A pilot study on some critical immune elements in HBV infection: evidence of Alpha-1 Antitrypsin as an immunological biomarker.

Aim: This study is an attempt to screen the key immune elements that participate during HBV infection and the related pathways that are modulated. Background: The pathogenesis of Hepatitis B virus and the corresponding clinical manifestations in the host are primarily immune-mediated. Methods: This study utilizes a PCR array to screen immune-related genes that are differentially expressed in the presence of the virus in HBV replicating HepG2.2.15 cells as compared to control HepG2 cells. The significantly up-regulated genes were subjected to bioinformatic analysis utilizing GSEA and STRING. Additionally, ELISA was used to corroborate the levels of Alpha 1 antitrypsin (AAT) from patients' sera. Results: The expressions of 31% of the studied genes were significantly up-regulated (> 2-fold, p<0.05) in HepG2.2.15 cells compared to controls, and this included the SERPINA1, FN1, IL1R2, LBP, LY96, LYZ and PROC genes. When they were clustered based on biological processes, signaling pathways, and disease progression, the genes related to biotic stimulus, complement-coagulation cascades, and fibrosis, respectively, showed the highest (p<0.05) enrichment. Analysis of patients' sera by ELISA revealed that the serum AAT (SERPINA1) levels were significantly higher in asymptomatic carriers and in patients with chronic liver disease than in controls (p<0.05). Moreover, SERPINA1 levels were also positively correlated with the levels of serum ALT (r=0.4495, p<0.05) among HBV infected patients. Conclusion: The current study highlights the key immune elements and pathways that are modulated during HBV infection and proposes the possible use of AAT as a non-invasive immunological biomarker to follow the progression of liver disease.

Lamivudine plus tenofovir versus lamivudine plus adefovir for the treatment of hepatitis B virus in HIV-coinfected patients, starting antiretroviral therapy.

Background: Combination of tenofovir disoproxil fumarate (TDF), lamivudine (3TC) and efavirenz (EFV) is preferred in the treatment of HIV/hepatitis B virus (HBV) coinfection. We postulated that a HBV active nucleoside reverse transcriptase (RT) inhibitor/nucleotide RT inhibitor backbone of adefovir dipivoxil (ADV) +3TC would be as effective as TDF +3TC for the Indian population. Objective: ADV + 3TC could be an alternative option for these HIV/HBV coinfected individuals, preserving the dually active TDF + 3TC as second-line nucleoside backbone following failure of the first-line ART. Materials and Methods: This randomised control trial (CTRI/2012/03/002471) was carried out at the ART Centre of Calcutta School of Tropical Medicine, India. Seventy-eight (39 on each arm) treatment-naïve HIV/HBV coinfected patients were randomised to receive either the combination of lamivudine + tenofovir + EFV or lamivudine + adefovir + zidovudine + EFV and followed up for 120 weeks. Results: Median age of the study participants was 36 years (21-62), majority were male (61/78; 78.2%) and heterosexually (39/78; 50%) infected. Baseline characteristics were identical in both arms. There was no statistically significant difference in median aspartate aminotransferase (37 vs. 29.5 U/L), alanine aminotransferase (ALT) (36 vs. 34.5 U/L), ALT normalisation rate (80 vs. 70%), AST to platelet ratio index (0.45 vs. 0.33), CD4 count (508 vs. 427 cells/mm3), HBV DNA suppression (81.8 vs. 70%), hepatitis B e antigen loss (9 vs. 5%), hepatitis B surface antigen seroclearance rate (6.06 vs. 18.75%) and death (3 vs. 3) at 120 weeks between TDF (n = 33) and ADV (n = 32), respectively. Conclusions: Adefovir plus lamivudine is an effective alternative of tenofovir plus lamivudine in long-term HBV treatment outcome in HIV/HBV coinfected patients.

Host transcription factor Speckled 110 kDa (Sp110), a nuclear body protein, is hijacked by hepatitis B virus protein X for viral persistence.

Promyelocytic leukemia nuclear bodies (PML-NB) are sub-nuclear organelles that are the hub of numerous proteins. DNA/RNA viruses often hijack the cellular factors resident in PML-NBs to promote their proliferation in host cells. Hepatitis B virus (HBV), belonging to Hepadnaviridae family, remains undetected in early infection as it does not induce the innate immune response and is known to be the cause of several hepatic diseases leading to cirrhosis and hepatocellular carcinoma. The association of PML-NB proteins and HBV is being addressed in a number of recent studies. Here, we report that the PML-NB protein Speckled 110 kDa (Sp110) is SUMO1-modified and undergoes a deSUMOylation-driven release from the PML-NB in the presence of HBV. Intriguingly, Sp110 knockdown significantly reduced viral DNA load in the culture supernatant by activation of the type I interferon-response pathway. Furthermore, we found that Sp110 differentially regulates several direct target genes of hepatitis B virus protein X (HBx), a viral co-factor. Subsequently, we identified Sp110 as a novel interactor of HBx and found this association to be essential for the exit of Sp110 from the PML-NB during HBV infection and HBx recruitment on the promoter of these genes. HBx, in turn, modulates the recruitment of its associated transcription cofactors p300/HDAC1 to these co-regulated genes, thereby altering the host gene expression program in favor of viral persistence. Thus, we report a mechanism by which HBV can evade host immune response by hijacking the PML-NB protein Sp110, and therefore, we propose it to be a novel target for antiviral therapy.

Occult hepatitis B virus infection in HIV positive patients at a tertiary healthcare unit in eastern India.

Occult HBV infection (OBI), defined by the presence of HBV DNA in absence of hepatitis B surface antigen (HBsAg), is a significant concern in the HIV-infected population. Of 441 HIV+/HBsAg- patients analyzed, the overall prevalence of OBI was 6.3% (28/441). OBI was identified in 21 anti-HBc positives (17.8%), as well as among those who lacked any HBV-specific serological markers (2.2%). Comparison with HIV/HBV co-infection revealed that the levels of CD4, ALT, and HBV DNA were significantly lower during occult infection. Discrete differences were also observed with respect to quasispecies divergence. Additionally, subgenotype D1 was most frequent in occult infection, while D2 was widespread during chronic infection. The majority (~90%) of occult D1 sequences had the sQ129R mutation in the surface gene. This study highlights several distinct features of OBI in India and underscores the need for additional HBV DNA screening in HIV-positive individuals.

Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line.

BACKGROUND: Toll like receptors (TLRs) play an important role in innate immunity and various studies suggest that TLRs play a crucial role in pathogenesis of hepatitis B virus (HBV) infection. The present study aims in looking into the status of crucial host and viral gene expression on inciting TLR7. METHODS: The transcription of TLR7 pathway signaling molecules and HBV DNA viral load were quantified by Real Time-PCR after stimulation of TLR7 with its imiquimod based ligand, R837. Cell cycle analysis was performed using flow-cytometry. Expression of TLR7 and chief cell cycle regulator governing G1/S transition, p53 was also seen in liver biopsysss samples of CHB patients. HBV induced alteration in histone modifications in HepG2 cells and its restoration on TLR7 activation was determined using western blot. RESULTS: The TLR7 expression remains downregulated in HepG2.2.15 cells and in liver biopsy samples from CHB patients. Interestingly HBV DNA viral load showed an inverse relationship with the TLR7 expression in the biopsy samples. We also evaluated the anti-viral activity of R837, an agonist of TLR7. It was observed that there was a suppression of HBV replication and viral protein production upon TLR7 stimulation. R837 triggers the anti-viral action probably through the Jun N-terminal Kinase (JNK) pathway. We also observed a downregulation of histone H3K9Me3 repression mark upon R837 treatment in HBV replicating HepG2.2.15 cells, mimicking that of un-infected HepG2 cells. Additionally, the G1/S cell cycle arrest introduced by HBV in HepG2.2.15 cells was released upon ligand treatment. CONCLUSION: The study thus holds a close insight into the changes in hepatocyte micro-environment on TLR7 stimulation in HBV infection.

Role of RNA secondary structure in emergence of compartment specific hepatitis B virus immune escape variants.

AIM: To investigate the role of subgenotype specific RNA secondary structure in the compartment specific selection of hepatitis B virus (HBV) immune escape mutations. METHODS: This study was based on the analysis of the specific observation of HBV subgenotype A1 in the serum/plasma, while subgenotype A2 with G145R mutation in the peripheral blood leukocytes (PBLs). Genetic variability found among the two subgenotypes was used for prediction and comparison of the full length pregenomic RNA (pgRNA) secondary structure and base pairings. RNA secondary structures were predicted for 37 °C using the Vienna RNA fold server, using default parameters. Visualization and detailed analysis was done using RNA shapes program. RESULTS: In this analysis, using similar algorithm and conditions, entirely different pgRNA secondary structures for subgenotype A1 and subgenotype A2 were predicted, suggesting different base pairing patterns within the two subgenotypes of genotype A, specifically, in the HBV genetic region encoding the major hydrophilic loop. We observed that for subgenotype A1 specific pgRNA, nucleotide 358U base paired with 1738A and nucleotide 587G base paired with 607C. However in sharp contrast, in subgenotype A2 specific pgRNA, nucleotide 358U was opposite to nucleotide 588G, while 587G was opposite to 359U, hence precluding correct base pairing and thereby lesser stability of the stem structure. When the nucleotides at 358U and 587G were replaced with 358C and 587A respectively (as observed specifically in the PBL associated A2 sequences), these nucleotides base paired correctly with 588G and 359U, respectively. CONCLUSION: The results of this study show that compartment specific mutations are associated with HBV subgenotype specific alterations in base pairing of the pgRNA, leading to compartment specific selection and preponderance of specific HBV subgenotype with unique mutational pattern.

Baseline characteristics of HIV & hepatitis B virus (HIV/HBV) co-infected patients from Kolkata, India.

BACKGROUND & OBJECTIVES: Hepatitis B virus (HBV) and HIV co-infection has variable prevalence worldwide. In comparison to HBV mono-infection, the course of chronic HBV infection is accelerated in HIV/HBV co-infected patients. the present study was carried out to analyse the baseline characteristics (clinical, biochemical, serological and virological) of treatment naïve HIV/HBV co-infected and HIV mono-infected patients. METHODS: Between July 2011 and January 2013, a total number of 1331 HIV-seropositive treatment naïve individuals, enrolled in the ART Centre of Calcutta School of Tropical Medicine, Kolkata, India, were screened for hepatitis B surface antigen (HBsAg). A total of 1253 HIV mono-infected and 78 HIV/HBV co-infected patients were characterized. The co-infected patients were evaluated for HBeAg and anti-HBe antibody by ELISA. HIV RNA was quantified for all co-infected patients. HBV DNA was detected and quantified by real time-PCR amplification followed by HBV genotype determination. RESULTS: HIV/HBV co-infected patients had proportionately more advanced HIV disease (WHO clinical stage 3 and 4) than HIV mono-infected individuals (37.1 vs. 19.9%). The co-infected patients had significantly higher serum bilirubin, alanine aminotransferase (ALT), alkaline phosphatase and ALT/platelet ratio index (APRI). CD4 count was non-significantly lower in co-infected patients. Majority (61.5%) were HBeAg positive with higher HIV RNA (P<0.05), HBV DNA (p<0.001) and APRI (p<0.05) compared to those who were HBeAg negative. HBV/D was the predominant genotype (73.2%) and D2 (43.7%) was the commonest subgenotype. INTERPRETATION & CONCLUSIONS: HIV/HBV co-infected patients had significantly higher serum bilirubin, ALT, alkaline phosphatase and lower platelet count. HBeAg positive co-infected patients had higher HIV RNA and HBV DNA compared to HBeAg negative co-infected patients. Prior to initiation of antiretroviral treatment (ART) all patients should be screened for HBsAg to initiate appropriate ART regimen.

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

BACKGROUND: Hepatitis 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. METHODS: Expression 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. RESULTS: Expression 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. CONCLUSION: Our 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.

Enhancement of PCR Detection Limit by Single-Tube Restriction Endonuclease-PCR (RE-PCR).

BACKGROUND: Polymerase chain reaction (PCR) is widely used in biological research and diagnostics because of its high sensitivity and specificity. However, the sensitivity of PCR is strongly influenced by topological characteristics of the template. Supercoiled templates are known to inhibit PCR, whereas linearized forms of the same supercoiled templates facilitate PCR. OBJECTIVES: This study was conducted to compare the PCR efficiency of circular supercoiled DNA templates to their restriction endonuclease (RE)-mediated linearized forms. Additionally, we also evaluated the possibility of RE digestion of the circular supercoiled templates within the complete PCR buffer. METHODS: Following a systematic approach, we demonstrated that circular supercoiled templates could be efficiently linearized by RE in the complete PCR buffer itself. This allowed linearization of circular supercoiled templates and their subsequent amplification in the PCR buffer in a single-tube format. RESULTS: Using this extremely simple RE-PCR approach, we documented up to tenfold increases in detection efficiency of PCR with two different circular supercoiled templates of clinical origin, including an international calibration standard. CONCLUSIONS: This inexpensive and easy approach to increasing PCR sensitivity can be easily adapted to any standard PCR protocol aimed at amplifying circular supercoiled genomes. Apart from its application in the development of sensitive clinical diagnostic PCR assays for a large number of organisms, this method could also prove to be very useful in simplifying the existing protocols for other applications where pre-PCR restriction digestion is required, such as mutation detection, genotyping, and selective template amplification.

Anti-viral role of toll like receptor 4 in hepatitis B virus infection: An in vitro study.

AIM: Toll like receptors plays a significant anti-viral role in different infections. The aim of this study was to look into the role of toll like receptor 4 (TLR4) in hepatitis B virus (HBV) infection. METHODS: Real time PCR was used to analyze the transcription of TLR4 signaling molecules, cell cycle regulators and HBV DNA viral load after triggering the HepG2.2.15 cells with TLR4 specific ligand. Nuclear factor (NF)-κB translocation on TLR4 activation was analyzed using microscopic techniques. Protein and cell cycle analysis was done using Western Blot and FACS respectively. RESULTS: The present study shows that TLR4 activation represses HBV infection. As a result of HBV suppression, there are several changes in host factors which include partial release in G1/S cell cycle arrest and changes in host epigenetic marks. Finally, it was observed that anti-viral action of TLR4 takes place through the NF-κB pathway. CONCLUSION: The study shows that TLR4 activation in HBV infection brings about changes in hepatocyte microenvironment and can be used for developing a promising therapeutic target in future.

Virological Characteristics of Acute Hepatitis B in Eastern India: Critical Differences with Chronic Infection.

Hepatitis B Virus (HBV) manifests high genetic variability and is classifiable into ten genotypes (A-J). HBV infection can lead to variable clinical outcomes, ranging from self-limiting acute hepatitis to active chronic hepatitis, cirrhosis and hepatocellular carcinoma. The present study characterizes HBV strains circulating among patients with acute (AHB) and chronic HBV infection (CHB). Among a total of 653 HBsAg positive cases, 40 manifested acute infection. After sequencing the surface(S), basal core promoter/pre-core(BCP/PC) and the X gene regions, phylogenetic tree was constructed using MEGA4 by neighbor-joining method. Statistical robustness was established with bootstrap analysis. Nucleotide diversity was determined by Shannon entropy per site using the Entropy program of the Los Alamos National Laboratories. Analyses of acute patients revealed that HBV/D2 is the major circulating sub-genotype and commonly associated with sexual promiscuity and the age group between15-30 years. Comparison of AHB and CHB patients revealed that HBeAg positivity, ALT levels and genotype D were significantly high in AHB, whereas CHB patients were predominantly male, had a high viral load, and were commonly associated with genotype C. The frequencies of mutations in the S, BCP/PC, and X gene were low in AHB as compared to CHB. Drug resistant mutations were not detectable in the polymerase gene of AHB. Average nucleotide diversity in AHB was considerably low as compared to CHB. Further, the highest average ΔH (average difference in entropy between chronic and acute infection) was observed in the BCP/PC region implying that this region was most vulnerable to mutations upon HBV persistence, especially in case of genotype C. Additionally, among all substitutions, the A1762T and G1764A BCP mutations were the strongest indicators of chronicity. In conclusion, the study exhibits a general portrait of HBV strains circulating among acute hepatitis B patients in Eastern India and their intricate differences with chronic patients which should be useful from the clinical point of view.

Insights into human immunodeficiency virus-hepatitis B virus co-infection in India.

Shared routes of transmission lead to frequent human immunodeficiency virus (HIV)-hepatitis B virus (HBV) co-infection in a host which results in about 10% of HIV positive individuals to have chronic hepatitis B infection worldwide. In post-antiretroviral therapy era, liver diseases have emerged as the leading cause of morbidity and mortality in HIV-infected individuals and HBV co-infection have become the major health issue among this population particularly from the regions with endemic HBV infection. In setting of HIV-HBV co-infection, HIV significantly impacts the natural history of HBV infection, its disease profile and the treatment outcome in negative manner. Moreover, the epidemiological pattern of HBV infection and the diversity in HBV genome (genotypic and phenotypic) are also varied in HIV co-infected subjects as compared to HBV mono-infected individuals. Several reports on the abovementioned issues are available from developed parts of the world as well as from sub-Saharan African countries. In contrast, most of these research areas remained unexplored in India despite having considerable burden of HIV and HBV infections. This review discusses present knowledge from the studies on HIV-HBV co-infection in India and relevant reports from different parts of the world. Issues needed for the future research relevant to HIV-HBV co-infection in India are also highlighted here, including a call for further investigations on this field of study.

Hepatitis B Virus Infection, MicroRNAs and Liver Disease.

Hepatitis B virus (HBV) attacks the liver and can cause both acute as well as chronic liver diseases which might lead to liver cirrhosis and hepatocellular carcinoma. Regardless of the availability of a vaccine and numerous treatment options, HBV is a major cause of morbidity and mortality across the world. Recently,microRNAs (miRNAs) have emerged as important modulators of gene function. Studies on the role of miRNA in the regulation of hepatitis B virus gene expression have been the focus of modern antiviral research. miRNAs can regulate viral replication and pathogenesis in a number of different ways, which includefacilitation, direct or indirect inhibition, activation of immune response, epigenetic modulation, etc. Nevertheless, these mechanisms can appropriately be used with a diagnosticand/or therapeutic approach. The present review is an attempt to classify specific miRNAs that are reported to be associated with various aspects of hepatitis B biology, in order to precisely present the participation of individual miRNAs in multiple aspects relating to HBV.

High incidence of lamivudine-resistance-associated vaccine-escape HBV mutants among HIV-coinfected patients on prolonged antiretroviral therapy.

BACKGROUND: Worldwide, frequent emergence of lamivudine (3TC)-resistant HBV mutants has been reported in HIV-HBV-coinfected patients during long-term antiretroviral therapy (ART) that contains 3TC as the sole anti-HBV drug. Three major patterns of mutations in HBV polymerase gene, namely single (rtM204V), double (rtL180M+rtM204V) and triple (rtV173L+rtL180M+rtM204V) mutations, are associated with 3TC-resistance; additionally, the triple mutation has vaccine-escape potential due to a corresponding change in overlapping surface gene. Data from India, a major reservoir for HIV and HBV infection, is lacking. Here we investigated the effect of long-term 3TC treatment on virological response for HBV and characterized the 3TC-resistant HBV mutations in a cohort of HIV-HBV-coinfected patients from eastern India. METHODS: A cross-sectional study was performed in HIV-infected patients (n=563) receiving 3TC-containing ART for ≥6 months from the major ART centre of eastern India during 2011-2012. The hepatitis B surface antigen-positive HIV-infected patients (n=62) were categorized into four groups with comparable sample size according to the 3TC exposure for ≥6-<12 months (group I; n=15), ≥12-<24 months (group II; n=20), ≥24-<48 months (group III; n=13) and ≥48 months (group IV; n=14). Patients' plasma samples were examined for hepatitis B e antigen (HBeAg), HBV DNA, viral load and covalently closed circular DNA (cccDNA). HBV reverse transcriptase region was sequenced. RESULTS: With a longer period of 3TC exposure, the frequency of HIV-HBV-coinfected patients having HBV DNA suppression decreased. The prevalence of HBeAg-positivity, serum HBV DNA load >2,000 IU/ml and 3TC-resistant mutations simultaneously increased. Remarkably, the 3TC-resistant triple mutation predominated over the double mutation in this cohort (32.26% versus 19.34%) and prevailed in significantly higher frequency among HBV viraemic patients experiencing 3TC for ≥48 months (60% versus 10%; P=0.03). Patients with 3TC-resistant triple mutants had HBV genotype-D, high serum HBV DNA load and elevated alanine aminotransferase level, and presence of cccDNA in their serum. CONCLUSIONS: Considering this alarmingly high incidence of 3TC-resistant triple mutation and its possible clinical/public health implications, proper management of 3TC-resistance among HIV-HBV-coinfected patients is an urgent necessity in 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.

BACKGROUND: Hepatitis 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. METHODS: Expressions 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. RESULTS: The 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. CONCLUSION: Thus, 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 characterization of HBV strains circulating among the treatment-naive HIV/HBV co-infected patients of eastern India.

Previously we reported that the exposure to hepatitis B virus (HBV) infection serves as a major threat among the treatment naive HIV infected population of eastern India. Hence, molecular characterization of these strains is of utmost importance in order to identify clinically significant HBV mutations. A total of 85 treatment naive HIV/HBV co-infected participants were included of whom the complete basal core promoter/precore region, the core and the whole envelope gene could be successfully sequenced for 59, 57 and 39 isolates respectively. Following phylogenetic analysis, it was found that HBV/D was the predominant genotype with HBV/D2 (38.5%) being the most prevalent subgenotype followed by HBV/A1. The major mutations affecting HBeAg expression includes the A1762T/G1764A (13.6%), G1896A (22%) and G1862T mutation (33.9%) which was predominantly associated with HBV/A1. Moreover, the prevalence of G1896A was considerably high among the HBeAg negative HIV/HBV co-infected subjects compared to HBV mono-infection. The main amino acid substitutions within the MHC class II restricted T-cell epitope of HBcAg includes the T12S (15.8%) and T67N (12.3%) mutation and the V27I (10.5%) mutation in the MHC class I restricted T-cell epitope. PreS1/S2 deletion was detected in 3 isolates with all harboring the BCP double mutation. Furthermore, the frequently occurring mutations in the major hydrophilic loop of the S gene include the T125M, A128V and M133I/L. Therefore, this study is the first from India to report useful information on the molecular heterogeneity of the HBV strains circulating among the treatment naive HIV/HBV co-infected population and is thus clinically relevant.

Association of TNF-α Promoter Polymorphism with HBV Associated Disease Outcome Among HBV Infected Patients from Orissa, Southern Part of East India.

BACKGROUND: TNF-α promoter polymorphism has been known to be a potential predictive factor in patients with HBV infection. We therefore tried to investigate whether the TNF-α promoter polymorphism at position -238, -857 and -863 was associated with the outcome of HBV infection in a population from Orissa, southern part of East India. METHODS: A total of 195 patients recruited for the study were classified into 85 controls and 110 HBV infected cases, which included 34 IC, 30 CLD, 32 LC and 14 HCC patients. The polymorphisms at the respective sites were detected by a PCR-RFLP followed by statistical analysis. RESULTS: The frequency of the genotype -238 GG and the allele -238G in the cases (89.0% and 92.7% respectively) was significantly higher than that in the controls (68.2% and 82.2% respectively) (P < 0.001, OR = 3.8 and P = 0.001, OR = 2.73). Whereas the -238 GA genotype was significantly high in the control group (28.2%) when compared to the cases (7.2%) (P < 0.001, OR = 0.2). Similarly, the frequency of -863CC and the allele -863C was significantly higher among the cases (24.5% and 49.5%) compared to controls (1.17% and 34.7%), (P < 0.001, OR = 27.32 and P = 0.003, OR = 1.85), whereas the -863CA genotype was significantly high in the controls (67.0%) when compared to the cases (50.0%) (P = 0.01, OR = 0.49). Haplotype -863C/-857C/-238G in cases was significantly higher than controls (P = 0.002). Multivariate logistic regression analysis indicates that the genotype -863CC bears a negative association with liver disease progression. CONCLUSION: The present study established an association of polymorphisms at site -238 and -863 of the TNF-α promoter with the outcome HBV infection and disease progression.

Characterization of the occult hepatitis B virus variants circulating among the blood donors from eastern India.

A previous study from West Bengal documented very high rate of occult HBV infection (OBI) among the HBsAg negative blood donors. This study was aimed to characterize the OBI strains circulating among the blood donors and to estimate the risk associated with the prevailing viral variants/mutants. Blood samples from 2195 voluntary blood donors were included in the study. HBsAg, HBeAg, anti-HBc, and anti-HBs statuses of the samples were done by ELISA based detection. PCR amplification and sequencing were done to determine HBV genotypes, basal core promoter (BCP), and precore (Pre-C) mutations. Among the study samples, 268 were anti-HBc positive/HBsAg negative, among which 65 (24.25%) were HBV DNA positive. Phylogenetic analysis revealed the presence of HBV/D (87.23%), HBV/A (8.51%), and HBV/C (4.26%) (P < 0.0001). HBV/D3 (65.85%) was the significantly prevalent subgenotype over HBV/D2 (26.83%) and HBV/D1 (7.31%) (P = 0.0003). Considerable prevalence of differential BCP (1752C, 1753C, 1762T/1764A, 1753C+1762T/1764A, 1773C, and 1814C) and reverse transcriptase (rt) gene (rtI91L, rtL93P, rtS106C, rtR110G, rtN118T, rtS119T, rtY126H, rtG127W/R, rtC136R, and rtY158H) mutations was identified. Association of specific HBV subgenotypes with OBI was interesting and needs further study. Clinically relevant mutations were prevalent among the OBI strains which are of serious concern.

Characterization of treatment-naive HIV/HBV co-infected patients attending ART clinic of a tertiary healthcare centre in eastern India.

OBJECTIVE: The study was designed to assess the hepatitis B virus (HBV) and hepatitis C virus (HCV) co-infection scenario among the human immunodeficiency virus (HIV) infected patients attending a tertiary healthcare unit in eastern India. Additionally, clinical and virological characterization of these viruses, prior to antiretroviral therapy (ART) initiation was also done for better understanding of the disease profile. METHODS: Pool of ART-naive HIV/HBV co-infected and HIV mono-infected patients, participating in two different studies, were included in this study. HBV DNA was detected by nested-PCR amplification followed by HBV genotype determination and HBV reverse transcriptase (RT) region amplification and direct sequencing for detecting drug resistance. RESULTS: The prevalence of HBsAg (11.3%) was higher compared to anti-HCV (1.9%) among the HIV infected ART-naive patients. Moreover, majority of the HBeAg positive HIV/HBV co-infected patients (87.7%) had HBV DNA ≥20,000 IU/ml with median HBV DNA significantly higher than that of HBeAg negative subjects (5.7 log10 IU/ml vs. 4.2 log10 IU/ml; p<0.0001). Multivariate analysis also showed that HBeAg-positive status was independently associated with higher HBV DNA level (p = <0.001). Notably, 60.9% of the HBeAg negative co-infected subjects had HBV DNA ≥2,000 IU/ml of which 37.0% had HBV DNA ≥20,000 IU/ml. Genotype HBV/D (68.2%) was the predominant genotype followed by HBV/A (24.3%) and HBV/C (7.5%). Anti-HBV drug resistant mutations were detected in two (3.8%) of the ART-naive patients. CONCLUSION: The prevalence of HIV/HBV co-infection was relatively higher in our study subjects. HBeAg testing might provide clue for early treatment initiation. Furthermore, HBeAg negative patients are also associated with high HBV DNA levels and therefore require appropriate medical attention. Pre-treatment screening for anti-HBV drug resistant mutations is not necessary before ART initiation.

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.