Runu Chakravarty

Hepatitis B virus subgenotypes D1 and D3 are prevalent in Pakistan

BackgroundAs the hepatitis B genotyping is important for assessing its clinical implications and geographical distribution, the sub-genotypes have been found useful for determination of specific genomic markers related to hepatocarcinogenesis. In Pakistan, there is no reported data on molecular evolutionary analysis of HBV. A study was, therefore, much needed to evaluate the spectra of mutations present in the strains prevalent here.Findingsto confirm specificity of PCR typing, phylogenetic analysis of the pre-S1 region and the divergence was studied through 13 sequences of 362 bp (accession number EF432765 – EF432777). A total of 315 serum samples, selected from HBsAg positive patients representing the major ethnic groups, residing in Karachi, Sindh were tested for genotyping. Genotype D (219/315) was found to be the most prevalent (70%) amongst our patients. The rest of the genotypes A and a mixture of A and D (AD) were distributed as 20%, and 10% respectively. Phylogenetic tree demonstrated clustering of 11 samples with subgenotype D1 sequences and the remaining two strains on a branch within D3 samples. All samples intermixed with strains from other countries and were found to be closely related to Indian, Iranian and Egyptian HBV strains with 98.7 – 99.0% homology.ConclusionThis study confirms the predominance of genotype D in southeastern Asia and presence of subgenotypes DI and D3 in the Pakistani infected patients. More studies are required to investigate the reason for fewer inclusions of D3 compared to the D1 in Pakistani HBV strains.

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.

Presence of hepatitis B surface antigen mutant G145R DNA in the peripheral blood leukocytes of the family members of an asymptomatic carrier and evidence of its horizontal transmission

An asymptomatic carrier and all six of his family members were detected positive for HBV DNA in their peripheral blood leukocytes (PBL), by polymerase chain reaction. Direct sequencing of the amplified DNA revealed that the HBV DNA from the carrier and his wife was of subtype ayw. Interestingly, the amplified HBV DNA from the five other members of the family was found to be not only of subtype adw but also contained G to A mutation at nucleotide position 587. This indicates the presence of established vaccine escape mutant of the virus (G145R) and suggests two different sources of infection within the family. Southern blot hybridization of EcoR1 digested DNA from PBL indicated presence of HBV DNA, integrated into cellular DNA and also in the form of free viral DNA. The study not only establishes the persistence of surface mutant G145R HBV DNA, within the PBL of HBsAg negative individuals from the non-vaccinated random population, but also suggests possible horizontal transmission of the mutant among the family members although none of the family members has received immunoprophylaxis against HBV or had clinically apparent disease or any other known risk factors of HBV infection. As all of them were seronegative for HBsAg/antiHBc, the presence of G145R mutant in the PBL signaled possibility of spread of the vaccine escape mutant virus by blood transfusion, unsafe injection practices or through sexual root.

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.

Nucleic Acid Sequence Analysis of Basal Core Promoter/Precore/Core Region of Hepatitis B Virus Isolated from Chronic Carriers of the Virus from Kolkata, Eastern India: Low Frequency of Mutation in the Precore Region

Objective: The aim of the present study was to characterize the predominant hepatitis B virus (HBV) strains and their molecular variants present in the HBV isolates of the different genotypes found among the chronic carriers of the virus in our community. Methods: Precore/core and core promoter regions of HBV DNA were amplified by polymerase chain reaction and then subjected to direct sequencing. Of the 64 hepatitis B surface antigen (HBsAg)-positive chronic HBV carriers investigated, 44 were HBeAg negative and 20 were HBeAg positive. Results: In addition to genotype D, which was the predominant genotype, 12 genotype C (18.7%) and 6 genotype A (9.4%) were also detected. Presence of T at nt 1858 has often been related to the development of precore stop mutation at nt 1896, while that of C has been related to the development of 1762–1764 double mutation. In our study group, 39 of the 44 HBeAg-negative samples have T1858. The precore stop codon mutation was found in only 8 (18%) of the HBeAg-negative samples. More than half of the HBeAg-negative samples had wild-type sequence in the precore region. The core promoter region could be sequenced from 40 samples, and 1762–1764 double mutation was detected in 13 (32.5%) of them. No significant changes could be detected in the core amino acid sequence of these isolates. Conclusion: The pattern of core promoter and precore mutation of HBV isolates in the present study is atypical and not in accordance with reports from other parts of the world, where genotype D and genotype C with T at codon 1858 are common.

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.

G1862T Mutation among Hepatitis B Virus-Infected Individuals: Association with Viral Genotypes and Disease Outcome in Kolkata, Eastern India

Objective: To study the prevalence of G1862T mutation in hepatitis B virus (HBV) isolates among Eastern Indian patients and its relationship with genotypes, HBeAg status and disease manifestation. Methods: HBV DNA was isolated from patients, amplified by nested PCR and sequenced directly. Results: Of the 102 patients, 32 were HBeAg positive and 70 HBeAg negative; 55, 24 and 23 isolates were infected with genotypes D, A and C, respectively. G1862T was detected in 18 samples, 15 (83%) of them belonged to genotype A (subgenotype HBV/A1), 3 (17%) to genotype D. This mutation was more frequent in HBeAg-negative than in HBeAg-positive patients (21 vs. 9%), whereas in HBV/A1 it was as common in HBeAg-positive as in HBeAg-negative patients and significantly associated with T1762/A1764 mutation. The mean viral load was lower in patients with G1862T mutation. Furthermore, this mutation was common in various clinical outcomes. Conclusion: In our community, G1862T mutation was predominantly found in HBV/A1 isolates irrespective of HBeAg status. Moreover this mutation could not be correlated to the clinical outcome. These findings indicate that the G1862T mutation is probably a part of the natural variability of HBV/A1.

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.