Paslahepevirus balyani in Suidae coming from the Monti Sibillini National Park (2020-2024)

Paslahepevirus balayani or hepatitis E virus (HEV) represents one the most diffused viral zoonotic foodborne pathogens in Europe, and genotype 3 results one of the most identified in Italy among Suidae. For this reason, the aim of this work was to carry out biomolecular and phylogenetic evaluations of the persistence and evolution of HEV-3 in wild boars (Sus scrofa) and domestic swines (Sus scrofa domesticus) from the Monti Sibillini National Park (Ascoli Piceno province) in the period between 2020 and 2024. More in detail, from December 2020 to January 2024, the sampling of n. 1054 Suidae was performed and two aliquots (15±2 g) of tissue were taken from each of them: one from the liver and one from muscle tissue (diaphragm pillar) obtained by plucks conferred at the slaughterhouse level in Ascoli Piceno (EU Reg. No. 624/2019, No. 625/2017, and R.L. Marche No. 13/2012). In the tested population, 818/1054 (77.61%) were wild boars and 236/1054 (22.39%) were domestic pigs raised with extensive methods for domestic consumption. Tissues followed the same standardized laboratory procedures: homogenization, RNA extraction by the Trizol LS method, and finally biomolecular tests: real time RT-qPCR and nested RT-PCR of the codifying regions ORF-1, ORF-2, and ORF-3 of HEV. Sequencing of the positive ones was followed by: nucleotide similarity, alignment and evolutionary analysis (phylogenetic tree construction using Neighboring-Joining) using MEGA X software. Finally, they have been deposited and published on the GenBank platform. The results showed that HEV RNA was amplified by the liver of 114/1054 (10.81%) (102 GE/ μL) subjects tested, and 26/114 (22.81%) of them also presented positive muscle tissues too. None of them harbored HEV RNA in the muscle only. From a comparative and species point of view, wild boars presented a prevalence of 72/114 (63.16%), and domestic pigs of 42/114 (36.84%). The ORF-1 region was mostly amplified by 97/114 (85.08%) (103 GE/μL) of Suidae, while ORF-2 (102 GE/μL) only in the wild boar populations 72/114 (63.16%). In the period between 2020-2024, the loaded sequences on the GenBank platform were as follow: MN20202101, MN20202102, MN20202103, MN20202104, OR933697, OR93398, MN2020210201, MN2020210208, MN2020210212, ON364349, ON364350, ON364351, ON364352. In all positive cases, nucleotide similarity (over 98.0% average), alignments and phylogenetic analyses have demonstrated the involvement of HEV-3c during the five years 2020-2024. The radicalization of the presence and persistence of HEV-3c (from 2020 to 2024) in the Ascoli Piceno province confirms that the Suidae have a fundamental role as reservoirs in urban and wild cycles. The sharing of areas between extensive domestic pigs’ farms and the coexistence with wild boars within the Monti Sibillini National Park have allowed possible cross species -infection, as supported by the analysis of the discovered sequences. It is therefore clear that biomolecular surveillance on the territory has a fundamental role in terms of food safety and prevention for the final consumer.