POLYMERASE CHAIN-REACTION TO MONITOR REPAIR OF THE HBV GENOME, THE FIRST STEP IN VIRAL REPLICATION

A method based on the polymerase chain reaction (PCR) for evidencing r epair of the hepatitis B virus (HBV) genome is described. Hepadnavirus es have a partially double-stranded relaxed circular genome (RC-DNA) w hich is converted into a covalently closed circular DNA (CCC-DNA) afte r entry of the virus into a target cell. Our aim was to set up a techn ique enabling us to determine whether possible in vitro replication of the virus in non-hepatic cells is initiated by formation of CCC-DNA. The relevant part of the strategy used for this PCR consisted of primi ng the HBV-DNA template with the same forward primer and with a revers e primer located either downstream or upstream from the minus strand g ap. The CCC-DNA form was found, as expected, in cells in which the vir us was known to be actively replicating; although most sera contained only the RC-DNA form, it was also possible to evidence the CCC form. S uch PCR amplification led to detection of 50-500 copies of the viral D NA. The method described should be useful in studying the biological f ate of HBV in non-hepatic cells (considered as non-permissive for viru s replication), and in exploring the clinical significance of the pres ence of CCC-DNA in sera.