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Hepatitis B virus infection in microcarrier-attached immortalized human hepatocytes cultured in molecularporous membrane bags: a model for long-term episomal replication of HBV. 1998

Z J Gong, and S De Meyer, and T Roskams, and J F van Pelt, and A Soumillion, and T Crabbé, and S H Yap
Department of Liver and Pancreatic Diseases, University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Belgium.

Studies on the pathobiology of chronic (long-term) hepatitis B virus (HBV) infection and in vitro drug testing have been hampered by the lack of appropriate systems for culturing susceptible cells chronically infected with HBV. Most of the in vitro studies of HBV replication have been performed with HBV genome-transduced cell lines. In this system, viral production is mainly the result of chromosomal replication. In an in vitro infection system, owing to medium refreshment (which leads to the removal of infectious particles necessary for the perpetuation of infection) and to trypsinization for cell passages, it is difficult, if not impossible, to maintain chronic HBV infection, despite the use of susceptible cells. To circumvent these unfavourable factors for chronic HBV infection in vitro, we cultured microcarrier-attached immortalized human hepatocytes, infected with HBV, in molecularporous (MW 12,000-14,000) membrane (dialysis) bags for a duration of 2 months. HBV covalently-closed-circular (ccc) DNA, HBV precore/core and X mRNAs were detected in the cells cultured in this system following infection until the end of the experiment (day 58), while in classical culture conditions (monolayer), markers of HBV replication were also detected. Production of hepatitis B surface antigen (HBsAg) and HBV DNA was detected and their levels in culture medium (collected at the end of experiments from the molecularporous membrane bags) were increased 2.86- and 3.28-fold respectively. Using Southern blot analysis, HBV replicative intermediates could also be demonstrated throughout the experiments. However, integrated HBV DNA was not present. In contrast, HBV ccc DNA, HBV precore/core and X mRNAs, and replicative intermediates were not demonstrable in FTO 2B rat hepatoma cells infected in the same manner in parallel experiments. This in vitro infection system, using susceptible, immortalized human hepatocytes, therefore provides a new tool for studying the long-term effect of HBV infection, mainly involving episomal replication in hepatocytes, and for drug testing.

UI MeSH Term Description Entries
D007150 Immunohistochemistry Histochemical localization of immunoreactive substances using labeled antibodies as reagents. Immunocytochemistry,Immunogold Techniques,Immunogold-Silver Techniques,Immunohistocytochemistry,Immunolabeling Techniques,Immunogold Technics,Immunogold-Silver Technics,Immunolabeling Technics,Immunogold Silver Technics,Immunogold Silver Techniques,Immunogold Technic,Immunogold Technique,Immunogold-Silver Technic,Immunogold-Silver Technique,Immunolabeling Technic,Immunolabeling Technique,Technic, Immunogold,Technic, Immunogold-Silver,Technic, Immunolabeling,Technics, Immunogold,Technics, Immunogold-Silver,Technics, Immunolabeling,Technique, Immunogold,Technique, Immunogold-Silver,Technique, Immunolabeling,Techniques, Immunogold,Techniques, Immunogold-Silver,Techniques, Immunolabeling
D008099 Liver A large lobed glandular organ in the abdomen of vertebrates that is responsible for detoxification, metabolism, synthesis and storage of various substances. Livers
D002214 Capsules Hard or soft soluble containers used for the oral administration of medicine. Capsule,Microcapsule,Microcapsules
D002478 Cells, Cultured Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others. Cultured Cells,Cell, Cultured,Cultured Cell
D003470 Culture Media Any liquid or solid preparation made specifically for the growth, storage, or transport of microorganisms or other types of cells. The variety of media that exist allow for the culturing of specific microorganisms and cell types, such as differential media, selective media, test media, and defined media. Solid media consist of liquid media that have been solidified with an agent such as AGAR or GELATIN. Media, Culture
D004270 DNA, Circular Any of the covalently closed DNA molecules found in bacteria, many viruses, mitochondria, plastids, and plasmids. Small, polydisperse circular DNA's have also been observed in a number of eukaryotic organisms and are suggested to have homology with chromosomal DNA and the capacity to be inserted into, and excised from, chromosomal DNA. It is a fragment of DNA formed by a process of looping out and deletion, containing a constant region of the mu heavy chain and the 3'-part of the mu switch region. Circular DNA is a normal product of rearrangement among gene segments encoding the variable regions of immunoglobulin light and heavy chains, as well as the T-cell receptor. (Riger et al., Glossary of Genetics, 5th ed & Segen, Dictionary of Modern Medicine, 1992) Circular DNA,Circular DNAs,DNAs, Circular
D004279 DNA, Viral Deoxyribonucleic acid that makes up the genetic material of viruses. Viral DNA
D006512 Hepatitis B Core Antigens The hepatitis B antigen within the core of the Dane particle, the infectious hepatitis virion. HBcAg,Hepatitis B Core Antigen
D006514 Hepatitis B Surface Antigens Those hepatitis B antigens found on the surface of the Dane particle and on the 20 nm spherical and tubular particles. Several subspecificities of the surface antigen are known. These were formerly called the Australia antigen. Australia Antigen,HBsAg,Hepatitis B Surface Antigen,Antigen, Australia
D006515 Hepatitis B virus The type species of the genus ORTHOHEPADNAVIRUS which causes human HEPATITIS B and is also apparently a causal agent in human HEPATOCELLULAR CARCINOMA. The Dane particle is an intact hepatitis virion, named after its discoverer. Non-infectious spherical and tubular particles are also seen in the serum. Dane Particle,Hepatitis Virus, Homologous Serum,B virus, Hepatitis,Hepatitis B viruses,Particle, Dane,viruses, Hepatitis B

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