ACE Score Identifies HBeAg-negative Inactive Carriers at a Single-point Evaluation, Regardless of HBV Genotype.

Background and Aims: Hepatitis B virus (HBV) biomarkers have been used for a better categorization of patients, even though the lack of simple algorithms and the impact of genotypes limit their application. Our aim was to assess the usefulness of noninvasive markers for the identification of HBV inactive carriers (ICs) in a single-point evaluation and to design a predictive model for their identification. Methods: This retrospective-prospective study included 343 consecutive HBeAg-negative individuals. Clinical, analytical, and virological data were collected, and a liver biopsy was performed if needed. Subjects were classified at the end of follow-up as ICs, chronic hepatitis B and gray zone.A predictive model was constructed, and validated by 1000-bootstrap samples. Results: After 39 months of follow-up, 298 subjects were ICs, 36 were chronic hepatitis B CHB, and nine were gray zone. Eighty-nine (25.9%) individuals required a liver biopsy. Baseline HBV DNA hazard ratio (HR) 6.0, p<0.001), HBV core-related antigen (HBcrAg) (HR 6.5, p<0.001), and elastography (HR 4.6, p<0.001) were independently associated with the IC stage. The ACE score (HBV DNA, HBcrAg, elastography), obtained by bootstrapping, yielded an area under the receiver operating characteristics (AUROC) of 0.925 (95% CI: 0.880-0.970, p<0.001) for identification of ICs. The AUROC for genotype D was 0.95, 0.96 for A, 0.90 for E, and 0.88 for H/F. An ACE score of <1 had a positive predictive value of 99.5%, and a score ≤12 points had a diagnostic accuracy of 93.8%. Conclusions: Low baseline HBV DNA, HBcrAg, and liver stiffness were independently associated with the IC phase. A score including those variables identified ICs at a single-point evaluation, and might be applied to implement less intensive follow-up strategies.

Hepatitis B Virus Variants with Multiple Insertions and/or Deletions in the X Open Reading Frame 3' End: Common Members of Viral Quasispecies in Chronic Hepatitis B Patients.

Deletions in the 3' end region of the hepatitis B virus (HBV) X open reading frame (HBX) may affect the core promoter (Cp) and have been frequently associated with hepatocellular carcinoma (HCC). The aim of this study was to investigate the presence of variants with deletions and/or insertions (Indels) in this region in the quasispecies of 50 chronic hepatitis B (CHB) patients without HCC. We identified 103 different Indels in 47 (94%) patients, in a median of 3.4% of their reads (IQR, 1.3-8.4%), and 25% (IQR, 13.1-40.7%) of unique sequences identified in each quasispecies (haplotypes). Of those Indels, 101 (98.1%) caused 44 different altered stop codons, the most commonly observed were at positions 128, 129, 135, and 362 (putative position). Moreover, 39 (37.9%) Indels altered the TATA-like box (TA) sequences of Cp; the most commonly observed caused TA2 + TA3 fusion, creating a new putative canonical TATA box. Four (8%) patients developed negative clinical outcomes after a median follow-up of 9.4 (8.7-12) years. In conclusion, we observed variants with Indels in the HBX 3' end in the vast majority of our CHB patients, some of them encoding alternative versions of HBx with potential functional roles, and/or alterations in the regulation of transcription.

Sophisticated viral quasispecies with a genotype-related pattern of mutations in the hepatitis B X gene of HBeAg-ve chronically infected patients.

Patients with HBeAg-negative chronic infection (CI) have not been extensively studied because of low viremia. The HBx protein, encoded by HBX, has a key role in viral replication. Here, we analyzed the viral quasispecies at the 5' end of HBX in CI patients and compared it with that of patients in other clinical stages. Fifty-eight HBeAg-negative patients were included: 16 CI, 19 chronic hepatitis B, 16 hepatocellular carcinoma and 6 liver cirrhosis. Quasispecies complexity and conservation were determined in the region between nucleotides 1255 and 1611. Amino acid changes detected were tested in vitro. CI patients showed higher complexity in terms of mutation frequency and nucleotide diversity and higher quasispecies conservation (p < 0.05). A genotype D-specific pattern of mutations (A12S/P33S/P46S/T36D-G) was identified in CI (median frequency, 81.7%), which determined a reduction in HBV DNA release of up to 1.5 log in vitro. CI patients showed a more complex and conserved viral quasispecies than the other groups. The genotype-specific pattern of mutations could partially explain the low viremia observed in these patients.

Conservation and variability of hepatitis B core at different chronic hepatitis stages.

BACKGROUND: Since it is currently not possible to eradicate hepatitis B virus (HBV) infection with existing treatments, research continues to uncover new therapeutic strategies. HBV core protein, encoded by the HBV core gene (HBC), intervenes in both structural and functional processes, and is a key protein in the HBV life cycle. For this reason, both the protein and the gene could be valuable targets for new therapeutic and diagnostic strategies. Moreover, alterations in the protein sequence could serve as potential markers of disease progression. AIM: To detect, by next-generation sequencing, HBC hyper-conserved regions that could potentially be prognostic factors and targets for new therapies. METHODS: Thirty-eight of 45 patients with chronic HBV initially selected were included and grouped according to liver disease stage [chronic hepatitis B infection without liver damage (CHB, n = 16), liver cirrhosis (LC, n = 5), and hepatocellular carcinoma (HCC, n = 17)]. HBV DNA was extracted from patients' plasma. A region between nucleotide (nt) 1863 and 2483, which includes HBC, was amplified and analyzed by next-generation sequencing (Illumina MiSeq platform). Sequences were genotyped by distance-based discriminant analysis. General and intergroup nt and amino acid (aa) conservation was determined by sliding window analysis. The presence of nt insertion and deletions and/or aa substitutions in the different groups was determined by aligning the sequences with genotype-specific consensus sequences. RESULTS: Three nt (nt 1900-1929, 2249-2284, 2364-2398) and 2 aa (aa 117-120, 159-167) hyper-conserved regions were shared by all the clinical groups. All groups showed a similar pattern of conservation, except for five nt regions (nt 1946-1992, 2060-2095, 2145-2175, 2230-2250, 2270-2293) and one aa region (aa 140-160), where CHB and LC, respectively, were less conserved (P < 0.05). Some group-specific conserved regions were also observed at both nt (2306-2334 in CHB and 1935-1976 and 2402-2435 in LC) and aa (between aa 98-103 in CHB and 28-30 and 51-54 in LC) levels. No differences in insertion and deletions frequencies were observed. An aa substitution (P79Q) was observed in the HCC group with a median (interquartile range) frequency of 15.82 (0-78.88) vs 0 (0-0) in the other groups (P < 0.05 vs CHB group). CONCLUSION: The differentially conserved HBC and HBV core protein regions and the P79Q substitution could be involved in disease progression. The hyper-conserved regions detected could be targets for future therapeutic and diagnostic strategies.

Detection of hyper-conserved regions in hepatitis B virus X gene potentially useful for gene therapy.

AIM: To detect hyper-conserved regions in the hepatitis B virus (HBV) X gene (HBX) 5' region that could be candidates for gene therapy. METHODS: The study included 27 chronic hepatitis B treatment-naive patients in various clinical stages (from chronic infection to cirrhosis and hepatocellular carcinoma, both HBeAg-negative and HBeAg-positive), and infected with HBV genotypes A-F and H. In a serum sample from each patient with viremia > 3.5 log IU/mL, the HBX 5' end region [nucleotide (nt) 1255-1611] was PCR-amplified and submitted to next-generation sequencing (NGS). We assessed genotype variants by phylogenetic analysis, and evaluated conservation of this region by calculating the information content of each nucleotide position in a multiple alignment of all unique sequences (haplotypes) obtained by NGS. Conservation at the HBx protein amino acid (aa) level was also analyzed. RESULTS: NGS yielded 1333069 sequences from the 27 samples, with a median of 4578 sequences/sample (2487-9279, IQR 2817). In 14/27 patients (51.8%), phylogenetic analysis of viral nucleotide haplotypes showed a complex mixture of genotypic variants. Analysis of the information content in the haplotype multiple alignments detected 2 hyper-conserved nucleotide regions, one in the HBX upstream non-coding region (nt 1255-1286) and the other in the 5' end coding region (nt 1519-1603). This last region coded for a conserved amino acid region (aa 63-76) that partially overlaps a Kunitz-like domain. CONCLUSION: Two hyper-conserved regions detected in the HBX 5' end may be of value for targeted gene therapy, regardless of the patients' clinical stage or HBV genotype.

Analysis of hepatitis B virus preS1 variability and prevalence of the rs2296651 polymorphism in a Spanish population.

AIM: To determine the variability/conservation of the domain of hepatitis B virus (HBV) preS1 region that interacts with sodium-taurocholate cotransporting polypeptide (hereafter, NTCP-interacting domain) and the prevalence of the rs2296651 polymorphism (S267F, NTCP variant) in a Spanish population. METHODS: Serum samples from 246 individuals were included and divided into 3 groups: patients with chronic HBV infection (CHB) (n = 41, 73% Caucasians), patients with resolved HBV infection (n = 100, 100% Caucasians) and an HBV-uninfected control group (n = 105, 100% Caucasians). Variability/conservation of the amino acid (aa) sequences of the NTCP-interacting domain, (aa 2-48 in viral genotype D) and a highly conserved preS1 domain associated with virion morphogenesis (aa 92-103 in viral genotype D) were analyzed by next-generation sequencing and compared in 18 CHB patients with viremia > 4 log IU/mL. The rs2296651 polymorphism was determined in all individuals in all 3 groups using an in-house real-time PCR melting curve analysis. RESULTS: The HBV preS1 NTCP-interacting domain showed a high degree of conservation among the examined viral genomes especially between aa 9 and 21 (in the genotype D consensus sequence). As compared with the virion morphogenesis domain, the NTCP-interacting domain had a smaller proportion of HBV genotype-unrelated changes comprising > 1% of the quasispecies (25.5% vs 31.8%), but a larger proportion of genotype-associated viral polymorphisms (34% vs 27.3%), according to consensus sequences from GenBank patterns of HBV genotypes A to H. Variation/conservation in both domains depended on viral genotype, with genotype C being the most highly conserved and genotype E the most variable (limited finding, only 2 genotype E included). Of note, proline residues were highly conserved in both domains, and serine residues showed changes only to threonine or tyrosine in the virion morphogenesis domain. The rs2296651 polymorphism was not detected in any participant. CONCLUSION: In our CHB population, the NTCP-interacting domain was highly conserved, particularly the proline residues and essential amino acids related with the NTCP interaction, and the prevalence of rs2296651 was low/null.

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.

Effectiveness and Safety of Entecavir or Tenofovir in a Spanish Cohort of Chronic Hepatitis B Patients: Validation of the Page-B Score to Predict Hepatocellular Carcinoma.

BACKGROUND: Long-term antiviral therapy has resulted in viral suppression and biochemical response in chronic hepatitis B, although the risk of hepatocellular carcinoma has not been abolished. The Page-B score could be useful to estimate the probability of HCC. AIMS: To analyze the effectiveness and safety of entecavir or tenofovir for more than 4 years and the usefulness of Page-B score in the real-world setting. METHODS: Analysis of Caucasian chronic hepatitis B subjects treated with entecavir or tenofovir from the prospective, multicenter database CIBERHEP. RESULTS: A total of 611 patients were enrolled: 187 received entecavir and 424 tenofovir. Most were men, mean age 50 years, 32% cirrhotic and 16.5% HBeAg-positive. Mean follow-up was 55 (entecavir) and 49 (tenofovir) months. >90% achieved HBV DNA <69 IU/mL and biochemical normalization by months 12 and 36, respectively. Cumulative HBeAg loss and anti-HBe seroconversion were achieved by 33.7 and 23.8%. Four patients lost HBsAg; three HBeAg-positive. Renal function remained stable on long-term follow-up. Fourteen (2.29%) developed HCC during follow-up all of them with baseline Page-B ≥10. Nine were diagnosed within the first 5 years of therapy. This contrasts with the 27 estimated by Page-B, a difference that highlights the importance of regular HCC surveillance even in patients with virological suppression. CONCLUSIONS: Entecavir and tenofovir achieved high biochemical and virological response. Renal function remained stable with both drugs. A Page-B cut-off ≥10 selected all patients at risk of HCC development.

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.

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.

Complex Genotype Mixtures Analyzed by Deep Sequencing in Two Different Regions of Hepatitis B Virus.

This study assesses the presence and outcome of genotype mixtures in the polymerase/surface and X/preCore regions of the HBV genome in patients with chronic hepatitis B virus (HBV) infection. Thirty samples from ten chronic hepatitis B patients were included. The polymerase/surface and X/preCore regions were analyzed by deep sequencing (UDPS) in the first available sample at diagnosis, a pre-treatment sample, and a sample while under treatment. HBV genotype was determined by phylogenesis. Quasispecies complexity was evaluated by mutation frequency and nucleotide diversity. The polymerase/surface and X/preCore regions were validated for genotyping from 113 GenBank reference sequences. UDPS yielded a median of 10,960 sequences per sample (IQR 16,645) in the polymerase/surface region and 11,595 sequences per sample (IQR 14,682) in X/preCore. Genotype mixtures were more common in X/preCore (90%) than in polymerase/surface (30%) (p<0.001). On X/preCore genotyping, all samples were genotype A, whereas polymerase/surface yielded genotypes A (80%), D (16.7%), and F (3.3%) (p = 0.036). Genotype changes in polymerase/surface were observed in four patients during natural quasispecies dynamics and in two patients during treatment. There were no genotype changes in X/preCore. Quasispecies complexity was higher in X/preCore than in polymerase/surface (p = 0.004). The results provide evidence of genotype mixtures and differential genotype proportions in the polymerase/surface and X/preCore regions. The genotype dynamics in HBV infection and the different patterns of quasispecies complexity in the HBV genome suggest a new paradigm for HBV genotype classification.

Quasispecies dynamics in main core epitopes of hepatitis B virus by ultra-deep-pyrosequencing.

AIM: To investigate the variability of the main immunodominant motifs of hepatitis B virus (HBV) core gene by ultra-deep-pyrosequencing (UDPS). METHODS: Four samples (2 genotype A and 2 genotype D) from 4 treatment-naïve patients were assessed for baseline variability. Two additional samples from one patient (patient 4, genotype D) were selected for analysis: one sample corresponded to a 36-mo treatment-free period from baseline and the other to the time of viral breakthrough after 18 mo of lamivudine treatment. The HBV region analyzed covered amino acids 40 to 95 of the core gene, and included the two main epitopic regions, Th50-69 and B74-84. UDPS was carried out in the Genome Sequencer FLX system (454 Life Sciences, Roche). After computer filtering of UDPS data based on a Poisson statistical model, 122,813 sequences were analyzed. The most conserved position detected by UDPS was analyzed by site-directed mutagenesis and evaluated in cell culture. RESULTS: Positions with highest variability rates were mainly located in the main core epitopes, confirming their role as immune-stimulating regions. In addition, the distribution of variability showed a relationship with HBV genotype. Patient 1 (genotype A) presented the lowest variability rates and patient 2 (genotype A) had 3 codons with variability higher than 1%. Patient 3 and 4 (both genotype D) presented 5 and 8 codons with variability higher than 1%, respectively. The median baseline frequencies showed that genotype A samples had higher variability in epitopic positions than in the other positions analyzed, approaching significance (P = 0.07, sample 1 and P = 0.05, sample 2). In contrast, there were no significant differences in variability between the epitopic and other positions in genotype D cases. Interestingly, patient 1 presented a completely mutated motif from amino acid 64 to 67 (E64LMT67), which is commonly recognized by T helper cells. Additionally, the variability observed in all 4 patients was particularly associated with the E64LMT67 motif. Codons 78 and 79 were highly conserved in all samples, in keeping with their involvement in the interaction between the HBV virion capsid and the surface antigens (HBsAg). Of note, codon 76 was even more conserved than codons 78 and 79, suggesting a possible role in HBsAg interactions or even in hepatitis B e antigen conformation. Sequential analysis of samples from patient 4 (genotype D) illustrated the dynamism of the HBV quasispecies, with strong selection of one minor baseline variant coinciding with a decrease in core variability during the treatment-free and lamivudine-treated period. The drop in variability seemed to result from a "steady state" situation of the HBV quasispecies after selection of the variant with greatest fitness. CONCLUSION: Host immune pressure seems to be the main cause of HBV core evolution. UDPS analysis is a useful technique for studying viral quasispecies.

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.