Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients

AbstractHepatitis B virus (HBV) covalently closed circular (ccc)DNA is the key genomic form responsible for viral persistence and virological relapse after treatment withdrawal. The assessment of residual intrahepatic cccDNA levels and activity after long-term nucleos(t)ide analogues therapy still represents a technical challenge. Quantitative (q)PCR, rolling circle amplification (RCA) and droplet digital (dd)PCR assays were used to quantify residual intrahepatic cccDNA in liver biopsies from 56 chronically HBV infected patients after 3 to 5 years of telbivudine treatment. Activity of residual cccDNA was evaluated by quantifying 3.5 kB HBV RNA (preC/pgRNA) and by assessing cccDNA-associated histone tails post-transcriptional modifications (PTMs) by micro-chromatin immunoprecipitation. Long-term telbivudine treatment resulted in serum HBV DNA suppression, with most of the patients reaching undetectable levels. Despite 38 out of 56 patients had undetectable cccDNA when assessed by qPCR, RCA and ddPCR assays detected cccDNA in all-but-one negative samples. Low preC/pgRNA level in telbivudine-treated samples was associated with enrichment for cccDNA histone PTMs related to repressed transcription. No difference in cccDNA levels was found according to serum viral markers evolution. This panel of cccDNA evaluation techniques should provide an added value for the new proof-of-concept clinical trials aiming at a functional cure of chronic hepatitis B.

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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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01318-07 ◽

2008 ◽

Vol 52 (9) ◽

pp. 3068-3073 ◽

Cited By ~ 40

Author(s):

Séverine Margeridon ◽

Sandra Carrouée-Durantel ◽

Isabelle Chemin ◽

Luc Barraud ◽

Fabien Zoulim ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Liver Biopsy ◽

Rolling Circle Amplification ◽

Full Length ◽

Lamivudine Resistance ◽

Circular Dna ◽

Rolling Circle ◽

Covalently Closed Circular Dna ◽

B Virus

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.

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Voltammetric determination of attomolar levels of a sequence derived from the genom of hepatitis B virus by using molecular beacon mediated circular strand displacement and rolling circle amplification

Microchimica Acta ◽

10.1007/s00604-018-2744-3 ◽

2018 ◽

Vol 185 (3) ◽

Cited By ~ 14

Author(s):

Shan Huang ◽

Mengmeng Feng ◽

Jiawen Li ◽

Yi Liu ◽

Qi Xiao

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Molecular Beacon ◽

Rolling Circle Amplification ◽

Voltammetric Determination ◽

Strand Displacement ◽

Rolling Circle ◽

B Virus

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Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV)

10.1101/470633 ◽

2018 ◽

Cited By ~ 1

Author(s):

Anna L McNaughton ◽

Hannah E Roberts ◽

David Bonsall ◽

Mariateresa de Cesare ◽

Jolynne Mokaya ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Large Scale ◽

Point Of Care ◽

Rolling Circle Amplification ◽

Genome Structure ◽

Whole Genome ◽

Nanopore Sequencing ◽

Rolling Circle ◽

B Virus

AbstractAdvancing interventions to tackle the huge global burden of hepatitis B virus (HBV) infection depends on improved insights into virus epidemiology, transmission, within-host diversity, drug resistance and pathogenesis, all of which can be facilitated by the large-scale generation of full-length virus genome data. Here we describe advances to a protocol to exploit the circular HBV genome structure, using isothermal rolling-circle amplification to enrich for HBV DNA and to generate concatemeric amplicons containing multiple successive copies of the same genome. We show that this product is suitable for Nanopore sequencing as single reads, as well as for generating short-read Illumina sequences. Nanopore reads can be used to implement a straightforward method for error correction that reduces the per-read error rate, by comparing multiple genome copies combined into a single concatemer and by comparing reads generated from plus and minus strands. Thus we can achieve improved consensus sequencing accuracy of 99.7% and resolve intra-sample sequence variants to form whole-genome haplotypes. The combination of isothermal amplification and Nanopore sequencing offers the longer-term potential to develop point-of-care tests for HBV, which could also be adapted for other viruses.

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