A case report on rare hepatitis B viral subgenotype from a tertiary care center in Chennai

Hepatitis B virus (HBV) is a global health concern with 350 million chronic carriers. With respect to HBV India is classified as an intermediate endemic country. The disease progression may be due to many viral factors including HBV viral load, HBe antigen, genotype, mutations in polymerase gene, and X gene. In this case, the individual was a treatment naïve chronic HBV carrier. The reverse transcriptase gene and X gene were sequenced and mutations were analyzed. The individual had D3 subgenotype. rt80I was identified in reverse transcriptase and A102V in HBx protein. Identification of genotype and mutations in reverse transcriptase/X gene may help in predicting and improving the clinical outcomes.

ATM-Dependent Phosphorylation of Hepatitis B Core Protein in Response to Genotoxic Stress

Chronic hepatitis caused by infection with the Hepatitis B virus is a life-threatening condition. In fact, 1 million people die annually due to liver cirrhosis or hepatocellular carcinoma. Recently, several studies demonstrated a molecular connection between the host DNA damage response (DDR) pathway and HBV replication and reactivation. Here, we investigated the role of Ataxia-telangiectasia-mutated (ATM) and Ataxia telangiectasia and Rad3-related (ATR) PI3-kinases in phosphorylation of the HBV core protein (HBc). We determined that treatment of HBc-expressing hepatocytes with genotoxic agents, e.g., etoposide or hydrogen peroxide, activated the host ATM-Chk2 pathway, as determined by increased phosphorylation of ATM at Ser1981 and Chk2 at Thr68. The activation of ATM led, in turn, to increased phosphorylation of cytoplasmic HBc at serine-glutamine (SQ) motifs located in its C-terminal domain. Conversely, down-regulation of ATM using ATM-specific siRNAs or inhibitor effectively reduced etoposide-induced HBc phosphorylation. Detailed mutation analysis of S-to-A HBc mutants revealed that S170 (S168 in a 183-aa HBc variant) is the primary site targeted by ATM-regulated phosphorylation. Interestingly, mutation of two major phosphorylation sites involving serines at positions 157 and 164 (S155 and S162 in a 183-aa HBc variant) resulted in decreased etoposide-induced phosphorylation, suggesting that the priming phosphorylation at these serine-proline (SP) sites is vital for efficient phosphorylation of SQ motifs. Notably, the mutation of S172 (S170 in a 183-aa HBc variant) had the opposite effect and resulted in massively up-regulated phosphorylation of HBc, particularly at S170. Etoposide treatment of HBV infected HepG2-NTCP cells led to increased levels of secreted HBe antigen and intracellular HBc protein. Together, our studies identified HBc as a substrate for ATM-mediated phosphorylation and mapped the phosphorylation sites. The increased expression of HBc and HBe antigens in response to genotoxic stress supports the idea that the ATM pathway may provide growth advantage to the replicating virus.

HBV Core Promoter Inhibition by Tubulin Polymerization Inhibitor (SRI-32007)

Approximately 257 million people chronically infected with hepatitis B virus (HBV) worldwide are at risk of developing hepatocellular carcinoma (HCC). However, despite the availability of potent nucleoside/tide inhibitors, currently there are no curative therapies for chronic HBV infections. To identify potential new antiviral molecules, a select group of compounds previously evaluated in clinical studies were tested against 12 different viruses. Amongst the compounds tested, SRI-32007 (CYT997) demonstrated antiviral activity against HBV (genotype D) in HepG2.2.2.15 cell-based virus yield assay with 50% effective concentration (EC50) and selectivity index (SI) of 60.1 nM and 7.2, respectively. Anti-HBV activity of SRI-32007 was further confirmed against HBV genotype B in huh7 cells with secreted HBe antigen endpoint (EC50 40 nM and SI 250). To determine the stage of HBV life cycle inhibited by SRI-32007, time of addition experiment was conducted in HepG2-NTCP cell-based HBV infectious assay. Results indicated that SRI-32007 retained anti-HBV activity even when added 72 hours postinfection (72 h). Additional mechanism of action studies demonstrated potent inhibition of HBV core promoter activity by SRI-32007 with an EC50 of 40 nM and SI of >250. This study demonstrates anti-HBV activity of a repurposed compound SRI-32007 through inhibition of HBV core promoter activity. Further evaluation of SRI-32007 in HBV animal models is needed to confirm its activity in vivo. Our experiments illustrate the utility of repurposing strategy to identify novel antiviral chemical leads. HBV core promoter inhibitors such as SRI-32007 might enable the development of novel therapeutic strategies to combat HBV infections.

Case 44

Healthcare workers can transmit blood-borne virus (BBV) infections to their patients during the course of performing exposure prone procedures. To reduce the risk of this happening, healthcare workers entering the National Health Service who wish to perform EPPs in the UK must be tested for BBV infection. Hepatitis B- infected healthcare workers may perform EPPs providing they are HBE antigen negative and have a viral load below 200 IU/ml, subject to annual viral load checks. If their viral load is >200 but < 20,000 IU/ml, then they may suppress HBV replication using nucleos(t)ide analogue antiviral therapy, subject to 3-monthly viral load measurement. Healthcare workers known to be currently infected with hepatitis C virus are not allowed to perform EPPs but may do so if they are clear of HCV RNA 12 weeks after antiviral therapy. HIV-infected healthcare workers may perform EPPs if their viral load is suppressed to < 200 copies/ml whilst on combination antiretroviral therapy, subject to 3-monthly monitoring.

The treatment outcome of chronic HBV infection among HBV/HIV co-infected and HBV mono-infected patients

The aim of this study was to analyze the treatment response of hepatitis B virus (HBV) to lamivudine and tenofovir disoproxil fumarate in HBV/HIV co-infected patients in comparison to HBV mono-infected patients. This study was conducted at the University Hospital for Infectious and Tropical Diseases in Belgrade from January 2000 until December 2017 and included all patients with chronic hepatitis B who received antiviral therapy. All patients initially treated with lamivudine were switched to tenofovir if lamivudine failure occurred. A patient was considered to have achieved a full treatment response if the level of HBV DNA was lower than 20 IU/ml or undetectable. HBs and HBe antigen loss and HBs seroconversion were also monitored. After a mean duration of lamivudine-containing antiretroviral therapy (ART) of 4.87 ± 3.48 years and lamivudine mono-therapy of 4 ± 2.52 years, failure was recorded in 82.1% and 79.3% of patients, respectively. HBV viral loads were 20 ± 32 IU/ml and 3 ± 13 IU/ml after 2.49 ± 1.56 years of tenofovir-containing ART and 1.9 ± 1.13 years of tenofovir mono-therapy, respectively. Overall mean treatment duration, taking both lamivudine- and tenofovir-based regimens into account, was 4.18 ± 2.72 and 6.17 ± 3.63 years in the mono- and co-infected patients, respectively ( p = 0.02).