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.