Hepatitis e virus in wild ungulates: serological evidence in chamois and red deer in the Alps and genetic assessment of viral variants in Europe

In recent years there has been an increasing interest in hepatitis E virus (HEV) because of its zoonotic ature. The different ecological interactions among its numerous host species contribute to make HEV pidemiology unclear and the actual reservoirs and spill-over hosts have to be still defined. Domestic pig and wild boar are considered as the main reservoirs of zoonotic HEV in Europe, while wild ruminants are supposed as spill-over hosts. This study aims to serologically investigate HEV infection in chamois and red deer in order to define their HEV seroprevalence and to clarify the epidemiology of this infection in alpine free-living wild ruminants. Moreover a genetic assessment of European HEV wild ungulates sequences retrieved from Genbank has been performed to identify the frequency of genotypes and subtypes, while European HEV sequences isolated from humans, domestic and wild animals have been checked to identify viral mutations associated with treatment failure and virus replication fitness in human. Sampling of sera was carried out from 2013 to 2015 during autumnal hunting seasons and depopulation plans, from three study areas: i) 173 red deer from Stelvio National Park (Valfurva, Sondrio Province, Central Italian Alps), ii) 44 chamois from Orobic Alps, iii) 128 chamois, 81 red deer and 32 roe deer from Lepontine Alps (North-West Italian lps).Sera were tested by a species-independent immune-enzymatic assay (HEV ELISA 4.0v, MP Diagnostics Biomedicals, Singapore). All publicly available European HEV sequences from ruminant species (n=7) have been aligned with HEV reference and field strains, phylogeny was estimated by the maximum likelihood method and the percentage of nucleotide similarity was calculated. The presence of nucleotide variations have been checked in ORF1 available sequences of ungulates (n=18), humans (n=11) and other species (n=3). Chamois and red deer showed a HEVseroprevalence of 1.16% (2/172; CI 95%:1.03-1.28) and 0.79% (2/254; CI 95%:0-1.87), respectively. Roe deer resulted HEV seronegative. A high level of nucleotide identity ( 6597%) has been observed among ruminants\u2019 genotype 3 sequences (ORF1 or ORF2) and HEV reservoirs or human ones isolated in the same country: i) a goat and red deer with a wild boar and swine respectively in Italy; ii) two red deer with a swine and human in Spain; iii) a roe deer and reed deer with swine and wild boar and also a roe deer with human in Hungary. As regard to nucleotide variations, G1634 codon showed substitutions in 16 out of 32 available sequences (6 humans, 6 swine, 2 rabbits, 1 elk and 1 polecat) and V1479 codon in 23/32 sequences (6 humans, 12 swine, 2 rabbits, 1 wild boar, 1 elk and 1 polecat). In 5 humans, 7 swine, 2 rabbits, 1 elk and 1 polecat that mutations occurred together. Sporadic HEV infection emerged in chamois, firstly highlighting the involvement of this species in virus transmission, and red deer from North-Western and Central Alps. These results, together with the increasing density and overlapping distribution of wild ungulates in the Alps, have to be considered in relation to virus spread in free-ranging animals. As this regards, although wild boar is known to be reservoir of HEV, this species shows a wide range of seroprevalences in Italy, ranging from 0% to 49% with the lowest observed in Alpine areas confirming the sporadic infection of wild ruminants. On the contrary, red deer from central Apennines, where this species shares its habitat with abundant wild boar populations, show the highest prevalence of 13.9 %. Therefore, data emerged support the hypothesis of a relation between HEV spread and the abundance of wild boar populations. Phylogenetic analysis showed significant clusters including ruminant and wild boar, swine or human sequences obtained in the same country, supporting ruminant as spillover hosts but also highlighting the wide host range of zoonotic HEV. Moreover different mutations, supposed to increase virus replication and associated with treatment failure in human, have been identified in HEV sequences isolated not only from humans, but also from domestic and wild animals, such as polecat, elk and wild boar. These results and the increasing density and overlapping distribution of wild ungulates on the Alps have to be considered for the risk of virus spread among animals and infections in humans