Virological Methods

The frozen plasma samples collected at the Belgian and French reference centers from suspected HEV infected patients were analyzed in Toulouse or Brussels. HEV RNA was extracted from 500 μl plasma using the MagnaPure 96 instrument (France, elution volume 50 μl), or from 140 μl plasma with the EasyMag instrument (Brussels, elution volume 60 μl). The HEV RNA concentration in 25 μl aliquots of extract was measured using the RealStar HEV RNA 2.0 kit from Altona (Abravanel et al., 2013). The HEV genotype and subtype were determined by population sequencing of open reading frame 2 (ORF2) (Legrand-Abravanel et al., 2009; Suin et al., 2019) and by phylogenetic analysis based on the reference sequences proposed by Smith et al. (2020). A 345 nt fragment of ORF2 (nucleotides 5,994–6,294 of the {"type":"entrez-nucleotide","attrs":{"text":"M73218","term_id":"330023","term_text":"M73218"}}M73218 genome) was amplified with a hemi-nested PCR protocol (Legrand-Abravanel et al., 2009; Suin et al., 2019). For phylogenetic analysis, nested RT-PCR was performed, targeting the ORF2 region (structural proteins), using primers HEV5944S (5′-GTGGCYGAGGAGGAGGCKAC-3′) and HEV-6484AS (5′-CCCTTRTCCTGCTGNGCATTCTCGACAGA-3′) in the first round and HEV-5930S (5′-CCCTTRTCCTGCTGNGCATTCT CGACAGA-3′) and HEV-6320AS (5′-TGYTGGTTRTCAT AATCCTG-3′) in the second round. The PCR products of these amplifications were purified and sequenced using the fluorescent dye terminator method for Big DyeTerminator cycle sequencing (Applied Biosystems, Paris, France) with the same primers as those used for amplification on an Applied Biosystems ABI 3130 XL analyzer. The nucleotide alignments were performed using ClustalX v2.0¹. The tree was constructed using the MEGA v5.0 software² with the neighbor-joining method. Reference sequences of avian HEV were included as an outgroup to root the tree. The final tree was prepared using the bootstrap method (bootstrapped with 1,000 replicates). The sequences have been submitted to Genbank (accession numbers: MW655496 to MW655522).