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.