south-eastern Portuguese border. Although recombination events were identified in several HEV-3 strains, none were detected in the sequence obtained in this study or in other HEV-3m strains. This study provides the first molecular evidence of HEV-3m circulation in wild boars in Portugal, offering valuable insight into the HEV strain circulation in European wildlife populations. The zoonotic potential of HEV and the likelihood of interspecies transmission highlight the need for coordinated cross-border surveillance and integrated One Health strategies.