Circulation of Hepatitis E virus in wild boar population in Central Italy

Introduction: Hepatitis E virus (HEV) is an RNA virus causing an acute generally self-limited disease in humans. Over the last 10 years, in Europe an increasing number of autochthonous cases linked to foodborne transmission of HEV genotype 3 have been reported. Pigs and wild boar are the main reservoirs. Human cases have been frequently linked to the consumption of raw or undercooked pork products and wild boar meat. In this study, the presence of HEV-RNA was investigated in 92 livers and 116 paired liver and muscles from wild boar (Sus scrofa) collected during two hunting seasons 2016 -2017 and 2017-2018, respectively, in Central Italy. Materials and Methods: Fifty mg of liver and muscle samples were used for RNA extraction (RNeasy Mini Kit, Qiagen). The HEV genome was detected and quantified by quantitative Real-Time RT-PCR (RTqPCR) (QuantiFast Pathogen +IC Kits). The RNA of HEV positive liver and muscle samples was analysed by nested RT-PCR amplifying a 348 bp fragment in the ORF2. The 800 bp fragment within the Methyltransferase region (ORF1) was also amplified from paired liver and muscle samples by nested RTPCR (ORF1). The phylogenetic analysis was performed using MEGA7 software. Results: HEV RNA was detected in 62 livers with prevalence ranging from 30% to 65.7% depending on the hunting area. All but one group of hunted animals was negative for HEV. Five animals (8%, 5/62) were positive for HEV in both muscle and liver while none of the animals was positive in muscle only. The ORF2 and ORF1 partial viral sequences were obtained from all five paired livers and muscles. The sequenced strains obtained from paired livers and muscles belonging to the same animal were identical (100% nt.id.). Phylogenetic analysis showed that strains clustered within the different subtypes HEV-3c, HEV-3f and other clusters not assignable to any subtypes described so far. Discussion: The main result of sequencing was the wide heterogeneity of circulating HEV strains that belonged to different subtypes: HEV‐3c, HEV‐3f and novel unclassified strains. Animals possibly belonging to the same family group hunted by the same team were infected with a unique strain (100% nucleotide identity). HEV-3 RNAs have been detected in muscles, probably caused by viremia, leaving open the question of precaution measures to be taken during slaughtering, including a better bleeding of animals which could reduce the risk of cross-contamination. Since wild animals are a source of HEV transmission to humans, results obtained underlined the risk of consuming raw or undercooked wild boar meat suggesting that this topic deserves further investigation.