Hepatitis E virus genotype 3 microbiological surveillance by the Spanish Reference Laboratory: geographic distribution and phylogenetic analysis of subtypes from 2009 to 2019.

BackgroundHepatitis E virus genotype 3 (HEV-3) is widely distributed throughout Europe, with incidence of infections increasing in many countries. Belgium, Bulgaria, France, Germany, Italy, the Netherlands and the United Kingdom have reported the distribution of HEV-3 subtypes in cohorts of patients with hepatic disease.AimTo describe the distribution of the HEV-3 subtypes in Spain at national and autonomous community (AC) levels between 2009 and 2019. The study was also extended to Andorra.MethodsOf 5,197 samples received by the National Reference Laboratory during the study, 409 were HEV-RNA-positive. Among these, 294 (71.9%) were further typed based on an ORF2 sequence fragment, or, for a subset of 74, based on the full-coding genome sequence.ResultsHEV-3 was detected in 291 samples. The dominant subtype in Spain was HEV-3f (88.3%; 257/291), which occurred in all ACs, with no change in detection level over time. Within this subtype, three subclusters were characterised: HEV-3f-B, HEV-3f-A1 and HEV-3f-A2. The second most common HEV subtype was the recently described HEV-3m (7%; 21/291), with two subclusters identified: HEV-3m-A, which has been known since 2010, and HEV-3m-B, since 2014. The third most encountered subtype was HEV-3c (4.1%; 12/291), with a frequency not increasing over time, unlike observations in some European countries.ConclusionThe importance of the surveillance of HEV-3 subtype and subcluster circulation is yet to be assessed. This surveillance together with the comprehensive epidemiological characterisation of clinical cases, could support the identification of sources of transmission and the establishment of control measures nationally and internationally.

Hepatitis E genotype 3 genome: A comprehensive analysis of entropy, motif conservation, relevant mutations, and clade-associated polymorphisms.

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.

Proline-Rich Hypervariable Region of Hepatitis E Virus: Arranging the Disorder.

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.