Rajesh Panigrahi

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.

Anti-hepatitis B core antigen testing with detection and characterization of occult hepatitis B virus by an in-house nucleic acid testing among blood donors in Behrampur, Ganjam, Orissa in southeastern India: implications for transfusion.

BackgroundOccult hepatitis B virus (HBV) infection might transmit viremic units into the public blood supply if only hepatitis B surface antigen (HBsAg) testing is used for donor screening. Our aim was to evaluate the prevalence of occult HBV infection among the HBsAg negative/antiHBc positive donations from a highly HIV prevalent region of India.MethodsA total of 729 HBsAg negative donor units were included in this study. Surface gene and precore region were amplified by in house nucleic acid test (NAT) for detection of occult HBV infection and surface gene was analyzed after direct sequencing.ResultsA total of 220 (30.1%) HBsAg negative donors were antiHBc positive, of them 66 (30%) were HBV DNA positive by NAT. HBV DNA positivity among 164 antiHBc only group, was 27.1% and among 40 antiHBs positive group was 30.0%. HBV/D (93.3%) was predominant and prevalence of both HBV/C and HBV/A was 3.3%. Single or multiple amino acids substitutions were found in 95% samples.ConclusionThus, a considerable number of HBV infected donors remain undiagnosed, if only HBsAg is used for screening. Addition of antiHBc testing for donor screening, although will lead to rejection of a large number of donor units, will definitely eliminate HBV infected donations and help in reducing HBV transmission with its potential consequences, especially among the immunocompromised population. The HBV genetic diversity found in this donor population are in accordance with other parts of India.

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.

Variations in the Functional Domain of Basal Core Promoter of Hepatitis B Virus Among Eastern Indian Patients With Prevalence of Genotypes A, C, and D Among the Same Ethnic Population

Mutations in the basal core promoter (BCP) and precore (PC) regions are associated with persistent and intermittently high hepatitis B virus (HBV) replication in several patients. The variability in the functional domains of BCP and PC region of HBV and their association with disease progression and clinical outcome were assessed in Eastern India, an unique region where three HBV genotypes, A, D, and C are prevalent among the same ethnic group. PCR amplification and direct sequencing of BCP and PC region was done on sera obtained from 130 HBsAg positive subjects with different clinical presentations. Associations of the apparent risk factors with clinical advancement were evaluated by statistical methods including multiple logistic regression analyses (MLR). HBV genotype A was present in 33.08%, C in 25.38%, and D in 41.54% cases. Genotypes A and C were associated with higher rate of T1762/A1764 mutations than the most predominant genotype D. HBeAg negative state was associated with considerably higher rate of C1753 mutation. T1762/A1764 along with C1753 was common among cirrhosis and T1762/A1764 without C1753 was frequent among chronic liver disease cases. No significant association was found between A1896 point mutation and clinical status. Multivariate analysis revealed that T1762/A1764 double mutation, HBV/A, age ≥25 years, C1753 and A1899 were critical factors for clinical advancement while age ≥25 years and C1753 as significant predictor for cirrhosis in comparison with chronic liver disease. In conclusion, the analysis of the BCP variability may help in monitoring the progression towards advanced liver disease in Eastern Indian patients. J. Med. Virol. 83:253–260, 2011.

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.

Impaired expression of type I and type II interferon receptors in HCV-associated chronic liver disease and liver cirrhosis.

Purpose Chronic Hepatitis C Virus (HCV)-infected patients with liver cirrhosis (LC) respond poorly to interferon-alpha (IFN-α) and ribavirin (RBV) combination therapy, but the reason for this is unclear. We previously reported that HCV-infection induces endoplasmic reticulum (ER) stress and autophagy response that selectively down regulates the type I IFN-α receptor-1 (IFNAR1) and RBV transporters (CNT1 and ENT1), leading to IFN-α/RBV resistance. The goal of this study is to verify whether an increase in ER stress and autophagy response is also associated with the reduced expression of IFNAR1 and RBV transporters in chronic HCV-infected patients. Methods Primary human hepatocytes (PHH) were infected with cell culture grown HCV particles (JFH-ΔV3-Rluc). HCV replication was confirmed by the detection of viral RNA by RT-qPCR and HCV-core protein by Western blotting. The ER stress and autophagy response and expression of IFN receptors and RBV transporters in HCV infected PHH and liver tissues derived from patients were measured by Western blotting. Result HCV infection of PHH showed impaired expression of IFNAR1, IFNγR1 (Type II IFN receptor) and RBV transporters but not IL10Rβ (Type III IFN-λ receptor). ER stress markers (BiP, IRE1α and peIF2α) and autophagy response (LC3II, Beclin 1 and ATG5) were induced in HCV infected chronic liver disease (CLD) and LC patients. Liver biopsies (CLD) show a 50% reduced expression of IFNAR1 and RBV transporters. Furthermore, the expression of IFNAR1 and RBV transporters was impaired in almost all LC patients. Conclusion HCV infection induces ER stress and autophagy response in infected PHH and chronically infected liver tissues. The expression of IFNAR1, IFNγR1 and RBV transporters were significantly impaired in CLD and cirrhotic livers. Our study provides a potential explanation for the reduced response rate of IFN-α and RBV combination therapy in HCV infected patients with liver cirrhosis.

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.