title: Rolling circle amplification, a powerful tool for genetic and functional studies of complete hepatitis B virus genomes from low-level infections and for directly probing covalently closed circular DNA.
abstract: Complete characterization of the biological properties of hepatitis B virus (HBV) variants requires the generation of full-length genomes. The aim of this study was to develop new tools for the efficient full-length genome amplification of virus from samples with low viral loads. Rolling circle amplification (RCA) was used to amplify full-length HBV genomes from both sera and liver biopsy samples from chronic HBV carriers. Serum-derived relaxed circular HBV DNA could be amplified only after completion and ligation of plus-strand DNA. Covalently closed circular DNA (cccDNA) from liver biopsies could be amplified directly from as few as 13 copies, using RCA, followed by a full-length HBV PCR. Three serial liver biopsy samples were obtained from a lamivudine-resistant patient who cleared detectable serum HBV after adefovir dipivoxil was added to the lamivudine therapy and then seroconverted to anti-HBs. Only the genomes from the last biopsy specimen obtained after the emergence of lamivudine resistance contained the lamivudine resistance-associated mutations rtL180M and rtM204V ("rt" indicates reverse transcriptase domain). Defective genomes were also found in this biopsy sample. Genomes cloned from the liver biopsy specimens were transfected into HuH7 cells to study their replication competence and their susceptibility to lamivudine. RCA is a powerful tool for amplifying full-length HBV genomes and will be especially useful for the study of occult or inactive HBV infections and patients undergoing antiviral treatment. It can also be used to probe HBV cccDNA, the crucial intermediate in viral persistence and the archive of resistance mutations.
authors.affiliation: INSERM, U871, Lyon, France.
authors.forename: Séverine
authors.initials: S
authors.lastname: Margeridon
authors.forename: Sandra
authors.initials: S
authors.lastname: Carrouée-Durantel
authors.forename: Isabelle
authors.initials: I
authors.lastname: Chemin
authors.forename: Luc
authors.initials: L
authors.lastname: Barraud
authors.forename: Fabien
authors.initials: F
authors.lastname: Zoulim
authors.forename: Christian
authors.initials: C
authors.lastname: Trépo
authors.forename: Alan
authors.initials: A
authors.lastname: Kay
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology / Q000627:therapeutic use; D002353:Carrier State / Q000188:drug therapy / Q000821:virology; D045744:Cell Line, Tumor; D004270:DNA, Circular / Q000032:analysis* / Q000096:biosynthesis; D004279:DNA, Viral / Q000032:analysis* / Q000096:biosynthesis; D024882:Drug Resistance, Viral; D016679:Genome, Viral; D006515:Hepatitis B virus / Q000235:genetics* / Q000378:metabolism; D019694:Hepatitis B, Chronic / Q000188:drug therapy / Q000821:virology*; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology / Q000627:therapeutic use; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D017422:Sequence Analysis, DNA; D014162:Transfection; D014779:Virus Replication
chemical_list: D000998:Antiviral Agents; D004270:DNA, Circular; D004279:DNA, Viral; D019259:Lamivudine