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Antimicrobial Agents and Chemotherapy, September 2008, p. 3068-3073, Vol. 52, No. 9
0066-4804/08/$08.00+0     doi:10.1128/AAC.01318-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Séverine Margeridon,1,2,{dagger} Sandra Carrouée-Durantel,3 Isabelle Chemin,1,2 Luc Barraud,3 Fabien Zoulim,1,2,4 Christian Trépo,1,2,4 and Alan Kay1,2*

INSERM, U871, Lyon, France,1 Université Claude Bernard Lyon 1, Faculté de Médecine Laennec, IFR62, Lyon, France,2 Bioalliance Pharma, Paris, France,3 Hospices Civils de Lyon, Hôtel Dieu, Service d'Hépatologie, Lyon, France4

Received 12 October 2007/ Returned for modification 21 November 2007/ Accepted 1 July 2008

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.

* Corresponding author. Mailing address: INSERM U871, 151 cours A; Thomas, 69003 Lyon, France. Phone: 33 472681976. Fax: 33 472681971. E-mail: alan.kay{at}inserm.fr

{triangledown} Published ahead of print on 7 July 2008.

{dagger} Present address: Division of Infectious Diseases, Stanford University, Stanford, California.

Antimicrobial Agents and Chemotherapy, September 2008, p. 3068-3073, Vol. 52, No. 9
0066-4804/08/$08.00+0     doi:10.1128/AAC.01318-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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