Qi-Ming Gong

Evolutionary patterns of hepatitis B virus quasispecies under different selective pressures: correlation with antiviral efficacy

Objective To investigate the evolution of hepatitis B virus (HBV) quasispecies (QS) within the reverse transcriptase (RT) region during the early stage of entecavir treatment and its impact on virological response, and to compare evolutionary patterns under different selective pressures. Methods 31 patients with chronic hepatitis B receiving entecavir (17 responders and 14 partial responders according to the HBV DNA levels at week 48) and 25 patients receiving lamivudine (14 responders and 11 non-responders) as controls were included. An average of 26 clones (2892 total from both groups) spanning the RT region per sample was sequenced. Results QS complexity and diversity, in addition to alanine aminotransferase and HBV DNA levels, were comparable between responders and partial responders at baseline. However, QS complexity in responders at week 4 was statistically lower than that in partial responders at the nucleotide level (0.6494 vs 0.7723, p=0.039). Net changes in diversity as well as the viral nucleotide substitution rate of responders were higher than those of partial responders, and both correlated with virological responses at both week 48 and the final visit (mean: 28 months). A preliminary model of QS evolution variables predicted 16 of 17 responders and 13 of 14 partial responders in the entecavir group. Despite significant differences between responders to entecavir and responders to lamivudine at week 4, the characteristics of QS were quite similar between partial responders to entecavir and non-responders to lamivudine. Conclusions The evolutionary patterns of HBV RT QS differ between responders and partial responders during the early stage of entecavir treatment. Characteristics of HBV QS evolution during the first 4 weeks contribute to the prediction of long-term virological responses. The similar patterns of HBV RT QS in partial responders and non-responders receiving different nucleoside analogues may imply a novel mechanism of drug resistance, which warrants further investigation.

N-glycosylation mutations within hepatitis B virus surface major hydrophilic region contribute mostly to immune escape

BACKGROUND & AIMS HBV immune escape represents a challenge to prevention, diagnosis, and treatment of hepatitis B. Here, we analyzed the molecular and clinical characteristics of HBV immune escape mutants in a Chinese cohort of chronically infected patients. METHODS Two hundred sixteen patients with HBsAg and anti-HBs were studied, with one hundred eighty-two HBV carriers without anti-HBs as a control group. Recombinant HBsAg bearing the most frequent N-glycosylation mutations were expressed in CHO and HuH7 cells. After confirming N-glycosylation at the most frequent sites (129 and 131), together with inserted mutations, functional analysis were performed to study antigenicity and secretion capacity. RESULTS One hundred twenty-three patients had the wild-type HBs gene sequence, 93 patients (43%) had mutants in the major hydrophilic region (MHR), and 47 of the 93 patients had additional N-glycosylation mutations, which were transmitted horizontally to at least 2 patients, one of whom was efficiently vaccinated. The frequency of N-glycosylation mutation in the case group was much higher than that of the control group (47/216 vs. 1/182). Compared with wild-type HBsAg, HBsAg mutants reacted weakly with anti-HBs using a chemiluminescent microparticle enzyme immunoassay. Native gel analysis of secreted virion in supernatants of transfected HuH7 cells indicated that mutants had better virion enveloping and secretion capacity than wild-type HBV. CONCLUSIONS Our results suggest that specific HBsAg MHR N-glycosylation mutations are implicated in HBV immune escape in a high endemic area.

Clinical and molecular characterization of Wilson's disease in China: identification of 14 novel mutations

BackgroundWilsons disease (WND) is a rare autosomal recessive disorder. Here we have evaluated 62 WND cases (58 probands) from the Chinese Han population to expand our knowledge of ATP7B mutations and to more completely characterize WND in China.MethodsThe coding and promoter regions of the ATP7B gene were analyzed by direct sequencing in 62 Chinese patients (58 probands) with WND (male, n = 37; female, n = 25; age range, 2 ~ 61 years old).ResultsNeurologic manifestations were associated with older age at diagnosis (p < 0.0001) and longer diagnostic delay (p < 0.0001). Age at diagnosis was also correlated with urinary copper concentration (r = 0.58, p < 0.001). Forty different mutations, including 14 novel mutations, were identified in these patients. Common mutations included p.Arg778Leu (31.9%) and p.Pro992Leu (11.2%). Homozygous p.Arg778Leu and nonsense mutation/frameshift mutations were more often associated with primary hepatic manifestations (p = 0.0286 and p = 0.0383, respectively) and higher alanine transaminase levels at diagnosis (p = 0.0361 and p = 0.0047, respectively). Nonsense mutation/frameshift mutations were also associated with lower serum ceruloplasmin (p = 0.0065).ConclusionsWe identified 14 novel mutations and found that the spectrum of mutations of ATP7B in China is quite distinct from that of Western countries. The mutation type plays a role in predicting clinical manifestations. Genetic testing is a valuable tool to detect WND in young children, especially in patients younger than 8 years old. Four exons (8, 12, 13, and 16) and two mutations (p.Arg778Leu, p.Pro992Leu) should be considered high priority for cost-effective testing in China.

Association study of IFNAR2 and IL10RB genes with the susceptibility and interferon response in HBV infection.

Summary.  A recent genome‐wide association study discovered that two polymorphisms, interferon (IFN) alpha receptor 2 (IFNAR‐2) F8S and interleukin 10 receptor (IL10RB) K47E, were associated with susceptibility to hepatitis B virus (HBV) infection in Africa. Here, we reevaluate the effects of the two polymorphisms on HBV susceptibility in the Chinese Han population, and extended the study to look at their association with IFN response in chronic hepatitis B (CHB). We included 341 patients with CHB and 341 unrelated controls presenting with asymptotic HBV self‐limited infection, who were well matched in age and sex. In the CHB group, 101 patients had been treated with peg‐IFN‐alpha‐2a for 48 weeks and followed up for 24 weeks to determine the clinical response, resulting 34 individuals with sustained virological response (SVR) and 67 individuals with nonsustained response (NR). Subgroups in the CHB group were divided according to the viral loads, HBeAg and maternal HBsAg status. The association with the susceptibility to HBV infection was only observed for IL10RB K47E when we compared the individuals with persistent HBV infection through nonmaternal transmission to the controls with asymptomatic self‐limited HBV infection. Further, we found that the IFNAR2‐8SS genotype was associated with HBeAg negative patients (OR = 0.316, 95% CI: 0.121–0.825, P = 0.019) and that the IFNAR2‐8F allele was associated with the risk to high viral loads (OR = 1.667, 95% CI: 1.148–2.420, P = 0.007). In addition, the IFNAR2‐8FF genotype predisposed to higher MxA gene induction and correlated with sustained IFN response (OR = 0.348, 95% CI: 0.129–0.935, P = 0.036). Haplotype analysis based on polymorphisms of three single‐nucleotide polymorphisms, MxA −88 G/T, IFNAR‐2 F8S and IL10RB K47E showed that the haplotype distribution was significantly different between the SVR and NR groups (P = 0.040). This study suggests that IFNAR2 may play an important role in determining IFN response and clinical phenotypes of HBV infection in the Chinese Han population.

Comparison of Next-Generation Sequencing and Clone-Based Sequencing in Analysis of Hepatitis B Virus Reverse Transcriptase Quasispecies Heterogeneity

ABSTRACT We previously reported that, based on clone-based sequencing (CBS), hepatitis B virus (HBV) heterogeneity within the reverse transcriptase (RT) region was a predictor of antiviral efficacy. Here, by comparing ultradeep pyrosequencing (UDPS), i.e., next-generation sequencing (NGS), with CBS in characterizing the genetic heterogeneity of HBV quasispecies within the RT region, we evaluated the performance of UDPS in the analysis of HBV viral populations. HBV genomic DNA was extracted from serum samples from 31 antiviral treatment-naive patients with chronic hepatitis B. The RT region quasispecies were analyzed in parallel using CBS and UDPS. Characterization of quasispecies heterogeneity was conducted using bioinformatics analysis. Quasispecies complexity values were calculated with the formula Sn = −Σ i (pi lnpi )/lnN. The number of qualified strains obtained by UDPS was much larger than that obtained by CBS (P < 0.001). Pearson analysis showed that there was a positive correlation of quasispecies complexity values at the nucleotide level for the two methods (P < 0.05), while the complexity value derived from UDPS data was higher than that derived from CBS data (P < 0.001). Study of the prevalences of variations within the RT region showed that CBS detected an average of 9.7 ± 1.1 amino acid substitutions/sample and UDPS detected an average of 16.2 ± 1.4 amino acid substitutions/sample. The phylogenetic analysis based on UDPS data showed more genetic entities than did that based on CBS data. Viral heterogeneity determination by the UDPS technique is more sensitive and efficient in terms of low-abundance variation detection and quasispecies simulation than that by the CBS method, although imperfect, and thus sheds light on the future clinical application of NGS in HBV quasispecies studies.

Serum WFA+‐M2BP levels for evaluation of early stages of liver fibrosis in patients with chronic hepatitis B virus infection

Accurate evaluation of liver fibrosis is crucial for predicting progression of chronic hepatitis B virus (HBV) infection. We assessed the utility of a novel fibrosis glycobiomarker Wisteria floribunda agglutinin‐positive Mac‐2‐binding protein (WFA+‐M2BP) for evaluating liver fibrosis and disease progression in patients with chronic HBV infection.

Assessment of Specific Antibodies to F Protein in Serum Samples from Chinese Hepatitis C Patients Treated with Interferon plus Ribavarin

ABSTRACT The hepatitis C virus (HCV) alternate reading frame protein or F protein of the HCV 1b genotype is a double-frameshift product of the HCV core protein. In order to assess the presence of antibodies specific for F protein and their clinical relevance in sera from HCV patients, we produced recombinant F protein and core protein of the HCV 1b genotype in Escherichia coli. An enzyme-linked immunosorbent assay was developed using purified recombinant HCV core, F protein, and a 99-residue synthetic F peptide (F99). The seroprevalences of anticore, anti-F protein, and anti-F99 synthetic peptide were 95%, 68%, and 36%, respectively, in 168 HCV patients. The prevalence of anti-F antibodies did not correlate with viral load, genotype, or alanine aminotransferase level. Interferon combination therapy induced a decline in the level of anti-F antibodies in 55 responders (P < 0.01). Thirteen responders (24%) lost their anti-F recombinant protein antibodies, and 17 (31%) lost their anti-F synthetic peptide antibodies, whereas no decrease was observed for the 17 nonresponders. These changes were significant between responders and nonresponders (P < 0.05). Meanwhile, no change was found in the anticore antibody titer of the 72 treated patients. The percentage of anti-F-protein-negative patients (15/15 [100%]) who achieved a sustained virological response (SVR) was higher than that of the anti-F-positive patients (70%) (P < 0.05). Based on these findings, HCV F protein elicits a specific antibody response other than the anticore protein response. Our data also suggest that the presence and level of anti-F antibody responses might be influenced by the treatment (interferon plus ribavirin) and associated with an SVR in Chinese hepatitis C patients.

Evolution of wild type and mutants of the YMDD motif of hepatitis B virus polymerase during lamivudine therapy

Background and Aim:  Long‐term lamivudine treatment for chronic hepatitis B virus (HBV) infection induces the emergence of lamivudine resistant HBV YMDD mutant strains. The aim of the present study was to observe the clone evolution of YMDD wild type and mutant strains in pretreatment and post‐treatment samples during lamivudine therapy and analyze their clinical significance.

Prediction of virological response by pretreatment hepatitis B virus reverse transcriptase quasispecies heterogeneity: the advantage of using next-generation sequencing

Prediction of antiviral efficacy prior to treatment remains largely unavailable. We have previously demonstrated the clinical value of on-treatment hepatitis B virus (HBV) reverse transcriptase (RT) quasispecies (QS) evolution patterns. In this study, we aimed to elucidate the relevance for prediction of pretreatment HBV RT QS characteristics by comparing the performance of next-generation sequencing (NGS) and clone-based Sanger sequencing (CBS). Thirty-six lamivudine-treated patients were retrospectively studied, including 18 responders and 18 non-responders. CBS and NGS data of pretreatment serum HBV were used to generate RT QS genetic complexity and diversity scores, according to our previous studies. The ability of both methods to predict responsiveness was evaluated with receiver operating characteristic (ROC) curves. A cut-off value was generated on the basis of prediction ability. Responders had significantly higher pretreatment RT QS genetic complexity and diversity (in the first two parts, which overlapped with the S gene, at both the nucleotide and amino acid levels) than non-responders by NGS-based testing. NGS-based algorithms predicted response better than CBS in the ROC curve analysis. The mean distance of the second contig had the highest area under the curve (AUC) value. When the cut-off value was set to 0.007186, the difference between survival curves was significant (p 0.0090). Pretreatment HBV RT QS heterogeneity in the overlapping region of the RT and S genes could be a predictor of antiviral efficacy. NGS improves the predictions of virological outcomes relative to CBS algorithms. This may have important implications for the clinical management of subjects chronically infected with HBV.

Early serum hepatitis B virus large surface protein level: a stronger predictor of virological response to peginterferon alfa-2a than that to entecavir in HBeAg-positive patients with chronic hepatitis B.

BACKGROUND The response rate to antiviral therapy varies greatly among individuals, and its prediction is still very challenging. OBJECTIVES To evaluate the usefulness of serum hepatitis B virus large surface protein (LHBs) levels compared with HBsAg in prediction of the antiviral treatment effect. STUDY DESIGN Quantification of LHBs, HBsAg and HBV DNA was carried out at baseline and during antiviral therapy (weeks 4, 12, 24, 36 and 48) in HBeAg-positive patients treated with peginterferon alfa-2a (n = 21) or entecavir (n = 41). RESULTS The serum LHBs concentration was correlated positively with HBV DNA and HBsAg (r = 0.635 and 0.588, respectively). LHBs and HBV DNA levels decreased significantly in a biphasic manner and HBsAg level tended to decrease slowly in both treatment groups. In peginterferon alfa-2a group, the cutoff of 88.46 ng/ml in serum LHBs at week 4 gave the best AUC ( = 0.96) with positive and negative predictive values of 88.9% and 100%, in association with virological response (VR). Serum LHBs level at week 4 also showed an association with VR in entecavir group (AUC 0.78). The predictive model incorporating LHBs, HBsAg and HBV DNA could discriminate VR at baseline (AUC 0.79) and showed an association with serological response (SR) at week 12 (AUC 0.80) in peginterferon alfa-2a group. CONCLUSIONS On-treatment quantification of serum LHBs may be a more useful parameter for predicting VR in patients on peginterferon alfa-2a than those on entecavir. Combining LHBs, HBsAg and HBV DNA can predict VR and SR more effectively and earlier.