Phylogenetic analysis of eight complete HEV-3 genomes from wild boar
The tree-puzzle analysis showed that the percentage of unresolved quartets was lower than 33% for all datasets providing a phylogenetic signal sufficient for analysis. The Maximum Likelihood tree (dataset I; Figure 1) showed the 8 complete genomes to cluster amongst five different HEV-3 subtypes: HEV-3c (n=1), HEV-3f (n=4), HEV-3a (n=1), the recently proposed subtype HEV-3n (n=1), and the novel unclassified subtype (HEV-3*; n=1) (Table 1). The wild boar strain (WB119VT2017) that clustered within subtype HEV-3c, showed a 93.5% nt. id. with the HEV isolate HEPAC-96 (MF444114) obtained from a human patient in France in 2008. Several strains of HEV-3c have been reported and sequenced from Italian wild boars. Despite this, only a few full genomes are available for comparison (n=6) (last sequence download from NCBI database February 2022); all these sequences were detected in wild boar hunted in Abruzzo (e.g: 2018.AZ.6050.8.2, MT840359), a region in Central Italy, and show ≈90.0% nt. id. with the aforementioned WB119VT2017 (Figure 1).
In the absence of complete genome sequences for Italian swine strains of HEV-3c, a BLASTn analysis was conducted based on the short 290bp diagnostic fragment contained within ORF2 and involving sequences available in the NCBI database. This comparison highlighted a nucleotide identity of 95.2% with the HEV-3c swine strain SWHEV82BO2012 (KF888276) detected in the north of Italy in 2012.
As noted above, four wild boar strains (WB03VT2016, WB110VT2017, 55863/2018 and 47272/2017) clustered within the HEV-3f subtype (Figure 1); however, their sequences were extremely diverse, separating into two sub-clusters (>87.0% nt. id.). The first sub-cluster includes a strain (WB03VT2016) that shared the highest nt. id. of 91.2% with a swine strain obtained in France in 2008, namely HEV strain FR-SHEV3f (JQ953666); this strain showed a lower nucleotide identity (≈87.0%) with strains of the second sub-cluster.
The second sub-cluster of HEV-3f includes strains WB110VT2017, 55863/2018 and 47272/2017 and the HEV complete genome of another previously reported strain (2019.AZ.5758.1.5, MT840365) obtained from a wild boar in the region of Abruzzo in 2019. Amongst these, strain WB110VT2017 was distant from other strains and its nucleotide identity was the lowest (<90.0%) in comparison to all other HEV-3f strains, including Italian strains (Figure 1). Two wild boar strains (WB03VT2016 and WB110VT2017) showed an epidemiological connection (>97% nt. id.) to HEV-3f strains isolated from patients hospitalized in the regions of Lazio and Abruzzo (the former with epidemiological link with Abruzzo): isolate ISS_ID_201/2017 (MZ274237), isolate ISS_ID_340/2019 (MZ274263), and ISS_ID_258/2018 (MZ274246), INMI_1902_2019 (MN537876), respectively. Two strains (55863/2018 and 47272/2017) showed the highest nucleotide identity (96%) with a swine strain HEV/13RS985-10 (KF939868) and a human strain ISS_ID_305/2019 (MZ274253). Lower identities (<93.5%) have been reported with other human strains isolated in Central Italy from patients hospitalized in Rome (INMI_1825_2018 MN444850; INMI_1823_2018 MN444849; INMI_1809_2018 MN444853; INMI_1203_2012 MN50947) and with several additional Italian swine strains detected in other regions (13RS1494-12, MK532924; SwHEVE12ITNA2012, KP965762; 13RS1494-11, MK532923; 13RS1494-9, MK532927; SwHEVPIR71ITA18, MN546867; HEV/13RS985-10, KF939868.1; 14RS333-8_50549-4, MK532935; ITswHEV1331, KF891380). Interestingly, all human strains correlated with wild boar strains, came from patients hospitalized in the regions of Abruzzo and Lazio, not far from the area where wild boar were hunted.
The complete genome of WB01VT2016 strain belonged to subtype HEV-3a with a 91.1% nt. id. with the HEV isolate_GiSw (KF303502) and 90.9% nt. id. with the HEV isolate HEV_34_Newcastle_UK_221214 (MH504130) reported in 2006 from a pig in Germany and from a human case in 2014 from the UK (Figure 1). In Italy, HEV-3a is not frequently encountered. The single wild boar HEV-3a strain reported in this study showed by BLASTn, comparing short ORF2 fragment, a 96.5% nt. id. with the HEV-3a human strain INMI_1736_2017 (MN444837) and 92.0% nt. id. with several strains reported from wild boar in Northern Italy (e.g.: IZSLER_B6_32, MW263036).
The strain132, isolated from a wild boar in 2012 (Lazio), clustered within a novel subtype of HEV-3* (reference strain MK390970) (Nicot et al., 2021; Smith et al., 2020); this novel subtype remains unassigned for the lack of the three full genomes needed for definitive classification. The cluster included three full genomes, all from wild boar, and detected in Italy: 132 and strains 17RS1920 (MK390971) proposed as references for the classification (Smith et al., 2020) and 17RS2551-4 (MK390970), showing a low 88.5% nt. id. (Figure 1).
Based on nucleotide sequences of short genome ORF2 fragment, strain 132 was similar (>93.0% nt. id.) to 5 other Italian strains from wild boar hunted in the same area (Di Pasquale et al., 2019) and with the human strain 122.16_F reported in 2012 from north-eastern Italy (KC782933). No higher identity was observed even by comparing the short ORF2 diagnostic fragments with the whole database of NCBI.
The strain WB171VT2017 formed a cluster (96.0 % nt.id.) with strains detected in 2016-2017 in wild boar hunted in the neighbouring Umbria region (e.g.: 54563/UM/2016 LR777864) and includes a human strain reported in 2018 from France (92.3% nt. id., HESQL059; MW355317) and the reference strain of the recently proposed novel subtype HEV-3n (WB/HEV/NA17ITA15, MF959764) first detected in southern Italy (Figure 1). Furthermore, using ORF2, the wild boar strain 47270.213/UM/2017 (LR777866) from Umbria and the human strain HESQL053 (MW355383) from France, together, supported the existence of tentatively named HEV-3n.