Sp Singh

Consensus Statement of HCV Task Force of the Indian National Association for Study of the Liver (INASL). Part I: Status Report of HCV Infection in India

Globally, around 150 million people are infected with hepatitis C virus (HCV). India contributes a large proportion of this HCV burden. The prevalence of HCV infection in India is estimated at between 0.5% and 1.5%. It is higher in the northeastern part, tribal populations and Punjab, areas which may represent HCV hotspots, and is lower in western and eastern parts of the country. The predominant modes of HCV transmission in India are blood transfusion and unsafe therapeutic injections. There is a need for large field studies to better understand HCV epidemiology and identify high-prevalence areas, and to identify and spread awareness about the modes of transmission of this infection in an attempt to prevent disease transmission.

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.

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.

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.

Frequency of Hepatitis B Virus Surface Gene Variant Circulating Among HBsAg Negative Individuals in Coastal Eastern India: A Preliminary Report

© 2011, INASL 8 Results: The IC50 of the monoherbal preparations found out to be 1 mg/mL, 20 uM and 10 uM for Phyllanthus amarus, Andrographis paniculata and Silybum marianum, respectively as obtained from the decline in viral load in the soups collected from HepG2 2.2.15 cell lines. Conclusion: The above monoherbal preparations found to have a significant decline in viral proliferating markers. In comparison with the control, herbal treatment proved to have an efficacious effect and IC50 was calculated for them. Thus the herbs could be beneficial against HBV with least side effects in comparison to synthetic drugs. However it’s a preliminary data and further studies are needed to prove it. Conflict of Interest: None Clinical Spectrum of Hepatitis B Infection in Southern India B Siva Subramaniam, V Arul Selvan, K Arun Kumar, V Jayanthi Department of Gastroenterology and Hepatology, Stanley Medical College and Hospital, Chennai Background: Hepatitis B is a global problem, affecting over 400 million populations. Large numbers of cases are referred to our center. Aim: To determine the spectrum of hepatitis B infection amongst patients attending the Liver Clinic at a tertiary GI and Hepatology center. Method: All patients who were diagnosed as HBsAg positive were included in the study. Baseline evaluation included LFT, ultrasound, alphafetoprotein estimation and virological profile for hepatitis B (HBeAg, quantification of HBsAg, HBV DNA), hepatitis C (anti-HCV antibody) and HIV status. Family members were also screened for the above markers if found to be HBsAg positive. Based on the viral profile, transaminase levels, patients were categorized into immunotolerant, inactive carriers, immune clearance and reactivation phase and risk factors were also looked for. Patients with incomplete virological profile were excluded from the study. Results: One hundred and thirteen patients were fulfilled the criteria. Among the 113 patients 38 (33.6%) patients were in immunotolerant phase, 15 (13.2%) patients in immune clearance phase, 57 (50.4%) patients were inactive carriers and 3 (2.6%) patients were in reactivation phase. Twenty patients had cirrhosis, 2 patients were presented with hepatocellular carcinoma and 7 patients were pregnant. HBV DNA levels are low in patients with cirrhosis and HCC. Table Different phases of HBV infection Variables Immunotolerant Immune clearance Inactive carrier Reactivation phase (n = 38) (33.6%) (n = 15) (13.2%) (n = 57) (50.4%) (n = 3) (2.6%) Mean age 32 (median) 40 (mean) 36 (mean) 55.33 (mean) Sex ratio 24:14 (M:F) 13:2 (M:F) 19:38 (M:F) 3 males ALT 52.4 137.3 34.7 174.6 HBsAg quantification 10905.4 3206.567 5885.958 9368.615 HBV DNA 2672419 856432 313.9963 4491218 Conclusion: The spectrum of hepatitis B infection observed in Southern India was similar to the spectrum seen in West. The immunotolerant phase extends up to 32 years and virus starts clearing in between 32 and 40 years. So far, no similar data is available from South India. Conflict of Interest: None Frequency of Hepatitis B Virus Surface Gene Variant Circulating Among HBsAg Negative Individuals in Coastal Eastern India: A Preliminary Report MK Panigrahi, SK Kar, B Misra, SP Singh, HS Das, C Panda Department of Gastroenterology, SCB Medical College, Cuttack, Orissa, India Background: Genetic mutation might account for the presence of hepatitis B virus (HBV) DNA among HBsAg negative individuals. The aim of the study was to assess the prevalence and significance of surface gene mutations among HBsAg negative chronic liver disease cases (CLD) who attended the Gastroenterology OPD at SCB Medical College, Cuttack. Methods: Twenty HBsAg negative CLD sera of adults (mean age 44.8 ± 12.1; range 28–70 years) were included in this study. Surface gene region were amplified and surface gene was analyzed after direct sequencing. Results: Out of the total 20 CLD cases studied, only 5 samples were found to be HBV DNA positive (5/20; 25.0%). Genotype D was the prevalent genotype found in the study with no prevalence of other genotypes. In all 5 patients, substitutions within as well as outside 03_JCEH-Abstract.indd 8 3/18/2011 11:13:03 AM

Characterization of S Gene Region Among HBV-infected Patients from Orissa

© 2011, INASL 12 Characterization of S Gene Region Among HBV-infected Patients from Orissa R Panigrahi*, A Biswas*, MK Panigrahi**, A Pal*, M Bandopadhyay*, SP Singh**, HS Das**, S Chakrabarti†, R Chakravarty* *ICMR Virus Unit, Kolkata, ID and BG Hospital Campus, Kolkata **SCB Medical College, Cuttack, Orissa †National Institute of Cholera and Enteric Diseases, Kolkata, India Background: Genetic mutation might account for the presence of hepatitis B virus (HBV) DNA among liver disease individuals. The aim of this work was to describe the clinical characteristics associated with the HBV genotypes circulating in south-eastern India. Materials: A total of 36 HBsAg patients were included in the study. Surface gene regions were amplified by PCR for detection of HBV infection and surface gene was analyzed after direct sequencing. Analysis of MHR region was analyzed by using the BLOSUM scores. Results: HBV genotypes observed in HBV-infected patients were as follows: 8 patients with genotype A (22.2%), 1 with C (2.7%), 27 with D (75.0%). Male patients predominated in this study (91.6%). Among the 18 patients infected with sub-genotype D5, a considerably higher prevalence was found among LC patients (55.5%), CHB patients (27.7%), and acute patients (16.6%). No prevalence of HBV/D2 was found in the present study. The distribution of MHR mutations among the clinical groups was found in acute patients (0%), CHB patients (10%), and LC patients (17.6%). Conclusion: HBV sub-genotypes D5 were found in a significant proportion in our study population as compared with other parts of India. Interestingly, HBV D5 was found to be more frequently associated with liver cirrhosis than other clinical groups. Mutations in the immunodominant domains of S gene were found to increase with increasing disease severity. Thus our study will add to the knowledge of HBV genotype distribution and characterization in India. Conflict of Interest: None 03_JCEH-Abstract.indd 12 3/18/2011 11:13:03 AM