In-Host HEV Quasispecies Evolution Shows the Limits of Mutagenic Antiviral Treatments.

Here, we report the in-host hepatitis E virus (HEV) quasispecies evolution in a chronically infected patient who was treated with three different regimens of ribavirin (RBV) for nearly 6 years. Sequential plasma samples were collected at different time points and subjected to RNA extraction and deep sequencing using the MiSeq Illumina platforms. Specifically, we RT-PCR amplified a single amplicon from the core region located in the open-reading frame 2 (ORF2). At the nucleotide level (genotype), our analysis showed an increase in the number of rare haplotypes and a drastic reduction in the frequency of the master (most represented) sequence during the period when the virus was found to be insensitive to RBV treatment. Contrarily, at the amino acid level (phenotype), our study revealed conservation of the amino acids, which is represented by a high prevalence of the master sequence. Our findings suggest that using mutagenic antivirals concomitant with high viral loads can lead to the selection and proliferation of a rich set of synonymous haplotypes that express the same phenotype. This can also lead to the selection and proliferation of conservative substitutions that express fitness-enhanced phenotypes. These results have important clinical implications, as they suggest that using mutagenic agents as a monotherapy treatment regimen in the absence of sufficiently effective viral inhibitors can result in diversification and proliferation of a highly diverse quasispecies resistant to further treatment. Therefore, such approaches should be avoided whenever possible.

HBsAg protein composition and clinical outcomes in chronic hepatitis D and variations across HBeAg-negative chronic HBsAg carriers.

Background & Aims: HBsAg proteins are useful to identify HBV inactive carriers (ICs), but data on chronic hepatitis D (CHD) are scarce. This study aimed to describe HBsAg composition in CHD, its changes during the evolution, and the potential association with clinical outcomes. In addition, we assess the composition of HBsAg across different HBV genotypes and validate previous results on HBsAg proteins in an independent HBV cohort. Methods: Quantitative HBsAg, medium HBsAg proteins (MHBs), and large HBsAg proteins (LHBs) were measured in two cohorts. The first cohort consisted of patients with CHD. A cross-sectional study of samples from two European institutions (N = 46) was conducted. Outcomes were assessed in a retrospective-prospective study of those patients with a follow-up of >1 year (n = 36), and the longitudinal evolution of HBsAg proteins in those with samples >5 years apart (n = 12) was analysed. The second cohort consisted of patients with HBeAg-negative HBV, and a cross-sectional study was performed (N = 141). Results: Forty-one (89%) patients with CHD had detectable HDV-RNA, and the presence of HDV-RNA was associated with higher LHBs proportion (p = 0.010). Baseline MHBs (p = 0.051) and MHBs proportion (p = 0.086) tended to be higher in those developing clinical outcomes (9/36, 25%) after a median follow-up of 5.9 years. Patients in which HDV-RNA became spontaneously undetectable during follow-up (5/31, 16.1%) tended to present lower MHBs proportion (p = 0.085). In the longitudinal study, changes in LHBs proportion were observed (p = 0.041), whereas MHBs proportion remained stable (p = 0.209). Regarding HBV, ICs showed lower LHBs proportion (p = 0.027). LHBs and MHBs differed significantly according to HBV genotype, regardless of the HBV phase. Conclusions: Patients with CHD with detectable HDV-RNA presented higher LHBs proportion than those with undetectable HDV-RNA. A trend toward having higher baseline MHBs proportion was observed in patients who developed clinical outcomes or remained with detectable HDV-RNA. This study validates the different HBsAg composition in HBV ICs and reveals the HBV-genotype influence in HBsAg composition. Impact and implications: The composition of HBsAg in chronic hepatitis D differs in patients with detectable and undetectable HDV viral load and may help predict the likelihood of achieving undetectable HDV viraemia and the development of clinical events such as decompensation. The composition of the surface antigen is also useful to distinguish inactive carriers of HBV, and it varies according to HBV genotype.

Quasispecies Fitness Partition to Characterize the Molecular Status of a Viral Population. Negative Effect of Early Ribavirin Discontinuation in a Chronically Infected HEV Patient.

The changes occurring in viral quasispecies populations during infection have been monitored using diversity indices, nucleotide diversity, and several other indices to summarize the quasispecies structure in a single value. In this study, we present a method to partition quasispecies haplotypes into four fractions according to their fitness: the master haplotype, rare haplotypes at two levels (those present at <0.1%, and those at 0.1−1%), and a fourth fraction that we term emerging haplotypes, present at frequencies >1%, but less than that of the master haplotype. We propose that by determining the changes occurring in the volume of the four quasispecies fitness fractions together with those of the Hill number profile we will be able to visualize and analyze the molecular changes in the composition of a quasispecies with time. To develop this concept, we used three data sets: a technical clone of the complete SARS-CoV-2 spike gene, a subset of data previously used in a study of rare haplotypes, and data from a clinical follow-up study of a patient chronically infected with HEV and treated with ribavirin. The viral response to ribavirin mutagenic treatment was selection of a rich set of synonymous haplotypes. The mutation spectrum was very complex at the nucleotide level, but at the protein (phenotypic/functional) level the pattern differed, showing a highly prevalent master phenotype. We discuss the putative implications of this observation in relation to mutagenic antiviral treatment.

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.

Standardized Hepatitis B Virus RNA Quantification in Untreated and Treated Chronic Patients: a Promising Marker of Infection Follow-Up.

The measurement and interpretation of HBV DNA and RNA levels in HBV infected patients treated with antiviral therapy supports the objective of HBV disease management. Here, we quantified circulating HBV RNA through a standardized and sensitive assay in follow-up samples from both naive and treated patients as a marker of infection evolution. HBV DNA (HBV DNA for use in Cobas 6800/8800 Automated Roche Molecular Systems), RNA (Roche HBV RNA Investigational Assay for use in the Cobas 6800/8800; Roche), HBeAg and HBsAg (Elycsys HBsAg chemiluminescence immunoassay by Cobas 8000; Roche), and core-related antigen (Lumipulse G chemiluminescence assay; Fujirebio) levels were measured in cohorts of untreated or nucleos(t)ide treated, HBV-infected subjects in an outpatient hospital setting. HBV DNA levels in untreated people were 3.6 log10 higher than corresponding RNA levels and were stable over 5 years of observation. While only five of 52 treated patients had DNA levels below the lower limit of quantification (10 IU/mL) at the end of follow-up, 13 had HBV RNA levels persistently above this limit, including eight with undetectable DNA. In samples with undetectable core-related antigen we observed a median HBsAg titer 2.7-fold higher than in samples with undetectable RNA (adjusted P = 0.012). Detectable HBV RNA with undetectable HBV DNA was a negative predictor of HBsAg decrease to a level ≤100 IU/mL (P = 0.03). In naive patients the difference between HBV DNA and RNA was higher than previously reported. HBV RNA rapidly decreased during treatment. However, in some cases, it was detectable even after years of effective therapy, being a negative predictor of HBsAg decrease. The investigational RNA assay for use on the Cobas 6800/8800 instruments is a sensitive and standardized method that could be applied in general management of HBV infection. IMPORTANCE This study focused on the quantification of circulating HBV RNA by using a standardized and sensitive assay. Thanks to this system we observed a higher difference between circulating HBV DNA and RNA than previously reported. In treated patients, HBV RNA decreased together with DNA, although some patients presented detectable levels even after years of successful antiviral treatment, suggesting a persistent viral transcription. Of note, the detection of viral RNA when HBV DNA is undetectable was a negative predictor of HBsAg decrease to a level ≤100 IU/mL. This assay could be extremely helpful in HBV patients management to study viral transcription and to identify those treated patients that may achieve sustained viral suppression.

Next-Generation Sequencing for Confronting Virus Pandemics.

Virus pandemics have happened, are happening and will happen again. In recent decades, the rate of zoonotic viral spillover into humans has accelerated, mirroring the expansion of our global footprint and travel network, including the expansion of viral vectors and the destruction of natural spaces, bringing humans closer to wild animals. Once viral cross-species transmission to humans occurs, transmission cannot be stopped by cement walls but by developing barriers based on knowledge that can prevent or reduce the effects of any pandemic. Controlling a local transmission affecting few individuals is more efficient that confronting a community outbreak in which infections cannot be traced. Genetic detection, identification, and characterization of infectious agents using next-generation sequencing (NGS) has been proven to be a powerful tool allowing for the development of fast PCR-based molecular assays, the rapid development of vaccines based on mRNA and DNA, the identification of outbreaks, transmission dynamics and spill-over events, the detection of new variants and treatment of vaccine resistance mutations, the development of direct-acting antiviral drugs, the discovery of relevant minority variants to improve knowledge of the viral life cycle, strengths and weaknesses, the potential for becoming dominant to take appropriate preventive measures, and the discovery of new routes of viral transmission.

Inspecting the Ribozyme Region of Hepatitis Delta Virus Genotype 1: Conservation and Variability.

The hepatitis delta virus (HDV) genome has an autocatalytic region called the ribozyme, which is essential for viral replication. The aim of this study was to use next-generation sequencing (NGS) to analyze the ribozyme quasispecies (QS) in order to study its evolution and identify highly conserved regions potentially suitable for a gene-silencing strategy. HDV RNA was extracted from 2 longitudinal samples of chronic HDV patients and the ribozyme (nucleotide, nt 688-771) was analyzed using NGS. QS conservation, variability and genetic distance were analyzed. Mutations were identified by aligning sequences with their specific genotype consensus. The main relevant mutations were tested in vitro. The ribozyme was conserved overall, with a hyper-conserved region between nt 715-745. No difference in QS was observed over time. The most variable region was between nt 739-769. Thirteen mutations were observed, with three showing a higher frequency: T23C, T69C and C64 deletion. This last strongly reduced HDV replication by more than 1 log in vitro. HDV Ribozyme QS was generally highly conserved and was maintained during follow-up. The most conserved portion may be a valuable target for a gene-silencing strategy. The presence of the C64 deletion may strongly impair viral replication, as it is a potential mechanism of viral persistence.

Microorganisms as Shapers of Human Civilization, from Pandemics to Even Our Genomes: Villains or Friends? A Historical Approach.

Universal history is characterized by continuous evolution, in which civilizations are born and die. This evolution is associated with multiple factors, among which the role of microorganisms is often overlooked. Viruses and bacteria have written or decisively contributed to terrible episodes of history, such as the Black Death in 14th century Europe, the annihilation of pre-Columbian American civilizations, and pandemics such as the 1918 Spanish flu or the current COVID-19 pandemic caused by the coronavirus SARS-CoV-2. Nevertheless, it is clear that we could not live in a world without these tiny beings. Endogenous retroviruses have been key to our evolution and for the regulation of gene expression, and the gut microbiota helps us digest compounds that we could not otherwise process. In addition, we have used microorganisms to preserve or prepare food for millennia and more recently to obtain drugs such as antibiotics or to develop recombinant DNA technologies. Due to the enormous importance of microorganisms for our survival, they have significantly influenced the population genetics of different human groups. This paper will review the role of microorganisms as "villains" who have been responsible for tremendous mortality throughout history but also as "friends" who help us survive and evolve.

Cross-sectional evaluation of circulating hepatitis B virus RNA and DNA: Different quasispecies?

BACKGROUND: Different forms of pregenomic and other hepatitis B virus (HBV) RNA have been detected in patients' sera. These circulating HBV-RNAs may be useful for monitoring covalently closed circular DNA activity, and predicting hepatitis B e-antigen seroconversion or viral rebound after nucleos(t)ide analog cessation. Data on serum HBV-RNA quasispecies, however, is scarce. It is therefore important to develop methodologies to thoroughly analyze this quasispecies, ensuring the elimination of any residual HBV-DNA. Studying circulating HBV-RNA quasispecies may facilitate achieving functional cure of HBV infection. AIM: To establish a next-generation sequencing (NGS) methodology for analyzing serum HBV-RNA and comparing it with DNA quasispecies. METHODS: Thirteen untreated chronic hepatitis B patients, showing different HBV-genotypes and degrees of severity of liver disease were enrolled in the study and a serum sample with HBV-DNA > 5 Log10 IU/mL and HBV-RNA > 4 Log10 copies/mL was taken from each patient. HBV-RNA was treated with DNAse I to remove any residual DNA, and the region between nucleotides (nt) 1255-1611 was amplified using a 3-nested polymerase chain reaction protocol, and analyzed with NGS. Variability/conservation and complexity was compared between HBV-DNA and RNA quasispecies. RESULTS: No HBV-DNA contamination was detected in cDNA samples from HBV-RNA quasispecies. HBV quasispecies complexity showed heterogeneous behavior among patients. The Rare Haplotype Load at 1% was greater in DNA than in RNA quasispecies, with no statistically significant differences (P = 0.1641). Regarding conservation, information content was equal in RNA and DNA quasispecies in most nt positions [218/357 (61.06%)]. In 102 of the remaining 139 (73.38%), HBV-RNA showed slightly higher variability. Sliding window analysis identified 4 hyper-conserved sequence fragments in each quasispecies, 3 of them coincided between the 2 quasispecies: nts 1258-1286, 1545-1573 and 1575-1604. The 2 hyper-variable sequence fragments also coincided: nts 1311-1344 and 1461-1485. Sequences between nts 1519-1543 and 1559-1587 were only hyper-conserved in HBV-DNA and RNA, respectively. CONCLUSION: Our methodology allowed analyzing HBV-RNA quasispecies complexity and conservation without interference from HBV-DNA. Thanks to this, we have been able to compare both quasispecies in the present study.

Next-generation sequencing for the diagnosis of hepatitis B: current status and future prospects.

INTRODUCTION: Hepatitis B virus (HBV) causes a complex and persistent infection with a major impact on patients health. Viral-genome sequencing can provide valuable information for characterizing virus genotype, infection dynamics and drug and vaccine resistance. AREAS COVERED: This article reviews the current literature to describe the next-generation sequencing progress that facilitated a more comprehensive study of HBV quasispecies in diagnosis and clinical monitoring. EXPERT OPINION: HBV variability plays a key role in liver disease progression and treatment efficacy. Second-generation sequencing improved the sensitivity for detecting and quantifying mutations, mixed genotypes and viral recombination. Third-generation sequencing enables the analysis of the entire HBV genome, although the high error rate limits its use in clinical practice.

Resistance-associated substitutions after sofosbuvir/velpatasvir/voxilaprevir triple therapy failure.

Direct-acting antivirals (DAAs) resolve chronic HCV infection in >95% of patients, but a small percentage do not respond to DAA-based therapy. These may be difficult to treat because of resistance-associated substitutions (RAS) emerging after treatment failure. Triple therapy with sofosbuvir (SOF)/velpatasvir (VEL)/voxilaprevir (VOX) is the recommended retreatment after DAA-based failure. However, in rare cases, failure to triple therapy occurs, and there is little information characterizing the viruses that relapse. To determine the RAS profile after failing SOF/VEL/VOX, and seek suitable alternatives for retreatment, samples from 5 patients were analysed using MiSeq Illumina deep sequencing before and after triple therapy. All patients were men, aged 59-78 years, 2 HCV genotype (G) 1b and 3 G3a. The most prevalent NS3 substitutions after SOF/VEL/VOX failure were Y56F and A166T. Four patients had the NS5A RAS, Y93H, after triple failure, and Y93H was observed in both G1b patients before retreatment and after SOF/ledipasvir failure. In 2 G3a patients, Y93H appeared at triple failure, and on the other G3a, A30K persisted in 100% of viral genomes. Finally, G1b patients showed C316N in NS5B, associated with SOF failure, but G3a patients had no known NS5B substitutions. HCV RAS analysis identified the following substitutions present at higher rates after triple failure: Y56F in NS3 (G1b), A166T in NS3 (G3a), A30K or Y93H in NS5A, and C316N in NS5B (G1b). A RAS-based salvage treatment (SOF + glecaprevir/pibrentasvir + RBV) was successfully used in one G3a patient.

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.

Reflex viral load testing in dried blood spots generated by plasma separation card allows the screening and diagnosis of chronic viral hepatitis.

Dried blood spots (DBS) have been proposed as an alternative diagnostic technique for chronic viral hepatitis. The aim of this observational study was to correlate serologic HBV, HCV, and HDV status and reflex the respective viral load testing by PSC-DBS samples from capillary blood vs conventional plasma samples in patients with chronic viral hepatitis. Besides, we apply these tests in a prospective study for chronic viral hepatitis diagnosis in a rural region of sub-Saharan Africa. In total, 124 HBsAg-positive patients, 75 anti-HCV positive, 2 with HBV-HCV coinfection, and 13 anti-HDV positive were included. PSC-DBS sensitivity/specificity was 98.4 %/96.2 % for HBsAg detection, 98.7 %/100 % for anti-HCV, and 84.6 %/100 % for anti-HDV. HCV-RNA was quantified in all viremic patients using DBS. Only 42 of 78 (53.8 %) samples with HBV-DNA viremia were quantifiable by DBS. Sensitivity increased to 95.7 % in patients with HBV-DNA levels >2000 IU/mL. There was a high correlation between DBS and venous blood. The prevalence of HBsAg among the 93 individuals tested in Angola was 11 %, and 60 % of cases had detectable HBV-DNA viremia. As a conclusion, PSC-DBS is useful for chronic viral hepatitis screening and reflex molecular diagnosis showing globally high sensitivities and correlation with conventional blood samples.

Application of EASL 2017 criteria for switching hepatitis B patients from tenofovir disoproxil to entecavir or tenofovir alafenamide.

BACKGROUND: To overcome safety limitations of tenofovir-disoproxil, EASL guidelines proposed switching chronic hepatitis B patients older than 60 years or with bone or renal disease to tenofovir-alafenamide or entecavir. AIMS: To estimate the number of patients who would benefit from a treatment switch in a real-life setting. METHODS: Consecutive hepatitis B patients receiving tenofovir-disoproxil before 31 December 2017 were enrolled in a cross-sectional study in two European hospitals. Clinical and virological data were recorded; renal function was assessed by estimated glomerular filtrate rate, serum phosphate and creatinine, proteinuria, and albuminuria; bone involvement by spine and femur DEXA scan. RESULTS: In total, 565 patients included: 62 (18-91) years, 75% males, 92% Caucasian, 92% HBeAg-negative, 40% cirrhotic. Fifty-five percent of patients fulfilled age criterion (>60 years). Older patients had higher rates of cirrhosis (51% vs 26%, p<0.001), cardiovascular disease, and renal impairment. Thirty-six percent of patients met renal criteria, more commonly NA-experienced individuals (35% vs 21%, p=0.001); 17% had bone disease. Overall, 66% of patients had at least one criterion (71% if NA-experienced), 8% all three criteria, 28% age and renal criteria. CONCLUSIONS: Approximately two-thirds of patients receiving long-term tenofovir-disoproxil are candidates for an entecavir or tenofovir-alafenamide switch according to EASL recommendations.

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.

Deep-sequencing reveals broad subtype-specific HCV resistance mutations associated with treatment failure.

A percentage of hepatitis C virus (HCV)-infected patients fail direct acting antiviral (DAA)-based treatment regimens, often because of drug resistance-associated substitutions (RAS). The aim of this study was to characterize the resistance profile of a large cohort of patients failing DAA-based treatments, and investigate the relationship between HCV subtype and failure, as an aid to optimizing management of these patients. A new, standardized HCV-RAS testing protocol based on deep sequencing was designed and applied to 220 previously subtyped samples from patients failing DAA treatment, collected in 39 Spanish hospitals. The majority had received DAA-based interferon (IFN) α-free regimens; 79% had failed sofosbuvir-containing therapy. Genomic regions encoding the nonstructural protein (NS) 3, NS5A, and NS5B (DAA target regions) were analyzed using subtype-specific primers. Viral subtype distribution was as follows: genotype (G) 1, 62.7%; G3a, 21.4%; G4d, 12.3%; G2, 1.8%; and mixed infections 1.8%. Overall, 88.6% of patients carried at least 1 RAS, and 19% carried RAS at frequencies below 20% in the mutant spectrum. There were no differences in RAS selection between treatments with and without ribavirin. Regardless of the treatment received, each HCV subtype showed specific types of RAS. Of note, no RAS were detected in the target proteins of 18.6% of patients failing treatment, and 30.4% of patients had RAS in proteins that were not targets of the inhibitors they received. HCV patients failing DAA therapy showed a high diversity of RAS. Ribavirin use did not influence the type or number of RAS at failure. The subtype-specific pattern of RAS emergence underscores the importance of accurate HCV subtyping. The frequency of "extra-target" RAS suggests the need for RAS screening in all three DAA target regions.

Role of Biomarkers in Guiding Cure of Viral Hepatitis B.

Chronic hepatitis B (CHB) virus infection is a global public health threat affecting approximately 257 million individuals worldwide. Hepatitis B surface antigen (HBsAg) loss has been the classic endpoint to define functional cure and decrease the large pool of individuals with CHB. Current treatments with nucleos(t)ide analogues persistently suppress hepatitis B virus (HBV) deoxyribonucleic acid in most CHB patients, but rarely achieve functional cure. New viral biomarkers, such as quantitative HBsAg, hepatitis B core-related antigen, and HBV ribonucleic acid, and combinations of these markers have the potential role of guiding HBV cure by enabling selection of the best candidates for therapy, identifying individuals with a higher likelihood of achieving HBsAg loss, and helping in the design of studies with new drugs. However, some of the assays used to analyze these markers require standardization and improvements in the level of detection. Analysis of host biomarkers to assess the host immune response to HBV is also important, as the natural course of CHB is determined by the interplay between viral replication and the immune response. These biomarkers are, however, in an early phase of development.

Characterization of hepatitis B virus X gene quasispecies complexity in mono-infection and hepatitis delta virus superinfection.

BACKGROUND: Hepatitis delta virus (HDV) seems to strongly suppress hepatitis B virus (HBV) replication, although little is known about the mechanism of this interaction. Both these viruses show a dynamic distribution of mutants, resulting in viral quasispecies. Next-generation sequencing is a viable approach for analyzing the composition of these mutant spectra. As the regulatory hepatitis B X protein (HBx) is essential for HBV replication, determination of HBV X gene (HBX) quasispecies complexity in HBV/HDV infection compared to HBV mono-infection may provide information on the interactions between these two viruses. AIM: To compare HBV quasispecies complexity in the HBX 5' region between chronic hepatitis delta (CHD) and chronic HBV mono-infected patients. METHODS: Twenty-four untreated patients were included: 7/24 (29.2%) with HBeAg-negative chronic HBV infection (CI, previously termed inactive carriers), 8/24 (33.3%) with HBeAg-negative chronic hepatitis B (CHB) and 9/24 (37.5%) with CHD. A serum sample from each patient was first tested for HBV DNA levels. The HBX 5' region [nucleotides (nt) 1255-1611] was then PCR-amplified for subsequent next-generation sequencing (MiSeq, Illumina, United States). HBV quasispecies complexity in the region analyzed was evaluated using incidence-based indices (number of haplotypes and number of mutations), abundance-based indices (Hill numbers of order 1 and 2), and functional indices (mutation frequency and nucleotide diversity). We also evaluated the pattern of nucleotide changes to investigate which of them could be the cause of the quasispecies complexity. RESULTS: CHB patients showed higher median HBV-DNA levels [5.4 logIU/mL, interquartile range (IQR) 3.5-7.9] than CHD (3.4 logIU/mL, IQR 3-7.6) (P = n.s.) or CI (3.2 logIU/mL, IQR 2.3-3.5) (P < 0.01) patients. The incidence and abundance indices indicated that HBV quasispecies complexity was significantly greater in CI than CHB. A similar trend was observed in CHD patients, although only Hill numbers of order 2 showed statistically significant differences (CHB 2.81, IQR 1.11-4.57 vs CHD 8.87, 6.56-11.18, P = 0.038). There were no significant differences in the functional indices, but CI and CHD patients also showed a trend towards greater complexity than CHB. No differences were found for any HBV quasispecies complexity indices between CHD and CI patients. G-to-A and C-to-T nucleotide changes, characteristic of APOBEC3G, were higher in CHD and CI than in CHB in genotype A haplotypes, but not in genotype D. The proportion of nt G-to-A vs A-to-G changes and C-to-T vs T-to-C changes in genotype A and D haplotypes in CHD patients showed no significant differences. In CHB and CI the results of these comparisons were dependent on HBV genotype. CONCLUSION: The lower-replication CHD and CI groups show a trend to higher quasispecies complexity than the higher-replication CHB group. The mechanisms associated with this greater complexity require elucidation.