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

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 appro priate systems for culturing susceptible cells chronically infected with HB V, Most of the in vitro studies of HBV replication have been performed with HBV genome-transduced cell lines. In this system, viral production is main ly the result of chromosomal replication. In an in vitro infection system, owing to medium refreshment (which leads to the removal of infectious parti cles 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 unfav ourable factors for chronic HBV infection in vitro, we cultured microcarrie r-attached immortalized human hepatocytes, infected with HBV, in molecularp orous (MW 12 000-14 000) membrane (dialysis) bags for a duration of 2 month s. HBV covalently-closed-circular (ccc) DNA, HBV precore/core and X mRNAs w ere 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 h epatitis B surface antigen (HBsAg) and HBV DNA was detected and their level s in culture medium (collected at the end of experiments from the molecular porous membrane bags) were increased 2.86- and 3.28-fold respectively. Usin g Southern blot analysis, HBV replicative intermediates could also be demon strated throughout the experiments. However, integrated HBV DNA was not pre sent. In contrast, HBV ccc DNA, HBV precore/core and X mRNAs, and replicati ve intermediates were not demonstrable in FTO 2B rat hepatoma cells infecte d in the same manner in parallel experiments. This in vitro infection syste m,lsing susceptible, immortalized human hepatocytes, therefore provides a n ew tool for studying the long-term effect of HBV infection, mainly involvin g episomal replication in hepatocytes, and for drug testing.