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HEV antibody detection constitutes the main screening test for HEV infection. The aim of this study is to compare the sensitivity and specificity of four techniques: LIAISON® MUREX DiaSorin anti-HEV IgG and anti-HEV IgM assays, Hepatitis E VIRCLIA® IgM and IgG monotests, WANTAI HEV-IgM and IgG ELISA and VIDAS® anti-HEV IgM and IgG tests in five panels of samples configurated according to the immunoblot (RecomLine, Mikrogen, Neuss, Germany). Anti-HEV IgM sensitivity in the acute phase was 100% in all techniques, while sensitivity, including the immediate convalescence phase, was 96.74% for LIAISON®, 83.14% for VIRCLIA®, 84.78% for WANTAI and 88.04% for VIDAS®. Anti-HEV IgM specificity was 100% for both LIAISON® and VIRCLIA®. Anti-HEV IgM WANTAI agreed with VIRCLIA® with a good Kappa coefficient (κ = 0.71). Anti-HEV IgG post-infection sensitivity was 100% for LIAISON®, VIDAS® and VIRCLIA® and 99% for WANTAI. Anti-HEV IgG specificity reached 97.17% for LIAISON and 88.68% for VIRCLIA®. Our results demonstrated a better capacity of LIAISON® MUREX anti-HEV IgM than that of competitors for detecting acute infections as well as accurate anti-HEV IgG results and in how to resolve them.

Hepatitis E virus genotype 3 (HEV-3) is an EU/EEA emergent zoonosis. HEV-3 clades/subtypes have been described. Its genome contains ORF1, which encodes nonstructural proteins for virus replication, ORF2, the capsid protein, and ORF3, a multifunctional protein involved in virion pathogenesis. The study aims with respect to HEV-3 are to: (1) calculate genome entropy (excluding hypervariable region); (2) analyze the described motifs/mutations; (3) characterize clade/subtype genome polymorphisms. Seven hundred and five sequences from the GenBank database were used. The highest entropies were identified in zoonotic genotypes (HEV-3 and HEV-4) with respect to HEV-1 in X domain, RdRp, ORF2, and ORF3. There were statistically significant differences in the entropy between proteins, protease and ORF3 being the most variable and Y domain being the most conserved. Methyltransferase and Y domain motifs were completely conserved. By contrast, essential protease H581 residue and catalytic dyad exhibited amino acid changes in 1.8% and 0.4% of sequences, respectively. Several X domain amino acids were associated with clades. We found sequences with mutations in all helicase motifs except number IV. Helicase mutations related to increased virulence and/or fulminant hepatitis were frequent, the 1,110 residue being a typical HEV-3e and HEV-3f-A2 polymorphism. RdRp motifs III, V, VII also had high mutation rates. Motif III included residues that are polymorphisms of HEV-3e (F1449) and HEV-3 m (D1451). RdRp ribavirin resistance mutations were frequent, mainly 1479I (67.4, 100% in HEV-3efglmk) and 1634R/K (10.0%, almost 100% in HEV-3e). With respect to ORF2, 19/27 neutralization epitopes had mutations. The S80 residue in ORF3 presented mutations in 3.5% of cases. Amino acids in the ORF3-PSAP motif had high substitution rates, being more frequent in the first PSAP (44.8%) than in the second (1.5%). This is the first comprehensive analysis of the HEV-3 genome, aimed at improving our knowledge of the genome, and establishing the basis for future genotype-to-phenotype analysis, given that viral features associated with severity have not been explored in depth. Our results demonstrate there are important genetic differences in the studied genomes that sometimes affect significant viral structures, and constitute clade/subtype polymorphisms that may affect the clinical course or treatment efficacy.

The hepatitis E virus (HEV) hypervariable region (HVR) presents the highest divergence of the entire HEV genome. It is characteristically rich in proline, and so is also known as the “polyproline region” (PPR). HEV genotype 3 (HEV-3) exhibits different PPR lengths due to insertions, PPR and/or RNA-dependent RNA polymerase (RdRp) duplications and deletions. A total of 723 PPR-HEV sequences were analyzed, of which 137 HEV-3 sequences were obtained from clinical specimens (from acute and chronic infection) by Sanger sequencing. Eight swine stool/liver samples were also analyzed. N- and C-terminal fragments were confirmed as being conserved, but they harbored differences between genotypes and were not proline-plentiful regions. The genuine PPR is the intermediate region between them. HEV-3 PPR contains a higher percentage (30.4%) of prolines than other genotypes. We describe for the first time: (1) the specific placement of HEV-3 PPR rearrangements in sites 1 to 14 of the PPR, noting that duplications are more frequently attached to sites 11 and 12 (AAs 74–79 and 113–118, respectively); (2) the cadence of repetitions follows a circular-like pattern of blocks A to J, with F, G, H, and I being the most frequent; (3) a previously unreported insertion homologous to apolipoprotein C1; and (4) the increase in frequency of potential N-glycosylation sites and differences in AAs composition related to duplications.