Discussion
Four studies were conducted over five years with the aim of detecting
HEV in wild boar living in bordering areas located in central Italy
(Lazio and Umbria regions) (De Sabato et al., 2018a; Di Pasquale et al.,
2019; De Sabato et al., 2020a; Pierini et al., 2021). The retrieved
strains were widely heterogeneous and based on ORF2, belong to multiple
HEV-3 subtypes. Following these preliminary results, complete genome
sequencing and characterization of the strains was undertaken to further
explore viral diversity, the evolutionary relationships amongst
subtypes, and geographical distribution.
The complete genomes for 8 HEV-3 strains, which displayed high
p-distance values between each other and other HEV reference sequences,
were obtained and subjected to phylogenetic analysis. The 13 subtype
reference strains, along with 6 reference strains representing
unassigned novel HEV subtypes, were used for this analysis, following
subtype assignment criteria recommended by Smith et al (2020).
Based on this, the maximum likelihood tree assigned the 8 complete HEV
genomes to 5 subtypes, namely HEV-3f, HEV-3c, HEV-3a, and two novel
recently recognized subtypes (Nicot et al., 2018; Smith et al., 2020;
Pierini et al., 2021). These novel subtypes remain unnumbered in the
continued absence of three full genomes needed to achieve definitive
classification (Smith et al., 2018). Only recently as it emerged that
these strains group with two full genome strains obtained from wild boar
(Perrini et al 2021) and for which subtype HEV-3n was proposed.
In Italy, HEV-3f is the subtype most frequently encountered in wild
boar, in order followed by HEV-3c, HEV-3e, and other rarer subtypes (De
Sabato et al., 2020b). The most common subtype in humans is HEV-3f,
followed by HEV-3e (De Sabato et al., 2020b; Garbuglia et al., 2021) and
last HEV3c; this last-mentioned subtype apparently is more prevalent
elsewhere in Europe (Lhomme et al., 2015; Nicot et al., 2018).
A different subtype distribution is observed in pigs, where the main
subtypes are HEV-3f, followed by HEV-3e and, less frequently, HEV-3c and
HEV-3l (De Sabato et al., 2020b).
In this study, the estimated mean value of the evolutionary rate is
2.72×10-3 substitutions/site/year for the HEV complete
genome (95% HPD:
1.51×10-3-4.06×10-3) and
2.87×10-3-9.9×10-3 for the HEV ORF2
capsid gene, which is similar in both analyses and to results reported
upon previously (Nakano et al., 2012; Zehender et al., 2014; Montesano
et al., 2016).
Bayesian trees built with the diagnostic fragment within the ORF2 capsid
gene, the region most sequenced and represented in the NCBI database,
revealed strains within subtypes HEV-3e and HEV-3f to form geographical
clusters. Some clusters involved strains sequenced in a single study,
and in terms of space and time, may introduce bias into Bayesian
geographical analyses. Nevertheless, within HEV-3f some clusters
included strains from pigs, wild boar and humans suggesting that
correlated strains circulate in different regions and hosts in parts of
Italy, and that transmission or co-circulation amongst swine and wild
boar may occur. This possibility underlines the importance of
biosecurity in terms of minimizing contact between wild boar and
domestic pig populations. Furthermore, in the HEV-3f cluster, some
groups that comprised both human and wild boar strains, showed
geographical correlation. This result supports the hypothesis that human
cases are definitively linked to the consumption of local produced wild
boar foods. Besides this, the four HEV-3f sequences included in this
study, though assigned to one and the same subtype, clearly fall within
different clades, revealing HEV-3f subtype sequences to be genetically
heterogeneous (Smith et al., 2020).
Within subtype HEV-3a, based on ORF2, the wild boar strain WB01VT2016
showed a high nucleotide identity (96.0%) with the only human HEV-3a
strain (MN444837) and obtained from a patient hospitalized in the same
region (Lazio) (De Sabato et al., 2020b) and which a year later (in
2017) was detected also in wild boar. Without corroborative
epidemiological data, this percentage of nucleotide identity is
insufficient for definitively correlating the two strains; however, the
result merits further investigation by sequencing and comparing complete
genomes, since HEV-3a in Italy is rare in both wild boar (Di Pasquale et
al., 2019) and humans (De Sabato et al., 2020b).
In this study, two of the full genomes obtained were assigned to novel
subtypes HEV-3n and HEV-3*. Only a limited number of sequences are
available for these recently recognized subtypes (Smith et al., 2020;
Nicot et al., 2021; Pierini et al., 2021).
By comparing full genomes, the HEV-3n sequences, from this and previous
studies, formed a group including strains only detected in wild boar in
Italy. By comparing ORF2 fragments, an additional human strain, detected
in France in 2018 (Kozyra et al., 2021a), was also included in the
HEV-3n subtype cluster. Since, according to the Bayesian tree, the
HEV-3n may have originated only recently (2014), the results suggest
that the subtype may have spread to other countries, where it could be
present, however, still with a low prevalence. The HEV-3n strains of
this study were detected in Italian wild boar hunted in two neighbouring
areas, the Lazio (Viterbo province) and Umbria regions (Perugia) in 2016
and 2017, indicating animal movement to occur between the two areas.
However, the first identification of this novel subtype, subsequently
established as reference strain (Smith et al., 2018), occurred in a wild
boar hunted in southern Italy (Aprea et al., 2018), suggesting that this
strain is widespread in Italy, circulating in more distant and
non-adjoining regions.
The other novel subtype detected in this study is still unclassified
(HEV-3*) and included the strain 132 and two wild boar strains from
northern Italy, collected in 2017 (17RS1920, 17RS2551-4) (Zecchin et
al., 2019). In the previous study (Di Pasquale et al., 2019), the strain
132 clustered with eleven other wild boar strains from the same
geographical area. It is noteworthy that this unclassified strain shares
a low percentage (<89.0%) of nucleotide identity with all
other HEV sequences in the NCBI database, representing either complete
genomes or the short ORF2 fragment. Based on phylogenetic and sequence
analysis, strains correlated to strain 132 have only been described from
Italian wild boar. This evidence suggests that this subtype could have
originated recently in Italy and may not have spread yet to other
countries. However, due to the absence of a supportive temporal signal,
a phylogenetic analysis was not conducted.
The strain132 is to some extent related (91%) to an Italian human
strain reported in 2012 (KC782933) (Giordani et al., 2013) but whose
source is uncertain, possibly linked to wild boar. The patient had
butchered a wild boar hunted in Tuscany region, not far from the area
where strain 132 was obtained in wild boar. Although these two strains
are not identical and related epidemiological data are missing, the
result may suggest the zoonotic transmission, due to the consumption of
raw or undercooked wild boar products (Rivero-Juarez et al., 2017;
Montone et al., 2019).
Within the same geographical area, and over a number of years, the local
wild boar population acts as host to a highly heterogeneous group of
HEV-3 strains, some of which belong to rare subtypes or have been
reported from Italy only. This picture around strain diversity possibly
is incomplete due to the limited number of full genome sequences
available. Nevertheless, the same result is obtained when analysing
ORF2, the fragment that is most abundantly represented in the NCBI
database. Since no intensive pig farms are present in the investigated
areas, contact and transmission between pigs and wild boar is greatly
limited; therefore, the wide heterogeneity observed confirms wild boar
to be a significant reservoir of HEV-3.
Within the subtypes for which whole genomes are available, such as
HEV-3c or HEV-3f (Zecchin et al., 2019; Aprea et al., 2020), no
sequences that could be considered identical were obtained by us in this
study. The absence of identical sequences may be explained by limited
intermixing of wild boar populations in recent time, and which deals
with an animal host that does not move far from its breeding grounds of
choice, thereby ensuring the maintenance of intra-subtype variability.
The occurrence of common subtypes such as HEV-3c and HEV-3f but also
less common strains such as HEV-3a and HEV-3n (detected only in wild
boar in Italy), supports the boar’s autochthonous reservoir role in the
maintenance of local strain heterogeneity. Among the subtypes analysed
in this study some are rare or not detected in humans (HEV-3a, HEV-3n
and HEV-3*) but are common in wild boar. This could be due to specific
host adaptation for some subtypes or could be associated to a lower
probability of transmission by wild boar derived food, which is not
consumed frequently.
In wild reservoir confined in an area with no or limited contacts with
farmed animals the variability of HEV-3 strains within the confined
population is kept. This wild boar scenario is in sharp contrast to the
situation on pig farms where the same HEV strain is identified
repeatedly over the years, even after stamping out (Widen et al., 2011;
Ianiro et al., 2021).
Our results show that whole genome sequencing and sharing of sequences
in public databases is essential to accurate analysis to track the
evolutionary history and geographic spread of HEV-3 subtypes and their
role in human and animal infection. Comparing only short gene fragments
may introduce bias into a classification. To date, only few Italian HEV
whole genomes are available in the NCBI database, 17 from wild boar, 3
from swine and none from humans, making it difficult to correlate
strains. The implementation of systematic whole genome sequencing for
human and veterinary samples is therefore of paramount importance for a
better understanding of virus circulation and evolution.