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. Chronic hepatitis B virus (HBV) infection is widespread,with an estimated 370 million carriers worldwide (1). Despite constraints imposed by a complex genetic architecture involving overlapping genes, the virus is highly variable, with eight known genotypes designated A to H (20).The complete molecular and biological characterization of HBV variants requires the isolation of full-length genomes. This has been done classically by amplifying overlapping fragments from serum-derived relaxed circular genomes (RC-DNA) (4, 21). However, the discontinuous structure of RC-DNA means that the amplification of at least one of the fragments can be problematical. Increasing the number of PCRs ineluctably augments the probability of incorporating PCR errors, and further steps are necessary to construct a full-length HBV genome. Günther et al. have developed an elegant method for amplifying full-length HBV genomes in a one-step PCR (7). However, this method requires serum with viral loads of Ͼ10 4 copies/ml of HBV DNA. In typical chronic HBV infections, viral loads usually range from 10 6 to 10 10 copies/ml. However, for inactive carriers (individuals who are hepatitis B surface antigen positive [HBsAg ϩ ]) with minimal virus replication and for occult HBV-infected patients (HBsAg Ϫ ), serum HBV DNA levels are usually very much lower. Consequently, few occult HBV infections have been studi...

Identification of novel recombinants of hepatitis B virus genotypes F and G in human immunodeficiency virus-positive patients from Argentina and Brazil

“…HBV DNA was extracted from 0.2 ml serum using a High Pure Viral Nucleic Acid kit (Roche Diagnostics). The full-length HBV genome was amplified as described previously (Günther et al, 1995), with or without prior rolling-circle amplification (RCA) (Margeridon et al, 2008). The precore/ core region (positions 1824-2467) of the Argentinian patient was amplified by subgenomic PCR.…”

“…To enrich for full-length non-G strains, especially HBV/ F1b strains in the Argentinian patient, serum extracts were first subjected to a modified RCA (Margeridon et al, 2008;N. Martel, C. Trepo & A. Kay, unpublished data).…”

HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B

“…Rolling circle amplification is a potential alternative as it should depend on a circular template 80. However, our own data using pure DHBV RC-DNA suggest that this dependence is, again, not absolute.…”

Recurrence of occult hepatitis B virus infection in a recipient of a liver transplant for HCV-related cirrhosis: full length genome, mutations analysis and literature review

“…For a complete characterization of the HBV genome, we use Rolling Circle Amplifiaction (RCA) 12 that can amplify of full-length HBV genomes from samples with low HBV viral loads.…”