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.

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.

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.

Host-dependent editing of SARS-CoV-2 in COVID-19 patients.

A common trait among RNA viruses is their high capability to acquire genetic variability due to viral and host mechanisms. Next-generation sequencing (NGS) analysis enables the deep study of the viral quasispecies in samples from infected individuals. In this study, the viral quasispecies complexity and single nucleotide polymorphisms of the SARS-CoV-2 spike gene of coronavirus disease 2019 (COVID-19) patients with mild or severe disease were investigated using next-generation sequencing (Illumina platform). SARS-CoV-2 spike variability was higher in patients with long-lasting infection. Most substitutions found were present at frequencies lower than 1%, and had an A → G or T → C pattern, consistent with variants caused by adenosine deaminase acting on RNA-1 (ADAR1). ADAR1 affected a small fraction of replicating genomes, but produced multiple, mainly non-synonymous mutations. ADAR1 editing during replication rather than the RNA-dependent RNA polymerase (nsp12) was the predominant mechanism generating SARS-CoV-2 genetic variability. However, the mutations produced are not fixed in the infected human population, suggesting that ADAR1 may have an antiviral role, whereas nsp12-induced mutations occurring in patients with high viremia and persistent infection are the main source of new SARS-CoV-2 variants.

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.

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.

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.

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.

Different kinetics of viral replication and DNA integration in the main HIV-1 cellular reservoirs in the presence and absence of integrase inhibitors.

To compare the kinetics of integration, p24 production and equilibrium of the different HIV-DNA forms in human primary cells in the presence/absence of integrase-inhibitors (INIs) in vitro. Monocyte-derived-macrophages (MDMs), CD4+ T-cells and peripheral blood mononuclear cells (PBMCs) were infected with HIV-1 in the presence/absence of raltegravir and dolutegravir. HIV-DNA levels and p24 production were measured by qPCR and ELISA assays, respectively. In the absence of INIs, levels of HIV-DNA forms were initially very low, with an increase in the integration process starting at 3 dpi. HIV-DNA increased more slowly in MDMs than it did in CD4+ T-cells and PMBCs peaking at 21 dpi with a mean of 1580 (±890) and 615 (±37) copies/103 cells for proviral and unintegrated HIV-DNA, and 455,972 (±213,255) pg/mL of p24 at the same time point. In CD4+ T-cells the proviral HIV-DNA increased together with unintegrated HIV-DNA peaking at 7 dpi (583 ±â€¯261 and 338 ±â€¯254 copies/103 cells) when the p24 was 218,000 (±75,600) pg/mL. A similar trend was observed in PBMCs (494 ±â€¯361 and 350 ±â€¯123 copies/103 cells for proviral and unintegrated HIV-DNA, and p24 production of 149,400 ±â€¯131,800 pg/mL). Both INIs inhibited viral replication and integration in all the cell types that were tested, especially starting at 3 dpi. However, a small but measurable amount of HIV-DNA (<5 copies/103 cells) was still observed in treated-MDMs up to 30 dpi. In conclusion, our study showed differences in HIV-DNA kinetic integration between CD4+ T-cells and MDMs, which could explain the divergent kinetics of viral-replication. Both INIs inhibited HIV-1 integration and replication with no difference found between CD4+ T-cells and MDMs. However, residual HIV-DNA remained detectable up to 30 dpi in INI-treated MDMs although complete inhibition of HIV replication was achieved. The clinical significance of this minor DNA persistence deserves further investigation considering the role of macrophages as reservoirs.

HDV evolution-will viral resistance be an issue in HDV infection?

Hepatitis D virus (HDV) is a hepatotropic subviral infectious agent, obligate satellite of the Hepatitis B virus (HBV) and is highly related to viroids. HDV affects around 5% of the 257 million chronic HBV-carriers worldwide, leading to the most severe form of chronic viral hepatitis. Interferon alpha is the only approved treatment for chronic hepatitis D, albeit with low response rates (around 20%-30%). New antiviral strategies are currently under study. Due to the high viral evolution rates (10-3 to 10-4 substitutions/site/year) HDV forms an extremely complex viral population (quasispecies) that can be studied by Next-Generation Sequencing. Therefore, although specific viral resistance in HDV infection has not been reported, it cannot be completely discarded.

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.

Novel HBsAg mutations correlate with hepatocellular carcinoma, hamper HBsAg secretion and promote cell proliferation in vitro.

BACKGROUND: An impaired HBsAg-secretion can increase HBV oncogenic-properties. Here, we investigate genetic-determinants in HBsAg correlated with HBV-induced hepatocellular carcinoma (HCC), and their impact on HBsAg-secretion and cell-proliferation. METHODS: This study included 128 chronically HBV-infected patients: 23 with HCC (73.9% D; 26.1% A HBV-genotype), and 105 without cirrhosis/HCC (72.4% D, 27.6% A) as reference-group. The impact of mutations on HBsAg-secretion was assessed by measuring the ratio [secreted/intracellular HBsAg] until day 5 post-transfection. The impact of mutations on cell-cycle advancement was assessed by flow-cytometry. RESULTS: Two HBsAg mutations significantly correlated with HCC: P203Q (17.4% [4/23] in HCC vs 1.0% [1/105] in non-HCC, P=0.004); S210R (34.8% [8/23] in HCC vs 3.8% [4/105] in non-HCC, P <0.001); P203Q+S210R (17.4% [4/23] in HCC vs 0% [0/110] in non-HCC, P=0.001). Both mutations reside in trans-membrane C-terminal domain critical for HBsAg-secretion. In in-vitro experiments, P203Q, S210R and P203Q+S210R significantly reduced the ratio [secreted/intracellular HBsAg] compared to wt at each time-point analysed (P <0.05), supporting an impaired HBsAg-secretion. Furthermore, P203Q and P203Q+S210R increased the percentage of cells in S-phase compared to wt, indicating cell-cycle progression (P203Q:26±13%; P203Q+S210R:29±14%; wt:18%±9, P <0.01. Additionally, S210R increased the percentage of cells in G2/M-phase (26±8% for wt versus 33±6% for S210R, P <0.001). CONCLUSIONS: Specific mutations in HBsAg C-terminus significantly correlate with HBV-induced HCC. They hamper HBsAg-secretion and are associated with increased cellular proliferation, supporting their involvement in HCC-development. The identification of viral genetic markers associated with HCC is critical to identify patients at higher HCC-risk that may deserve intensive liver monitoring, and/or early anti-HBV therapy.