Whole Genome Sequencing Characterization of HEV3-e and HEV3-f Subtypes among the Wild Boar Population in the Abruzzo Region, Italy: First Report

Abstract

Hepatitis E virus (HEV) is an emergent zoonotic pathogen, causing worldwide acute and chronic hepatitis in humans. HEV comprises eight genotypes and several subtypes. HEV genotypes 3 and 4 (HEV3 and HEV4) are zoonotic. In Italy, the most part of HEV infections (80%) is due to autochthonous HEV3 circulation of the virus, and the key role played by wild animals is generally accepted. Abruzzo is an Italian region officially considered an HEV “hot spot”, with subtype HEV3-c being up to now the only one reported among wild boars. During the year 2018–2019, a group of wild boars in Abruzzo were screened for HEV; positive RNA liver samples were subjected to HEV characterization by using the whole genome sequencing (WGS) approach methodology. This represents the first report about the detection of HEV-3 subtypes e and f in the wild boar population in this area. Since in Italy human infections from HEV 3-e and f have been associated with pork meat consumption, our findings deserve more in-depth analysis with the aim of evaluating any potential correlation between wild animals, the pork chain production and HEV human infections.

Molecular and Pathological Detection of Hepatitis E Virus in Roe Deer (Capreolus capreolus) and Fallow Deer (Dama dama) in Central Italy

“…No. MT840367.1) by Aprea et al [ 33 ] with an Expect (E) value of 1 × 10 −12 , confirming the specificity of the molecular analysis.…”

West Nile Virus Lineage 1 in Italy: Newly Introduced or a Re-Occurrence of a Previously Circulating Strain?

“…Briefly, total RNA was treated with TURBO DNase (Thermo Fisher Scientific, Waltham, MA, USA) at 37 • C for 20 min, and then purified by an RNA Clean & Concentrator™-5 Kit (Zymo Research, Irvine, CA, USA). The purified RNA was used for the assessment of sequencing independent single primer amplification protocol (SISPA) [62,63]. In detail, viral RNA underwent cDNA synthesis using 200 units of the SuperScript ® IV Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA), in the presence of 5X SSIV Buffer, 50 µM of the random hexamer FR26RV-N 5 -GCCGGAGCTCTGCAGATATCNNNNNN-3 , 10 mM of dNTPs mix, 100 mM of DTT, and 40U of RNAse OUT RNase inhibitor (Thermo Fisher Scientific, Waltham, MA).…”

“…The reads obtained were trimmed using a Trimmomatic script (Trimmomatic v0.36) to remove low quality and short reads [65]. Furthermore, the reads were quality controlled by using FastQC v0.11.5 [63,66]. The resulting 2,149,990 reads were de novo assembled using SPADES v3.11.1 [67].…”

“…The resulting 2,149,990 reads were de novo assembled using SPADES v3.11.1 [67]. Based on genome assemblies, a de novo filtering for the scaffolds with a minimum length of 200 nucleotides, and a matching for the best reference for each assembly using ABRicate was carried out [63]. Finally, a mapping with the references found in the previous step using Bowtie2 (v.2.1.0) was performed [68].…”

Detection of Hepatitis E Virus Infections in Wild Boars in Southwest Germany Using a Stepwise Laboratory Diagnostic Approach

“…Samples yielding Cq values in RT-qPCR <30 proved suitable for providing sufficient quantities of HEV nucleic acid suitable for successful DNA sequencing. Likewise, other researchers have come to similar conclusions and recommended samples with Cq values <31 in qPCR for reliable success in whole genome sequencing (WGS) [53].…”