Abstract
The awareness of hepatitis E virus (HEV) increased significantly in the last decade due to its unexpectedly high prevalence in high-income countries. There, infections with HEV-genotype 3 (HEV-3) are predominant which can progress to chronicity in immunocompromised individuals. Persistent infection and antiviral therapy can select HEV-3 variants; however, the spectrum and occurrence of HEV-3 variants is underreported. To gain in-depth insights into the viral population and to perform detailed characterization of viral genomes, we used a new approach combining long-range PCR with next-generation and third-generation sequencing which allowed near full-length sequencing of HEV-3 genomes. Furthermore, we developed a targeted ultra-deep sequencing approach to assess the dynamics of clinically relevant mutations in the RdRp-region and to detect insertions in the HVR-domain in the HEV genomes. Using this new approach, we not only identified several insertions of human (AHNAK, RPL18) and viral origin (RdRp-derived) in the HVR-region isolated from an exemplary sample but detected a variant containing two different insertions simultaneously (AHNAK- and RdRp-derived). This finding is the first HEV-variant recognized as such showing various insertions in the HVR-domain. Thus, this molecular approach will add incrementally to our current knowledge of the HEV-genome organization and pathogenesis in chronic hepatitis E.
Highlights
The awareness of hepatitis E virus (HEV) increased significantly in the last decade due to its unexpectedly high prevalence in high-income countries
To capture the heterogeneity of the HEV quasispecies, including recombination events and Single-nucleotide polymorphisms (SNP) that occur with very low frequencies, we developed new sequencing approaches for HEV genotype 3 based on the amplification of the near full-length genome of HEV by long-range PCR followed by subsequent next-generation sequencing (NGS) and third-generation sequencing
We were able to detect in sample 2 five Oxford Nanopore Technologies (ONT) reads showing both the AHNAK-derived and the RdRp-derived insertions, separated by a 132 bp HEV specific connecting sequence (Fig. 2)
Summary
The awareness of hepatitis E virus (HEV) increased significantly in the last decade due to its unexpectedly high prevalence in high-income countries. To capture the heterogeneity of the HEV quasispecies, including recombination events and SNP that occur with very low frequencies, we developed new sequencing approaches for HEV genotype 3 based on the amplification of the near full-length genome of HEV by long-range PCR (lrPCR) followed by subsequent next-generation sequencing (NGS) and third-generation sequencing. These methods allow the identification of insertions and SNPs in the HEV genome. Amplicon-based NGS was performed to detect multiple insertions that coexist in the viral population and to determine the dynamic of SNPs in the RdRp region of the HEV genome
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