Shekhar Chakrabarti

Subgenotypes of hepatitis B virus genotype D (D1, D2, D3 and D5) in India: differential pattern of mutations, liver injury and occult HBV infection

Summary.  Hepatitis B genotype D (HBV/D) is the most widespread genotype and exists as at least five subgenotypes (HBV/D1–D5). However, little is known about the association of virological characteristics with clinical differences among HBV/D subgenotypes. To investigate the virological characteristics of these subgenotypes and their clinical implications, we selected a cohort of 109 genotype D infected individuals from the state of West Bengal, India, including 68 HBsAg positive patients and 41 with occult HBV infection. Among the HBsAg positive subjects 28 had chronic hepatitis B virus infection, 40 were asymptomatic carriers based on clinical examination, liver function test and ultrasonograph results. Overall, HBV/D1 was found in 17%, HBV/D2 in 29%, HBV/D3 in 34% and HBV/D5 in 20% of the cases. HBV/D1 was significantly associated with chronic liver disease (P = 0.01), and in this subgenotype A1896 (PreC mutations) were most common. Although BCP mutations (A/C1753 and T1762/A1764) were found to be frequently associated with HBV/D2 (33% and 33%) and D5 (47% and 59%), no apparent clinical correlation was observed. On the other hand, occult HBV infection was significantly associated with HBV/D3 infection, along with low level of BCP and PreC mutations and several non‐synonymous substitutions in the catalytic reverse transcriptase (RT) domain of polymerase gene. Similar nucleotide substitutions in the surface (S) gene region were observed from both northern and eastern Indian HBV/D3 isolates. In conclusion, HBV/D subgenotypes differ in their mutational patterns in the S, polymerase and the BCP/PreC regions that may influence their clinical outcomes.

Genetic Characterization of Hepatitis B Virus in Peripheral Blood Leukocytes: Evidence for Selection and Compartmentalization of Viral Variants with the Immune Escape G145R Mutation

ABSTRACT The compartmentalization of viral variants in distinct host tissues is a frequent event in many viral infections. Although hepatitis B virus (HBV) classically is considered hepatotropic, it has strong lymphotropic properties as well. However, unlike other viruses, molecular evolutionary studies to characterize HBV variants in compartments other than hepatocytes or sera have not been performed. The present work attempted to characterize HBV sequences from the peripheral blood leukocytes (PBL) of a large set of subjects, using advanced molecular biology and computational methods. The results of this study revealed the exclusive compartmentalization of HBV subgenotype Ae/A2-specific sequences with a potent immune escape G145R mutation in the PBL of the majority of the subjects. Interestingly, entirely different HBV genotypes/subgenotypes (C, D, or Aa/A1) were found to predominate in the sera of the same study populations. These results suggest that subgenotype Ae/A2 is selectively archived in the PBL, and the high prevalence of G145R indicates high immune pressure and high evolutionary rates of HBV DNA in the PBL. The results are analogous to available literature on the compartmentalization of other viruses. The present work thus provides evidence in favor of the compartment-specific abundance, evolution, and emergence of the potent immune escape mutant. These findings have important implications in the field of HBV molecular epidemiology, transmission, transfusion medicine, organ transplantation, and vaccination strategies.

Frequency and distribution of hepatitis B virus genotypes among eastern Indian voluntary blood donors: Association with precore and basal core promoter mutations

Aim:  To screen hepatitis B virus (HBV) genotypes and associated basal core promoter (BCP; T1762/A1764) and precore (PreC; A1896) mutations among the HBV surface antigen (HBsAg) positive voluntary blood donors in eastern India.

Tumor suppressor micro RNA miR-145 and onco micro RNAs miR-21 and miR-222 expressions are differentially modulated by Hepatitis B virus X protein in malignant hepatocytes

BackgroundHepatitis B Virus (HBV) X protein (HBx) is known to be involved in the initiation and progression of hepatocellular carcinoma (HCC) through modulation of host gene response. Alterations in miRNA expressions are frequently noted in HCC. This study is aimed to examine the role of HBx protein in the modulation of oncogenic miRNA-21, miRNA-222 and tumor suppressor miRNA-145 in malignant hepatocytes.MethodsExpressions of miRNA-21, miRNA-222 and miRNA-145 were measured in HepG2 cells transfected with HBx-plasmid (genotype D) and with full length HBV genome (genotype D) and also in stably HBV producing HepG2.2.15 cells using real time PCR. Their target mRNAs and proteins - PTEN, p27 and MAP3K - were analyzed by real time PCR and western blot respectively. miRNA expressions were measured after HBx/D mRNA specific siRNA treatment. The expressions of these miRNAs were analyzed in liver cirrhosis and HCC patients also.ResultsThe study revealed a down-regulation of miRNA-21 and miRNA-222 expressions in HBx transfected HepG2 cells, pUC-HBV 1.3 plasmid transfected HepG2 cells as well as in HepG2.2.15 cells. Down regulation of miRNA-21 and miRNA-222 expression was observed in patient serum samples. Down regulation of miRNA-145 expression was observed in HepG2 cells transiently transfected with HBx and pUC-HBV1.3 plasmid as well as in patient samples but the expression of miRNA-145 was increased in HepG2.2.15 cells. Target mRNA and protein expressions were modulated in HepG2 cells and in HepG2.2.15 cell line consistent with the modulation of miRNA expressions.ConclusionThus, HBx protein differentially modulated the expression of miRNAs. The study throws light into possible way by which HBx protein acts through microRNA and thereby regulates host functioning. It might suggest new therapeutic strategies against hepatic cancer.

Molecular Epidemiology and Clinical Significance of Hepatitis B Virus Genotypes, Core Promoter and Precore Mutations in Eastern India

Objectives: This unmatched case-control study aimed at determining the molecular epidemiology and clinical significance of HBV genotypes, core promoter (CP) and precore (PC) mutations in Eastern India. Methods: Serological, biochemical and molecular assays were used to examine antigens, ALT, genotypes, mutations and viremia among 106 inactive carriers and 183 chronic liver disease (CLD) patients. Results: Male gender (p < 0.001), HBeAg positivity (p = 0.050), high ALT (p < 0.001), high viremia (p < 0.001), CP mutations (p < 0.001), and genotypes A (p < 0.001) and C (p = 0.027) were significantly associated with CLD. Subjects infected with genotypes A and C had significantly higher prevalence of BCP mutations (p < 0.001), and low incidence of PC mutation (p < 0.001 and p = 0.047, respectively). Prevalence of genotype D was significantly higher among subjects with history of familial/childhood jaundice, while genotypes A and C were frequent among subjects with possible percutaneous exposure. Conclusions: Significant differences in risk factors and disease manifestation do exist among patients infected with different HBV genotypes. Genotypes A and C are frequently found among chronic liver disease patients, while genotype D is associated with inactive HBeAg-negative infections. This evaluation of clinical relevance of HBV genotypes, mutations and risk factors may be useful in disease prognosis, management and prevention strategies.

Analysis of hepatitis B virus X gene phylogeny, genetic variability and its impact on pathogenesis: Implications in Eastern Indian HBV carriers

HBx genetic variability was explored in the Eastern Indian population with low HCC incidence. DNase I sensitive HBV DNA was detected in 53% samples, which differed significantly between clinical groups (P<0.001). HBV genotypes A (Aa/A1), C (Cs/C1) and D (D1, D2, D3, D5) were detected in 37.5%, 18.7% and 43.7% samples respectively. Population specific signature HBx residues A(36), V(88), S(101) in Aa/A1 and residues P(41), Q(110) in D5 were detected. Mutations T(127), M(130) and I(131) were detected in 66.7%, 91% and 75% of genotype A, C and D5 samples respectively. Very low occurrence of HCC associated mutations (V(5)M/L, P(38)S, and H(94)Y) and absence of C-terminal deletions were observed. Our study shows that HBV genotype associated clinically important HBx variations may evolve and act distinctly in different geo-ethnic populations. Further studies on HBx functions from the perspective of genetic variability are essential for the better understanding of the clinical significance of HBV.

Expression of microRNA-155 correlates positively with the expression of Toll-like receptor 7 and modulates hepatitis B virus via C/EBP-β in hepatocytes

Effective recognition of viral infection and successive activation of antiviral innate immune responses are vital for host antiviral defence, which largely depends on multiple regulators, including Toll‐like receptors (TLRs) and microRNAs. Several early reports suggest that specific TLR‐mediated immune responses can control hepatitis B virus (HBV) replication and express differentially with disease outcome. Considering the versatile function of miR‐155 in the TLR‐mediated innate immune response, we aimed to study the association between miR‐155 and TLRs and their subsequent impact on HBV replication using both a HBV‐replicating stable cell line (HepG2.2.15) and HBV‐infected liver biopsy and serum samples. Our results showed that miR‐155 was suppressed during HBV infection and a subsequent positive correlation of miR‐155 with TLR7 activation was noted. Further, ectopic expression of miR‐155 in vitro reduced HBV load as evidenced from reduced viral DNA, mRNA and subsequently reduced level of secreted viral antigens (HBsAg and HBeAg). Our results further suggested that CCAAT/enhancer‐binding protein‐β (C/EBP‐β), a positive regulator of HBV transcription, was inhibited by miR‐155. Taken together, our study established a correlation between miR‐155 and TLR7 during HBV infection and also demonstrated in vitro that increased miR‐155 level could help to reduce HBV viral load by targeting C/EBP‐β.

Shift in the hepatitis B virus genotype distribution in the last decade among the HBV carriers from eastern India: Possible effects on the disease status and hbv epidemiology

In a previous study from eastern India, the prevalence of HBV/C has been increasing among the blood donors. In order to analyze whether there has been any shift in HBV genotype distributions in recent years, the HBV genotypes prevalent during the periods 2000–2002 (Group‐I; n = 176) and 2007–2009 (Group‐II; n = 203) were compared, with special attention to changes in the proportion of HBV/C. The rate of prevalence of the three HBV genotypes (A, C, and D; percent prevalence 19.9/21.6/58.5 in Group‐I vs. 31.0/28.6/40.4 in Group‐II) underwent significant changes with increases in HBV/A and HBV/C among the HBV carriers (0.002). Among the asymptomatic carriers, the prevalence of these two genotypes (P = 0.021 for HBV/A and P = 0.005 for HBV/C) was significantly high. A notable increase was also observed among the chronic liver disease cases. HBV/A increased significantly among the older age Groups (≥51 years), whereas the increase of HBV/C was significant among the younger age Groups (≤20 years). With the increase of HBV/A and HBV/C, the rates of basal core promoter double mutation (1762T/1764A) also increased considerably. Binary logistic regression analysis revealed that both HBV/A and 1762T/1764A mutations are predictors of chronic liver disease state over asymptomatic carrier state. Thus, this study highlights the possible influence of HBV genotype shift on the changing scenario of HBV epidemiology and disease in the population. J. Med. Virol. 85:1340–1347, 2013.

Hepatitis B virus X protein mediated suppression of miRNA-122 expression enhances hepatoblastoma cell proliferation through cyclin G1-p53 axis

BackgroundHepatitis B virus (HBV) X protein (HBx) reported to be associated with pathogenesis of hepatocellular carcinoma (HCC) and miR-122 expression is down regulated in HCC. Previous studies reported miR-122 targets cyclin G1 (CCNG1) expression and this in turn abolishes p53-mediated inhibition of HBV replication. Here we investigated the involvement of HBx protein in the modulation of miR-122 expression in hepatoblastoma cells.MethodsExpression of miR-122 was measured in HepG2 cells transfected with HBx plasmid (HBx-HepG2), full length HBV genome (HBV-HepG2) and in constitutively HBV synthesizing HepG2.2.15 cells. CCNG1 mRNA (a direct target of miR-122) and protein expressions were also measured in both HBx-HepG2, HBV-HepG2 cells and in HepG2.2.15 cells. miR-122 expressions were analyzed in HBx-HepG2, HBV-HepG2 and in HepG2.2.15 cells after treatment with HBx mRNA specific siRNA. Expressions of p53 mRNA and protein which is negatively regulated by CCNG1 were analyzed in HBx transfected HepG2 cells; X silenced HBx-HepG2 cells and X silenced HepG2.2.15 cells. HBx induced cell proliferation in HepG2 cells was measured by cell proliferation assay. Flow cytometry was used to evaluate changes in cell cycle distribution. Expression of cell cycle markers were measured by real time PCR.ResultsExpression of miR-122 was down regulated in HBx-HepG2, HBV-HepG2 and also in HepG2.2.15 cell line compared to control HepG2 cells. CCNG1 expression was found to be up regulated in HBx-HepG2, HBV-HepG2 cells and in HepG2.2.15 cells. Following siRNA mediated silencing of HBx expression; increased miR-122 levels were documented in HBx-HepG2, HBV-HepG2 and in HepG2.2.15 cells. HBx silencing in HBx-HepG2 and HepG2.2.15 cells also resulted in increased p53 expression. FACS analysis and assessment of expressions of cell cycle markers revealed HBx induced a release from G1/S arrest in HepG2 cells. Further, cell proliferation assay showed HBx promoted proliferation of HepG2 cell.ConclusionOur study revealed that HBx induced down regulation of miR-122 expression that consequently increased CCNG1 expression. This subsequently caused cell proliferation and release from G1/S arrest in malignant hepatocytes. The study provides the potential to utilize the HBx-miR-122 interaction as a therapeutic target to limit the development of HBV related HCC.

49 HEPATITIS B VIRUS X PROTEIN INDUCED MODULATION IN THE EXPRESSION OF MIR-222 IN MALIGNANT HEPATOCYTES IN VITRO

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