Introduction
Hepatitis E infection is recognized worldwide as an emerging public health issue (Kamar et al., 2017). The virus (HEV) is a quasi-enveloped RNA virus with a single-stranded positive-sense genome with an extensive genomic diversity, which determines the classification into two genera:Orthohepevirus and Piscihepevirus . TheOrthohepevirus is divided into four species (A, B, C, D) of which only two infect humans (A, C). Orthohepevirus A includes genetically distinct genotypes (HEV 1–8): HEV-1 and HEV-2 only infect humans; HEV-3 and HEV-4 are zoonotic. The infections are globally distributed in low-income and developed countries, causing large outbreaks mostly waterborne in the former and foodborne sporadic cases and small outbreaks in the latter. The two different epidemiological settings are associated with different genotypes of Orthohepevirus A species: HEV-1 and HEV-2 circulate in low-income countries; HEV-3 and HEV-4 mainly circulate in developed countries (Kamar et al., 2017; Dalton et al., 2018). HEV-3 and HEV-4 can be transmitted from animal to animal via the faecal-oral route or from animal to human by consumption of contaminated food of animal origin. Pigs and wild boar are the main reservoirs of HEV-3 and HEV-4. In Europe, HEV-3 is the most frequently detected genotype (Adlhoch et al., 2016). Due to the nucleotide diversity, the genotypes have been further classified into subtypes, named using alphabetical letters, based on their shared nucleotide and amino acid identity (Lu et al., 2006; Smith et al., 2013; Smith et al., 2018).
HEV-3 genotype strains are classified into 11 subtypes (a-m) and novel subtypes are constantly proposed. At least three full genomes clustering together and showing a high p-distance with all known subtypes by phylogenetic analyses are required for the classification into novel subtype (Smith et al., 2016; Smith et al., 2018). Some sequence strains have not been assigned yet, due to a limited number of fully sequenced genomes (less than 3), necessary to assign any novel subtype (Smith et al., 2020). In Europe, the subtypes HEV-3c, HEV-3e and HEV-3f are those most commonly detected in both humans and animals (Lapa et al., 2015; Adlhoch et al., 2016; Dorn-In et al., 2017; Nicot et al., 2018; Oeser et al., 2019; Suin et al., 2019) while the others are rarely detected (Reuter et al., 2009; Kozyra et al., 2021b; Nicot et al., 2021). To date, few studies aimed at understanding if any differences such as host adaptation, infectious dose and symptoms caused among subtypes exist. There is evidence to suggest that subtypes may have a clinical significance and enable adaptation to new hosts (Subissi et al., 2019; Abravanel et al., 2020). To some extent, the subtypes also show a geographical and temporal distribution, therefore their identification helps in evolutionary studies of HEV-3 and could also be useful in tracing time-space movement of strains.
In Italy, a recent study reported that HEV-3f is the most common subtype affecting humans and pigs, followed by HEV-3c and HEV-3e (De Sabato et al., 2020b). A few subtypes have also been reported less frequently, such as HEV-3a, and others that remain unclassified (De Sabato et al., 2020b). Elsewhere in Europe, HEV-3c has recently emerged as predominant in humans, pigs and wild boar (Izopet et al., 2019; Wang et al., 2019).
Heterogeneity of HEV-3 viral strains is the greatest in wild boar. HEV-3c and HEV-3i are widespread in wild boar in Central Europe (Oliveira-Filho et al., 2014; Anheyer-Behmenburg et al., 2017) and several other subtypes have been detected over European countries (Okano et al., 2014; Vina-Rodriguez et al., 2015; Kukielka et al., 2016; Mesquita et al., 2016; Dorn-In et al., 2017). In Italy, several studies have reported HEV-3 to circulate in wild boar throughout the country (Caruso et al., 2015; Martinelli et al., 2015; Mazzei et al., 2015; Aprea et al., 2018; Di Pasquale et al., 2019; De Sabato et al., 2020a; Forzan et al., 2021; Pierini et al., 2021). Just as in pigs and humans, the wild boar strains belong to subtypes HEV-3f and HEV-3e but include also subtypes HEV-3c, HEV-3a and several unclassified subtypes (Caruso et al., 2015; Serracca et al., 2015; Aprea et al., 2018; De Sabato et al., 2018a; Di Pasquale et al., 2019; Zecchin et al., 2019; Aprea et al., 2020; Lo Presti et al., 2020; Arnaboldi et al., 2021; Pierini et al., 2021). Four recent studies have revealed the high prevalence of HEV-3 in wild boar hunted in central Italy (De Sabato et al., 2018a; Di Pasquale et al., 2019; De Sabato et al., 2020a; Pierini et al., 2021); in addition, short genome region sequencing has revealed the genetic diversity of these HEV-3 strains to be also higher than expected. To more fully explore this variability, and to better understand the evolution of HEV-3 in this subpopulation of wild boar, 8 of the HEV-3 strains, collected in Central Italy (Lazio and Umbria, two neighbouring regions), were selected and subjected to full genome sequencing and detailed phylogenetic analysis.