Recombinant strains of hepatitis E virus (HEV) with insertions of human genomic fragments or HEV sequence duplications in the sequence encoding the polyproline region (PPR) were previously described in chronically infected patients. Such genomic rearrangements confer a replicative advantage in vitro but little is known about their frequency, location, or origin. As the sequences of only a few virus genomes are available, we analyzed the complete genomes of 114 HEV genotype 3 strains from immunocompromised (n = 85) and immunocompetent (n = 29) patients using the single molecular real-time sequencing method to determine the frequency, location, and origin of inserted genomic fragments, plus the proportions of variants with genomic rearrangements in each virus quasispecies. We also examined the amino acid compositions and post-translational modifications conferred by these rearrangements by comparing them to sequences without human gene insertions or HEV gene duplications. We found genomic rearrangements in 7/114 (6.1%) complete genome sequences (4 HEV-3f, 1 HEV-3e, 1 HEV-3 h, and 1 HEV-3chi-new), all from immunocompromised patients, and 3/7 were found at the acute phase of infection. Six of the seven patients harbored virus-host recombinant variants, including one patient with two different recombinant variants. We also detected three recombinant variants with genome duplications of the PPR or PPR + X domains in a single patient. All the genomic rearrangements (seven human fragment insertions of varying origins and three HEV genome duplications) occurred in the PPR. The sequences with genomic rearrangements had specific characteristics: increased net load ( p < 0.001) and more ubiquitination ( p < 0.001), phosphorylation ( p < 0.001), and acetylation ( p < 0.001) sites. The human fragment insertions and HEV genome duplications had slightly different characteristics. We believe this is the first description of HEV strains with genomic rearrangements in patients at the acute phase of infection; perhaps these strains are directly transmitted. Clearly, genomic rearrangements produce a greater net load with duplications and insertions having different features. Further studies are needed to clarify the mechanisms by which such modifications influence HEV replication.

In this recommendation, we update our 2016 table of reference sequences of subtypes of hepatitis E virus (HEV; species Orthohepevirus A, family Hepeviridae) for which complete genome sequences are available (Smith et al., 2016). This takes into account subsequent publications describing novel viruses and additional proposals for subtype names; there are now eight genotypes and 36 subtypes. Although it remains difficult to define strict criteria for distinguishing between virus subtypes, and is not within the remit of the International Committee on Taxonomy of Viruses (ICTV), the use of agreed reference sequences will bring clarity and stability to researchers, epidemiologists and clinicians working with HEV.

Hepatitis C virus (HCV) is a major cause of liver disease. Knowledge of HCV variability is crucial for clinical and epidemiological analysis. HCV genotype 4 (HCV-4) has become increasingly prevalent in European countries, including France, in recent years. The present study investigates the heterogeneity of HCV-4 in south-western France by phylogenetic analysis of NS5B sequences from 166 patients. The E2 region of 90 strains was also analysed. Genotype 4 accounts for 7?4 % of HCV infections in this area. Analysis of the NS5B region revealed 12 subtypes and the NS5B and E2 phylogeny data were congruent, except for one strain. The epidemiological data indicated two main groups of patients. One included intravenous drug users (IVDUs) of French origin, who were infected by homogeneous strains of subtypes 4a or 4d. The second group comprised non-IVDU patients who were infected with heterogeneous strains. This group was subdivided into patients of French origin, who were infected with eight subtypes, and patients from non-European countries (Central Africa or the Middle East), who were mainly infected with 4f, 4k, 4r and other subtypes; they showed the greatest genetic heterogeneity. This study of a large cohort of patients shows the great diversity of HCV-4 strains, and that these subtypes have spread differently.

Hepatitis E virus (HEV) genotype 3 is the most common genotype linked to HEV infections in Europe and America. Three major clades (HEV-3.1, HEV-3.2, and HEV-3.3) have been identified but the overlaps between intra-subtype and inter-subtype p-distances make subtype classification inconsistent. Reference sequences have been proposed to facilitate communication between researchers and new putative subtypes have been identified recently. We have used the full or near full-length HEV-3 genome sequences available in the Genbank database (April 2020; n = 503) and distance analyses of clades HEV-3.1 and HEV-3.2 to determine a p-distance cut-off (0.093 nt substitutions/site) in order to define subtypes. This could help to harmonize HEV-3 genotyping, facilitate molecular epidemiology studies and investigations of the biological and clinical differences between HEV-3 subtypes.

m Hepatitis E virus (HEV) can lead to chronic infection in solid-organ transplant patients. Ribavirin is efficient for treatment of chronically infected patients. Recently, the1634R mutation in the HEV polymerase has been associated with treatment failure. However, it is unclear if this mutation can be used as a prognostic marker of treatment outcome. We studied the prevalence of the 1634R mutation in the HEV polymerase of patients starting ribavirin therapy, the influence of the 1634R variants on the viral response, the frequency of the 1634R mutation in patients whose treatment failed, and its impact on ribavirin retreatment. We analyzed pretreatment samples from 63 solid-organ transplant patients with chronic hepatitis E using deep sequencing; 42 patients had a sustained virologic response (SVR), and 21 were non-SVR patients. We detected the 1634R variant by deep sequencing in 36.5% (23/63) of the patients (proportions, 1.3 to 100%). The 1634R variant was detected in 31.0% (13/42) of baseline plasma samples from patients with SVR and in 47.6% (10/21) in the other patients (P ‫؍‬ 0.2). The presence of this mutation did not influence the initial decrease in viral RNA. Lastly, a second prolonged ribavirin treatment led to SVR in 70% of the patients who initially did not have SVR, despite the presence of the 1634R variant. We conclude that the presence of the 1634R variant at ribavirin initiation does not lead to absolute ribavirin resistance. Although its proportion increased in patients whose treatment failed, the presence of the 1634R variant did not compromise the response to a second ribavirin treatment.

A 9.2-kb sequence from a hepatitis C virus (HCV) strain found in southwest France was compared to sequences from reference strains in HCV sequence databases. We found a recombinant virus with genotype 2 at the 5 end and genotype 5 at the 3 end. The crossover point was located between genes NS2 and NS3. Recombination between HCV genotypes must now be considered in studies on HCV epidemiology and evolution and in predictions of the virus response to antiviral therapy. Knowing the location of the recombination point may also be useful for constructing infectious chimeric viruses.Studies of the nucleotide sequences of hepatitis C virus (HCV) variants from different individuals and geographical regions have revealed at least six genotypes and many subtypes (15,16). The genotype is presently a major factor in the choice of treatment and the prognosis (2, 7). Knowledge of the distributions of genotypes in different parts of the world may help us to understand the epidemiology and evolution of HCV. Until 2001, the evolution of HCV was thought to proceed in a clonal manner, with diversity generated through the accumulation of mutations. However, homologous recombinations have been demonstrated in places as far apart as St. Petersburg (5), Vietnam (10), the Philippines (4), and Peru (1). We have now identified a new natural intergenotypic recombinant. Fullgenome sequencing indicated that it was produced by recombination between genotypes 2 and 5.Identification of a potential recombinant strain of HCV. We previously analyzed the responses to treatment of 26 patients infected with HCV genotype 5 (HCV-5) in southwest France and the associated epidemiology (6). Phylogenetic analysis of the NS5B genes of the 26 corresponding strains pointed out a particular strain, R1. Twenty-five local strains formed a cluster with the genotype 5a reference sequences, while the R1 strain formed a distinct branch in the tree (Fig. 1A). The NS5B sequence with accession number AY373486 corresponds to the strain called R1 in the present study. The E2 region of the genome was sequenced to further characterize this strain (12). The MEGA program showed that the R1 sequence belonged to the genotype 2 in the E2 phylogenetic tree (Fig. 1B), but the strain could not be subtyped. Although these preliminary analyses were compatible with a recombinant strain of HCV, they could also indicate a mixed infection with two genotypes. Two genome regions were cloned and sequenced using the Invitrogen TA cloning kit. Phylogenetic analysis indicated that all the clones from the 5Ј untranslated region (UTR; n ϭ 19) clustered with genotype 2 and all the clones from the NS5B region (n ϭ 35) clustered with genotype 5 (data not shown). This result excluded a double infection.Determination of the entire sequence of the R1 strain. cDNA was synthesized with reverse transcriptase (Superscript II; Invitrogen) and an RNase inhibitor (RNaseOUT; Invitrogen). A 7,400-nucleotide (nt) fragment was amplified and cloned using the Expand long-template PCR system (Roche Diagnostics, F...

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