D. Tabernero

Ultra-Deep Pyrosequencing Detects Conserved Genomic Sites and Quantifies Linkage of Drug-Resistant Amino Acid Changes in the Hepatitis B Virus Genome

Background Selection of amino acid substitutions associated with resistance to nucleos(t)ide-analog (NA) therapy in the hepatitis B virus (HBV) reverse transcriptase (RT) and their combination in a single viral genome complicates treatment of chronic HBV infection and may affect the overlapping surface coding region. In this study, the variability of an overlapping polymerase-surface region, critical for NA resistance, is investigated before treatment and under antiviral therapy, with assessment of NA-resistant amino acid changes simultaneously occurring in the same genome (linkage analysis) and their influence on the surface coding region. Methodology/Principal Findings Serum samples obtained from chronic HBV-infected patients at pre-treatment and during sequential NA treatment with lamivudine, adefovir, and entecavir were analyzed by ultra-deep pyrosequencing (UDPS) using the GS-FLX platform (454 Life Sciences-Roche). The pre-treatment HBV quasispecies was not enriched with NA-resistant substitutions. The frequencies of this type of substitutions at pre-treatment did not predict the frequencies observed during lamivudine treatment. On linkage analysis of the RT region studied, NA-resistant HBV variants (except for rtA181T) were present in combinations of amino acid substitutions that increased in complexity after viral breakthrough to entecavir, at which time the combined variant rtL180M-S202G-M204V-V207I predominated. In the overlapping surface region, NA-resistant substitutions caused selection of stop codons in a significant percentage of sequences both at pre-treatment and during sequential treatment; the rtA181T substitution, related to sW172stop, predominated during treatment with lamivudine and adefovir. A highly conserved RT residue (rtL155), even more conserved than the essential residues in the RT catalytic motif YMDD, was identified in all samples. Conclusions UDPS methodology enabled quantification of HBV quasispecies variants, even those harboring complex combinations of amino acid changes. The high percentage of potentially defective genomes, especially in the surface region, suggests effective trans-complementation of these variants.

A comparative study of ultra-deep pyrosequencing and cloning to quantitatively analyze the viral quasispecies using hepatitis B virus infection as a model.

In this study, the reliability and reproducibility of viral quasispecies quantification by three ultra-deep pyrosequencing (UDPS) methods (FLX+, FLX, and Junior) were investigated and results compared with the conventional cloning technique. Hepatitis B virus (HBV) infection was selected as the model. The preCore/Core region, the least overlapped HBV region, was analyzed in samples from a chronic hepatitis B patient by cloning and by UDPS. After computation filtering of the UDPS results, samples A1 and A2 (FLX+) and sample B (FLX) yielded the same 20 polymorphic positions. Junior yielded 18 polymorphic positions that coincided with the FLX results. In contrast, 50 polymorphic positions were detected by cloning. Quasispecies complexity plotted on graphs showed superimposed patterns and the quantitative parameters were similar between FLX+, FLX, Junior, and the cloning sequences. Twenty-two haplotypes were detected by Junior, and 37, 40, and 39 were detected by FLX A1, A2, and B, respectively. These differences may be attributable to methodological differences between FLX and Junior. By cloning, 47 haplotypes were detected. Eight clones with insertions and deletions that induced de novo stop codons were not observed by UDPS because the UDPS filter discarded them. Our results indicate that UDPS is an optimal alternative to molecular cloning for quantitative study of the viral quasispecies. Nonetheless, specific mutations, such as insertions and deletions, were only detected by cloning. A filter should be designed to analyze cloning sequences, and UDPS filters should be improved to include the specific mutations.

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

Hepatitis B virus (HBV) pregenomic RNA contains a hairpin structure (ϵ) located in the preCore region, essential for viral replication. ϵ stability is enhanced by the presence of preCore variants and ϵ is recognized by the HBV polymerase (Pol). Mutations in the retrotranscriptase domain (YMDD) of Pol are associated with treatment resistance. The aim of this study was to analyze the preCore region and YMDD motif by ultra-deep pyrosequencing (UDPS). To evaluate the UDPS error rate, an internal control sequence was inserted in the amplicon. A newly developed technique enabled simultaneous analysis of the preCore region and Pol in the same viral genome, as well as the conserved sequence of the internal control. Nucleotide errors in HindIII yielded a UDPS error rate <0.05%. UDPS study confirmed the possibility of simultaneous detection of preCore and YMDD mutations, and demonstrated the complexity of the HBV quasispecies and cooperation between viruses. Thermodynamic stability of the ϵ signal was found to be the main constraint for selecting main preCore mutations. Analysis of ϵ-signal variability suggested the essential nature of the ϵ structural motif and that certain nucleotides may be involved in ϵ signal functions.

Clinical application of estimating hepatitis B virus quasispecies complexity by massive sequencing: correlation between natural evolution and on-treatment evolution.

Aim To evaluate HBV quasispecies (QA) complexity in the preCore/Core regions in relation to HBeAg status, and explore QA changes under natural evolution and nucleoside analogue (NUC) treatment. Methods Ultra-deep pyrosequencing of HBV preCore/Core regions in 30 sequential samples (baseline [diagnosis], treatment-free, and treatment-nonresponse) from 10 retrospectively selected patients grouped according to HBeAg status over time: HBeAg+ (N = 4), HBeAg- (N = 2), and fluctuating HBeAg (transient seroreversion/seroconversion pattern) (N = 4). QA complexity was defined by Shannon entropy, mutation frequency, nucleotide diversity, and mutation frequency of amino acids (MfAA) in preCore and Core. Results The QA was less complex in HBeAg+ than in HBeAg- or fluctuating HBeAg. High complexity in preCore was associated with decreased viral replication (preCore MfAA negatively correlated with HBV-DNA, p = 0.005). QA complexity in the treatment-free period negatively correlated with values seen during treatment. Specific variants were mainly selected in the Core region in HBeAg- and fluctuating HBeAg patients, suggesting higher immune pressure than in HBeAg+. Conclusions The negative correlation between QA natural evolution and on-treatment evolution indicates the importance of pre-treatment QA study to predict QA changes in NUC nonresponders. Study of QA complexity could be useful for managing HBV infection.

Selection of the highly replicative and partially multidrug resistant rtS78T HBV polymerase mutation during TDF-ETV combination therapy

BACKGROUND & AIMS Patients chronically infected with the hepatitis B virus (HBV) and receiving long-term treatment with nucleoside or nucleotide analogues are at risk of selecting HBV strains with complex mutational patterns. We herein report two cases of HBV-infected patients with insufficient viral suppression, despite dual antiviral therapy with entecavir (ETV) and tenofovir (TDF). One patient died from aggressive hepatocellular carcinoma (HCC). METHODS Serum samples from the two patients at different time points were analyzed using ultra-deep pyrosequencing analysis. HBV mutations were identified and transiently transfected into hepatoma cells in vitro using replication-competent HBV vectors, and functionally analyzed. We assessed replication efficacy, resistance to antivirals and potential impact on HBV secretion (viral particles, exosomes). RESULTS Sequencing analyses revealed the selection of the rtS78T HBV polymerase mutation in both cases that simultaneously creates a premature stop codon at sC69 and thereby deletes almost the entire small HBV surface protein. One of the patients had an additional 261bp deletion in the preS1/S2 region. Functional analyses of the mutations in vitro revealed that the rtS78T/sC69∗ mutation, but not the preS1/S2 deletion, significantly enhanced viral replication and conferred reduced susceptibility to ETV and TDF. The sC69∗ mutation caused truncation of HBs protein, leading to impaired detection by commercial HBsAg assay, without causing intracellular HBsAg retention or affecting HBV secretion. CONCLUSIONS The rtS78T/sC69∗ HBV mutation, associated with enhanced replication and insufficient response to antiviral treatment, may favor long-term persistence of these isolates. In addition to the increased production of HBV transcripts and the sustained secretion of viral particles in the absence of antigenic domains of S protein, this HBV mutation may predispose patients to carcinogenic effects. LAY SUMMARY Long-term treatment with antiviral drugs carries the risk of selecting mutations in the hepatitis B virus (HBV). We herein report two cases of patients with insufficient response to dual tenofovir and entecavir therapy. Molecular analyses identified a distinct mutation, rtS78T/sC69∗, that abolishes HBsAg detection, enhances replication, sustains exosome-mediated virion secretion and decreases susceptibility to antivirals, thereby representing a potentially high-risk mutation for HBV-infected individuals.

Tenofovir discontinuation after long-term viral suppression in HBeAg negative chronic hepatitis B. Can HBsAg levels be useful?

BACKGROUND Recent studies have shown that antiviral treatment discontinuation is safe and associated with virologic remission in HBeAg-negative patients. However, the period of viral suppression and follow-up in these studies was relatively short. OBJECTIVES To investigate whether continuous viral suppression with tenofovir disoproxil fumarate for more than 7 years is associated with HBsAg loss and sustained response after treatment discontinuation and receiving a full course of hepatitis B vaccination. STUDY DESIGN Patients with HBeAg-negative chronic HBV infection and more than 7 years of persistent viral suppression with tenofovir therapy were selected for treatment discontinuation and HBV vaccination. Follow-up with monthly ALT, HBV-DNA, and HBsAg determinations lasted 72 weeks. In patients with viral relapse, the viral quasispecies in the overlapping reverse transcriptase and small surface protein regions was analysed by ultra-deep pyrosequencing. RESULTS Eight of 17 HBeAg-negative patients accepted tenofovir discontinuation: 5 patients achieved sustained response (persistent HBV-DNA levels <2000IU/mL and normal ALT) despite an initial virologic relapse, one lost HBsAg, and two needed re-treatment. All patients with an on-treatment HBsAg level decline >5000IU/mL achieved sustained response. Patients with HBsAg level <100IU/mL during an ALT flare after antiviral discontinuation achieved sustained response. Significant changes were seen in the composition of the HBV quasispecies, and half the patients showed changes in HBV genotype. CONCLUSIONS Even though the majority of patients presented an initial relapse with selection of HBV variants, most achieved sustained response. Changes in HBsAg levels on and off treatment may be useful for predicting the likelihood of virologic remission.

Evidence of an Exponential Decay Pattern of the Hepatitis Delta Virus Evolution Rate and Fluctuations in Quasispecies Complexity in Long-Term Studies of Chronic Delta Infection

Chronic HDV infection can cause a severe form of viral hepatitis for which there is no specific treatment. Characterization of the hepatitis B or C viral quasispecies has provided insight into treatment failure and disease recurrence following liver transplantation, has proven useful to understand hepatitis B e antigen seroconversion, and has helped to predict whether hepatitis C infection will resolve or become chronic. It is likely that characterization of the hepatitis delta virus (HDV) quasispecies will ultimately have similar value for the management of this infection. This study sought to determine the RNA evolution rates in serum of chronic hepatitis delta (CHD) treatment-naïve patients, using next-generation sequencing methods. The region selected for study encompassed nucleotide positions 910 to 1270 of the genome and included the amber/W codon. Amber/W is a substrate of the editing process by the ADAR1 host enzyme and is essential for encoding the 2 delta antigens (HDAg). The amber codon encodes the small (unedited) HDAg form and the W codon the large (edited) HDAg form. The evolution rate was analyzed taking into account the time elapsed between samples, the percentage of unedited and edited genomes, and the complexity of the viral population. The longitudinal studies included 29 sequential samples from CHD patients followed up for a mean of 11.5 years. In total, 121,116 sequences were analyzed. The HDV evolution rate ranged from 9.5x10-3 to 1.2x10-3 substitutions/site/year and showed a negative correlation with the time elapsed between samples (p<0.05). An accumulation of transition-type changes was found to be responsible for higher evolution rates. The percentages of unedited and edited genomes and the quasispecies complexity showed no relationships with the evolution rate, but the fluctuations in the percentages of genomes and in complexity suggest continuous adaptation of HDV to the host conditions.

Deep sequencing in the management of hepatitis virus infections

The hepatitis viruses represent a major public health problem worldwide. Procedures for characterization of the genomic composition of their populations, accurate diagnosis, identification of multiple infections, and information on inhibitor-escape mutants for treatment decisions are needed. Deep sequencing methodologies are extremely useful for these viruses since they replicate as complex and dynamic quasispecies swarms whose complexity and mutant composition are biologically relevant traits. Population complexity is a major challenge for disease prevention and control, but also an opportunity to distinguish among related but phenotypically distinct variants that might anticipate disease progression and treatment outcome. Detailed characterization of mutant spectra should permit choosing better treatment options, given the increasing number of new antiviral inhibitors available. In the present review we briefly summarize our experience on the use of deep sequencing for the management of hepatitis virus infections, particularly for hepatitis B and C viruses, and outline some possible new applications of deep sequencing for these important human pathogens.

Assessment of a Novel Automatic Real-Time PCR Assay on the Cobas 4800 Analyzer as a Screening Platform for Hepatitis C Virus Genotyping in Clinical Practice: Comparison with Massive Sequencing

ABSTRACT The unequivocal identification of hepatitis C virus (HCV) subtypes 1a/1b and genotypes 2 to 6 is required for optimizing the effectiveness of interferon-free, direct-acting antiviral therapies. We compared the performance of a new real-time HCV genotyping assay used on the Cobas 4800 system (C4800) with that of high-resolution HCV subtyping (HRCS). In total, 502 samples were used, including 184 samples from chronic HCV patients (from routine laboratory activity during April 2016), 5 stored samples with double HCV genotype infections for testing the limitations of the method, and 313 samples from a screening protocol implemented in our hospital (from May to August 2016) based on the new method to further determine its genotyping accuracy. A total of 282 samples, including 171 from April 2016 (the 13 remaining had too low of a viral load for HRCS), 5 selected with double infections, and 106 from screening, were analyzed by both methods, and 220 were analyzed only by the C4800. The C4800 correctly subtyped 125 of 126 1a/1b samples, and the 1 remaining sample was reported as genotype 1. The C4800 correctly genotyped 38 of 45 non-1a/1b samples (classified by HRCS), and it reported the remaining 7 samples as indeterminate. One hundred two of 106 non-1a/1b genotype samples that were identified using the C4800 for screening were confirmed by HRCS. In the 4 remaining samples, 3 were correctly reported as genotype 1 (without defining the subtype) and 1 was reported as indeterminate. None of the samples were misgenotyped. Four of 7 samples with double HCV infections were correctly genotyped by the C4800. Excluding the 5 selected double-infected samples, the C4800 showed 95.7% concordant results for genotyping HCVs 2 to 6 and 1a/1b subtyping, and 99.2% concordance for subtyping 1a/1b single infections in clinical samples. To improve laboratory workflow, we propose using the C4800 as a first-line test for HCV genotyping and 1a/1b classification, followed by transferring non-1a/1b samples to a center where HRCS is available, if further characterization is needed.

Quantitative characterization of hepatitis delta virus genome edition by next-generation sequencing

AIM To determine the capacity of next-generation sequencing (NGS) for quantifying edited and unedited HDV populations, and to confirm if edition is a general phenomenon taking place along the entire HDV region analyzed, as we previously reported (Homs M et al. PLoS One 2016, 11, e0158557). METHODS Four serum samples from 4 patients with chronic HDV/HBV infection were included in the study. The region selected for analysis covered 360 nucleotides (nt), positions 910-1270 of the HDV genome, which included the HDAg ORF editing site (nt 1014 within codon 196). Quantification of edited and unedited genomes was performed by molecular cloning and Sanger sequencing and by NGS. To evaluate the reliability of the NGS values obtained, we combined a clone with an edited codon and one with an unedited codon in known percentages in a series of artificial mixtures, which were then analyzed by NGS. In addition, we determined the nt changes occurring over the complete amplified region after excluding the editing codon (196) to evaluate edition along it. RESULTS In total, 11,208 quality-filtered sequences were obtained in the 4 samples. The 95% confidence intervals for the proportions of unedited populations by molecular cloning and NGS were overlapping, and those of cloning were wider, indicating that they are comparable and that NGS is more precise than cloning. Unedited genomes predominated over edited ones in all 4 samples analyzed by NGS and in 3 of the 4 samples analyzed by molecular cloning. In total, 83,276 quality-filtered sequences were obtained from the artificial mixtures. Percentages of the two viral populations detected by NGS in these mixtures were comparable to the expected percentages. Evaluation of edition along the HDV coding region showed that transitions were more frequent than transversions, accounting for 63.09% and 36.91%, respectively. Interestingly, among the 4 possible transition-type changes, G:A and A:G accounted for 73.86% of the total. CONCLUSION Next-generation sequencing proved useful to quantify edited and unedited HDV genomes, and provided relevant information on the HDV quasispecies.