Hepatitis B Virus Quasispecies Research Articles

The Characteristic of HBV Quasispecies Is Related to Occult HBV Infection of Infants Born to Highly Viremic Mothers.

Although a combination of immunoprophylaxis and antiviral therapy can effectively prevent mother-to-child transmission (MTCT) of hepatitis B virus (HBV), a considerable number of infants born to highly viremic mothers still develop occult HBV infection (OBI). To uncover the virological factor and risk predictor for OBI in infants, we found that the diversity and complexity of maternal HBV quasispecies in the case group were lower than those in the control group. Mutations with significant differences between the two groups were most enriched in the NTCPbd and PreC regions. Genetic distance at the amino-acid level of the PreC region, especially the combination of three amino-acid mutations in the PreC region, could strongly predict the risk of OBI in infants. HBV quasispecies in OBI infants were highly complex, and the non-synonymous substitutions were mainly found in the RT and HBsAg regions. The sK47E (rtQ55R) and sP49L mutations in OBI infants might contribute to OBI through inhibiting the production of HBV DNA and HBsAg, respectively. This study found the potential virological factors and risk predictors for OBI in infants born to highly viremic mothers, which might be helpful for controlling OBI in infants.

Genetic variability of hepatitis B virus in acute and in different phases of chronic infection in Brazil

The selection pressure imposed by the host immune system impacts on hepatitis B virus (HBV) variability. This study evaluates HBV genetic diversity, nucleos(t)ide analogs resistance and HBsAg escape mutations in HBV patients under distinct selective pressures. One hundred and thirteen individuals in different phases of HBV infection were included: 13 HBeAg-positive chronic infection, 9 HBeAg-positive chronic hepatitis, 47 HBeAg-negative chronic infection (ENI), 29 HBeAg-negative chronic hepatitis (ENH) and 15 acute infected individuals. Samples were PCR amplified, sequenced and genetically analyzed for the overlapping POL/S genes. Most HBV carriers presented genotype A (84/113; 74.3%), subgenotype A1 (67/84; 79.7%), irrespective of group, followed by genotypes D (20/113; 17.7%), F (8/113; 7.1%) and E (1/113; 0.9%). Clinically relevant mutations in polymerase (tL180M/M204V) and in the Major Hydrophilic Region of HBsAg (sY100C, T118A/M, sM133T, sD144A and sG145R) were observed. Our findings, however, indicated that most polymorphic sites were located in the cytosolic loops (CYL1-2) and transmembrane domain 4 (TMD4) of HBsAg. Lower viral loads and higher HBV genetic diversity were observed in ENI and ENH groups (p < 0.001), suggesting that these groups are subjected to a higher selective pressure. Our results provide information on the molecular characteristics of HBV in a diverse clinical setting, and may guide future studies on the balance of HBV quasispecies at different stages of infection.

The central nervous system is a potential reservoir and possible origin of drug resistance in hepatitis B infection

BackgroundThe significance of hepatitis B virus (HBV) in cerebrospinal fluid (CSF) is unclear. MethodsSynchronous serum and CSF samples were collected from 13 patients. HBV DNA, full-length genome, quasispecies, phylogenetic tree, compartmentalization and mutation of the reverse transcriptase (RT) region were performed based on PCR and sequencing methods. ResultsHBV DNA was detected in the CSF of 3 antiviral-naïve individuals and 1 individual after successful antiviral therapy. Complete full-length HBV genomes were isolated from the CSF of 5 individuals, including 2 with undetectable serum HBV DNA. Ten individuals exhibited distinct CSF-serum quasispecies, 8 harbored independent CSF-serum genetic compartmentalization and phylogenetic trees, and 5 lamivudine/entecavir-associated resistance mutations only in the CSF. The frequencies of rtL180M and rtM204I/V mutations in both serum and CSF were higher in HIV-HBV-coinfected individuals than in the HBV-monoinfected ones (serum: rtL180M: 3.9% vs. 0, P = 0.004; rtM204I/V: 21.3% vs. 0, P < 0.001; CSF: rtL180M: 7.6% vs. 0, P = 0.026; rtM204I/V 7.6% vs. 1.6%, P = 0.097). ConclusionCSF is a potential HBV reservoir, and HBV in CSF harbors distinct evolution and mutation characteristics from those in serum. HIV infection increases the possibility of HBV rtL180M and rtM204I/V mutations in both serum and CSF.

Low host immune pressure may be associated with the development of hepatocellular carcinoma: a longitudinal analysis of complete genomes of the HBV 1762T, 1764A mutant.

It has been reported that hepatitis B virus (HBV) double mutations (A1762T, G1764A) are an aetiological factor of hepatocellular carcinoma (HCC). However, it is unclear who is prone to develop HCC, among those infected with the mutant. Exploring HBV quasispecies, which are strongly influenced by host immune pressure, may provide more information about the association of viral factors and HCC. Nine HCC cases and 10 controls were selected from the Long An cohort. Serum samples were collected in 2004 and 2019 from subjects with HBV double mutations and the complete genome of HBV was amplified and sequenced using next-generation sequencing (NGS). The Shannon entropy values increased from 2004 to 2019 in most cases and controls. There was no significant difference in mean intrahost quasispecies genetic distances between cases and controls. The change in the values of mean intrahost quasispecies genetic distances of the controls between 2004 and 2019 was significantly higher than that of the cases (P<0.05). The viral loads did not differ significantly between cases and controls in 2004(p=0.086) but differed at diagnosed in 2019 (p=0.009). Three mutations occurring with increasing frequency from 2004 to 2019 were identified in the HCC cases, including nt446 C→G, nt514 A→C and nt2857T→C. Their frequency differed significantly between the cases and controls (P<0.05). The change in the values of mean intrahost quasispecies genetic distances in HCC was smaller, suggesting that HBV in HCC cases may be subject to low host immune pressure. Increasing viral loads during long-term infection are associated with the development of HCC. The novel mutations may increase the risk for HCC.

Compartmentalisation of Hepatitis B virus X gene evolution in hepatocellular carcinoma microenvironment and the genotype-phenotype correlation of tumorigenicity in HBV-related patients with hepatocellular carcinoma

ABSTRACT Hepatitis B virus (HBV) exists as quasispecies (QS). However, the evolutionary characteristics of haplotypes of HBV X gene in the hepatocellular carcinoma (HCC) microenvironment remain unclear. Mutations across X gene are essential for the tumorigenicity of HBV X protein (HBx). However, the functional phenotypes of many mutant HBx remain unknown. This study aims to compare the characteristics of X gene evolution between tumour and non-tumour tissues in HCC patients and investigate the tumorigenic phenotype of HBx harbouring mutation T81P/S101P/L123S. This study included 24 HCC patients. Molecular cloning of X gene was performed to analyse characteristics of haplotypes in liver tissues. HCC cell lines stably expressing wild-type or mutant HBx and subcutaneous tumour xenograft mouse model were used to assess HBx-T81P/S101P/L123S tumorigenicity. The mean heterogeneity of HBV QS across X gene in tumour tissues was lower than that in non-tumour tissues. A location bias was observed in X gene clones with genotype C or D in tumour tissues compared to those with genotype B. Mutations in genotype-C or – D clones were mainly clustered in the dimerization region and aa110-aa140 within the transactivation region. A novel mutation combination at residues 81, 101 and 123 was identified in tumour tissues. Further, HBx-T81P/S101P/L123S promotes cell proliferation and increases genomic instability, which was mediated by MYC. This study elucidates the compartmentalized evolution patterns of HBV X gene between intra tumour and non-tumour tissues in HCC patients and provides a new mechanism underlying HBV-driven hepatocarcinogenesis, suggesting a potential viral marker for monitoring HCC.

Sparse logistic regression revealed the associations between HBV PreS quasispecies and hepatocellular carcinoma

BackgroundChronic infection with hepatitis B virus (HBV) has been proved highly associated with the development of hepatocellular carcinoma (HCC).AimsThe purpose of the study is to investigate the association between HBV preS region quasispecies and HCC development, as well as to develop HCC diagnosis model using HBV preS region quasispecies.MethodsA total of 104 chronic hepatitis B (CHB) patients and 117 HBV-related HCC patients were enrolled. HBV preS region was sequenced using next generation sequencing (NGS) and the nucleotide entropy was calculated for quasispecies evaluation. Sparse logistic regression (SLR) was used to predict HCC development and prediction performances were evaluated using receiver operating characteristic curves.ResultsEntropy of HBV preS1, preS2 regions and several nucleotide points showed significant divergence between CHB and HCC patients. Using SLR, the classification of HCC/CHB groups achieved a mean area under the receiver operating characteristic curve (AUC) of 0.883 in the training data and 0.795 in the test data. The prediction model was also validated by a completely independent dataset from Hong Kong. The 10 selected nucleotide positions showed significantly different entropy between CHB and HCC patients. The HBV quasispecies also classified three clinical parameters, including HBeAg, HBVDNA, and Alkaline phosphatase (ALP) with the AUC value greater than 0.6 in the test data.ConclusionsUsing NGS and SLR, the association between HBV preS region nucleotide entropy and HCC development was validated in our study and this could promote the understanding of HCC progression mechanism.

Comparative analysis of HBV basic core promoter/pre-core gene mutations and viral quasispecies diversity in HIV/HBV co-infected and HBV mono-infected patients.

Human immunodeficiency virus (HIV)/hepatitis B virus (HBV) co-infection accelerates the progression of HBV-related liver diseases. HBV basic core promoter (BCP)/pre-core (preC) gene mutations may be one of the most important risk factors. In this study, atotal of 230 patients were recruited, and 199 patients whose HBV BCP/preC gene were successfully amplified and sequenced, including 99 HIV/HBV co-infected and 100 HBV mono-infected patients. Next-generation sequencing was used for detection of BCP/preC mutations which were then compared in patients with different HBV genotypes and different HBeAg statuses, and 1% and 20% cutoff values were defined to evaluate the mutations. HBV quasispecies diversity was also compared in HIV/HBV co-infected and HBV mono-infected patients. Among the patients infected with HBV genotype C and HBeAg-negative status, the frequency of A1762T/G1764A double mutations was significantly lower in HIV/HBV co-infected patients than in HBV mono-infected patients (53.3% vs. 100.0%, P = 0.008) regardless of the 1% or 20% cutoff value level. However, A1762T/G1764A double mutations did not differ in the other groups (P >0.05). Viral quasispecies diversity was lower in HIV/HBV co-infected patients than in HBV mono-infected patients (P Keywords: human immunodeficiency virus, hepatitis B virus; mutations; viral quasispecies; next-generation sequencing.

The dynamic changes of HBV quasispecies diversity in infancy after immunoprophylaxis failure: a prospective cohort study

BackgroundPrevious works have observed that younger infants with chronic hepatitis B virus (HBV) infection are more responsive to antiviral treatment. However, the underlying mechanism remains unclear. In this study, the dynamic changes of HBV quasispecies in infants with immunoprophylaxis failure were investigated to provide virological explanations for clinical management on infantile antiviral therapy.MethodsThirteen 7-month-old infants with immunoprophylaxis failure and their mothers were enrolled from a prospective cohort, and 8 of them were followed up to 3 years old. The sequences of HBV quasispecies were analyzed by the full-length genome clone-based sequencing, and compared among mothers and their infants at different ages.ResultsThe results revealed that the complexity, mutation frequency and genetic distance of HBV quasispecies decreased significantly at full-length, partial open reading frames and regulatory regions of HBV genome at nucleotide level in 7-month-old infants comparing with their mothers, whereas increased significantly to near the maternal level when infants grew up to 3 years old. Furthermore, similar changes were also found in Core, PreS2, RT and P regions of HBV genome at amino acid level, especially for potential NAs-resistant mutants in RT region and immune-escape mutants in Core and PreS2 regions.ConclusionsThis study uncovered the evolution of HBV quasispecies in infancy after mother-to-child transmission, which may provide the virological evidence for explaning that younger children are more responsive to antiviral therapy.

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

BACKGROUNDDifferent 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.AIMTo establish a next-generation sequencing (NGS) methodology for analyzing serum HBV-RNA and comparing it with DNA quasispecies.METHODSThirteen 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.RESULTSNo 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.CONCLUSIONOur 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.

Increased hepatitis B virus quasispecies diversity is correlated with liver fibrosis progression.

Host immune response and viral factors are involved in disease progression in patients with chronic hepatitis B virus (HBV) infection. However, the relationship between HBV quasispecies and liver fibrosis progression remains unclear. In this study, 447 patients with chronic HBV infection, including 239 with chronic hepatitis B (CHB), 104 with liver cirrhosis (LC) and 104 with hepatocellular carcinoma (HCC) were enrolled. The 239 CHB patients were divided into groups F1, F2, and F3 according to liver fibrosis score. Four fragments of the HBV genome were determined and analyzed using next-generation sequencing. Specific mutations, such as A1762T, G1764A and G1896A, in the BCP/PC region were more common in patients with advanced liver disease and formed the majority of the viral quasispecies pool in patients with LC and HCC. The viral complexity and diversity increased as the fibrosis progressed, especially in patients with CHB who were comparable in age but at different stages of fibrosis. Patients with early-stage fibrosis experienced higher purifying selection pressure in the four sequenced regions, whereas different protein-coding region experienced different negative selection with disease progression. HBV quasispecies diversity may increase fibrosis progression in CHB patients with aging under immune selection.

Comparison and Correlation of Genetic Variability of the HBV Pre-S Region in HIV/HBV Co-Infected Patients: Quasispecies Perspective.

BackgroundHuman immunodeficiency virus (HIV)/hepatitis B virus (HBV) co-infection can accelerate HBV-induced liver disease. A previous study showed that variation in the HBV pre-S region and quasispecies heterogeneity (Sn, mean genetic distance, dS, dN, and dS/dN) are both related to HBV-induced terminal liver disease in HBV mono-infection. Currently, data are lacking on quasispecies variation of the HBV pre-S region in HIV/HBV co-infection. Investigating the quasispecies variation of the HBV pre-S region and its related factors in HIV/HBV co-infection will help to better explore the pathogenic mechanism of HIV/HBV co-infection.MethodsAccording to the HIV antibody results obtained before treatment, chronic HBV-infected patients were divided into HIV/HBV co-infected and HBV mono-infected groups. The clinical characteristics of all patients were collected, and DNA was extracted from the serum. The HBV pre-S region was amplified by nested PCR and was further TA cloned. BioEdit software 7.0 was used for sequence alignment with reference to the standard sequence of the matched HBV genotype. We used 1:1 propensity score matching (PSM) to control for baseline confounding factors between the two groups.ResultsAfter 1:1 PSM, we identified 100 patients with similar propensities: 50 HIV/HBV co-infected patients and 50 HBV mono-infected patients. HBV quasispecies indices were lower in the HIV/HBV co-infected group than those in the HBV mono-infected group. A significant correlation was observed between all quasispecies indices and soluble cluster of differentiation 163 (sCD163) and interleukin-18 (IL-18) in the HIV/HBV co-infected group; however, this phenomenon was not found in the HBV mono-infected group.ConclusionCombined HIV infection reduces quasispecies heterogeneity in the HBV pre-S region, and the quasispecies heterogeneity is related to the sCD163 and IL-18 levels.

Comparison of Serum Hepatitis B Virus RNA Levels and Quasispecies Evolution Patterns between Entecavir and Pegylated-Interferon Mono-treatment in Chronic Hepatitis B Patients.

Hepatitis B virus (HBV) RNA may independently predict virological and serological response. This study aimed to compare dynamic changes in serum HBV RNA levels and HBV quasispecies evolution patterns between entecavir and pegylated-interferon mono-treatment in chronic hepatitis B patients and to determine the clinical significance during treatment. TaqMan real-time PCR was used for quantitative analysis. HBV RNA levels were retrospectively determined in serial serum samples from 178 chronic hepatitis B patients who received either entecavir or pegylated-interferon treatment. Both serum HBV DNA and RNA quasispecies were analyzed via next-generation sequencing. Receiver operating characteristics (ROC) analysis was performed to evaluate the prediction value of individual biomarkers for hepatitis B e antigen (HBeAg) seroconversion. Patients who received pegylated-interferon treatment showed stronger declines in HBV RNA levels than did those who received entecavir treatment. Serum HBV RNA levels were lower in patients with subsequent HBeAg seroconversion. At baseline, the level of HBV RNA was better than other indicators in predicting HBeAg seroconversion. Moreover, the predictive value of serum HBV RNA levels was better in the entecavir group. Baseline HBV RNA exhibited a significantly higher genetic diversity than HBV DNA and had a significant decline after 4 weeks of entecavir treatment. Higher baseline genetic diversity may result in a better outcome in pegylated-interferon-treated patients. Serum HBV RNA levels showed different decline kinetics, and HBV RNA quasispecies showed different evolution patterns in entecavir and pegylated-interferon mono-treatment. Taken together, serum HBV RNA may serve as a promising biomarker of HBeAg seroconversion in patients during antiviral treatment.

Quasispecies characteristic in “a” determinant region is a potential predictor for the risk of immunoprophylaxis failure of mother-to-child-transmission of sub-genotype C2 hepatitis B virus: a prospective nested case–control study

ObjectiveThis study was performed to explore the correlation between the characteristics of hepatitis B virus (HBV) quasispecies in HBV-infected pregnant women and the risk of immunoprophylaxis failure for their infants.DesignIn...

Intra-host diversity of hepatitis B virus during mother-to-child transmission: the X gene may play a key role in virus survival in children after transmission.

Mother-to-child transmission of hepatitis B virus (HBV) is the main route of transmission in Asia, and characterization of HBV quasispecies is needed to further understand virus evolution and adaptation. To understand changes in HBV during mother-to-child transmission, we enrolled nine pairs of mothers and children in the study, including a set of twins. Three groups were infected with HBV genotype C, and six groups were infected with HBV genotype B. The full-length HBV genome was amplified by PCR from serum samples before antiviral treatment, the whole viral genomes from each pair were sequenced, and the complexity and diversity of the quasispecies were analyzed. The entropy of transmitted HBV in children was found to be lower than their mothers, suggesting that there was a bottleneck effect during HBV transmission from the mother to the child. Selective evolution was shown by calculating πN and πS in the whole genomes, and the highest values were obtained for the X gene, which plays a role in viral replication and immune escape. All genotype C patients and only one genotype B pair had a πN/πS greater than 1 ratio, indicating that positive selection had occurred. In addition, quasispecies were found to be different between the twin children despite having the same mother, indicating that virus evolution is host-specific.

Machine-learning based patient classification using Hepatitis B virus full-length genome quasispecies from Asian and European cohorts

Chronic infection with Hepatitis B virus (HBV) is a major risk factor for the development of advanced liver disease including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The relative contribution of virological factors to disease progression has not been fully defined and tools aiding the deconvolution of complex patient virus profiles is an unmet clinical need. Variable viral mutant signatures develop within individual patients due to the low-fidelity replication of the viral polymerase creating ‘quasispecies’ populations. Here we present the first comprehensive survey of the diversity of HBV quasispecies through ultra-deep sequencing of the complete HBV genome across two distinct European and Asian patient populations. Seroconversion to the HBV e antigen (HBeAg) represents a critical clinical waymark in infected individuals. Using a machine learning approach, a model was developed to determine the viral variants that accurately classify HBeAg status. Serial surveys of patient quasispecies populations and advanced analytics will facilitate clinical decision support for chronic HBV infection and direct therapeutic strategies through improved patient stratification.

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

BACKGROUNDHepatitis 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.AIMTo compare HBV quasispecies complexity in the HBX 5’ region between chronic hepatitis delta (CHD) and chronic HBV mono-infected patients.METHODSTwenty-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.RESULTSCHB 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.CONCLUSIONThe 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.

Viral quasispecies of hepatitis B virus in patients with YMDD mutation and lamivudine resistance may not predict the efficacy of lamivudine/adefovir rescue therapy.

The association between hepatitis B virus (HBV) quasispecies (QS) and the efficacy of nucleos(t)ide analog therapy is currently not well defined, particularly in the case of lamivudine (LAM)/adefovir (ADV) combination rescue therapy for patients with chronic HBV infection (CHB) presenting with LAM resistance. In the present study, 16 CHB patients with the rtM204I/V mutation in the tyrosine-methionine-aspartate-aspartate motif of the C domain of the polymerase gene who switched to LAM/ADV treatment due to LAM resistance were assessed. HBV DNA was isolated from these patients and the reverse transcriptase (RT) region was sequenced. The QS heterogeneity and distribution was analyzed, the mutation sites were recorded and the phylogenetic trees were constructed. The results indicated that QS heterogeneity and distribution in the RT and S regions were not significantly different between responders (RS) and non-RS (NRS) at baseline (P>0.05), except for the higher frequency of a dominant strain in the RT region at the nucleotide level in the RS group (P=0.039). In addition, in NRS, no significant difference in QS heterogeneity or distribution in these regions was identified at six months vs. the baseline. Furthermore, although in the non-responder group the frequency of the LAM resistance-associated mutations (rtM204V/I) decreased at 6 months compared with the baseline, it did not disappear in any of the patients after six months of treatment. Analysis of individual patients did not indicate any consistent selection of specific HBV mutants during LAM/ADV rescue therapy. In conclusion, the baseline HBV QS within the RT and S regions may not be a valid predictor of the response to LAM/ADV rescue treatment in CHB patients with LAM resistance.

Early changes in quasispecies variant after antiviral therapy for chronic hepatitis B.

Hepatitis B virus (HBV) polymerase gene is targeted by nucleos(t)ide analogues (NUC), but it is unclear how HBV quasispecies of whole genome changes during early period of NUC treatment. To understand the unknown region of drug sensitivity and treatment resistance, HBV quasispecies of whole genome during early period of NUC treatment was examined using ultra‑deep sequencing. Eleven patients with chronic HBV infection who received NUC treatment were enrolled in the current study. Viral DNA was extracted from serum samples before and early period of NUC treatment. Polymerase chain reaction analysis was subsequently performed on the DNA products. The viral quasispecies of the entire genome was analyzed by ultra‑deep sequencing. The regions and positions corresponding to the changes in the quasispecies were investigated before and early period of NUC treatment. The secondary structure changes were predicted by mutations/substitutions detected using Lasergene Protean v14.1 software. The frequency of quasispecies variants increased significantly in the polymerase domain from before to early period of NUC treatment (3.08±1.28 vs.3.51±1.47%, P<0.008), particularly the reverse transcription (RT) domain (3.76±1.25 vs.4.52±1.37%, P<0.012). In addition, increased variation detected from HBsAg domain showed statistically significant during NUC treatment (6.81±3.26 vs.7.81±3.26%, P<0.040). The amino acid (aa) mutations/substitutions were detected and compared from before to early period of treatment. Interestingly, most of them were located in the RT region (RT1 motif: aa21‑aa51) and small S region in the early duration of NUC treatment. Furthermore, several mutation patterns, such as cI97L and cP130T showed alterations in the secondary structure and predicted antigenicity of HBV protein. Although the HBV whole genome can be affected by NUC treatment, RT 1 motif region and small S region are more sensitive to the early period of NUC treatment. This study suggested the initial changes of HBV quasispecies might affect the long‑term drug sensitivity and resistance to NUC treatment.

Quasispecies variant of pre-S/S gene in HBV-related hepatocellular carcinoma with HBs antigen positive and occult infection

BackgroundHepatocellular carcinoma (HCC) can develop in patients who are negative for the hepatitis B surface antigen (HBsAg) in serum but positive for hepatitis B virus (HBV) DNA in the liver, referred to as occult HBV infection (OBI). Previous reports showed that HBV variants in OBI-related HCC are different from those in HBsAg-positive HCC. In the present study, HBV quasispecies based on the pre-S/S gene in OBI-related HCC patients were examined by high throughput sequencing and compared with those in HBsAg-positive HCC.MethodsNineteen tissue samples (9 OBI-related and 10 HBsAg-positive non-cancerous tissues) were collected at the time of surgery at Kobe University Hospital. The quasispecies with more than 1% variation in the pre-S/S region were isolated and analysed by ultra-deep sequencing.ResultsThere were no significant differences in the major HBV populations, which exhibit more than 20% variation within the entire pre-S/S region, between OBI-related HCC and HBsAg-positive HCC. However, the prevalences of major populations with pre-S2 region mutations and of minor populations with polymerized human serum albumin-binding domain mutations were significantly higher in OBI-related HCC than in HBsAg-positive HCC. Moreover, the major variant populations associated with the B-cell epitope, located within the pre-S1 region, and the a determinant domain, located in the S region, were detected frequently in HBsAg-positive HCC. The minor populations of variants harbouring the W4R, L30S, Q118R/Stop, N123D and S124F/P mutations in the pre-S region and the L21F/S and L42F/S mutations in the S region were detected more frequently in OBI-related HCC than in HBsAg-positive HCC.ConclusionsUltra-deep sequencing revealed that the B-cell epitope domain in the pre-S1 region and alpha determinant domain in the S region were variable in HBsAg-positive HCC, although the quasispecies associated with the pre-S2 region were highly prevalent in OBI-related HCC.Trial registrationRef: R000034382/UMIN000030113; Retrospectively registered 25 November 2017.

Evolution of full-length genomes of HBV quasispecies in sera of patients with a coexistence of HBsAg and anti-HBs antibodies

Although the evolutionary changes of viral quasispecies are correlated to the pathological status of a disease, little is known in the coexistence of hepatitis B surface antigen (HBsAg) and antibodies to these antigens (anti-HBs). To examine evolutionary changes in hepatitis B virus (HBV) and their relationship to the coexistence of HBsAg and anti-HBs antibodies, HBV genomes in patients with a coexistence of HBsAg and anti-HBs antibodies (experimental group) and HBsAg positive without anti-HBs (control group) were assessed. Our results showed that quasispecies diversity was significantly higher in the experimental group for large HBsAg (LHBsAg), middle HBsAg (MHBsAg), and HBsAg genes. LHBsAg harbored dN/dS values eight times higher in the experimental group; however, the mean dN/dS ratios in genes HbxAg, Pol and PreC/C of the experimental patients had an opposite trend. Phylogenetic trees in the experimental group were more complex than the control group. More positive selection sites, mutations and deletions were observed in the experimental group in specific regions. Furthermore, several amino acid variants in epitopes were potentially associated with the immune evasion. In conclusion, cumulative evolutionary changes in HBV genome that facilitate immune evasion provide insights into the genetic mechanism of a coexistence of HBsAg and anti-HBs antibodies.