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 repair of the hepatitis B virus (HBV) genome is described. Hepadnaviruses have a partially double-stranded relaxed circular genome (RC-DNA) which is converted into a covalently closed circular DNA (CCC-DNA) after entry of the virus into a target cell. Our aim was to set up a technique 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 priming the HBV-DNA template with the same forward primer and with a reverse primer located either downstream or upstream from the minus strand gap. The CCC-DNA form was found, as expected, in cells in which the virus was known to be actively replicating; although most sera contained only the RC-DNA form, it was also possible to evidence the CCC form. Such PCR amplification led to detection of 50-500 copies of the viral DNA. The method described should be useful in studying the biological fate of HBV in non-hepatic cells (considered as non-permissive for virus replication), and in exploring the clinical significance of the presence of CCC-DNA in sera.
Maillard
P
P
Institut Pasteur, Unité d'Immunologie Microbienne, Paris.
Pillot
J
J
D017088:AIDS-Related Opportunistic Infections / Q000097:blood / Q000150:complications / Q000821:virology*; D001483:Base Sequence; D017931:DNA Primers; D004260:DNA Repair; D004279:DNA, Viral / Q000032:analysis*; D006509:Hepatitis B / Q000097:blood / Q000150:complications / Q000473:pathology / Q000821:virology*; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D006801:Humans; D008099:Liver / Q000473:pathology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D014779:Virus Replication
D017931:DNA Primers; D004279:DNA, Viral