Molecular detection and phylogenetic analysis of HEV-3 in wild boars in Central Italy

Background: Hepatitis E virus (HEV) causes acute hepatitis in humans. Over the last decade, in Europe, the zoonotic HEV-3 genotype has caused an increasing number of autochthonous cases linked to the consumption of raw or undercooked pork products and wild boar meat. In Italy, HEV-3 is widespread in both pigs and wild boar. In this study, 207 wild boar hunted in central Italy were tested for HEV. Methods: Liver samples from 207 wild boars (Sus scrofa) were collected from 2016 to 2018. The HEV RNA was extracted from 100 milligrams of liver and detected by quantitative RT-qPCR. A 348-bp fragment within the ORF2 was amplified by nested RT-PCR. Full genome of 4 strains was obtained by NGS multiplex PCRs (Ion Personal Genome Machine). Results: HEV RNA was detected in 29.5% (61/207) of liver. The phylogenetic analysis conducted on short genomic sequences showed the circulation of -3f (n=2), -3c (n=5) subtype strains and two clusters of HEV-3* unclassified strains (n=18). Four strains were fully sequenced by NGS and classified into the -3f, -3c, -3i and one HEV-3*. The latter subtype forms a cluster with other European swine and human strains and was recently proposed. The sequenced strains were related (94-99% nt.id.) to wild boar, human and pig strains identified in Italy. Conclusion: In this study, a high heterogeneity of HEV-3 strains was revealed in wild boar. Strains are more correlated to other wild boar strains detected in Europe than to Italian pig strains. Only three wild boar strains were correlated (94% nt. id.) to an Italian human strain detected in a patient who declared consumption of figatelli (liver sausage) in 2011. Wild boar plays an important role as a source of infection of HEV-3 to breeding pig populations and to humans in those regions where wild boar meat is consumed frequently.