In situ and blot hybridization techniques have been used with strand- and region-specific probes to characterize the forms of hepatitis B virus (HBV) DNA in the liver of a patient with chronic active hepatitis B. The hepatocytes contain a heterogeneous population of rapidly migrating DNA species in the 0.5-1.4 kb position that are localized predominantly in the cytoplasm and are of minus-strand polarity. The findings indicate that the replication is asymmetric, with separate pathways for plus- and minus-strand synthesis of HBV DNA; that viral DNA synthesis is initiated at a site near the nick in the minus strand of virion DNA; and that actively replicating forms of HBV DNA can be identified at the cellular level by in situ hybridization.
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We thank Dr. J. Summers for providing the HBV DNA clone, Dr. W. J. Rutter for the initial supply of PLC/PRF/B cells, and J. Expose-Spencerf or propagating them in continuous culture. H.E.B. is a recipient of a Heisenberg Award from the Deutsche ForschungsgemeinschaftT. he Liver Center is supported by NIH Grant AM-26743.A .T.H. was a Medical Investigator of the Veterans Administration. This work was supported in part by basic institutional mechanismso f the Veterans Administration and grants from the USPHS, the National Multiple Sclerosis Society, the American Cancer Society, and by the Alpha Therapeutic Corporation.