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In situ analyses of intrahepatic virological events in chronic hepatitis B

Xiaonan Zhang (张小楠), Wei Lu(陆伟), Ye Zheng(郑叶), Weixia Wang(王伟霞),Lu Bai (白露),Liang Chen(陈良), Yanling Feng(冯艳玲), Zhanqing Zhang(张占卿), and Zhenghong Yuan(袁正宏)
Shanghai Public Health Clinical Center, Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University


Persistent hepatitis B virus (HBV) infection is established by the formation of an intranuclear pool of covalently closed circular DNA (cccDNA) in the liver. Very little is known about the intrahepatic distribution of HBV cccDNA in infected patients, particularly at the single-cell level. Here, we established a highly sensitive and specific ISH assay for the detection of HBV RNA, DNA, and cccDNA. The specificity of our cccDNA probe set was confirmed by its strict intranuclear signal and by a series of Southern blot analyses. Use of our in situ assay in conjunction with IHC or immunofluorescence uncovered a surprisingly mosaic distribution of viral antigens and nucleic acids. Most strikingly, a mutually exclusive pattern was found between HBV surface antigen–positive (HBsA-positive) and HBV DNA– and cccDNA-positive cells. A longitudinal observation of patients over a 1-year period of adeforvir therapy confirmed the persistence of a nuclear reservoir of viral DNA, although cytoplasmic DNA was effectively depleted in these individuals. In conclusion, our method for detecting viral nucleic acids, including cccDNA, with single-cell resolution provides a means for monitoring intrahepatic virological events in chronic HBV infection.  More important, our observations unravel the complexity of the HBV life cycle in vivo.









Typical staining pattern of HBV DNA(left) and cccDNA (purple blue,right)   with HBsAg (Brown) in liver sections of chronic hepatitis B patients


Original Paper

Image Collections

Disease phase
  • HBeAg(-)
  • HBeAg(+)
  • HBV
Type of Staining
  • HBV DNA
  • HBVDNA-HBsAg
  • HBsAg-HBcAg
  • S-Core-HBVDNA
  • S-core-cccDNA
  • pgRNA-S
  • HBVRNA-S
Case No.