Molecular Characterization of HEV Genotype 3 in Italy at Human/Animal Interface

Abstract

Hepatitis E virus (HEV) is an emerging public health issue in industrialized countries. In the last decade the number of autochthonous human infections has increased in Europe. Genotype 3 (HEV-3) is typically zoonotic, being foodborne the main route of transmission to humans, and is the most frequently detected in Europe in both humans and animals (mainly pigs and wild boars). In Italy, the first autochthonous human case was reported in 1999; since then, HEV-3 has been widely detected in both humans and animals. Despite the zoonotic characteristic of HEV-3 is well established, the correlation between animal and human strains has been poorly investigated in Italy. In the present study, we compared the subtype distribution of HEV-3 in humans and animals (swine and wild boar) in the period 2000-2018 from Italy. The dataset for this analysis included a total of 96 Italian ORF2 sequences (300 nt long), including both NCBI databasederived (n = 64) and recent sequences (2016-2018, n = 32) obtained in this study. The results show that subtype 3f is the most frequent in humans and pigs, followed by the HEV-3e, HEV-3c and other unassignable HEV-3 strains. Diversely, in wild boar a wider group of HEV-3 subtypes have been detected, including HEV-3a, which has also been detected for the first time in a human patient in Central Italy in 2017, and a wide group of unassignable HEV-3 strains. The phylogenetic analysis including, besides Italian strains, also sequences from other countries retrieved from the NCBI database, indicated that human Italian sequences, in particular those of HEV-3f and HEV-3e, form significant clusters mainly with sequences of animal origin from the same country. Nevertheless, for HEV-3c, rarely detected in Italian pigs, human sequences from Italy are more correlated to human sequences from other European countries. Furthermore, clusters of nearidentical human strains identified in a short time interval in Lazio Region (Central Italy) can be recognized in the phylogenetic tree, suggesting that multiple infections originating from a common source have occurred, and confirming the importance of sequencing support to HEV surveillance.

Clinical Characteristics of Acute Hepatitis E and Their Correlation with HEV Genotype 3 Subtypes in Italy

“…Most sequences ( n = 44) clustered with clade 3efg: 29 were GT3f and 15 were GT3e. Nine sequences clustered with clade 3abkchij: 7 were GT3c, 1 was a previously described GT3a sequence (INMI_1736_2017) [ 26 ] and 1 strain was unsubtyped (INMI_1909_2019) ( Figure 5 A). No GT3b, GT3g, GT3h, GT3k, GT3i or GT3j subtypes were identified among study patient-derived sequences.…”

“…The prevalence of HEV GT3e acute infections was high in 2019, attributable to an outbreak in people vacationing in the Abruzzo region. GT3c represented the third most abundant subtype (9 cases), while only a single GT3a case [ 26 ] was observed. Our GT3f patient-derived sequences were most similar to French human sequences, KC166968 (96.1% with INMI_1719_2017) and MN646689 (97.3%); a Belgian human sequence, MN614143 (93.2%); and one Italian pig sequence, KF891380 (94.4%).…”

“…In samples collected from January 2011 to December 2018, reverse transcription and first round amplification were performed by one-step RT-PCR using ORF2 primers. Resultant product (5 µL) was used in a second round of PCR, as previously described [ 26 ]. From January 2019 onwards, a more sensitive detection protocol was employed.…”

Anti-HEV IgG Avidity Testing: Utility for Diagnosing Acute and Resolved Genotype 3 Infections

“…HEV RNA was isolated from 400 µL of serum using the QIASYMPHONY automated instrument (QIAGEN, Hilden, Germany). All samples collected before 2019 were retro-transcribed employing the QIAGEN Onestep RT-PCR kit with primers for the HEV ORF2 region as previously described [ 29 ]. For samples obtained since 2019, RT-PCR was performed by Super Script IV PCR with random primers (SSIV) (Thermofisher Scientific, Paisley, UK), according to the manufacturer’s instructions.…”

“…For samples obtained since 2019, RT-PCR was performed by Super Script IV PCR with random primers (SSIV) (Thermofisher Scientific, Paisley, UK), according to the manufacturer’s instructions. Amplification of cDNA was achieved by a nested polymerase chain reaction protocol (PCR) using TaqGold polymerase and two sets of primers encompassing a 457 bp fragment within the ORF2 gene, as used in the OneStep protocol [ 29 ]. Cycling conditions for first and second round PCR were as follows: (1) 95 °C (15 min), (2) 94 °C (30 s), (3) 56 °C (30 s) and (4) 72 °C (45 s); steps 2–4 were repeated 35 times.…”