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Serum viral duplex-linear DNA proportion increases with the progression of liver disease in patients infected with HBV
  1. Xing-Liang Zhao1,
  2. Jian-Rong Yang2,
  3. Sheng-Zhang Lin3,
  4. Hui Ma1,
  5. Fang Guo1,4,
  6. Rui-Feng Yang1,
  7. Heng-Hui Zhang1,
  8. Jin-Chao Han1,
  9. Lai Wei1,
  10. Xiao-Ben Pan1
  1. 1Peking University People's Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing, P.R. China
  2. 2Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China
  3. 3Department of Hepato-Biliary-Pancreatic Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, P.R. China
  4. 4Department of Microbiology and Immunology, Drexel Institute for Biotechnology and Virology Research, Drexel University College of Medicine, Doylestown, Pennsylvania, USA
  1. Correspondence to Dr Xiao-Ben Pan, Peking University People's Hospital, Peking University Hepatology Institute; 11# Xizhimeng South Street, Xicheng District, Beijing 100044, P.R. China; panxiaoben{at}


Objective HBV has two forms of genomic DNA, relaxed-circular DNA (rcDNA) and duplex-linear DNA (dlDNA). Compared to rcDNA, dlDNA has been demonstrated to integrate more frequently into host cellular chromosomes, which may have oncogenic consequences. However, the dlDNA proportion relative to total HBV DNA and its clinical significance in patients remain to be investigated.

Design Based on the structural difference between rcDNA and dlDNA, we developed a peptide nucleic acid (PNA)-mediated quantitative real-time PCR (qPCR) clamping assay to measure the proportions of dlDNA in total HBV DNA in sera obtained from patients with chronic hepatitis B (CHB), liver cirrhosis (LC) or LC-developed hepatocellular carcinoma (HCC). The factors that influence the proportion of dlDNA were also investigated.

Results The average dlDNA proportion was approximately 7% in the sera of chronic HBV-infected patients and was elevated in CHB patients with abnormal levels of alanine aminotransferase. The sera dlDNA proportions increased to approximately 14% and 20% in the patients with LC and HCC, respectively. Interferon-α treatment slightly increased the dlDNA proportion in the responders; and nucleotide analogue therapy spuriously elevated the proportion. Moreover, treatment of human hepatoma cells supporting HBV replication with inflammatory cytokines significantly altered the dlDNA proportion in vitro.

Conclusions Using a novel PNA-mediated qPCR clamping assay, we first showed that serum dlDNA proportions progressively increased during the development of HBV-related liver diseases. The dlDNA proportion can be regulated by inflammatory cytokines, suggesting an association among inflammation, increased production of HBV dlDNA and development of HCC.


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