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Case Report: Application of hepatitis B virus (HBV) deep sequencing to distinguish between acute and chronic infection.

ABSTRACT: Deep sequencing of the full-length hepatitis B virus (HBV) genome provides the opportunity to determine the extent to which viral diversity, genotype, polymorphisms, insertions and deletions may influence presentation and outcomes of disease. Increasing experience with analysis of HBV genomic data opens up the potential for using these data to inform insights into pathophysiology of infection and to underpin decision making in clinical practice. We here set out to undertake whole genome HBV sequencing from an adult who presented acutely unwell with a new diagnosis of HBV infection, and tested positive for both HBV anti-core IgM and IgG, possibly representing either acute hepatitis B infection (AHB) or chronic hepatitis B with an acute reactivation (CHB-AR). The distinction between these two scenarios may be important in predicting prognosis and underpinning treatment decisions, but can be challenging based on routine laboratory tests. Through application of deep whole-genome sequencing we typed the isolate as genotype-D1, and identified several minority variants including G1764A and G1986A substitutions in the pre-core promoter and pre-core regions, which support CHB-AR rather than AHB. In the longer term, enhanced deep sequencing data for HBV may provide improved evidence to distinguish between acute and chronic infection, to predict outcomes and to stratify treatment.

SUBMITTER: Downs LO

PROVIDER: S-EPMC7802106 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Case Report: Application of hepatitis B virus (HBV) deep sequencing to distinguish between acute and chronic infection.

Downs Louise O LO McNaughton Anna L AL de Cesare Mariateresa M Ansari M Azim MA Martin Jacqueline J Woodrow Charles C Bowden Rory R Collier Jane J Barnes Eleanor E Matthews Philippa C PC

Wellcome open research 20200101

Deep sequencing of the full-length hepatitis B virus (HBV) genome provides the opportunity to determine the extent to which viral diversity, genotype, polymorphisms, insertions and deletions may influence presentation and outcomes of disease. Increasing experience with analysis of HBV genomic data opens up the potential for using these data to inform insights into pathophysiology of infection and to underpin decision making in clinical practice. We here set out to undertake whole genome HBV sequ ...[more]

PMID: 33458253

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Project description:Background & aimsHBV infects over 257 million people worldwide and is associated with the development of hepatocellular carcinoma (HCC). Integration of HBV DNA into the host genome is likely a key driver of HCC oncogenesis. Here, we utilise targeted long-read sequencing to determine the structure of HBV DNA integrations as well as full isoform information of HBV mRNA with more accurate quantification than traditional next generation sequencing platforms.MethodsDNA and RNA were isolated from fresh frozen liver biopsies collected within the GS-US-174-0149 clinical trial. A pan-genotypic panel of biotinylated oligos was developed to enrich for HBV sequences from sheared genomic DNA (∼7 kb) and full-length cDNA libraries from poly-adenylated RNA. Samples were sequenced on the PacBio long-read platform and analysed using a custom bioinformatic pipeline.ResultsHBV-targeted long-read DNA sequencing generated high coverage data spanning entire integrations. Strikingly, in 13 of 42 samples (31%) we were able to detect HBV sequences flanked by 2 different chromosomes, indicating a chromosomal translocation associated with HBV integration. Chromosomal translocations were unique to each biopsy sample, suggesting that each originated randomly, and in some cases had evidence of clonal expansion. Using targeted long-read RNA sequencing, we determined that upwards of 95% of all HBV transcripts in patients who are HBeAg-positive originate from cccDNA. In contrast, patients who are HBeAg-negative expressed mostly HBsAg from integrations.ConclusionsTargeted lso-Seq allowed for accurate quantitation of the HBV transcriptome and assignment of transcripts to either cccDNA or integration origins. The existence of multiple unique HBV-associated inter-chromosomal translocations in non-HCC CHB patient liver biopsies suggests a novel mechanism with mutagenic potential that may contribute to progression to HCC.Lay summaryFresh frozen liver biopsies from patients infected with HBV were subjected to targeted long-read RNA and DNA sequencing. Long-read RNA sequencing captures entire HBV transcripts in a single read, allowing for resolution of overlapping transcripts from the HBV genome. This resolution allowed us to quantify the burden of transcription from integrations vs. cccDNA origin in individual patients. Patients who were HBeAg-positive had a significantly larger fraction of the HBV transcriptome originating from cccDNA compared with those who were HBeAg-negative. Long-read DNA sequencing captured entire integrated HBV sequences including multiple kilobases of flanking host sequence within single reads. This resolution allowed us to describe integration events flanked by 2 different host chromosomes, indicating that integrated HBV DNA are associated with inter-chromosomal translocations. This may lead to significant transcriptional dysregulation and drive progression to HCC.

Dataset Information

Case Report: Application of hepatitis B virus (HBV) deep sequencing to distinguish between acute and chronic infection.

Publications

Case Report: Application of hepatitis B virus (HBV) deep sequencing to distinguish between acute and chronic infection.

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