Genomic Investigation of Hepatitis E Virus Genotype 3 Circulation in the animal reservoir in Central Italy by a One Health approach

Background: Hepatitis E virus genotype 3 (HEV-3) is an emerging zoonotic pathogen in Europe, spreading predominantly among wild boars and domestic pigs and causing infections to humans. Accurate genomic characterization is essential for understanding the diversity of circulating genotypes, transmission dynamics and tracing infection sources.Methods: The authors developed and optimized an HEV-3-specific AmpliSeq panel for near-complete genome sequencing, enabling sensitive detection in samples with low viral loads. Whole-Genome Sequences were obtained from a total of 103 samples related to human cases, wild boars, domestic pigs, and food, collected in Central Italy. Phylogenetic analyses were performed to assess subtype distribution, genetic correlation and infer potential transmission pathways.Results: The AmpliSeq approach successfully recovered 97 complete HEV genomes, covering all three open reading frames, even in partially degraded RNA samples. Subtype 3c predominated in wild boars, whereas human infections were more frequently associated with subtype 3f, with additional detection of 3e and sporadic subtypes. Phylogenetic comparisons revealed clusters of closely related sequences across humans, animals, and food, suggesting indirect transmission through wild boars/domestic pigs and pork products. High-resolution of Whole Genome Sequencing (WGS) allowed the comparison of approximately 97% of whole genome, providing a highly reliable framework for source attribution in foodborne outbreaks and for studying the subtype distribution among humans and animals.Conclusions: This study provides a comprehensive picture of HEV-3 circulation in Central Italy in the animal reservoir, supported by direct comparison with HEV-3 in human cases. The results demonstrate the utility of high-resolution WGS for tracing infection sources and for analysing the subtype distribution. By integrating human, animal, and food-derived sequences, the findings underscore the value of a One Health approach in providing a comprehensive overview of the context, useful to implement prevention and control measures.