Chinmoy Kumar Panda

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.

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.

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.

Genetic variants of MAOB affect serotonin level and specific behavioral attributes to increase autism spectrum disorder (ASD) susceptibility in males

Serotonergic system participates in various developmental processes and modulation of behaviour. Autism Spectrum Disorder (ASD) is characterized by a range of behavioral symptoms scaling from mild to severe. Abnormal 5-HT synthesis and signalling, platelet hyperserotonemia and amelioration of repetitive behaviours by SSRI are some of the key findings, which reinforced the hypothesis that serotonergic genes might act as ASD susceptible genes. Therefore, genes encoding monoamine oxidases A/B (MAOA/MAOB) received special attention as these genes are located on the X-chromosome and the gene products are responsible for 5-HT degradation. In the present study, we conducted population-based association analysis of eight markers of MAOB with ASD in a study cohort of 203 cases and 236 controls form India and examined its effect on platelet 5-HT content and behaviour. Gender-specific changes were observed for the contrasting LD between pair of markers among cases and controls. Case-control analysis demonstrated over-distribution of major C allele of rs2283728 and rs2283727 in male and female ASD cases respectively. Haplotypic distribution and interaction among markers showed more robust effect in male cases. Interestingly, male ASD cases displayed higher platelet 5-HT content in comparison to the respective controls. Quantitative trait analysis revealed significant correlation of genetic variants and haplotypes of MAOB markers, rs1799836 and rs6324 with increased platelet 5-HT level and CARS scores for specific behavioral symptoms respectively in males. This study suggests that MAOB increases ASD risk in males, possibly through its sex-specific regulatory effect on 5-HT metabolism and behavior.