Ultra-deep pyrosequencing analysis of the hepatitis B virus preCore region and main catalytic motif of the viral polymerase in the same viral genome

Homs, María; Buti, Marı́a; Quer, Josep; Jardí, Rosendo; Schaper, Melanie; Tabernero, David; Ortega, Israel Díaz; Sánchez-Pla, Álex; Esteban, Rafael; Rodríguez‐Frías, Francisco

doi:10.1093/nar/gkr451

Cited by 44 publications

(39 citation statements)

References 45 publications

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“…Several nucleotide deletions and insertions at the region overlap the CP, and the precore region results in truncated HBxAg or additional amino acids, which potentially alters HBx functionality (46), was detected in our study (Table 4). Hot spots include the precore/core genes, preS region, and the region of the X gene overlapping the core promoter (47), which were more frequent and complex in CHB and ACLF patients than in AHB and IT patients.…”

Section: Discussionmentioning

confidence: 52%

Characterization of Full-Length Genomes of Hepatitis B Virus Quasispecies in Sera of Patients at Different Phases of Infection

et al. 2015

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Hepatitis B virus (HBV) infection results in different clinical presentation due to different levels of immune response. Our study aimed to characterize HBV full-length genome quasispecies (QS) in patients with different phases of infection to better understand its pathogenesis. Forty treatment-naive HBV-infected patients were enrolled, including 10 cases of acute hepatitis B (AHB), 9 cases of immunotolerant (IT) HBV carriers, 11 cases of chronic hepatitis B (CHB), and 10 cases of acute-on-chronic liver failure (ACLF). The present study was conducted by clone-based sequencing. QS heterogeneity within each open reading frame was calculated. The mutation frequency index (MFI) and amino acid variations within the large HBsAg, HBcAg, and HBxAg regions were analyzed based on the different infection phases. In total, 606 HBV full-length sequences were obtained. HBV QS had higher heterogeneity in ACLF and CHB than that in IT among chronically infected individuals. AHB patients had the lower QS heterogeneity at onset than those with chronic infection. ACLF patients had the highest frequency of mutations in the core promoter and precore region. A triple mutation (A1762T/G1764A/G1896A) was observed more frequently in genotype C than in genotype B. The MFI indicated that specific peptides of the studied regions had more frequent mutations in ACLF. Furthermore, several amino acid variations, known as T-and B-cell epitopes, were potentially associated with the immunoactive phase of infection. More HBV genome mutations and deletions were observed in patients with more severe diseases, particularly in specific regions of the core and preS regions, the clinical significance and mechanism of which need to be further investigated.

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Section: Discussionmentioning

confidence: 52%

Characterization of Full-Length Genomes of Hepatitis B Virus Quasispecies in Sera of Patients at Different Phases of Infection

et al. 2015

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“…For reads data processing a demultiplexing step where the reads were assigned to samples and amplicons was followed by a quality filter and repair step, and by the intersection of haplotypes in the forward and reverse strands, and an abundance filter for these consensus haplotypes above 0.25% [152]. Accuracy validations were performed using published procedures [150], [153]–[155]; the same conclusions on Rib mutagenesis were obtained with a cut-off value of 1.0%.…”

Section: Methodsmentioning

confidence: 99%

Extinction of Hepatitis C Virus by Ribavirin in Hepatoma Cells Involves Lethal Mutagenesis

et al. 2013

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Lethal mutagenesis, or virus extinction produced by enhanced mutation rates, is under investigation as an antiviral strategy that aims at counteracting the adaptive capacity of viral quasispecies, and avoiding selection of antiviral-escape mutants. To explore lethal mutagenesis of hepatitis C virus (HCV), it is important to establish whether ribavirin, the purine nucleoside analogue used in anti-HCV therapy, acts as a mutagenic agent during virus replication in cell culture. Here we report the effect of ribavirin during serial passages of HCV in human hepatoma Huh-7.5 cells, regarding viral progeny production and complexity of mutant spectra. Ribavirin produced an increase of mutant spectrum complexity and of the transition types associated with ribavirin mutagenesis, resulting in HCV extinction. Ribavirin-mediated depletion of intracellular GTP was not the major contributory factor to mutagenesis since mycophenolic acid evoked a similar decrease in GTP without an increase in mutant spectrum complexity. The intracellular concentration of the other nucleoside-triphosphates was elevated as a result of ribavirin treatment. Mycophenolic acid extinguished HCV without an intervening mutagenic activity. Ribavirin-mediated, but not mycophenolic acid-mediated, extinction of HCV occurred via a decrease of specific infectivity, a feature typical of lethal mutagenesis. We discuss some possibilities to explain disparate results on ribavirin mutagenesis of HCV.

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“…In particular, its high coverage provides the sensitivity to detect very rare variants with the frequency of 1% or even less in quasispecies [8]. In HBV studies, such a strategy has been applied to analyse minor antiviral resistance mutations [9,10], G to A hypermutations in reverse transcriptase [11], and mutation features in preC and YMDD motif [12]. Thus far, these studies all focus on targeted HBV genes in specific clinical groups, lacking of whole genome investigation, and quasispecies evolution analysis in different stages of liver diseases.…”

Section: Introductionmentioning

confidence: 99%

Whole genome characterization of hepatitis B virus quasispecies with massively parallel pyrosequencing

Zhang

et al. 2015

Clinical Microbiology and Infection

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Viral quasispecies analysis is important for basic and clinical research. This study was designed to detect hepatitis B virus (HBV) genome-wide mutation profiling with detailed variant composition in individual patients, especially quasispecies evolution correlating with liver disease progression. We characterized viral populations by massively parallel pyrosequencing at whole HBV genome level in 17 patients with advanced liver disease (ALD) and 30 chronic carriers (CC). An average sequencing coverage of 2047× and 687× in ALD and CC groups, respectively, were achieved. Deep sequencing data resolved the landscapes of HBV substitutions and a more complicated quasispecies composition than previously observed. The values of substitution frequencies in quasispecies were clustered as either more than 80% or less than 20%, forming a unique U-shaped distribution pattern in both clinical groups. Furthermore, quantitative comparison of mutation frequencies of each site between two groups resulted in a spectrum of substitutions associated with liver disease progression, and among which, C2288A/T, C2304A, and A/G2525C/T were novel candidates. Moreover, distinct deletion patterns in preS, X, and C regions were shown between the two groups. In conclusion, pyrosequencing of the whole HBV genome revealed a panorama of viral quasispecies composition, characteristics of substitution distribution, and mutations correlating to severe liver disease.

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Ultra-deep pyrosequencing analysis of the hepatitis B virus preCore region and main catalytic motif of the viral polymerase in the same viral genome

Cited by 44 publications

References 45 publications

Characterization of Full-Length Genomes of Hepatitis B Virus Quasispecies in Sera of Patients at Different Phases of Infection

Characterization of Full-Length Genomes of Hepatitis B Virus Quasispecies in Sera of Patients at Different Phases of Infection

Extinction of Hepatitis C Virus by Ribavirin in Hepatoma Cells Involves Lethal Mutagenesis

Whole genome characterization of hepatitis B virus quasispecies with massively parallel pyrosequencing

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