Comprehensive Molecular Approach for Characterization of Hepatitis E Virus Genotype 3 Variants

Abstract

Autochthonous hepatitis E virus genotype 3 (HEV-3) infections in industrialized countries are more frequent than previously assumed. HEV-3 is zoonotic and the causal pathogen of chronic hepatitis E. According to the latest classification of the family , 10 designated HEV-3 subtypes (HEV-3a to HEV-3j) and 7 unassigned HEV-3 subtypes are proposed. In order to identify and characterize the HEV-3 variants in circulation, we developed a molecular approach combining a sensitive HEV-specific real-time reverse transcription-PCR (RT-PCR) targeting the overlapping region of HEV ORF2 and ORF3 (the ORF2/3 region) and two newly designed consensus nested RT-PCRs targeting the HEV ORF1 and ORF2 genes, respectively. Since complete genome sequences are required for new HEV-3 subtype assignment, we implemented a straightforward approach for full-length HEV-3 genome amplification. Twenty-nine human serum samples and six human feces samples from chronic hepatitis E patients were selected for evaluation of the system. Viral loads ranged from 1 × 10 to 1.9 × 10 copies/ml of serum and from 1.8 × 10 to 1 × 10 copies/g of feces. Sequence and phylogenetic analyses of partial ORF1 and ORF2 sequences showed that HEV strains had considerable genetic diversity and clustered into the HEV-3c (29/35), HEV-3e (2/35), HEV-3f (2/35), and unassigned HEV-3 (2/35) subtypes. Moreover, from these strains, three full-length HEV-3 genome sequences were generated and characterized. DE/15-0030 represents a typical HEV-3c strain (95.7% nucleotide identity to wbGER27), while DE/15-0031 and SW/16-0282 have <89.2% homology to known HEV-3 strains and are phylogenetically divergent, indicating novel HEV-3 subtypes. In summary, our approach will significantly facilitate the detection, quantification, and determination of HEV-3 strains and will thus help to improve molecular diagnostics and our knowledge of HEV diversity and evolution.

Drastic decline of hepatitis E virus detection in domestic pigs after the age of 6 months, Corsica, France

“…The remaining 12 farms in which HEV RNA positive samples were detected are disseminated in different regions of Corsica without clustering into geographically defined hotspots. The vast majority of HEV RNA positive samples (83/85) contained sequences corresponding to genotype 3c, which has been associated with human infections (Koot, Hogema, Koot, Molier, & Zaaijer, ; Lhomme et al, ; Wang et al, ), and found highly prevalent in wild boars (Pavio et al, ), domestic pigs, and pork meat products (Boxman et al, ). The two remaining samples contained sequences of genotype 3f, which has also been reported in human infections in Europe (Fogeda, Avellon, Cilla, & Echevarria, ; La Rosa et al, ; Lhomme et al, ; Wang et al, ).…”

“…The vast majority of HEV RNA positive samples (83/85) contained sequences corresponding to genotype 3c, which has been associated with human infections (Koot, Hogema, Koot, Molier, & Zaaijer, ; Lhomme et al, ; Wang et al, ), and found highly prevalent in wild boars (Pavio et al, ), domestic pigs, and pork meat products (Boxman et al, ). The two remaining samples contained sequences of genotype 3f, which has also been reported in human infections in Europe (Fogeda, Avellon, Cilla, & Echevarria, ; La Rosa et al, ; Lhomme et al, ; Wang et al, ). Interestingly, the shape of the phylogram did not support the clonality of the 83 genotype 3c sequences; these 83 sequences belonged to multiple genetic clusters that also included sequences corresponding to strains identified in human cases and in ficatellu (Pavio et al, ).…”

Detection and Characterization of Hepatitis E Virus Genotype 3 in Wastewater and Urban Surface Waters in Germany

“…Obtained HEV sequences were aligned to 41 HEV-subtype reference sequences (or a subset of 19 genotype 3 reference subtype sequences), as recommended by Smith et al (2016b). In addition, sequences were aligned to the HEV-3c positive control (isolate 47832c from Johne et al 2014) and 17 sequences from HEV infected patients from the Charité Hospital in Berlin (Wang et al 2018a).…”

Patterns of mutation within an emerging endemic lineage of HEV‐3a

“…The definition and classification of HEV‐3 subtypes are continually being updated, and new subtypes are proposed as sequences emerge. Around 10 subtypes have been named so far within the HEV‐3 genotype, with G3abcdhij belonging to one group that is thought to be Asian in origin, and G3efg falling into to a second group believed to be European in origin .…”