Analysis of antihepadnaviral activity of nucleoside analogs using hepatitis B virus recombinant baculovirus-HepG2 system

Hepadnaviruses are a family of small, enveloped DNA viruses that include human hepatitis B virus (HBV), woodchuck hepatitis virus (WHV), ground squirrel hepatitis virus (GSHV), and several avian hepatitis viruses, including duck hepatitis B virus (DHBV). Recently, a deoxycytidine analog (lamivudine, L-2′,3′-dideoxythiacytidine, 3TC) was approved for HBV treatment.; Treatment with 3TC is tolerable and effective in reducing or eliminating HBV DNA from the sera of chronically infected patients. The HBV baculovirus/HepG2 system is the only in vitro system in which it is possible to readily detect and quantify nuclear nonprotein-bound HBV DNA. We also investigated whether the HBV baculovirus system could be utilized for antiviral testing and showed that the HBV baculovirus-HepG2 system made it possible for the first time to systematically observe the effects of 3TC on CCC DNA in vitro.; The second chapter focuses on the use of the HBV recombinant baculovirus/HepG2 system to study the rebound phenomenon that has been observed in human and woodchucks after cessation of 3TC therapy. Our goals were to (1) quantify the relative time course and magnitude of disappearance of HBV DNA species involved in replication after drug therapy and (2) quantify the time course and relative magnitude of reappearance of HBV DNA species after cessation of drug treatment.; In the third chapter study, the HBV recombinant baculovirus/HepG2 system was used to measure the time course of antiviral mediated inhibition of HBV replication as well as the time course and magnitude of HBV production after release from antiviral treatment. The results showed that the levels of HBV replicative intermediates and extracellular DNA decreased in a concentration dependent fashion following antiviral treatment. The data clearly indicated that (1) nuclear HBV DNAs are more resistant to antiviral therapy than cytoplasmic or extracellular HBV DNAs and (2) nuclear HBV CCC DNA is more resistant than the nuclear relaxed circular form.; The fourth chapter describes the use of the HBV recombinant baculovirus/HepG2 system to deliver HBV genome into primary human hepatocytes (PHH). We conclude the following regarding baculovirus-mediated gene transfer into PHH cells: (1) Recombinant baculovirus can be used to efficiently transfer genes to HFH cells. (2) Recombinant HBV baculovirus can be used to initiate HBV gene expression and HBV replication in normal human fetal/neonatal hepatocytes. (3) HFH are different from adult PHH with respect to HBV replication. (4) HBV x mRNA expression is considerably higher in HFH cells than in HepG2 cells. (5) Study of antihepadnaviral compound using fetal/neonatal hepatocytes infected with HBV recombinant baculovirus is feasible. (Abstract shortened by UMI.)...