Increased expression of long non-coding RNA FIRRE promotes hepatocellular carcinoma by HuR-CyclinD1 axis signaling.

There is a critical need to understand the disease processes and identify improved therapeutic strategies for hepatocellular carcinoma (HCC). The long noncoding RNAs (lncRNAs) display diverse effects on biological regulations. The aim of this study was to identify a lncRNA as a potential biomarker of HCC and investigate the mechanisms by which the lncRNA promotes HCC progression using human cell lines and in vivo. Using RNA-Seq analysis, we found that lncRNA FIRRE was significantly upregulated in hepatitis C virus (HCV) associated liver tissue and identified that lncRNA FIRRE is significantly upregulated in HCV-associated HCC compared to adjacent non-tumor liver tissue. Further, we observed that FIRRE is significantly upregulated in HCC specimens with other etiologies, suggesting this lncRNA has the potential to serve as an additional biomarker for HCC. Overexpression of FIRRE in hepatocytes induced cell proliferation, colony formation, and xenograft tumor formation as compared to vector-transfected control cells. Using RNA pull-down proteomics, we identified HuR as an interacting partner of FIRRE. We further showed that the FIRRE-HuR axis regulates cyclin D1 expression. Our mechanistic investigation uncovered that FIRRE is associated with an RNA-binding protein HuR for enhancing hepatocyte growth. Together, these findings provide molecular insights into the role of FIRRE in HCC progression.

Hepatitis B virus suppresses complement C9 synthesis by limiting the availability of transcription factor USF-1 and inhibits formation of membrane attack complex: implications in disease pathogenesis.

BACKGROUND: The complement system functions primarily as a first-line host defense against invading microbes, including viruses. However, the interaction of Hepatitis B virus (HBV) with the complement-components during chronic HBV infection remains largely unknown. We investigated the mechanism by which HBV inhibits the formation of cytolytic complement membrane-attack complex (MAC) and studied its impact on MAC-mediated microbicidal activity and disease pathogenesis. METHODS: Blood/liver tissues were collected from chronically HBV-infected patients and controls. HepG2hNTCP cells were infected with HBV particles and Huh7 cells were transfected with full-length linear HBV-monomer or plasmids containing different HBV-ORFs and expression of complement components or other host genes were evaluated. Additionally, ELISA, Real-time PCR, Western blot, bioinformatics analysis, gene overexpression/knock-down, mutagenesis, chromatin immunoprecipitation, epigenetic studies, immunofluorescence, and quantification of serum HBV-DNA, bacterial-DNA and endotoxin were performed. RESULTS: Among the MAC components (C5b-C9), significant reduction was noted in the expression of C9, the major constituent of MAC, in HBV-infected HepG2hNTCP cells and in Huh7 cells transfected with full-length HBV as well as HBX. C9 level was also marked low in sera/liver of chronic hepatitis B (CHB) and Immune-tolerant (IT) patients than inactive carriers and healthy controls. HBX strongly repressed C9-promoter activity in Huh7 cells but CpG-island was not detected in C9-promoter. We identified USF-1 as the key transcription factor that drives C9 expression and demonstrated that HBX-induced hypermethylation of USF-1-promoter is the leading cause of USF-1 downregulation that in turn diminished C9 transcription. Reduced MAC formation and impaired lysis of HBV-transfected Huh7 and bacterial cells were observed following incubation of these cells with C9-deficient CHB sera but was reversed upon C9 supplementation. Significant inverse correlation was noted between C9 concentration and HBV-DNA, bacterial-DNA and endotoxin content in HBV-infected patients. One-year Tenofovir therapy resulted in improvement in C9 level and decline in viral/bacterial/endotoxin load in CHB patients. CONCLUSION: Collectively, HBX suppressed C9 transcription by restricting the availability of USF-1 through hypermethylation of USF-1-promoter and consequently hinder the formation and lytic functions of MAC. Early therapy is needed for both CHB and IT to normalize the aberrant complement profile and contain viral and bacterial infection and limit disease progression.

Diverse facets of MDSC in different phases of chronic HBV infection: Impact on HBV-specific T-cell response and homing.

BACKGROUND AND AIMS: Chronic HBV infection (CHI) is associated with a diverse natural history that includes immune-tolerant (IT), HBeAg-positive chronic hepatitis B (CHB) (EP-CHB), inactive carrier, and HBeAg-negative CHB (EN-CHB) phases. A hallmark of CHI is impairment of HBV-specific T-cell response. Recently, myeloid-derived suppressor cells (MDSCs) have emerged as key regulator of T cells, and their properties are sculpted by their microenvironment. Here, we investigated the distinctive features of MDSCs during CHI, identified factors responsible for their functional discrepancies, and studied their impact on HBV-specific T-cell response and homing. Influence of antiviral therapy on MDSC profile and T-cell response was also assessed. APPROACH AND RESULTS: Flow cytometric analysis indicated that MDSCs in EP-CHB/EN-CHB patients had profound suppressive ability, expressing arginase 1 (Arg1)/inducible nitric oxide synthase (iNOS)/programmed death ligand 1 (PD-L1)/cytotoxic T lymphocyte-associated protein 4 (CTLA-4)/CD40 at significantly greater levels relative to healthy controls (HC). However, in IT, only Arg1+ MDSCs and in inactive carrier, iNOS+ and PD-L1+ MDSCs were higher than HC. In vitro assays demonstrated that high HBsAg titer in IT/CHB induced Arg1+ MDSC. Furthermore, elevated serum TNF-α and IL-4 in CHB potentiated Arg1/PD-L1/CD40/CTLA-4 expression, whereas increased IL-1ß in CHB/IC triggered the expansion of PD-L1+ MDSCs and iNOS+ MDSCs. MDSCs, sorted from CHB/IC, greatly attenuated IL-2/interferon gamma (IFN-γ) production by HBV-specific CD8+ /CD4+ T cells, the effect being more pronounced in CHB. However, MDSCs of IT minimally affected the cytokine production by T cells. Adding Arg1-/iNOS-inhibitor restored only IFN-γ production, while neutralizing PD-L1 recovered both IL-2 and IFN-γ secretion by T cells. Moreover, MDSCs from IT/CHB disrupted virus-specific T-cell trafficking by down-regulating chemokine receptor type 5 on them via TGF-ß signaling. One year of tenofovir therapy failed to normalize MDSC phenotype and HBV-specific T-cell response. CONCLUSIONS: Diversity of MDSCs during CHI affects HBV-specific T-cell response and homing. Hence, therapeutic targeting of MDSCs could boost anti-HBV immunity.

Variability in the Responses of Hepatitis B Virus D-Subgenotypes to Antiviral Therapy: Designing Pan-D-Subgenotypic Reverse Transcriptase Inhibitors.

Nucleos(t)ide analogues entecavir (ETV) and tenofovir disoproxil fumarate (TDF) are recommended as first-line monotherapies for chronic hepatitis B (CHB). Multiple HBV genotypes/subgenotypes have been described, but their impact on treatment response remains largely elusive. We investigated the effectiveness of ETV/TDF on HBV/D-subgenotypes, D1/D2/D3/D5, studied the structural/functional differences in subgenotype-specific reverse transcriptase (RT) domains of viral polymerase, and identified novel molecules with robust inhibitory activity on various D-subgenotypes. Transfection of Huh7 cells with full-length D1/D2/D3/D5 and in vitro TDF/ETV susceptibility assays demonstrated that D1/D2 had greater susceptibility to TDF/ETV while D3/D5 exhibited poorer response. Additionally, HBV load was substantially reduced in TDF-treated CHB patients carrying D1/D2 but minimally reduced in D3/D5-infected patients. Comparison of RT sequences of D-subgenotypes led to identification of unique subgenotype-specific residues, and molecular modeling/docking/simulation studies depicted differential bindings of TDF/ETV to the active site of their respective RTs. Replacement of signature residues in D3/D5 HBV clones with corresponding amino acids seen in D1/D2 improved their susceptibility to TDF/ETV. Using high throughput virtual screening, we identified N(9)-[3-fluoro-2-(phosphonomethoxy)propyl] (FPMP) derivatives of purine bases, including N6-substituted (S)-FPMP derivative of 2,6-diaminopurine (DAP) (OB-123-VK), as potential binders of RT of different D-subgenotypes. We synthesized (S)-FPMPG prodrugs (FK-381-FEE/FK-381-SEE/FK-382) and tested their effectiveness along with OB-123-VK. Both OB-123-VK and FK-381-FEE exerted similar antiviral activities against all D-subgenotypes, although FK-381-FEE was more potent. Our study highlighted the natural variation in therapeutic response of D1/D2/D3/D5 and emphasized the need for HBV subgenotype determination before treatment. Novel molecules described here could benefit future design/discovery of pan-D-subgenotypic inhibitors. IMPORTANCE Current treatment of chronic hepatitis B relies heavily on nucleotide/nucleoside analogs in particular, tenofovir disoproxil fumarate (TDF) and entecavir (ETV) to keep HBV replication under control and prevent end-stage liver diseases. However, it was unclear whether the therapeutic effects of TDF/ETV differ among patients infected with different HBV genotypes and subgenotypes. HBV genotype D is the most widespread of all HBV genotypes and multiple D-subgenotypes have been described. We here report that different subgenotypes of HBV genotype-D exhibit variable response toward TDF and ETV and this could be attributed to naturally occurring amino acid changes in the reverse transcriptase domain of the subgenotype-specific polymerase. Further, we identified novel molecules and also synthesized prodrugs that are equally effective on different D-subgenotypes and could facilitate management of HBV/D-infected patients irrespective of D-subgenotype.

Hepatitis C Virus Evades Interferon Signaling by Suppressing Long Noncoding RNA Linc-Pint Involving C/EBP-ß.

Hepatitis C virus (HCV) regulates many cellular genes in modulating the host immune system for benefit of viral replication and long-term persistence in a host for chronic infection. Long noncoding RNAs (lncRNAs) play an important role in the regulation of many important cellular processes, including immune responses. We recently reported that HCV infection downregulates lncRNA Linc-Pint (long intergenic non-protein-coding RNA p53-induced transcript) expression, although the mechanism of repression and functional consequences are not well understood. In this study, we demonstrate that HCV infection of hepatocytes transcriptionally reduces Linc-Pint expression through CCAAT/enhancer binding protein ß (C/EBP-ß). Subsequently, we observed that the overexpression of Linc-Pint significantly upregulates interferon alpha (IFN-α) and IFN-ß expression in HCV-replicating hepatocytes. Using unbiased proteomics, we identified that Linc-Pint associates with DDX24, which enables RIP1 to interact with IFN-regulatory factor 7 (IRF7) of the IFN signaling pathway. We furthermore observed that IFN-α14 promoter activity was enhanced in the presence of Linc-Pint. Together, these results demonstrated that Linc-Pint acts as a positive regulator of host innate immune responses, especially IFN signaling. HCV-mediated downregulation of Linc-Pint expression appears to be one of the mechanisms by which HCV may evade innate immunity for long-term persistence and chronicity. IMPORTANCE The mechanism by which lncRNA regulates the host immune response during HCV infection is poorly understood. We observed that Linc-Pint was transcriptionally downregulated by HCV. Using a chromatin immunoprecipitation (ChIP) assay, we showed inhibition of transcription factor C/EBP-ß binding to the Linc-Pint promoter in the presence of HCV infection. We further identified that Linc-Pint associates with DDX24 for immunomodulatory function. The overexpression of Linc-Pint reduces DDX24 expression, which in turn results in the disruption of DDX24-RIP1 complex formation and the activation of IRF7. The induction of IFN-α14 promoter activity in the presence of Linc-Pint further confirms our observation. Together, our results suggest that Linc-Pint acts as a positive regulator of host innate immune responses. Downregulation of Linc-Pint expression by HCV helps in escaping the innate immune system for the development of chronicity.

Exosomes from COVID-19 Patients Carry Tenascin-C and Fibrinogen-ß in Triggering Inflammatory Signals in Cells of Distant Organ.

SARS-CoV-2 infection can cause cytokine storm and may overshoot immunity in humans; however, it remains to be determined whether virus-induced soluble mediators from infected cells are carried by exosomes as vehicles to distant organs and cause tissue damage in COVID-19 patients. We took an unbiased proteomic approach for analyses of exosomes isolated from plasma of healthy volunteers and COVID-19 patients. Our results revealed that tenascin-C (TNC) and fibrinogen-ß (FGB) are highly abundant in exosomes from COVID-19 patients' plasma compared with that of healthy normal controls. Since TNC and FGB stimulate pro-inflammatory cytokines via the Nuclear factor-κB (NF-κB) pathway, we examined the status of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and C-C motif chemokine ligand 5 (CCL5) expression upon exposure of hepatocytes to exosomes from COVID-19 patients and observed significant increase compared with that from healthy subjects. Together, our results demonstrate that TNC and FGB are transported through plasma exosomes and potentially trigger pro-inflammatory cytokine signaling in cells of distant organ.

Hepatitis C virus associated hepatocellular carcinoma.

Liver cancer is a global problem and hepatocellular carcinoma (HCC) accounts for about 85% of this cancer. In the USA, etiologies and risk factors for HCC include chronic hepatitis C virus (HCV) infection, diabetes, non-alcoholic steatohepatitis (NASH), obesity, excessive alcohol drinking, exposure to tobacco smoke, and genetic factors. Chronic HCV infection appears to be associated with about 30% of HCC. Chronic HCV infection induces multistep changes in liver, involving metabolic disorders, steatosis, cirrhosis and HCC. Liver carcinogenesis requires initiation of neoplastic clones, and progression to clinically diagnose malignancy. Tumor progression associates with profound exhaustion of tumor-antigen-specific CD8+T cells, and accumulation of PD-1hi CD8+T cells and Tregs. In this chapter, we provide a brief description of HCV and environmental/genetic factors, immune regulation, and highlight mechanisms of HCV associated HCC. We also underscore HCV treatment and recent paradigm of HCC progression, highlighted the current treatment and potential future therapeutic opportunities.

Inhibition of Long Noncoding RNA Linc-Pint by Hepatitis C Virus in Infected Hepatocytes Enhances Lipogenesis.

BACKGROUND AND AIMS: HCV often causes chronic infection in liver, cirrhosis, and, in some instances, HCC. HCV encodes several factors' those impair host genes for establishment of chronic infection. The long noncoding RNAs (lncRNAs) display diverse effects on biological regulations. However, their role in virus replication and underlying diseases is poorly understood. In this study, we have shown that HCV exploits lncRNA long intergenic nonprotein-coding RNA, p53 induced transcript (Linc-Pint) in hepatocytes for enhancement of lipogenesis. APPROACH AND RESULTS: We identified a lncRNA, Linc-Pint, which is significantly down-regulated in HCV-replicating hepatocytes and liver specimens from HCV infected patients. Using RNA pull-down proteomics, we identified serine/arginine protein specific kinase 2 (SRPK2) as an interacting partner of Linc-Pint. A subsequent study demonstrated that overexpression of Linc-Pint inhibits the expression of lipogenesis-related genes, such as fatty acid synthase and ATP-citrate lyase. We also observed that Linc-Pint significantly inhibits HCV replication. Furthermore, HCV-mediated enhanced lipogenesis can be controlled by exogenous Linc-Pint expression. Together, our results suggested that HCV-mediated down-regulation of Linc-Pint enhances lipogenesis favoring virus replication and liver disease progression. CONCLUSIONS: We have shown that SRPK2 is a direct target of Linc-Pint and that depletion of SRPK2 inhibits lipogenesis. Our study contributes to the mechanistic understanding of the role of Linc-Pint in HCV-associated liver pathogenesis.

A Model of Care Optimized for Marginalized Remote Population Unravels Migration Pattern in India.

BACKGROUND AND AIMS: Access to basic health needs remains a challenge for most of world's population. In this study, we developed a care model for preventive and disease-specific health care for an extremely remote and marginalized population in Arunachal Pradesh, the northeasternmost state of India. APPROACH AND RESULTS: We performed patient screenings, performed interviews, and obtained blood samples in remote villages of Arunachal Pradesh through a tablet-based data collection application, which was later synced to a cloud database for storage. Positive cases of hepatitis B virus (HBV) were confirmed and genotyped in our central laboratory. The blood tests performed included liver function tests, HBV serologies, and HBV genotyping. HBV vaccination was provided as appropriate. A total of 11,818 participants were interviewed, 11,572 samples collected, and 5,176 participants vaccinated from the 5 westernmost districts in Arunachal Pradesh. The overall hepatitis B surface antigen (HBsAg) prevalence was found to be 3.6% (n = 419). In total, 34.6% were hepatitis B e antigen positive (n = 145) and 25.5% had HBV DNA levels greater than 20,000 IU/mL (n = 107). Genotypic analysis showed that many patients were infected with HBV C/D recombinants. Certain tribes showed high seroprevalence, with rates of 9.8% and 6.3% in the Miji and Nishi tribes, respectively. The prevalence of HBsAg in individuals who reported medical injections was 3.5%, lower than the overall prevalence of HBV. CONCLUSIONS: Our unique, simplistic model of care was able to link a highly resource-limited population to screening, preventive vaccination, follow-up therapeutic care, and molecular epidemiology to define the migratory nature of the population and disease using an electronic platform. This model of care can be applied to other similar settings globally.

Seed Priming with Phytohormones: An Effective Approach for the Mitigation of Abiotic Stress.

Plants are often exposed to abiotic stresses such as drought, salinity, heat, cold, and heavy metals that induce complex responses, which result in reduced growth as well as crop yield. Phytohormones are well known for their regulatory role in plant growth and development, and they serve as important chemical messengers, allowing plants to function during exposure to various stresses. Seed priming is a physiological technique involving seed hydration and drying to improve metabolic processes prior to germination, thereby increasing the percentage and rate of germination and improving seedling growth and crop yield under normal and various biotic and abiotic stresses. Seed priming allows plants to obtain an enhanced capacity for rapidly and effectively combating different stresses. Thus, seed priming with phytohormones has emerged as an important tool for mitigating the effects of abiotic stress. Therefore, this review discusses the potential role of priming with phytohormones to mitigate the harmful effects of abiotic stresses, possible mechanisms for how mitigation is accomplished, and roles of priming on the enhancement of crop production.

Exogenous proline enhances antioxidant enzyme activities but does not mitigate growth inhibition by selenate stress in tobacco BY-2 cells.

Selenium (Se) causes oxidative damage to plants. Proline is accumulated as a compatible solute in plants under stress conditions and mitigates stresses. Selenate at 250 µM increased cell death and inhibited the growth of tobacco BY-2 cells while exogenous proline at 10 mM did not mitigate the inhibition by selenate. Selenate increased accumulation of Se and ROS and activities of antioxidant enzymes but not lipid peroxidation in the BY-2 cells. Proline increased Se accumulation and antioxidant enzyme activities but not either ROS accumulation or lipid peroxidation in the selenate-stressed cells. Glutathione (GSH) rather than ascorbic acid (AsA) mitigated the growth inhibition although both reduced the accumulation of ROS induced by selenate. These results indicate that proline increases both antioxidant enzyme activities and Se accumulation, which overall fails to ameliorate the growth inhibition by selenate and that the growth inhibition is not accounted for only by ROS accumulation. Abbreviations: APX: ascorbate peroxidase; AsA: ascorbic acid; BY-2: Bright Yellow-2; CAT: catalase; DAI: days after inoculation; DW: dry weight; FW: fresh weight; GSH: glutathione; ROS: reactive oxygen species.

Establishment of a Patient-Derived Xenograft Tumor From Hepatitis C-Associated Liver Cancer and Evaluation of Imatinib Treatment Efficacy.

BACKGROUND AND AIMS: Chronic hepatitis C virus (HCV) infection is one of the major causal factors for hepatocellular carcinoma (HCC). The treatment options for HCC are limited for lack of a convenient animal model for study in HCV infection and liver pathogenesis. This study aimed to develop a patient-derived xenograft (PDX) tumor in mice by using a tumor from a patient with HCV-associated HCC and evaluating this model's therapeutic potential. APPROACH AND RESULTS: After resection of the primary tumor from the patient liver, excess viable tumor was implanted into highly immunodeficient mice. A mouse xenograft tumor line was developed, and the tumor was successfully passaged for at least three rounds in immunodeficient mice. The patient's primary tumor and the mouse xenografts were histologically similar. Genetic profiling by short-tandem-repeat analysis verified that the HCC-PDX model was derived from the HCC clinical specimen. HCV RNA present in the patient liver specimen was undetectable after passage as xenograft tumors in mice. Human albumin, α1 -antitrypsin, glypican-3, α-smooth muscle actin, and collagen type 1A2 markers were detected in human original tumor tissues and xenograft tumors. Both the patient primary tumor and the xenograft tumors had a significantly higher level of receptor tyrosine kinase (c-Kit) mRNA. Treatment of HCC-PDX xenograft tumor-bearing mice with the c-Kit inhibitor imatinib significantly reduced tumor growth and phospho-Akt and cyclin D1 expression, as compared with untreated control tumors. CONCLUSIONS: Our results demonstrated establishment of an HCV-associated HCC-PDX model as a powerful tool for evaluating candidate drugs. Information on molecular changes in cancer-specific gene expression facilitates efficient targeted therapies and treatment strategies.

Mechanisms Underlying Hepatitis C Virus-Associated Hepatic Fibrosis.

Hepatitis C virus (HCV) infection often causes liver diseases, including fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Liver fibrosis is the outcome of the wound healing response to tissue damage caused by chronic HCV infection. This process is characterized by the excessive accumulation of extracellular matrix (ECM) proteins, such as collagen fibers secreted by activated hepatic stellate cells (HSCs). Activation of HSCs from the quiescent stage is mediated by different mechanisms, including pro-inflammatory cytokines and chemokines released from HCV-infected hepatocytes and liver macrophages. HCV infection modulates the expression of different microRNAs that can be transported and delivered to the HSCs via exosomes released from infected cells, also leading to the development of advanced disease pathogenesis. Although recent advancements in direct-acting antiviral (DAA) treatment can efficiently control viremia, there are very few treatment strategies available that can be effective at preventing pathogenesis in advanced liver fibrosis or cirrhosis in patients. Assessment of fibrosis is considered to be the major part of proper patient care and decision making in clinical practice. In this review, we highlighted the current knowledge of molecular mechanisms responsible for the progression of liver fibrosis in chronically HCV-infected patients, and currently available methods for evaluation of fibrosis in patients. A detailed understanding of these aspects at the molecular level may contribute to the development of new therapies targeting HCV-related liver fibrosis.

Distinctiveness in virological features and pathogenic potentials of subgenotypes D1, D2, D3 and D5 of Hepatitis B virus.

Distinct clinical features of HBV infection have been associated with different viral genotype/subgenotype. HBV Genotype-D comprised of 10 subgenotypes, D1-D10, whose clinical implications still remain elusive. We investigated for the first-time, the virologic characteristics and cytopathic effects of four non-recombinant D-subgenotypes, D1/D2/D3/D5. Expressions of viral/host genes were evaluated in Huh7 cells transfected with full-length, linear-monomers of HBV/D-subgenotypes or pGL3-Basic vector carrying subgenotype-specific HBx. Intracellular HBV-DNA and pregenomic-RNA levels were high in D1/D2 than D3/D5. Expressions of PreC-mRNA and HBx were highest for D2 and D1 respectively, whereas PreS2/S-transcript was significantly reduced in D5. Increased apoptotic cell death and marked upregulation in caspase-3/Bax/TNF-R1/FasR/TRAIL-R1/ROS/MCP-1/IP-10/MIP-1ß expression were noticed specifically in D2- and also in D3-transfected cells, while D5 resulted in over-expression of ER-stress-markers. D-subgenotype-transfected Huh7 cells were co-cultured with PBMC of healthy-donors or LX-2 cells and significant increase in pro-inflammatory cytokines in PBMC and fibrogenic-markers in LX-2 were noticed in presence of D2/D3. Further, Huh7 cells transfected with D1, in particular and also D5, displayed remarkable induction of EMT-markers and high proliferative/migratory abilities. Collectively, our results demonstrated that D2/D3 were more associated with hepatic apoptosis/inflammation/fibrosis and D1/D5 with increased risk of hepatocarcinogenesis and emphasize the need for determining HBV-subgenotype in clinical practice.

Synergistic impact of mutations in Hepatitis B Virus genome contribute to its occult phenotype in chronic Hepatitis C Virus carriers.

We characterized occult HBV (OHBV) from hepatitis B surface antigen (HBsAg)-negative chronic HCV carriers of Eastern India to explore the impact of genomic variability of HBV in causing undetectability of HBsAg and low viremia that define the occult phenomenon. Screening of sera samples revealed the presence of OHBV in 17.8% of HCV-infected patients. Determination of full-length OHBV sequences and comparison with that from HBsAg-positive carriers led to the detection of distinct substitutions/mutations in PreS2, S, P and X ORFs and in X-promoter and Enhancer-II of OHBV. These mutations were introduced in wild-type HBV and their effects were evaluated by transfection in Huh7 cells. In vitro assays demonstrated that S-substitutions resulted in antigenically modified HBsAg that escaped detection by immunoassays whereas those in ORF-P caused significant decline in viral replication. Impairment in Enhancer-II and X-promoter activities were noted due to occult-associated mutations that generated reduced pregenomic RNA and intracellular HBV-DNA. Additionally, Enhancer-II mutations altered the small to large surface protein ratio and diminished extracellular HBV-DNA and HBsAg secretion. Further, mutations in PreS2, X and enhancer-II increased Grp78-promoter activity, suggesting that OHBV could trigger endoplasmic reticulum stress. Thus viral mutations contribute synergistically towards the genesis of occult phenotype and disease progression.

HBV quasispecies composition in Lamivudine-failed chronic hepatitis B patients and its influence on virological response to Tenofovir-based rescue therapy.

The present study sought to evaluate the structure of HBV quasispecies in Lamivudine (LMV)-failed chronic hepatitis B (CHB) patients and its impact in defining the subsequent virological responses to Tenofovir (TDF)-based rescue-therapy. By analyzing HBV clones encompassing reverse transcriptase (RT) and surface (S) region from LMV-failed and treatment-naïve CHB patients, we identified 5 classical and 12 novel substitutions in HBV/RT and 9 substitutions in immune-epitopes of HBV/S that were significantly associated with LMV failure. In silico analysis showed spatial proximity of some of the newly-identified, mutated RT residues to the RT catalytic centre while most S-substitutions caused alteration in epitope hydrophobicity. TDF administration resulted in virological response in 60% of LMV-failed patients at 24-week but non-response in 40% of patients even after 48-weeks. Significantly high frequencies of 6 S-substitutions and one novel RT-substitution, rtH124N with 6.5-fold-reduced susceptibility to TDF in vitro, were noted at baseline in TDF non-responders than responders. Follow-up studies depicted greater evolutionary drift of HBV quasispecies and significant decline in frequencies of 3 RT and 6 S-substitutions in responder-subgroup after 24-week TDF-therapy while most variants persisted in non-responders. Thus, we identified the HBV-RT/S variants that could potentially predict unfavorable response to LMV/TDF-therapy and impede immune-mediated viral clearance.

A rare HBV subgenotype D4 with unique genomic signatures identified in north-eastern India--an emerging clinical challenge?

BACKGROUND/AIMS: HBV has been classified into ten genotypes (A-J) and multiple subgenotypes, some of which strongly influence disease outcome and their distribution also correlate with human migration. HBV infection is highly prevalent in India and its diverse population provides an excellent opportunity to study the distinctiveness of HBV, its evolution and disease biology in variegated ethnic groups. The North-East India, having international frontiers on three sides, is one of the most ethnically and linguistically diverse region of the country. Given the paucity of information on molecular epidemiology of HBV in this region, the study aimed to carry out an in-depth genetic characterization of HBV prevailing in North-East state of Tripura. METHODS: From sera of chronically HBV infected patients biochemical/serological tests, HBV DNA quantification, PCR-amplification, sequencing of PreS/S or full-length HBV genomes were done. HBV genotype/subgenotype determination and sequence variability were assessed by MEGA5-software. The evolutionary divergence times of different HBV subgenotypes were estimated by DNAMLK/PHYLIP program while jpHMM method was used to detect any recombination event in HBV genomes. RESULTS: HBV genotypes D (89.5%), C (6.6%) and A (3.9%) were detected among chronic carriers. While all HBV/A and HBV/C isolates belonged to subgenotype-A1 and C1 respectively, five subgenotypes of HBV/D (D1-D5) were identified including the first detection of rare D4. These non-recombinant Indian D4 (IndD4) formed a distinct phylogenetic clade, had 2.7% nucleotide divergence and recent evolutionary radiation than other global D4. Ten unique amino acids and 9 novel nucleotide substitutions were identified as IndD4 signatures. All IndD4 carried T120 and R129 in ORF-S that may cause immune/vaccine/diagnostic escape and N128 in ORF-P, implicated as compensatory Lamivudine resistance mutation. CONCLUSIONS: IndD4 has potential to undermine vaccination programs or anti-viral therapy and its introduction to North-East India is believed to be linked with the settlement of ancient Tibeto-Burman migrants from East-Asia.