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Integration of hepatitis B virus DNA into chromosomal DNA during acute hepatitis B.

ABSTRACT: AIM:To examine the serum from black African patients with acute hepatitis B to ascertain if integrants of viral DNA can be detected in fragments of cellular DNA leaking from damaged hepatocytes into the circulation. METHODS:DNA was extracted from the sera of five patients with uncomplicated acute hepatitis B and one with fulminant disease. Two subgenomic PCRs designed to amplify the complete genome of HBV were used and the resulting amplicons were cloned and sequenced. RESULTS:HBV and chromosomal DNA were amplified from the sera of all the patients. In one patient with uncomplicated disease, HBV DNA was integrated into host chromosome 7 q11.23 in the WBSCR1 gene. The viral DNA comprised 200 nucleotides covering the S and X genes in opposite orientation, with a 1 169 nucleotide deletion. The right virus/host junction was situated at nucleotide 1,774 in the cohesive overlap region of the viral genome, at a preferred topoisomerase I cleavage motif. The chromosomal DNA was not rearranged. The patient made a full recovery and seroconverted to anti-HBs- and anti-HBe-positivity. Neither HBV nor chromosomal DNA could be amplified from his serum at that time. CONCLUSION:Integration of viral DNA into chromosomal DNA may occur rarely during acute hepatitis B and, with clonal propagation of the integrant, might play a role in hepatocarcinogenesis.

SUBMITTER: Kimbi GC

PROVIDER: S-EPMC4355779 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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Integration of hepatitis B virus DNA into chromosomal DNA during acute hepatitis B.

Kimbi Gerald C GC Kramvis Anna A Kew Michael C MC

World journal of gastroenterology 20051101 41

<h4>Aim</h4>To examine the serum from black African patients with acute hepatitis B to ascertain if integrants of viral DNA can be detected in fragments of cellular DNA leaking from damaged hepatocytes into the circulation.<h4>Methods</h4>DNA was extracted from the sera of five patients with uncomplicated acute hepatitis B and one with fulminant disease. Two subgenomic PCRs designed to amplify the complete genome of HBV were used and the resulting amplicons were cloned and sequenced.<h4>Results< ...[more]

PMID: 16425409

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Project description:BackgroundHBV DNA integration into the host genome is frequently found in HBV-associated HCC tissues and is associated with hepatocarcinogenesis. Multiple detection methods, including hybrid capture-sequencing, have identified integration sites and provided clinical implications; however, each has advantages and disadvantages concerning sensitivity, cost, and throughput. Therefore, methods that can comprehensively and cost-effectively detect integration sites with high sensitivity are required. Here, we investigated the efficiency of RAISING (Rapid Amplification of Integration Site without Interference by Genomic DNA contamination) as a simple and inexpensive method to detect viral integration by amplifying HBV-integrated fragments using virus-specific primers covering the entire HBV genome.Methods and resultsIllumina sequencing of RAISING products from HCC-derived cell lines (PLC/PRF/5 and Hep3B cells) identified HBV-human junction sequences as well as their frequencies. The HBV-human junction profiles identified using RAISING were consistent with those determined using hybrid capture-sequencing, and the representative junctions could be validated by junction-specific nested PCR. The comparison of these detection methods revealed that RAISING-sequencing outperforms hybrid capture-sequencing in concentrating junction sequences. RAISING-sequencing was also demonstrated to determine the sites of de novo integration in HBV-infected HepG2-NTCP cells, primary human hepatocytes, liver-humanized mice, and clinical specimens. Furthermore, we made use of xenograft mice subcutaneously engrafted with PLC/PRF/5 or Hep3B cells, and HBV-human junctions determined by RAISING-sequencing were detectable in the plasma cell-free DNA using droplet digital PCR.ConclusionsRAISING successfully profiles HBV-human junction sequences with smaller amounts of sequencing data and at a lower cost than hybrid capture-sequencing. This method is expected to aid basic HBV integration and clinical diagnosis research.

Dataset Information

Integration of hepatitis B virus DNA into chromosomal DNA during acute hepatitis B.

Publications

Integration of hepatitis B virus DNA into chromosomal DNA during acute hepatitis B.

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