Distribution and characterization of zoonotic Hepatitis E virus in wild ungulates hunted in North-Eastern Italy.

Hepatitis E is an emerging zoonosis caused by a virus of the Hepeviridae family. In industrialized countries, pigs are the main reservoir for zoonotic genotypes 3 and 4, and a relevant role seems to be played by wild boar; moreover, the virus has been detected in cervids. Considering the increasing interest in wild game meat as a sustainable food, data are needed to elucidate HEV epidemiology. In this perspective, IZSVe started a research project (RC IZSVe 14/15) aimed at i) determining the Hepatitis E virus (HEV) prevalence and distribution in wild boar, red deer and roe deer in North-East Italy; ii) characterizing HEV genotypes in wildlife compared to domestic animals; iii) identifying factors associated with HEV occurrence; iv) assessing the risk of human HEV infection from large game. Serological and virological analyses were performed on animals hunted in 2016-2019: 117 red deer, 259 roe deer and 569 wild boars. Serology was investigated using an indirect commercial ELISA kit on meat juice from wild boar, and a commercial competitive ELISA kit on deer sera. RNA was extracted from livers, bile (wild boar only) and/or faeces, and it was analysed with an ORF-3 targeting rRT-PCR protocol. ORF regions of positive RNAs were sequenced and investigated trough a preliminary phylogenetic analysis. Positive samples were found only from wild boar of the Euganean Hills (serological prevalence on meat juice = 17.6; 11.8-23.4, 95% C.L – virological prevalence = 7.3; 3.5-11.1, 95% C.L.). The few serological positivities (4 wild boars and 1 red deer) detected in the other areas whithin Verona, Vicenza, Treviso and Belluno provinces and Friuli Venezia Giulia region, were probably due to the non-absolute Sp of the test; no virological positives were indeed detected. Phylogenetic analysis of two complete viral genomes and seven partial ORF1 and ORF2 sequences of HEV viruses, selected from the positive wild boar of the Euganean Hills, showed that the viruses grouped together within genotype 3 but clustered separately from previously identified subtypes, thus suggesting the identification of a novel genotype 3 subtype. The phylogenetic analysis of nine partial ORF2 sequences showed the closest similarity with wild boar/human viruses identified in centralnorthern Italy in 2012. Wild boar overpopulation is a well-known problem in the Euganean Hills, a small and clearly isolated area where high population density could have significantly favoured the circulation of HEVs and consequently the prevalence. On the other hand, the apparent absence of HEV in the other areas, where wild boar distribution can be thought as a continuum, is unexpected and probably referable to the lower sampling fraction. Finally, to investigate HEV risk for humans, a serological and epidemiological survey on potentially exposed people (hunters and gamekeepers) is also in progress in the Euganean Hills and in an apparently HEV-negative area which stands as a comparison.