title: In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection.
abstract: 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.
authors.forename: Xiaonan
authors.initials: X
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Lu
authors.forename: Ye
authors.initials: Y
authors.lastname: Zheng
authors.forename: Weixia
authors.initials: W
authors.lastname: Wang
authors.forename: Lu
authors.initials: L
authors.lastname: Bai
authors.forename: Liang
authors.initials: L
authors.lastname: Chen
authors.forename: Yanling
authors.initials: Y
authors.lastname: Feng
authors.forename: Zhanqing
authors.initials: Z
authors.lastname: Zhang
authors.forename: Zhenghong
authors.initials: Z
authors.lastname: Yuan
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology / Q000627:therapeutic use; D016022:Case-Control Studies; D015342:DNA Probes / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D056945:Hep G2 Cells; D006512:Hepatitis B Core Antigens / Q000378:metabolism; D006514:Hepatitis B Surface Antigens / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000175:diagnosis / Q000188:drug therapy / Q000378:metabolism / Q000821:virology*; D006801:Humans; D017403:In Situ Hybridization; D008099:Liver / Q000821:virology; D063065:Organophosphonates / Q000494:pharmacology / Q000627:therapeutic use; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D014779:Virus Replication
chemical_list: D015342:DNA Probes; D004279:DNA, Viral; D006512:Hepatitis B Core Antigens; D006514:Hepatitis B Surface Antigens; D063065:Organophosphonates; D012367:RNA, Viral; C053001:adefovir; D000225:Adenine