Conformational Changes in the Hepatitis B Virus Core Protein Are Consistent with a Role for Allostery in Virus Assembly

DOE Office of Scientific and Technical Information (OSTI.GOV)

Packianathan, Charles; Katen, Sarah P.; Dann, III, Charles E.

2010-01-12

In infected cells, virus components must be organized at the right place and time to ensure assembly of infectious virions. From a different perspective, assembly must be prevented until all components are available. Hypothetically, this can be achieved by allosterically controlling assembly. Consistent with this hypothesis, here we show that the structure of the hepatitis B virus (HBV) core protein dimer, which can spontaneously self-assemble, is incompatible with capsid assembly. Systematic differences between core protein dimer and capsid conformations demonstrate linkage between the intradimer interface and interdimer contact surface. These structures also provide explanations for the capsid-dimer selectivity of somemore » antibodies and the activities of assembly effectors. Solution studies suggest that the assembly-inactive state is more accurately an ensemble of conformations. Simulations show that allostery supports controlled assembly and results in capsids that are resistant to dissociation. We propose that allostery, as demonstrated in HBV, is common to most self-assembling viruses.« less

A broad investigation of the HBV-mediated changes to primary hepatocyte physiology reveals HBV significantly alters metabolic pathways.

PubMed

Lamontagne, R Jason; Casciano, Jessica C; Bouchard, Michael J

2018-06-01

As the leading risk factor for the development of liver cancer, chronic infection with hepatitis B virus (HBV) represents a significant global health concern. Although an effective HBV vaccine exists, at least 240 million people are chronically infected with HBV worldwide. Therapeutic options for the treatment of chronic HBV remain limited, and none achieve an absolute cure. To develop novel therapeutic targets, a better understanding of the complex network of virus-host interactions is needed. Because of the central metabolic role of the liver, we assessed the metabolic impact of HBV infection as a means to identify viral dependency factors and metabolic pathways that could serve as novel points of therapeutic intervention. Primary rat hepatocytes were infected with a control adenovirus, an adenovirus expressing a greater-than-unit-length copy of the HBV genome, or an adenovirus expressing the HBV X protein (HBx). A panel of 369 metabolites was analyzed for HBV- or HBx-induced changes 24 and 48 h post infection. Pathway analysis was used to identify key metabolic pathways altered in the presence of HBV or HBx expression, and these findings were further supported through integration of publically available gene expression data. We observed distinct changes to multiple metabolites in the context of HBV replication or HBx expression. Interestingly, a panel of 7 metabolites (maltotriose, maltose, myristate [14:0], arachidate [20:0], 3-hydroxybutyrate [BHBA], myo-inositol, and 2-palmitoylglycerol [16,0]) were altered by both HBV and HBx at both time points. In addition, incorporation of data from a transcriptome-based dataset allowed us to identify metabolic pathways, including long chain fatty acid metabolism, glycolysis, and glycogen metabolism, that were significantly altered by HBV and HBx. Because the liver is a central regulator of metabolic processes, it is important to understand how HBV replication and HBV protein expression affects the metabolic function of

Hepatitis B virus genotypes, precore and core promoter variants among predominantly Asian patients with chronic HBV infection in a Canadian center.

PubMed

Fung, Scott K; Wong, Florence S H; Wong, David K H; Hussain, Munira T; Lok, Anna S F

2006-09-01

The epidemiology of hepatitis B virus (HBV) infection in North America may be changing as a result of immigration from endemic countries. The purpose of this study was to determine the prevalence of HBV genotypes, precore (PC) and core promoter (CP) variants, and the proportion of patients meeting treatment criteria for HBV. A cross-sectional study of consecutive HBV patients attending a Canadian tertiary liver center was conducted. HBV DNA was quantified by polymerase chain reaction assay. HBV genotypes and variants were determined using a line probe assay. Two hundred and seventy-two patients were enrolled; 200 were not receiving treatment at enrollment, of whom 116 were men and 84 women with a mean age 42+/-14 years. Among this group, 177 (88%) patients were Asian and 19 (10%) were Caucasian and 69 (35%) patients were hepatitis B e antigen (HBeAg) positive. Genotypes B and C were found in 42% and 50% untreated patients, respectively; while CP and PC were detected in 52% and 43% patients, respectively. Approximately 20% patients not receiving treatment (29% HBeAg positive, 14% HBeAg negative) met AASLD guidelines for antiviral therapy. If lower cutoff values for alanine aminotransferase and HBV DNA levels were used, 49% patients would qualify for treatment. The vast majority of patients at a Canadian tertiary referral center were Asian. Virological and clinical characteristics of these patients reflect their country of origin. Our findings highlight the need to monitor the changing patterns of HBV infection in countries with large immigrant populations.

Monitoring of Hepatitis B Virus (HBV) DNA and Risk of HBV Reactivation in B-Cell Lymphoma: A Prospective Observational Study.

PubMed

Kusumoto, Shigeru; Tanaka, Yasuhito; Suzuki, Ritsuro; Watanabe, Takashi; Nakata, Masanobu; Takasaki, Hirotaka; Fukushima, Noriyasu; Fukushima, Takuya; Moriuchi, Yukiyoshi; Itoh, Kuniaki; Nosaka, Kisato; Choi, Ilseung; Sawa, Masashi; Okamoto, Rumiko; Tsujimura, Hideki; Uchida, Toshiki; Suzuki, Sachiko; Okamoto, Masataka; Takahashi, Tsutomu; Sugiura, Isamu; Onishi, Yasushi; Kohri, Mika; Yoshida, Shinichiro; Sakai, Rika; Kojima, Minoru; Takahashi, Hiroyuki; Tomita, Akihiro; Maruyama, Dai; Atsuta, Yoshiko; Tanaka, Eiji; Suzuki, Takayo; Kinoshita, Tomohiro; Ogura, Michinori; Mizokami, Masashi; Ueda, Ryuzo

2015-09-01

There is no standard management of reactivation of hepatitis B virus (HBV) infection in HBV-resolved patients without hepatitis B surface antigen (HBsAg), but with antibodies against hepatitis B core antigen and/or antibodies against HBsAg (anti-HBs). We conducted a prospective observational study to evaluate the occurrence of HBV reactivation by serial monthly monitoring of HBV DNA and to establish preemptive therapy guided by this monitoring in B-cell non-Hodgkin lymphoma (B-NHL) treated with rituximab plus corticosteroid-containing chemotherapy (R-steroid-chemo). The primary endpoint was the incidence of HBV reactivation defined as quantifiable HBV DNA levels of ≥ 11 IU/mL. With a median HBV DNA follow-up of 562 days, HBV reactivation was observed in 21 of the 269 analyzed patients. The incidence of HBV reactivation at 1.5 years was 8.3% (95% confidence interval, 5.5-12.4). No hepatitis due to HBV reactivation was observed in patients who received antiviral treatment when HBV DNA levels were between 11 and 432 IU/mL. An anti-HBs titer of <10 mIU/mL and detectable HBV DNA remaining below the level of quantification at baseline were independent risk factors for HBV reactivation (hazard ratio, 20.6 and 56.2, respectively; P < .001). Even in 6 patients with a rapid increase of HBV due to mutations, the monthly HBV DNA monitoring was effective at preventing HBV-related hepatitis. Monthly monitoring of HBV DNA is useful for preventing HBV reactivation-related hepatitis among B-NHL patients with resolved HBV infection following R-steroid-chemo (UMIN000001299). © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

HBV Genotypic Variability in Cuba

PubMed Central

Loureiro, Carmen L.; Aguilar, Julio C.; Aguiar, Jorge; Muzio, Verena; Pentón, Eduardo; Garcia, Daymir; Guillen, Gerardo; Pujol, Flor H.

2015-01-01

The genetic diversity of HBV in human population is often a reflection of its genetic admixture. The aim of this study was to explore the genotypic diversity of HBV in Cuba. The S genomic region of Cuban HBV isolates was sequenced and for selected isolates the complete genome or precore-core sequence was analyzed. The most frequent genotype was A (167/250, 67%), mainly A2 (149, 60%) but also A1 and one A4. A total of 77 isolates were classified as genotype D (31%), with co-circulation of several subgenotypes (56 D4, 2 D1, 5 D2, 7 D3/6 and 7 D7). Three isolates belonged to genotype E, two to H and one to B3. Complete genome sequence analysis of selected isolates confirmed the phylogenetic analysis performed with the S region. Mutations or polymorphisms in precore region were more common among genotype D compared to genotype A isolates. The HBV genotypic distribution in this Caribbean island correlates with the Y lineage genetic background of the population, where a European and African origin prevails. HBV genotypes E, B3 and H isolates might represent more recent introductions. PMID:25742179

Human hepatocytes apoptosis induced by replication of hepatitis B virus subgenotypes F1b and F4: Role of basal core promoter and preCore mutations.

PubMed

Elizalde, María Mercedes; Sevic, Ina; González López Ledesma, María Mora; Campos, Rodolfo Héctor; Barbini, Luciana; Flichman, Diego Martin

2018-01-01

In the context of pathogenesis of HBV infection, HBV genotypes and mutants have been shown to affect the natural course of chronic infection and treatment outcomes. In this work, we studied the induction of apoptosis by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. Both subgenotypes F1b and F4 HBV genome transfections induced cell death by apoptosis in human hepatocytes. The BCPdm (A1762T/G1764A) and preCore (G1896A) mutants induced higher levels of apoptosis than the wt virus. This increase in apoptosis was not associated with the enhanced viral replication of the variants. HBV-mediated apoptosis was independent of viral subgenotypes, and associated with the modulation of members of the regulatory Bcl-2 family proteins expression in the mitochondrial apoptotic pathway. Finally, the apoptosis induction increase observed for the preCore mutants suggests that HBeAg might have an anti-apoptotic effect in human hepatocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

Core promoter mutations 3 years after anti-hepatitis B e seroconversion in patients with chronic hepatitis B or hepatitis B and C infection and cancer remission.

PubMed

Zampino, Rosa; Marrone, Aldo; Karayiannis, Peter; Cirillo, Grazia; del Giudice, Emanuele Miraglia; Rania, Giovanni; Utili, Riccardo; Ruggiero, Giuseppe

2002-09-01

In this study, we aimed to evaluate the persistence of hepatitis B virus (HBV) DNA and the role of HBV core promoter and precore region mutations in 28 young cancer survivor patients with HBV or HBV and hepatitis C virus (HCV) infections, and persistently normal ALT levels, after spontaneous or interferon (IFN)-induced anti-hepatitis B e (HBe) seroconversion. Sera from 15 patients with HBV and 13 with dual HBV-HCV infection were analyzed for the presence of HBV-DNA and HCV-RNA by polymerase chain reaction 3 yr after anti-HBe seroconversion. A total of 21 patients had seroconverted spontaneously and seven did so after IFN treatment. The core promoter and the precore regions were amplified sequenced directly. Among patients with HBV infection, HBV-DNA was detected in five of nine (55%) with spontaneous anti-HBe and in all six treated patients (p = 0.092). In the coinfected patients, four had cleared both HBV-DNA and HCV-RNA, five were HBV-DNA negative/HCV-RNA positive and four had the reverse viral pattern. Among the 15 patients with persistence of HBV-DNA, a 7-base pair nucleotide deletion in the core promoter (1757-1763) was present in seven of 10 patients with spontaneous and in one of five patients with IFN-induced seroconversion (p = 0.033). The G1896A precore stop codon mutation was never observed. HBV-DNA levels were significantly lower in patients with the core promoter deletion (p = 0.011). The 7-base pair deletion generated a truncated X protein at amino-acid position 132. A core promoter deletion after anti-HBe seroconversion was associated with low HBV-DNA levels, probably because of downregulation of pregenomic RNA production and truncation of the X protein. HBV-DNA persistence was a frequent event, even in the absence of active liver disease.

Identification of p90 Ribosomal S6 Kinase 2 as a Novel Host Protein in HBx Augmenting HBV Replication by iTRAQ-Based Quantitative Comparative Proteomics.

PubMed

Yan, Li-Bo; Yu, You-Jia; Zhang, Qing-Bo; Tang, Xiao-Qiong; Bai, Lang; Huang, FeiJun; Tang, Hong

2018-05-01

The aim of this study was to screen for novel host proteins that play a role in HBx augmenting Hepatitis B virus (HBV) replication. Three HepG2 cell lines stably harboring different functional domains of HBx (HBx, HBx-Cm6, and HBx-Cm16) were cultured. ITRAQ technology integrated with LC-MS/MS analysis was applied to identify the proteome differences among these three cell lines. In brief, a total of 70 different proteins were identified among HepG2-HBx, HepG2-HBx-Cm6, and HepG2-HBx-Cm16 by double repetition. Several differentially expressed proteins, including p90 ribosomal S6 kinase 2 (RSK2), were further validated. RSK2 was expressed at higher levels in HepG2-HBx and HepG2-HBx-Cm6 compared with HepG2-HBx-Cm16. Furthermore, levels of HBV replication intermediates were decreased after silencing RSK2 in HepG2.2.15. An HBx-minus HBV mutant genome led to decreased levels of HBV replication intermediates and these decreases were restored to levels similar to wild-type HBV by transient ectopic expression of HBx. After silencing RSK2 expression, the levels of HBV replication intermediates synthesized from the HBx-minus HBV mutant genome were not restored to levels that were observed with wild-type HBV by transient HBx expression. Based on iTRAQ quantitative comparative proteomics, RSK2 was identified as a novel host protein that plays a role in HBx augmenting HBV replication. © 2018 The Authors. Proteomics - Clinical Application Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Natural history of acute and chronic hepatitis B: The role of HBV genotypes and mutants.

PubMed

Lin, Chih-Lin; Kao, Jia-Horng

2017-06-01

Molecular epidemiologic studies reveal remarkable differences in the geographical distribution of hepatitis B virus (HBV) genotypes. The frequency of mutants among HBV genotypes also varies. The role of HBV genotypes/mutants in the pathogenesis of HBV infection and natural history of HBV infection has been extensively investigated. The distribution of HBV genotypes in acute hepatitis B patients reflects the predominant genotypes in a given geographic area. In chronic hepatitis B patients, genotype C and D have a higher frequency of basal core promoter A1762T/G1764A mutations than genotype A and B. HBV genotypes C, D and F carry a higher lifetime risk of cirrhosis and HCC development than genotype A and B. HBV pre-S/S gene mutations were associated with immune escape of hepatitis B immunoglobulin or vaccine-induced immunity. Mutations in the pre-S, core promoter and X regions correlate with an increased risk of cirrhosis and HCC. In summary, HBV genotypes and mutants are associated with the disease progression and long-term outcome of HBV infection. They may serve as viral genetic markers for risk stratification of chronic hepatitis B patients in clinical practice. Copyright © 2017 Elsevier Ltd. All rights reserved.

Naturally occurring deletions/insertions in HBV core promoter tend to decrease in hepatitis B e antigen-positive chronic hepatitis B patients during antiviral therapy.

PubMed

Peng, Yaqin; Liu, Baoming; Hou, Jinlin; Sun, Jian; Hao, Ran; Xiang, Kuanhui; Yan, Ling; Zhang, Jiangbo; Zhuang, Hui; Li, Tong

2015-01-01

Mutations in HBV core promoter (CP) are suggested to affect viral replication and disease progression. We investigated CP deletion/insertion mutations (Del/Ins) in hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients before and during antiviral treatment. Direct and clone sequencings were used for detection of CP Del/Ins in 12 patients. The dynamic changes of CP Del/Ins were tracked in these cases until week 48 of treatment. The effects of Del/Ins on CP activities and hepatitis B X protein (HBx) were analysed using luciferase assay and sequence comparison, respectively. Furthermore, 292 untreated HBeAg-positive CHB cases were also analysed. Twelve cases with multi-peak PCR direct sequencing electropherograms at baseline were confirmed to have CP Del/Ins by clone sequencing, with detection rates varying from 14.8% to 93.3% of clones analysed. Follow-up studies showed the detection rates of CP Del/Ins in patients decreased from 100% (12/12) at baseline to 16.7% (2/12) at week 48 of treatment (P<0.001), in parallel with a decline in HBV DNA, hepatitis B surface antigen (HBsAg), alanine aminotransferase (ALT) and aspartate transaminase (AST) levels along with an increase in HBeAg loss. Luciferase assay results showed distinct promoter activities among Del/Ins-harbouring CP sequences. Importantly, 71.8% (148/206) of Del/Ins sequences potentially resulted in HBx carboxy-terminal truncations. CP Del/Ins mutations were also found in 27.4% (80/292) of untreated cases. Naturally occurring complex of CP Del/Ins mutants existed in untreated HBeAg-positive CHB patients. These mutations would affect HBV transcription activities and integrity of HBx, which might correlate with disease progression. Their prevalence decreases on antiviral therapy in parallel with the decline in HBV DNA, HBsAg and ALT and AST levels.

Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids.

PubMed

Schlicksup, Christopher John; Wang, Joseph Che-Yen; Francis, Samson; Venkatakrishnan, Balasubramanian; Turner, William W; VanNieuwenhze, Michael; Zlotnick, Adam

2018-01-29

Defining mechanisms of direct-acting antivirals facilitates drug development and our understanding of virus function. Heteroaryldihydropyrimidines (HAPs) inappropriately activate assembly of hepatitis B virus (HBV) core protein (Cp), suppressing formation of virions. We examined a fluorophore-labeled HAP, HAP-TAMRA. HAP-TAMRA induced Cp assembly and also bound pre-assembled capsids. Kinetic and spectroscopic studies imply that HAP-binding sites are usually not available but are bound cooperatively. Using cryo-EM, we observed that HAP-TAMRA asymmetrically deformed capsids, creating a heterogeneous array of sharp angles, flat regions, and outright breaks. To achieve high resolution reconstruction (<4 Å), we introduced a disulfide crosslink that rescued particle symmetry. We deduced that HAP-TAMRA caused quasi-sixfold vertices to become flatter and fivefold more angular. This transition led to asymmetric faceting. That a disordered crosslink could rescue symmetry implies that capsids have tensegrity properties. Capsid distortion and disruption is a new mechanism by which molecules like the HAPs can block HBV infection. © 2017, Schlicksup et al.

Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids

PubMed Central

Schlicksup, Christopher John; Wang, Joseph Che-Yen; Francis, Samson; Venkatakrishnan, Balasubramanian; Turner, William W; VanNieuwenhze, Michael

2018-01-01

Defining mechanisms of direct-acting antivirals facilitates drug development and our understanding of virus function. Heteroaryldihydropyrimidines (HAPs) inappropriately activate assembly of hepatitis B virus (HBV) core protein (Cp), suppressing formation of virions. We examined a fluorophore-labeled HAP, HAP-TAMRA. HAP-TAMRA induced Cp assembly and also bound pre-assembled capsids. Kinetic and spectroscopic studies imply that HAP-binding sites are usually not available but are bound cooperatively. Using cryo-EM, we observed that HAP-TAMRA asymmetrically deformed capsids, creating a heterogeneous array of sharp angles, flat regions, and outright breaks. To achieve high resolution reconstruction (<4 Å), we introduced a disulfide crosslink that rescued particle symmetry. We deduced that HAP-TAMRA caused quasi-sixfold vertices to become flatter and fivefold more angular. This transition led to asymmetric faceting. That a disordered crosslink could rescue symmetry implies that capsids have tensegrity properties. Capsid distortion and disruption is a new mechanism by which molecules like the HAPs can block HBV infection. PMID:29377794

TGF-β Suppression of HBV RNA through AID-Dependent Recruitment of an RNA Exosome Complex

PubMed Central

Kitamura, Kouichi; Wang, Zhe; Chowdhury, Sajeda; Monjurul, Ahasan Md; Wakae, Kousho; Koura, Miki; Shimadu, Miyuki; Kinoshita, Kazuo; Muramatsu, Masamichi

2015-01-01

Transforming growth factor (TGF)-β inhibits hepatitis B virus (HBV) replication although the intracellular effectors involved are not determined. Here, we report that reduction of HBV transcripts by TGF-β is dependent on AID expression, which significantly decreases both HBV transcripts and viral DNA, resulting in inhibition of viral replication. Immunoprecipitation reveals that AID physically associates with viral P protein that binds to specific virus RNA sequence called epsilon. AID also binds to an RNA degradation complex (RNA exosome proteins), indicating that AID, RNA exosome, and P protein form an RNP complex. Suppression of HBV transcripts by TGF-β was abrogated by depletion of either AID or RNA exosome components, suggesting that AID and the RNA exosome involve in TGF-β mediated suppression of HBV RNA. Moreover, AID-mediated HBV reduction does not occur when P protein is disrupted or when viral transcription is inhibited. These results suggest that induced expression of AID by TGF-β causes recruitment of the RNA exosome to viral RNP complex and the RNA exosome degrades HBV RNA in a transcription-coupled manner. PMID:25836330

A European multicientre study on the comparison of HBV viral loads between VERIS HBV assay and Roche COBAS® TAQMAN® HBV test, Abbott RealTime HBV assay, Siemens VERSANT HBV assay, and Qiagen artus HBV RG kit.

PubMed

Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Izopet, Jacques; Lombardi, Alessandra; Marcos, MaAngeles; Sauné, Karine; O'Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel

2017-10-01

Hepatitis B viral load testing is essential to treatment and monitoring decisions in patients with chronic Hepatitis B. Beckman Coulter has developed the VERIS HBV Assay (Veris) for use on the fully automated DxN VERIS Molecular Diagnostics System. 1 OBJECTIVES: To evaluate the clinical performance of the Veris HBV Assay at multiple EU laboratories STUDY DESIGN: Method comparison was performed with a total of 344 plasma specimens from HBV infected patients tested with Veris and COBAS ® TaqMan ® HBV Test (Cobas), 207 specimens tested with Veris and RealTime HBV Assay (RealTime), 86 specimens tested with Veris and VERSANT ® HBV Assay (Versant), and 74 specimens tested with Veris and artus ® HBV RG PCR kit (artus). Bland-Altman analysis showed average bias of -0.46 log 10 IU/mL between Veris and Cobas, -0.46 log 10 IU/mL between Veris and RealTime, -0.36 log 10 IU/mL between Veris and Versant, and -0.12 log 10 IU/mL between Veris and artus. Bias was consistent across the assay range. Patient monitoring results using Veris demonstrated similar viral load trends over time to Cobas, RealTime, and artus. The VERIS HBV Assay demonstrated comparable clinical performance, with varying degrees of negative bias, compared to other currently marketed assays for HBV DNA monitoring. This negative bias should be taken into consideration if switching monitoring methods to Veris. Copyright © 2017 Elsevier B.V. All rights reserved.

Novel pre-mRNA splicing of intronically integrated HBV generates oncogenic chimera in hepatocellular carcinoma.

PubMed

Chiu, Yung-Tuen; Wong, John K L; Choi, Shing-Wan; Sze, Karen M F; Ho, Daniel W H; Chan, Lo-Kong; Lee, Joyce M F; Man, Kwan; Cherny, Stacey; Yang, Wan-Ling; Wong, Chun-Ming; Sham, Pak-Chung; Ng, Irene O L

2016-06-01

Hepatitis B virus (HBV) integration is common in HBV-associated hepatocellular carcinoma (HCC) and may play an important pathogenic role through the production of chimeric HBV-human transcripts. We aimed to screen the transcriptome for HBV integrations in HCCs. Transcriptome sequencing was performed on paired HBV-associated HCCs and corresponding non-tumorous liver tissues to identify viral-human chimeric sites. Validation was further performed in an expanded cohort of human HCCs. Here we report the discovery of a novel pre-mRNA splicing mechanism in generating HBV-human chimeric protein. This mechanism was exemplified by the formation of a recurrent HBV-cyclin A2 (CCNA2) chimeric transcript (A2S), as detected in 12.5% (6 of 48) of HCC patients, but in none of the 22 non-HCC HBV-associated cirrhotic liver samples examined. Upon the integration of HBV into the intron of the CCNA2 gene, the mammalian splicing machinery utilized the foreign splice sites at 282nt. and 458nt. of the HBV genome to generate a pseudo-exon, forming an in-frame chimeric fusion with CCNA2. The A2S chimeric protein gained a non-degradable property and promoted cell cycle progression, demonstrating its potential oncogenic functions. A pre-mRNA splicing mechanism is involved in the formation of HBV-human chimeric proteins. This represents a novel and possibly common mechanism underlying the formation of HBV-human chimeric transcripts from intronically integrated HBV genome with functional impact. HBV is involved in the mammalian pre-mRNA splicing machinery in the generation of potential tumorigenic HBV-human chimeras. This study also provided insight on the impact of intronic HBV integration with the gain of splice sites in the development of HBV-associated HCC. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Diagnostic value of serum Golgi protein 73 for HBV-related primary hepatic carcinoma

PubMed Central

Gao, Guosheng; Dong, Feibo; Xu, Xiaozhen; Hu, Airong; Hu, Yaoren

2015-01-01

Background: Alpha-fetoprotein (AFP) levels are routinely used for diagnosis and monitoring of hepatic diseases, but it has a limited value. Golgi protein 73 (GP73) has been suggested as a new marker for hepatic diseases. Objective: To explore the clinical value of serum GP73 in different diseases associated with hepatitis B virus (HBV) infection. Method: Between January 2010 and August 2014, serum samples from 88 patients with chronic hepatitis B (CHB), 78 patients with HBV-related liver cirrhosis (LC), and 194 patients with HBV-related primary hepatic cancer (PHC) were collected. Serum samples from 30 healthy volunteers were used as controls. ELISA and microparticle enzyme immunoassay were used to measure serum GP73 and AFP levels. Receiver operating characteristic (ROC) curves were used to analyze the diagnostic value of serum GP73 and AFP for PHC. Results: For the diagnosis of PHC, GP73 showed a sensitivity of 65.5% and specificity of 66.3%, while AFP levels showed sensitivity of 64.4% and specificity of 76.5%. Serial testing (both tests are positive) could increase the specificity (sensitivity of 45.9% and specificity of 85.5%) while parallel testing (any single positive test result) could increase the sensitivity (sensitivity of 84.0% and specificity of 57.2%). Serum GP73 and AFP levels were significantly different between Child-Pugh grades (P<0.001 for GP73 and P=0.044 for AFP). Significant differences in serum GP73 and AFP were found between TNM stages (all P<0.001). Conclusion: Serum GP73 had limited diagnostic value for HBV-related PHC. The combined use of serum GP73 and AFP levels improved the diagnostic efficacy. PMID:26617863

Episomal HBV persistence within transcribed host nuclear chromatin compartments involves HBx.

PubMed

Hensel, Kai O; Cantner, Franziska; Bangert, Felix; Wirth, Stefan; Postberg, Jan

2018-06-22

In hepatocyte nuclei, hepatitis B virus (HBV) genomes occur episomally as covalently closed circular DNA (cccDNA). The HBV X protein (HBx) is required to initiate and maintain HBV replication. The functional nuclear localization of cccDNA and HBx remains unexplored. To identify virus-host genome interactions and the underlying nuclear landscape for the first time, we combined circular chromosome conformation capture (4C) with RNA-seq and ChIP-seq. Moreover, we studied HBx-binding to HBV episomes. In HBV-positive HepaRG hepatocytes, we observed preferential association of HBV episomes and HBx with actively transcribed nuclear domains on the host genome correlating in size with constrained topological units of chromatin. Interestingly, HBx alone occupied transcribed chromatin domains. Silencing of native HBx caused reduced episomal HBV stability. As part of the HBV episome, HBx might stabilize HBV episomal nuclear localization. Our observations may contribute to the understanding of long-term episomal stability and the facilitation of viral persistence. The exact mechanism by which HBx contributes to HBV nuclear persistence warrants further investigations.

Transmission of HBV DNA Mediated by Ceramide-Triggered Extracellular Vesicles.

PubMed

Sanada, Takahiro; Hirata, Yuichi; Naito, Yutaka; Yamamoto, Naoki; Kikkawa, Yoshiaki; Ishida, Yuji; Yamasaki, Chihiro; Tateno, Chise; Ochiya, Takahiro; Kohara, Michinori

2017-03-01

An extracellular vesicle (EV) is a nanovesicle that shuttles proteins, nucleic acids, and lipids, thereby influencing cell behavior. A recent crop of reports have shown that EVs are involved in infectious biology, influencing host immunity and playing a role in the viral life cycle. In the present work, we investigated the EV-mediated transmission of hepatitis B virus (HBV) infection. We investigated the EV-mediated transmission of HBV infection by using a HBV infectious culture system that uses primary human hepatocytes derived from humanized chimeric mice (PXB-cells). Purified EVs were isolated by ultracentrifugation. To analyze the EVs and virions, we used stimulated emission depletion microscopy. Purified EVs from HBV-infected PXB-cells were shown to contain HBV DNA and to be capable of transmitting HBV DNA to naive PXB-cells. These HBV-DNA-transmitting EVs were shown to be generated through a ceramide-triggered EV production pathway. Furthermore, we showed that these HBV-DNA-transmitting EVs were resistant to antibody neutralization; stimulated emission depletion microscopy showed that EVs lacked hepatitis B surface antigen, the target of neutralizing antibodies. These findings suggest that EVs harbor a DNA cargo capable of transmitting viral DNA into hepatocytes during HBV infection, representing an additional antibody-neutralization-resistant route of HBV infection.

Characterization of HBV integration patterns and timing in liver cancer and HBV-infected livers.

PubMed

Furuta, Mayuko; Tanaka, Hiroko; Shiraishi, Yuichi; Unida, Takuro; Imamura, Michio; Fujimoto, Akihiro; Fujita, Masahi; Sasaki-Oku, Aya; Maejima, Kazuhiro; Nakano, Kaoru; Kawakami, Yoshiiku; Arihiro, Koji; Aikata, Hiroshi; Ueno, Masaki; Hayami, Shinya; Ariizumi, Shun-Ichi; Yamamoto, Masakazu; Gotoh, Kunihito; Ohdan, Hideki; Yamaue, Hiroki; Miyano, Satoru; Chayama, Kazuaki; Nakagawa, Hidewaki

2018-05-18

Integration of Hepatitis B virus (HBV) into the human genome can cause genetic instability, leading to selective advantages for HBV-induced liver cancer. Despite the large number of studies for HBV integration into liver cancer, little is known about the mechanism of initial HBV integration events owing to the limitations of materials and detection methods. We conducted an HBV sequence capture, followed by ultra-deep sequencing, to screen for HBV integrations in 111 liver samples from human-hepatocyte chimeric mice with HBV infection and human clinical samples containing 42 paired samples from non-tumorous and tumorous liver tissues. The HBV infection model using chimeric mice verified the efficiency of our HBV-capture analysis and demonstrated that HBV integration could occur 23 to 49 days after HBV infection via microhomology-mediated end joining and predominantly in mitochondrial DNA. Overall HBV integration sites in clinical samples were significantly enriched in regions annotated as exhibiting open chromatin, a high level of gene expression, and early replication timing in liver cells. These data indicate that HBV integration in liver tissue was biased according to chromatin accessibility, with additional selection pressures in the gene promoters of tumor samples. Moreover, an integrative analysis using paired non-tumorous and tumorous samples and HBV-related transcriptional change revealed the involvement of TERT and MLL4 in clonal selection. We also found frequent and non-tumorous liver-specific HBV integrations in FN1 and HBV-FN1 fusion transcript. Extensive survey of HBV integrations facilitates and improves the understanding of the timing and biology of HBV integration during infection and HBV-related hepatocarcinogenesis.

Occult HBV infection in HIV-infected adults and evaluation of pooled NAT for HBV.

PubMed

Dinesha, T R; Boobalan, J; Sivamalar, S; Subashini, D; Solomon, S S; Murugavel, K G; Balakrishnan, P; Smith, D M; Saravanan, S

2018-06-01

The study aimed to determine the prevalence of occult hepatitis B virus infection among HIV-infected persons and to evaluate the use of a pooling strategy to detect occult HBV infection in the setting of HIV infection. Five hundred and two HIV-positive individuals were tested for HBV, occult HBV and hepatitis C and D with serologic and nucleic acid testing (NAT). We also evaluated a pooled NAT strategy for screening occult HBV infection among the HIV-positive individuals. The prevalence of HBV infection among HIV-positive individuals was 32 (6.4%), and occult HBV prevalence was 10%. The pooling HBV NAT had a sensitivity of 66.7% and specificity of 100%, compared to HBV DNA NAT of individual samples. In conclusion, this study found a high prevalence of occult HBV infection among our HIV-infected population. We also demonstrated that pooled HBV NAT is highly specific, moderately sensitive and cost-effective. As conventional HBV viral load assays are expensive in resource-limited settings such as India, pooled HBV DNA NAT might be a good way for detecting occult HBV infection and will reduce HBV-associated complications. © 2018 John Wiley & Sons Ltd.

The presence of HBV mRNA in the fertilized in vitro embryo of HBV patients confirms vertical transmission of HBV via the ovum.

PubMed

Ye, F; Jin, Y; Kong, Y; Shi, J Z; Qiu, H T; Zhang, X; Zhang, S L; Lin, S M

2013-05-01

This study aimed to confirm that vertical transmission of hepatitis B virus (HBV) can occur via the infected ovum. Specimens studied were obtained from discarded test-tube embryos from mothers with chronic HBV infection who had received in vitro fertilization treatment. Single-cell reverse transcriptase-polymerase chain reaction was used to detect HBV mRNA in the embryos. HBV mRNA was detected in the cleavage embryos of patients with chronic HBV infection, with a detection rate of 13.2% (5/38). The level of serum HBV DNA was not related to the HBV mRNA positivity rates in embryos. In this study, HBV mRNA was detected in test-tube embryos from HBV-infected mothers who had received in vitro fertilization treatment. This confirms the theory of vertical transmission of HBV via the ovum, thereby providing an important theoretical basis for further study on the mechanism of HBV vertical transmission, influencing factors and blocking measures.

Nuclear export of human hepatitis B virus core protein and pregenomic RNA depends on the cellular NXF1-p15 machinery.

PubMed

Yang, Ching-Chun; Huang, Er-Yi; Li, Hung-Cheng; Su, Pei-Yi; Shih, Chiaho

2014-01-01

Hepatitis B virus (HBV) core protein (HBc) can shuttle between nucleus and cytoplasm. Cytoplasm-predominant HBc is clinically associated with severe liver inflammation. Previously, we found that HBc arginine-rich domain (ARD) can associate with a host factor NXF1 (TAP) by coimmunoprecipitation. It is well known that NXF1-p15 heterodimer can serve as a major export receptor of nuclear mRNA as a ribonucleoprotein complex (RNP). In the NXF1-p15 pathway, TREX (transcription/export) complex plays an important role in coupling nuclear pre-mRNA processing with mRNA export in mammalian cells. Here, we tested the hypothesis whether HBc and HBV specific RNA can be exported via the TREX and NXF1-p15 mediated pathway. We demonstrated here that HBc can physically and specifically associate with TREX components, and the NXF1-p15 export receptor by coimmunoprecipitation. Accumulation of HBc protein in the nucleus can be induced by the interference with TREX and NXF1-p15 mediated RNA export machinery. HBV transcripts encodes a non-spliced 3.5 kb pregenomic RNA (pgRNA) which can serve as a template for reverse transcription. Cytoplasmic HBV pgRNA appeared to be reduced by siRNA treatment specific for the NXF1-p15 complex by quantitative RT-qPCR and Northern blot analyses. This result suggests that the pgRNA was also exported via the NXF1-p15 machinery. We entertain the hypothesis that HBc protein can be exported as an RNP cargo via the mRNA export pathway by hijacking the TREX and NXF1-p15 complex. In our current and previous studies, HBc is not required for pgRNA accumulation in the cytoplasm. Furthermore, HBc ARD can mediate nuclear export of a chimeric protein containing HBc ARD in a pgRNA-independent manner. Taken together, it suggests that while both pgRNA and HBc protein exports are dependent on NXF1-p15, they are using the same export machinery in a manner independent of each other.

A Longitudinal Hepatitis B Vaccine Cohort Demonstrates Long-lasting Hepatitis B Virus (HBV) Cellular Immunity Despite Loss of Antibody Against HBV Surface Antigen

PubMed Central

Simons, Brenna C.; Spradling, Philip R.; Bruden, Dana J. T.; Zanis, Carolyn; Case, Samantha; Choromanski, Tammy L.; Apodaca, Minjun; Brogdon, Hazel D.; Dwyer, Gaelen; Snowball, Mary; Negus, Susan; Bruce, Michael G.; Morishima, Chihiro; Knall, Cindy; McMahon, Brian J.

2016-01-01

Background. Long-lasting protection resulting from hepatitis B vaccine, despite loss of antibody against hepatitis B virus (HBV) surface antigen (anti-HBs), is undetermined. Methods. We recruited persons from a cohort vaccinated with plasma-derived hepatitis B vaccine in 1981 who have been followed periodically since. We performed serological testing for anti-HBs and microRNA-155 and assessed HBV-specific T-cell responses by enzyme-linked immunospot and cytometric bead array. Study subgroups were defined 32 years after vaccination as having an anti-HBs level of either ≥10 mIU/mL (group 1; n = 13) or <10 mIU/mL (group 2; n = 31). Results. All 44 participants, regardless of anti-HBs level, tested positive for tumor necrosis factor α, interleukin 10, or interleukin 6 production by HBV surface antigen–specific T cells. The frequency of natural killer T cells correlated with the level of anti-HBs (P = .008). The proportion of participants who demonstrated T-cell responses to HBV core antigen varied among the cytokines measured, suggesting some natural exposure to HBV in the study group. No participant had evidence of breakthrough HBV infection. Conclusions. Evidence of long-lasting cellular immunity, regardless of anti-HBs level, suggests that protection afforded by primary immunization with plasma-derived hepatitis B vaccine during childhood and adulthood lasts at least 32 years. PMID:27056956

An overview of molecular epidemiology of hepatitis B virus (HBV) in India.

PubMed

Datta, Sibnarayan

2008-12-19

Hepatitis B virus (HBV) is one of the major global public health problems. In India, HBsAg prevalence among general population ranges from 2% to 8%, placing India in intermediate HBV endemicity zone and the number of HBV carriers is estimated to be 50 million, forming the second largest global pool of chronic HBV infections. India is a vast country, comprised of multiracial communities with wide variations in ethnicity and cultural patterns, which is attributable to its geographical location, gene influx due to invasion and/or anthropological migrations in the past. Moreover, recent increase in trade, trafficking and use of illicit drugs has also considerably influenced the epidemiology of HBV, specifically in the eastern and north eastern parts of India. However, data on the molecular epidemiology of HBV in India is scanty. HBV genotypes A and D have been well documented from different parts of mainland India. Interestingly, in addition to genotypes A and D, genotype C having high nucleotide similarity with south East Asian subgenotype Cs/C1 strain, have been detected exclusively from eastern Indian HBV carriers, suggesting a recent introduction. Thus, compared to other parts of India, the molecular epidemiology of HBV is naturally distinct in eastern India. Very recently, taking the advantage of circulation of three distinct HBV genotypes within the population of eastern India, different aspects of HBV molecular epidemiology was studied that revealed very interesting results. In this study, the clinical significance of HBV genotypes, core promoter and precore mutations, possible routes of introduction of HBV genotype C in eastern India, the clinical implications of x gene variability, prevalence of the AFB1 induced p53 gene codon 249 mutation, the transmission potentiality of HBV among asymptomatic/inactive or occult HBV carriers and the genetic variability of HBV persisting in the PBL was investigated. In this manuscript, the information available on the

HBV-Derived Synthetic Long Peptide Can Boost CD4+ and CD8+ T-Cell Responses in Chronic HBV Patients Ex Vivo

PubMed Central

Dou, Yingying; van Montfoort, Nadine; van den Bosch, Aniek; de Man, Robert A; Zom, Gijs G; Krebber, Willem-Jan; Melief, Cornelis J M; Buschow, Sonja I; Woltman, Andrea M

2018-01-01

Abstract Background Vaccination with synthetic long peptides (SLP) is a promising new treatment strategy for chronic hepatitis B virus (CHB). SLP can induce broad T-cell responses for all HLA types. Here we investigated the ability of a prototype HBV-core (HBc)-sequence-derived SLP to boost HBV-specific T cells in CHB patients ex vivo. Methods HBc-SLP was used to assess cross-presentation by monocyte-derived dendritic cells (moDC) and BDCA1+ blood myeloid DC (mDC) to engineered HBV-specific CD8+ T cells. Autologous SLP-loaded and toll-like receptor (TLR)-stimulated DC were used to activate patient HBc-specific CD8+ and CD4+ T cells. Results HBV-SLP was cross-presented by moDC, which was further enhanced by adjuvants. Patient-derived SLP-loaded moDC significantly increased autologous HBcAg18-27-specific CD8+ T cells and CD4+ T cells ex vivo. HBV-specific T cells were functional as they synthesized tumor necrosis factor-alpha and interferon-gamma. In 6/7 of patients blockade of PD-L1 further increased SLP effects. Also, importantly, patient-derived BDCA1+ mDC cross-presented and activated autologous T-cell responses ex vivo. Conclusions As a proof of concept, we showed a prototype HBc-SLP can boost T-cell responses in patients ex vivo. These results pave the way for the development of a therapeutic SLP-based vaccine to induce effective HBV-specific adaptive immune responses in CHB patients. PMID:29220492

Packing in protein cores

NASA Astrophysics Data System (ADS)

Gaines, J. C.; Clark, A. H.; Regan, L.; O'Hern, C. S.

2017-07-01

Proteins are biological polymers that underlie all cellular functions. The first high-resolution protein structures were determined by x-ray crystallography in the 1960s. Since then, there has been continued interest in understanding and predicting protein structure and stability. It is well-established that a large contribution to protein stability originates from the sequestration from solvent of hydrophobic residues in the protein core. How are such hydrophobic residues arranged in the core; how can one best model the packing of these residues, and are residues loosely packed with multiple allowed side chain conformations or densely packed with a single allowed side chain conformation? Here we show that to properly model the packing of residues in protein cores it is essential that amino acids are represented by appropriately calibrated atom sizes, and that hydrogen atoms are explicitly included. We show that protein cores possess a packing fraction of φ ≈ 0.56 , which is significantly less than the typically quoted value of 0.74 obtained using the extended atom representation. We also compare the results for the packing of amino acids in protein cores to results obtained for jammed packings from discrete element simulations of spheres, elongated particles, and composite particles with bumpy surfaces. We show that amino acids in protein cores pack as densely as disordered jammed packings of particles with similar values for the aspect ratio and bumpiness as found for amino acids. Knowing the structural properties of protein cores is of both fundamental and practical importance. Practically, it enables the assessment of changes in the structure and stability of proteins arising from amino acid mutations (such as those identified as a result of the massive human genome sequencing efforts) and the design of new folded, stable proteins and protein-protein interactions with tunable specificity and affinity.

Cytotoxic T lymphocytes and CD4 epitope mutations in the pre-core/core region of hepatitis B virus in chronic hepatitis B carriers in Northeast Iran.

PubMed

Zhand, Sareh; Tabarraei, Alijan; Nazari, Amineh; Moradi, Abdolvahab

2017-07-01

Hepatitis B virus (HBV) is vulnerable to many various mutations. Those within epitopes recognized by sensitized T cells may influence the re-emergence of the virus. This study was designed to investigate the mutation in immune epitope regions of HBV pre-core/core among chronic HBV patients of Golestan province, Northeast Iran. In 120 chronic HBV carriers, HBV DNA was extracted from blood plasma samples and PCR was done using specific primers. Direct sequencing and alignment of the pre-core/core region were applied using reference sequence from Gene Bank database (Accession Number AB033559). The study showed 27 inferred amino acid substitutions, 9 of which (33.3%) were in CD4 and 2 (7.4%) in cytotoxic T lymphocytes' (CTL) epitopes and 16 other mutations (59.2%) were observed in other regions. CTL escape mutations were not commonly observed in pre-core/core sequences of chronic HBV carriers in the locale of study. It can be concluded that most of the inferred amino acid substitutions occur in different immune epitopes other than CTL and CD4.

Sleeping Beauty transposon-based system for rapid generation of HBV-replicating stable cell lines.

PubMed

Wu, Yong; Zhang, Tian-Ying; Fang, Lin-Lin; Chen, Zi-Xuan; Song, Liu-Wei; Cao, Jia-Li; Yang, Lin; Yuan, Quan; Xia, Ning-Shao

2016-08-01

The stable HBV-replicating cell lines, which carry replication-competent HBV genome stably integrated into the genome of host cell, are widely used to evaluate the effects of antiviral agents. However, current methods to generate HBV-replicating cell lines, which are mostly dependent on random integration of foreign DNA via plasmid transfection, are less-efficient and time-consuming. To address this issue, we constructed an all-in-one Sleeping Beauty transposon system (denoted pTSMP-HBV vector) for robust generation of stable cell lines carrying replication-competent HBV genome of different genotype. This vector contains a Sleeping Beauty transposon containing HBV 1.3-copy genome with an expression cassette of the SV40 promoter driving red fluorescent protein (mCherry) and self-cleaving P2A peptide linked puromycin resistance gene (PuroR). In addition, a PGK promoter-driven SB100X hyperactive transposase cassette is placed in the outside of the transposon in the same plasmid.The HBV-replicating stable cells could be obtained from pTSMP-HBV transfected HepG2 cells by red fluorescence-activated cell sorting and puromycin resistant cell selection within 4-week. Using this system, we successfully constructed four cell lines carrying replication-competent HBV genome of genotypes A-D. The replication and viral protein expression profiles of these cells were systematically characterized. In conclusion, our study provides a high-efficiency strategy to generate HBV-replicating stable cell lines, which may facilitate HBV-related virological study. Copyright © 2016. Published by Elsevier B.V.

A Longitudinal Hepatitis B Vaccine Cohort Demonstrates Long-lasting Hepatitis B Virus (HBV) Cellular Immunity Despite Loss of Antibody Against HBV Surface Antigen.

PubMed

Simons, Brenna C; Spradling, Philip R; Bruden, Dana J T; Zanis, Carolyn; Case, Samantha; Choromanski, Tammy L; Apodaca, Minjun; Brogdon, Hazel D; Dwyer, Gaelen; Snowball, Mary; Negus, Susan; Bruce, Michael G; Morishima, Chihiro; Knall, Cindy; McMahon, Brian J

2016-07-15

Long-lasting protection resulting from hepatitis B vaccine, despite loss of antibody against hepatitis B virus (HBV) surface antigen (anti-HBs), is undetermined. We recruited persons from a cohort vaccinated with plasma-derived hepatitis B vaccine in 1981 who have been followed periodically since. We performed serological testing for anti-HBs and microRNA-155 and assessed HBV-specific T-cell responses by enzyme-linked immunospot and cytometric bead array. Study subgroups were defined 32 years after vaccination as having an anti-HBs level of either ≥10 mIU/mL (group 1; n = 13) or <10 mIU/mL (group 2; n = 31). All 44 participants, regardless of anti-HBs level, tested positive for tumor necrosis factor α, interleukin 10, or interleukin 6 production by HBV surface antigen-specific T cells. The frequency of natural killer T cells correlated with the level of anti-HBs (P = .008). The proportion of participants who demonstrated T-cell responses to HBV core antigen varied among the cytokines measured, suggesting some natural exposure to HBV in the study group. No participant had evidence of breakthrough HBV infection. Evidence of long-lasting cellular immunity, regardless of anti-HBs level, suggests that protection afforded by primary immunization with plasma-derived hepatitis B vaccine during childhood and adulthood lasts at least 32 years. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

HBV life cycle is restricted in mouse hepatocytes expressing human NTCP.

PubMed

Li, Hanjie; Zhuang, Qiuyu; Wang, Yuze; Zhang, Tianying; Zhao, Jinghua; Zhang, Yali; Zhang, Junfang; Lin, Yi; Yuan, Quan; Xia, Ningshao; Han, Jiahuai

2014-03-01

Recent studies have revealed that human sodium taurocholate cotransporting polypeptide (SLC10A1 or NTCP) is a functional cellular receptor for hepatitis B virus (HBV). However, whether human NTCP can support HBV infection in mouse hepatocyte cell lines has not been clarified. Because an HBV-permissible mouse model would be helpful for the study of HBV pathogenesis, it is necessary to investigate whether human NTCP supports the susceptibility of mouse hepatocyte cell lines to HBV. The results show that exogenous human NTCP expression can render non-susceptible HepG2 (human), Huh7 (human), Hepa1-6 (mouse), AML-12 (mouse) cell lines and primary mouse hepatocyte (PMH) cells susceptible to hepatitis D virus (HDV) which employs HBV envelope proteins. However, human NTCP could only introduce HBV susceptibility in human-derived HepG2 and Huh7 cells, but not in mouse-derived Hepa1-6, AML-12 or PMH cells. These data suggest that although human NTCP is a functional receptor that mediates HBV infection in human cells, it cannot support HBV infection in mouse hepatocytes. Our study indicated that the restriction of HBV in mouse hepatocytes likely occurs after viral entry but prior to viral transcription. We have excluded the role of mouse hepatocyte nuclear factors in the restriction of the HBV life cycle and showed that knockdown or inhibition of Sting, TBK1, IRF3 or IRF7, the components of the anti-viral signaling pathways, had no effect on HBV infection in mouse hepatocytes. Therefore, murine restriction factors that limit HBV infection need to be identified before a HBV-permissible mouse line can be created.

Effects of transarterial chemoembolization combined with antiviral therapy on HBV reactivation and liver function in HBV-related hepatocellular carcinoma patients with HBV-DNA negative.

PubMed

Wang, Kai; Jiang, Guomin; Jia, Zhongzhi; Zhu, Xiaoli; Ni, Caifang

2018-06-01

The aim of this study was to investigate the reactivation of the hepatitis B virus (HBV) following transarterial chemoembolization (TACE) in primary hepatocellular carcinoma (HCC) patients with HBV-DNA negative and to evaluate the effects of TACE combined with antiviral therapy. This prospective study involved 98 patients with HBV-related and HBV-DNA negative HCC (HBV DNA < 10 copies/mL) underwent TACE procedures with serial HBV DNA tests. Patients were divided into the antiviral treatment group and the no-antiviral group. The antiviral group received entecavir antiviral therapy, and the other group received no antiviral therapy. Two groups of patients were compared in rate of HBV reactivation and liver function before and after only 1 session of TACE in average 1-month follow-up after operation. P < .05 indicated differences with a statistical significance. HBV reactivation occurred in 11 patients in the nonantiviral group (11/47, 23.4%) but only 3 patients in the antiviral group (3/51, 5.9%, P < .05). On multivariate analysis, HBeAg-positive status, number of tumors more than 3, and absence of antiviral therapy were the independent risk predictor of HBV reactivation. Liver function indicators did not differ significantly between the antiviral group and the nonantiviral group in 5 days after TACE. However, the level of alanine aminotransferase and bilirubin were raised and albumin was reduced at the HBV reactivation group compared with no HBV reactivation group (P < .05). At 1 month after TACE, liver function indicators did not differ significantly between the HBV reactivation group and without HBV reactivation group. HCC patients with HBV DNA negative still remain associated with risk of HBV reactivation after TACE. HBeAg-positive, number of tumors more than 3, and absence of antiviral therapy in HCC patients after TACE have a higher risk of HBV reactivation. Antiviral therapy can reduce the risk of reactivation, helping improve liver function

Core-shell microparticles for protein sequestration and controlled release of a protein-laden core.

PubMed

Rinker, Torri E; Philbrick, Brandon D; Temenoff, Johnna S

2017-07-01

Development of multifunctional biomaterials that sequester, isolate, and redeliver cell-secreted proteins at a specific timepoint may be required to achieve the level of temporal control needed to more fully regulate tissue regeneration and repair. In response, we fabricated core-shell heparin-poly(ethylene-glycol) (PEG) microparticles (MPs) with a degradable PEG-based shell that can temporally control delivery of protein-laden heparin MPs. Core-shell MPs were fabricated via a re-emulsification technique and the number of heparin MPs per PEG-based shell could be tuned by varying the mass of heparin MPs in the precursor PEG phase. When heparin MPs were loaded with bone morphogenetic protein-2 (BMP-2) and then encapsulated into core-shell MPs, degradable core-shell MPs initiated similar C2C12 cell alkaline phosphatase (ALP) activity as the soluble control, while non-degradable core-shell MPs initiated a significantly lower response (85+19% vs. 9.0+4.8% of the soluble control, respectively). Similarly, when degradable core-shell MPs were formed and then loaded with BMP-2, they induced a ∼7-fold higher C2C12 ALP activity than the soluble control. As C2C12 ALP activity was enhanced by BMP-2, these studies indicated that degradable core-shell MPs were able to deliver a bioactive, BMP-2-laden heparin MP core. Overall, these dynamic core-shell MPs have the potential to sequester, isolate, and then redeliver proteins attached to a heparin core to initiate a cell response, which could be of great benefit to tissue regeneration applications requiring tight temporal control over protein presentation. Tissue repair requires temporally controlled presentation of potent proteins. Recently, biomaterial-mediated binding (sequestration) of cell-secreted proteins has emerged as a strategy to harness the regenerative potential of naturally produced proteins, but this strategy currently only allows immediate amplification and re-delivery of these signals. The multifunctional, dynamic

[Impact of HIV/HBV infection and HIV/HBV co-infection on outcomes of pregnancy].

PubMed

Yang, Y; Cheng, W T; Zhou, Y B; Jiang, Q W

2017-06-10

Both HIV and HBV infection have become major health problems, of global concern, due to the high prevalence in the past few decades. Data from cumulated epidemiological surveys have shown the links between maternal HIV or HBV infection and adverse outcomes on pregnancy. Maternal HIV or HBV infection may also increase the mother-to-child (MTCT) transmission of the two diseases. However, association between HIV-HBV co-infection and adverse pregnancy is still inconclusive. Does maternal HIV-HBV co-infection have an impact on mother-to-child transmission on either HIV or HBV? Study on effective precautionary measures to promote both maternal and child's health is deemed necessary.

Evidence of susceptibility to lamivudine-based HAART and genetic stability of hepatitis B virus (HBV) in HIV co-infected patients: A South African longitudinal HBV whole genome study.

PubMed

Amponsah-Dacosta, Edina; Rakgole, J Nare; Gededzha, Maemu P; Lukhwareni, Azwidowi; Blackard, Jason T; Selabe, Selokela G; Mphahlele, M Jeffrey

2016-09-01

Reports on the concomitant impact of HIV co-infection and long term highly active anti-retroviral therapy (HAART) on the genetic stability and molecular evolution of HBV are limited in sub-Saharan Africa. This retrospective study investigated the molecular evolution of chronic HBV in HIV co-infected patients on lamivudine (3TC)-based HAART over a 5year period. Four HIV co-infected patients, consecutively recruited and followed-up, were screened for hepatitis B serological markers, and their viral loads determined. The HBV genome was amplified from longitudinal samples and characterized by Bayesian inference, mutational analysis, and identification of immune selection pressure. All patients exhibited persistent chronic HBV infection at baseline, as well as over the course of follow-up despite exposure to 3TC-based HAART. The polymerase gene in all isolates was relatively variable prior to HAART initiation at baseline and during the course of follow-up, although primary drug resistance mutations were not detected. All but one patient were infected with HBV subgenotype A1. The divergence rates between baseline and the last follow-up sequences ranged from 0 to 2.0×10(-3) substitutions per site per year (s/s/y). Positive selection pressure was evident within the surface and core genes. Despite persistent HBV infection in the HIV co-infected patients exposed to long term 3TC-based HAART, the molecular evolution of HBV over a 5year period was unremarkable. In addition, HBV exhibited minimal genetic variability overtime. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

Das, Dipanwita; Sengupta, Isha; Sarkar, Neelakshi; Pal, Ananya; Saha, Debraj; Bandopadhyay, Manikankana; Das, Chandrima; Narayan, Jimmy; Singh, Shivaram Prasad; Chakrabarti, Sekhar; Chakravarty, Runu

2017-01-14

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. 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. 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. The study thus holds a close insight into the changes in hepatocyte micro-environment on TLR7 stimulation in HBV infection.

Novel pH-sensitive multifunctional envelope-type nanodevice for siRNA-based treatments for chronic HBV infection.

PubMed

Yamamoto, Naoki; Sato, Yusuke; Munakata, Tsubasa; Kakuni, Masakazu; Tateno, Chise; Sanada, Takahiro; Hirata, Yuichi; Murakami, Shuko; Tanaka, Yasuhito; Chayama, Kazuaki; Hatakeyama, Hiroto; Hyodo, Mamoru; Harashima, Hideyoshi; Kohara, Michinori

2016-03-01

Antiviral agents including entecavir (ETV) suppress the replication of the hepatitis B virus (HBV) genome in human hepatocytes, but they do not reduce the abundance of viral proteins. The present study focused on effectively reducing viral protein levels. We designed siRNAs (HBV-siRNA) that target consensus sequences in HBV genomes. To prevent the emergence of escaped mutant virus, we mixed three HBV-siRNAs (HBV-siRNAmix); the mixture was encapsulated in a novel pH-sensitive multifunctional envelope-type nanodevice (MEND), a hepatocyte-specific drug delivery system. Coagulation factor 7 siRNA was used to assess delivery and knockdown efficiencies of MEND/siRNA treatments in mice. The potency of MEND/HBV-siRNAmix was evaluated in primary human hepatocytes and in chimeric mice with humanized liver persistently infected with HBV. Effective knockdown of targets, efficient delivery of siRNA, and liver-specific delivery were each observed with MEND. MEND/HBV-siRNA caused efficient reduction of HBsAg and HBeAg in vitro and in vivo. However, ETV treatment did not efficiently reduce HBsAg or HBeAg when compared with a single MEND/HBV-siRNAmix treatment. Furthermore, the suppressive effects of a single dose of MEND/HBV-siRNAmix persisted for 14days in vitro and in vivo. We demonstrated that MEND/HBV-siRNA controlled HBV more efficiently than did ETV. Furthermore, the effect of a single dose of MEND/HBV-siRNA persisted for a long time. These results indicated that MEND/HBV-siRNA may be a promising novel HBV treatment that is more effective than reverse transcriptase inhibitors. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Resveratrol enhances HBV replication through activating Sirt1-PGC-1α-PPARα pathway.

PubMed

Shi, Yixian; Li, Yongjun; Huang, Chenjie; Ying, Lixiong; Xue, Jihua; Wu, Haicong; Chen, Zhi; Yang, Zhenggang

2016-04-21

The population of hepatitis B combined with a number of metabolic disorders is increasing significantly. Resveratrol (RSV) has been used as a preclinical drug for the treatment of the metabolic disorders. However, the impact of RSV on HBV replication remains unknown. In this study, the HBV-expressing hepatocelluar carcinoma cell line and mouse model created by hydrodynamic injection of viral DNA were used. We found that RSV activates Sirt1, which in turn deacetylates PGC-1α and subsequently increases the transcriptional activity of PPARα, leading to the enhanced HBV transcription and replication in vitro and in vivo. In addition, we found that this pathway is also required for fasting-induced HBV transcription. Taken together, this study identifies that RSV enhances HBV transcription and replication especially acting on the core promoter, which depends on Sirt1-PGC-1α-PPARα pathway. We conclude that RSV may exacerbate the progression of hepatitis B and that patients with hepatitis B infection should be cautious taking RSV as a dietary supplement.

Presence of intrahepatic (total and ccc) HBV DNA is not predictive of HBV recurrence after liver transplantation.

PubMed

Hussain, Munira; Soldevila-Pico, Consuelo; Emre, Sukru; Luketic, Velimir; Lok, Anna S F

2007-08-01

Previous studies reported that hepatitis B virus (HBV) deoxyribonucleic acid (DNA) can be detected in livers of patients who received transplants for hepatitis B despite the absence of serological markers of HBV recurrence. Quantification of HBV DNA was not performed and presence of covalently closed circular (ccc) DNA was not analyzed in most studies. We aimed to quantify total and ccc HBV DNA in explant liver and post-orthotopic liver transplantation (OLT) biopsies and to correlate the values with HBV recurrence post-OLT. Frozen liver tissue from 34 patients (9 with explant liver only, 9 with explant liver and post-OLT liver biopsies, and 16 with post-OLT biopsies only) in the National Institutes of Health HBV-OLT study was examined using real-time polymerase chain reaction (PCR). Among the 18 patients with explant liver, 7 were hepatitis B e antigen (HBeAg)-positive, 8 had detectable serum HBV DNA, and 10 received antiviral therapy prior to OLT. Total and ccc HBV DNA was detected in explant livers of 17 and 16 patients, respectively. Of the 10 patients who received antiviral therapy pre-OLT, serum HBV DNA was undetectable in 8 at transplantation but 7 had detectable total and ccc HBV DNA in their explant liver. Of the 25 patients with post-OLT biopsies, total HBV DNA was detected in 83% and ccc DNA in 17% of 47 biopsies, although only 2 patients had HBV recurrence. In conclusion, total and ccc HBV DNA could be detected in explant livers of most patients despite antiviral therapy pre-OLT. Total but not ccc HBV DNA could be detected in post-OLT liver biopsies of most patients despite undetectable serum HBV DNA and hepatitis B surface antigen (HBsAg). Our findings suggest that occult HBV reinfection occurs in most HBV patients after OLT and continued administration of appropriate prophylactic therapy is important in preventing overt HBV recurrence. Copyright (c) 2007 AASLD.

Examining Hepatitis, A and B Vaccination, and HBV Reactivation Monitoring During Direct-Acting Antiviral Therapy for Hepatitis C.

PubMed

Davison, John; O'Shea, Amy; Waterbury, Nancee; Villalvazo, Yolanda

2018-05-30

The objective of this study was to examine Hepatitis A (HAV) and Hepatitis B (HBV) screening, and the risk of HBV reactivation during Hepatitis C (HCV) therapy with direct-acting antivirals (DAAs). A retrospective chart review was performed of patients treated with second generation DAA therapy from January 2014 to September 2016 at the Iowa City VA Healthcare System. In total 409 patients initiated HCV treatment, 308 (75%) and 241 (59%) were HAV and HBV vaccine eligible, respectively. Among those, 24 (8%) received a HAV vaccine, while only 20 (8%) received a HBV vaccine. Of these, 7 patients initiating an immunization in the clinic had record of completing the series. Further, 101 patients had a reactive Hepatitis B core Antibody indicating previous HBV infection, and 3 of these were tested for HBV reactivation during HCV therapy. Overall, the assessment found low rates of HAV and HBV vaccine administration, indicating missed opportunities for preventative care during HCV therapy. With the known risk of HBV reactivation with DAAs, the need for HAV and HBV screening is essential.

Performance evaluation of new automated hepatitis B viral markers in the clinical laboratory: two quantitative hepatitis B surface antigen assays and an HBV core-related antigen assay.

PubMed

Park, Yongjung; Hong, Duck Jin; Shin, Saeam; Cho, Yonggeun; Kim, Hyon-Suk

2012-05-01

We evaluated quantitative hepatitis B surface antigen (qHBsAg) assays and a hepatitis B virus (HBV) core-related antigen (HBcrAg) assay. A total of 529 serum samples from patients with hepatitis B were tested. HBsAg levels were determined by using the Elecsys (Roche Diagnostics, Indianapolis, IN) and Architect (Abbott Laboratories, Abbott Park, IL) qHBsAg assays. HBcrAg was measured by using Lumipulse HBcrAg assay (Fujirebio, Tokyo, Japan). Serum aminotransferases and HBV DNA were respectively quantified by using the Hitachi 7600 analyzer (Hitachi High-Technologies, Tokyo, Japan) and the Cobas AmpliPrep/Cobas TaqMan test (Roche). Precision of the qHBsAg and HBcrAg assays was assessed, and linearity of the qHBsAg assays was verified. All assays showed good precision performance with coefficients of variation between 4.5% and 5.3% except for some levels. Both qHBsAg assays showed linearity from 0.1 to 12,000.0 IU/mL and correlated well (r = 0.9934). HBsAg levels correlated with HBV DNA (r = 0.3373) and with HBcrAg (r = 0.5164), and HBcrAg also correlated with HBV DNA (r = 0.5198; P < .0001). This observation could provide impetus for further research to elucidate the clinical usefulness of the qHBsAg and HBcrAg assays.

[Distribution and clinical significance of HBV genotypes in patients with HBV infection in 30 regions of China].

PubMed

Zhang, Ai-min; Wang, Hui-fen; Wang, Hai-bin; Su, Hai-bin; Xin, Shao-jie; Hu, Jin-hua; You, Shao-li

2011-04-01

To explore the distribution and clinical significance of HBV genotypes in patients with HBV infection in China. Serum samples were collected from 2922 patients with HBV infection. HBV genotyping was performed with type-specific primers polymerase chain reaction, and the virological and biochemical markers were detected, which differences in the genotypes distribution between various regions and liver function and virological markers between various HBV genotyping were analyzed. The genotype B, C, B + C, D of 2922 patients with HBV infection accounted for 15.9%, 83.5%, 0.41%, 0.21% respectively. In Northern China, genotype C was most prevalent, accounting for 90% of all cases, while it was less common in Southern China; genotype C was present in Zhejiang and Jiangsu provinces, but genotype B was comparatively more common in Guangdong, Hunan, Hubei, and Jiangxi provinces. B, C genotype HBV infection patients in the sex difference was not statistically significant; B genotypes compared with C genotype HBV infection patients, the average age of is less (P < 0.001); HBeAg positive rate of C genotype HBV infection patients are higher than that of B genotype (P = 0.023); Viral load of genotype C HBV infection patients is higher than that of genotype B (P = 0.038); Cholinesterase and Albumin levels of genotype C HBV infection patients are lower than that of genotype B (P values were 0.016, <0.001). There were HBV genotype B, C, B + C and D in Chinese patients with HBV infection, with genotype B and C being the major ones. Mainly in northern regions of genotype C, C genotype significantly reduced the southern region, some of the southern region dominated by B genotype. Genotype C HBV infection patients are older, and their HBeAg-positive rate is higher, and their liver damage is more severe, but their viral load is less.

Hepatitis B (HBV)

MedlinePlus

... syringes for injecting drugs transmission from HBV-infected mothers to their newborn babies Who Is at Risk for Hepatitis B? In the United States, the most common way people get infected with HBV is through unprotected sex with someone who has the disease. People who ...

HBV endemicity in Mexico is associated with HBV genotypes H and G

PubMed Central

Roman, Sonia; Panduro, Arturo

2013-01-01

Hepatitis B virus (HBV) genotypes have distinct genetic and geographic diversity and may be associated with specific clinical characteristics, progression, severity of disease and antiviral response. Herein, we provide an updated overview of the endemicity of HBV genotypes H and G in Mexico. HBV genotype H is predominant among the Mexican population, but not in Central America. Its geographic distribution is related to a typical endemicity among the Mexicans which is characterized by a low hepatitis B surface antigen seroprevalence, apparently due to a rapid resolution of the infection, low viral loads and a high prevalence of occult B infection. During chronic infections, genotype H is detected in mixtures with other HBV genotypes and associated with other co-morbidities, such as obesity, alcoholism and co-infection with hepatitis C virus or human immunodeficiency virus. Hepatocellular carcinoma prevalence is low. Thus, antiviral therapy may differ significantly from the standard guidelines established worldwide. The high prevalence of HBV genotype G in the Americas, especially among the Mexican population, raises new questions regarding its geographic origin that will require further investigation. PMID:24023487

Performance of the cobas Hepatitis B virus (HBV) test using the cobas 4800 system and comparison of HBV DNA quantification ability between the COBAS AmpliPrep/COBAS TaqMan HBV test version 2.0 and cobas HBV test.

PubMed

Shin, Kyung-Hwa; Lee, Hyun-Ji; Chang, Chulhun L; Kim, Hyung-Hoi

2018-04-01

Hepatitis B virus (HBV) DNA levels are used to predict the response to therapy, determine therapy initiation, monitor resistance to therapy, and establish treatment success. To verify the performance of the cobas HBV test using the cobas 4800 system for HBV DNA quantification and to compare the HBV DNA quantification ability between the cobas HBV test and COBAS AmpliPrep/COBAS TaqMan HBV version 2.0 (CAP/CTM v2.0). The precision, linearity, and limit of detection of the cobas HBV test were evaluated using the 4th World Health Organization International Standard material and plasma samples. Clinical samples that yielded quantitative results using the CAP/CTM v2.0 and cobas HBV tests were subjected to correlational analysis. Three hundred forty-nine samples were subjected to correlational analysis, among which 114 samples showed results above the lower limit of quantification. Comparable results were obtained ([cobas HBV test] = 1.038 × [CAP/CTM v2.0]-0.173, r = 0.914) in 114 samples, which yielded values above the lower limit of quantification. The results for 86.8% of the samples obtained using the cobas HBV test were within 0.5 log 10 IU/mL of the CAP/CTM v2.0 results. The total precision values against the low and high positive controls were 1.4% (mean level: 2.25 log 10 IU/mL) and 3.2% (mean level: 6.23 log 10 IU/mL), respectively. The cobas HBV test demonstrated linearity (1.15-6.75 log 10 IU/mL, y = 0.95 × 6 + 0.17, r 2  = 0.994). The cobas HBV test showed good correlation with CAP/CTM v2.0, and had good precision and an acceptable limit of detection. The cobas HBV test using the cobas 4800 is a reliable method for quantifying HBV DNA levels in the clinical setting. Copyright © 2018. Published by Elsevier B.V.

Tears from children with chronic hepatitis B virus (HBV) infection are infectious vehicles of HBV transmission: experimental transmission of HBV by tears, using mice with chimeric human livers.

PubMed

Komatsu, Haruki; Inui, Ayano; Sogo, Tsuyoshi; Tateno, Akihiko; Shimokawa, Reiko; Fujisawa, Tomoo

2012-08-15

Body fluids such as saliva, urine, sweat, and tears from hepatitis B virus (HBV) carriers are potential sources of HBV transmission. Thirty-nine children and 8 adults who were chronically infected with HBV were enrolled. Real-time polymerase chain reaction was used for the quantification of HBV DNA. HBV DNA was detected in 73.7% of urine samples (14 of 19), 86.8% of saliva samples (33 of 38), 100% of tear samples (11 of 11), and 100% of sweat samples (9 of 9). Mean HBV DNA levels (±SD) in urine, saliva, tears, and sweat were 4.3 ± 1.1 log copies/mL, 5.9 ± 1.2 log copies/mL, 6.2 ± 0.7 log copies/mL, and 5.2 ± 0.6 log copies/mL, respectively. A statistically significant correlation was observed between the HBV DNA level in serum specimens and HBV DNA levels in saliva and tear specimens (r = 0.88; P < .001). Tear specimens from a child were injected intravenously into 2 human hepatocyte-transplanted chimeric mice. One week after inoculation, both chimeric mice had serum positive for HBV DNA. The levels of HBV DNA in tear specimens from young children were high. Tears were confirmed to be infectious, using chimeric mice. Strict precautions should be taken against direct contact with body fluids from HBV carriers with high-level viremia.

HBV-specific and global T-cell dysfunction in chronic hepatitis B

PubMed Central

Park, Jang-June; Wong, David K.; Wahed, Abdus S.; Lee, William M.; Feld, Jordan J.; Terrault, Norah; Khalili, Mandana; Sterling, Richard K.; Kowdley, Kris V.; Bzowej, Natalie; Lau, Daryl T.; Kim, W. Ray; Smith, Coleman; Carithers, Robert L.; Torrey, Keith W.; Keith, James W.; Levine, Danielle L.; Traum, Daniel; Ho, Suzanne; Valiga, Mary E.; Johnson, Geoffrey S.; Doo, Edward; Lok, Anna S. F.; Chang, Kyong-Mi

2015-01-01

Background & Aims T cells play a critical role in in viral infection. We examined whether T-cell effector and regulatory responses can define clinical stages of chronic hepatitis B (CHB). Methods We enrolled 200 adults with CHB who participated in the NIH-supported Hepatitis B Research Network from 2011 through 2013 and 20 uninfected individuals (controls). Peripheral blood lymphocytes from these subjects were analyzed for T-cell responses (proliferation and production of interferon-γ and interleukin-10) to overlapping hepatitis B virus (HBV) peptides (preS, S, preC, core, and reverse transcriptase), influenza matrix peptides, and lipopolysaccharide. T-cell expression of regulatory markers FOXP3, programmed death-1 (PD1), and cytotoxic T lymphocyte-associated antigen-4 (CTLA4) was examined by flow cytometry. Immune measures were compared with clinical parameters, including physician-defined immune-active, immune-tolerant, or inactive CHB phenotypes, in a blinded fashion. Results Compared to controls, patients with CHB had weak T-cell proliferative, interferon-γ, and interleukin-10 responses to HBV, with increased frequency of circulating FOXP3+CD127− regulatory T cells and CD4+ T-cell expression of PD1 and CTLA4. T-cell measures did not clearly distinguish between clinical CHB phenotypes, although the HBV core-specific T-cell response was weaker in HBeAg+ than HBeAg− patients (% responders: 3% vs 23%, P=.00008). Although in vitro blockade of PD1 or CTLA4 increased T-cell responses to HBV, the effect was weaker in HBeAg+ than HBeAg− patients. Furthermore, T-cell responses to influenza and lipopolysaccharide were weaker in CHB patients than controls. Conclusion HBV persists with virus-specific and global T-cell dysfunction mediated by multiple regulatory mechanisms including circulating HBeAg, but without distinct T-cell–based immune signatures for clinical phenotypes. These findings suggest additional T-cell independent or regulatory mechanisms of CHB

Discovery and Mechanistic Study of Benzamide Derivatives That Modulate Hepatitis B Virus Capsid Assembly.

PubMed

Wu, Shuo; Zhao, Qiong; Zhang, Pinghu; Kulp, John; Hu, Lydia; Hwang, Nicky; Zhang, Jiming; Block, Timothy M; Xu, Xiaodong; Du, Yanming; Chang, Jinhong; Guo, Ju-Tao

2017-08-15

Chronic hepatitis B virus (HBV) infection is a global public health problem. Although the currently approved medications can reliably reduce the viral load and prevent the progression of liver diseases, they fail to cure the viral infection. In an effort toward discovery of novel antiviral agents against HBV, a group of benzamide (BA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA were discovered. The initial lead optimization efforts identified two BA derivatives with improved antiviral activity for further mechanistic studies. Interestingly, similar to our previously reported sulfamoylbenzamides (SBAs), the BAs promote the formation of empty capsids through specific interaction with HBV core protein but not other viral and host cellular components. Genetic evidence suggested that both SBAs and BAs inhibited HBV nucleocapsid assembly by binding to the heteroaryldihydropyrimidine (HAP) pocket between core protein dimer-dimer interfaces. However, unlike SBAs, BA compounds uniquely induced the formation of empty capsids that migrated more slowly in native agarose gel electrophoresis from A36V mutant than from the wild-type core protein. Moreover, we showed that the assembly of chimeric capsids from wild-type and drug-resistant core proteins was susceptible to multiple capsid assembly modulators. Hence, HBV core protein is a dominant antiviral target that may suppress the selection of drug-resistant viruses during core protein-targeting antiviral therapy. Our studies thus indicate that BAs are a chemically and mechanistically unique type of HBV capsid assembly modulators and warranted for further development as antiviral agents against HBV. IMPORTANCE HBV core protein plays essential roles in many steps of the viral replication cycle. In addition to packaging viral pregenomic RNA (pgRNA) and DNA polymerase complex into nucleocapsids for reverse transcriptional DNA replication to take place, the core protein dimers, existing in several

Inhibition of hepatitis B virus replication via HBV DNA cleavage by Cas9 from Staphylococcus aureus.

PubMed

Liu, Yu; Zhao, Miaoxian; Gong, Mingxing; Xu, Ying; Xie, Cantao; Deng, Haohui; Li, Xueying; Wu, Hongkai; Wang, Zhanhui

2018-04-01

Chronic hepatitis B virus (HBV) infection is difficult to cure due to the presence of covalently closed circular DNA (cccDNA). Accumulating evidence indicates that the CRISPR/Cas9 system effectively disrupts HBV genome, including cccDNA, in vitro and in vivo. However, efficient delivery of CRISPR/Cas9 system to the liver or hepatocytes using an adeno-associated virus (AAV) vector remains challenging due to the large size of Cas9 from Streptococcus pyogenes (Sp). The recently identified Cas9 protein from Staphylococcus aureus (Sa) is smaller than SpCas9 and thus is able to be packaged into the AAV vector. To examine the efficacy of SaCas9 system on HBV genome destruction, we designed 5 guide RNAs (gRNAs) that targeted different HBV genotypes, 3 of which were shown to be effective. The SaCas9 system significantly reduced HBV antigen expression, as well as pgRNA and cccDNA levels, in Huh7, HepG2.2.15 and HepAD38 cells. The dual expression of gRNAs/SaCas9 in these cell lines resulted in more efficient HBV genome cleavage. In the mouse model, hydrodynamic injection of gRNA/SaCas9 plasmids resulted in significantly lower levels of HBV protein expression. We also delivered the SaCas9 system into mice with persistent HBV replication using an AAV vector. Both the AAV vector and the mRNA of Cas9 could be detected in the C3H mouse liver cells. Decreased hepatitis B surface antigen (HBsAg), HBV DNA and pgRNA levels were observed when a higher titer of AAV was injected, although this decrease was not significantly different from the control. In summary, the SaCas9 system accurately and efficiently targeted the HBV genome and inhibited HBV replication both in vitro and in vivo. The system was delivered by an AAV vector and maybe used as a novel therapeutic strategy against chronic HBV infection. Copyright © 2018 Elsevier B.V. All rights reserved.

HBV genotypes and drug resistance mutations in antiretroviral treatment-naive and treatment-experienced HBV-HIV-coinfected patients.

PubMed

Archampong, Timothy Na; Boyce, Ceejay L; Lartey, Margaret; Sagoe, Kwamena W; Obo-Akwa, Adjoa; Kenu, Ernest; Blackard, Jason T; Kwara, Awewura

2017-01-01

The presence of HBV resistance mutations upon initiation or during antiretroviral therapy (ART) in HIV-coinfected patients is an important determinant of treatment response. The main objective of the study was to determine the prevalence of HBV resistance mutations in antiretroviral treatment-naive and treatment-experienced HBV-HIV-coinfected Ghanaian patients with detectable HBV viraemia. HBV-HIV-coinfected patients who were ART-naive or had received at least 9 months of lamivudine (3TC)-containing ART were enrolled in a cross-sectional study. Demographic and clinical data were collected and HBV DNA quantified. Partial HBV sequences were amplified by PCR and sequenced bi-directionally to obtain a 2.1-2.2 kb fragment for phylogenetic analysis of HBV genotypes and evaluation of drug resistance mutations. Of the 100 HBV-HIV-coinfected study patients, 75 were successfully PCR-amplified, and 63 were successfully sequenced. Of these 63 patients, 27 (42.9%) were ART-experienced and 58 (92.1%) had HBV genotype E. No resistance mutations were observed in the 36 ART-naive patients, while 21 (77.8%) of 27 treatment-experienced patients had resistance mutations. All patients with resistance mutations had no tenofovir in their regimens, and 80% of them had HIV RNA <40 copies/ml. The 3TC resistance mutations rtL180M and rtM204V were observed in 10 (47.6%) of the 21 patients, while 5 patients (23.8%) had rtV173L, rtL180M and rtM204V mutations. A high proportion of HBV-HIV-coinfected patients with detectable viraemia on 3TC-containing ART had resistance mutations despite good ART adherence as determined by HIV RNA suppression. This study emphasizes the need for dual therapy as part of a fully suppressive ART in all HBV-HIV-coinfected patients in Ghana.

The synergistic transactivation of the hepatitis B viral (HBV) pregenomic promoter by the E6 protein of human papillomavirus type 16 (HPV-16 E6) with HBV X protein was mediated through the AP1 site of E element in the enhancer I (EnI) in human liver cell.

PubMed

Lee, D H; Choi, B H; Rho, H M

1999-11-01

Infection by HBV of a cell already infected with other viral species or vice versa has been suggested as being involved in hepatocellular carcinoma. Using the CAT assay method, we investigated the interactive roles of HBx and potentially oncogenic and transactivating viral early proteins such as Ad5 E1A, HPV-16 E6, and SV40 T ag. In the presence of HBx, only HPV-16 E6 showed significant synergistic transactivation of EnI. We further investigated the function of the HPV-16 E6 using deletion, heterologous promoter, and mutation analyses on the EnI promoter. The results showed that the synergistic effect was mediated through the AP1 site of the E element in EnI by the direct activation of AP1 and support the idea that the infection by HBV of the cell with other viral species such as HPV-16 could increase the transcription activity of the HBV and other oncogenes containing an AP1 site in the promoter. Copyright 1999 Academic Press.

Hepatitis B infection among HIV infected individuals in Gabon: Occult hepatitis B enhances HBV DNA prevalence

PubMed Central

Amougou-Atsama, Marie; Zoa-Assoumou, Samira; M’boyis Kamdem, Hervé; Nzengui-Nzengui, Guy Francis; Ndojyi-Mbiguino, Angélique; Njouom, Richard; François-Souquière, Sandrine

2018-01-01

In Gabon, a central African country, human immunodeficiency virus (HIV) and hepatitis B virus (HBV) are endemic. In a recent study, conducted in a semi-urban area (Franceville, Gabon), HBV infection was found to be more prevalent among HIV infected individuals. This study aims to investigate the prevalence and genetic diversity of hepatitis B virus infection among HIV infected individuals, predominantly under antiretroviral therapy, living in fully urbanized area: Libreville, capital of Gabon. Serological and molecular tests were performed to detect HBV infection among patients living with HIV/AIDS (PLHA). We used Monolisa HBsAg ULTRA, Anti-HBc Plus and Anti-HBs Plus EIA kits for serological analyses. HBV DNA viral load (HBV DNA VL) was determined by real time PCR and molecular characterization of HBV strains was performed by sequencing and phylogenetic analysis of partial HBV surface and core genes. At all, 70.2% of patients were under antiretroviral therapy. The prevalence of HBsAg was 8.8% (43/487). Detectable HBV DNA was found in 69.7% (30/43) of HBsAg positive patients and in 17.5% (24/137) HBsAg negative patients. HBV DNA VL was significantly higher among patient with CD4 cell counts less than 200 cells/mm3 than those with CD4 cell counts greater than 500 cells/mm3 (p = 0.008). We confirmed the presence of HBV sub-genotypes QS-A3 (40%), and A4 (20%) and HBV-E genotype (40%). The percentage of resistance to Lamivudine was high (40%) and varied according to the M204V/I motif. Occult hepatitis B infection (OBI) was found in patients with isolated HBcAb and among patients who had completed their HBsAg seroconversion. We detected HBV DNA for one patient without any HBV serological marker. This study provides a new landmark for the comprehension of HBV infection in PLHA in urban areas. OBI enhances HBV DNA prevalence and should be investigated in all HBsAg negative individuals. PMID:29315352

Hepatitis B infection among HIV infected individuals in Gabon: Occult hepatitis B enhances HBV DNA prevalence.

PubMed

Bivigou-Mboumba, Berthold; Amougou-Atsama, Marie; Zoa-Assoumou, Samira; M'boyis Kamdem, Hervé; Nzengui-Nzengui, Guy Francis; Ndojyi-Mbiguino, Angélique; Njouom, Richard; François-Souquière, Sandrine

2018-01-01

In Gabon, a central African country, human immunodeficiency virus (HIV) and hepatitis B virus (HBV) are endemic. In a recent study, conducted in a semi-urban area (Franceville, Gabon), HBV infection was found to be more prevalent among HIV infected individuals. This study aims to investigate the prevalence and genetic diversity of hepatitis B virus infection among HIV infected individuals, predominantly under antiretroviral therapy, living in fully urbanized area: Libreville, capital of Gabon. Serological and molecular tests were performed to detect HBV infection among patients living with HIV/AIDS (PLHA). We used Monolisa HBsAg ULTRA, Anti-HBc Plus and Anti-HBs Plus EIA kits for serological analyses. HBV DNA viral load (HBV DNA VL) was determined by real time PCR and molecular characterization of HBV strains was performed by sequencing and phylogenetic analysis of partial HBV surface and core genes. At all, 70.2% of patients were under antiretroviral therapy. The prevalence of HBsAg was 8.8% (43/487). Detectable HBV DNA was found in 69.7% (30/43) of HBsAg positive patients and in 17.5% (24/137) HBsAg negative patients. HBV DNA VL was significantly higher among patient with CD4 cell counts less than 200 cells/mm3 than those with CD4 cell counts greater than 500 cells/mm3 (p = 0.008). We confirmed the presence of HBV sub-genotypes QS-A3 (40%), and A4 (20%) and HBV-E genotype (40%). The percentage of resistance to Lamivudine was high (40%) and varied according to the M204V/I motif. Occult hepatitis B infection (OBI) was found in patients with isolated HBcAb and among patients who had completed their HBsAg seroconversion. We detected HBV DNA for one patient without any HBV serological marker. This study provides a new landmark for the comprehension of HBV infection in PLHA in urban areas. OBI enhances HBV DNA prevalence and should be investigated in all HBsAg negative individuals.

De novo activation of HBV with escape mutations from hepatitis B surface antibody after living donor liver transplantation.

PubMed

Ueda, Yoshihide; Marusawa, Hiroyuki; Egawa, Hiroto; Okamoto, Shinya; Ogura, Yasuhiro; Oike, Fumitaka; Nishijima, Norihiro; Takada, Yasutsugu; Uemoto, Shinji; Chiba, Tsutomu

2011-01-01

De novo activation of HBV occurs after liver transplantation from hepatitis B surface antigen (HBsAg)-negative and hepatitis B core antibody (anti-HBc)-positive donors, even under hepatitis B immunoglobulin (HBIG) prophylaxis. One reason for the activation of HBV is the emergence of HBV with escape mutations from hepatitis B surface antibody (anti-HBs). The aim of this study is to clarify the clinical features for de novo activation of HBV with anti-HBs escape mutations after liver transplantation. Clinical features of 75 patients who received HBIG prophylaxis >6 months after liver transplantation with liver grafts from anti-HBc-positive donors were retrospectively analysed. Among the 75 recipients, 19 (25%) developed de novo activation of HBV. Of the 19 recipients, the emergence of HBV with anti-HBs escape mutations was confirmed in 7 patients. The rate of de novo activation of HBV with anti-HBs escape mutations was 12% at 5 years. Sequence analysis revealed mutations in the common 'a' determinant region of the surface gene, including G145R, G145A and Q129P, in HBsAg. Administration of entecavir immediately after the occurrence of de novo HBV activation resolved hepatitis and induced clearance of serum HBsAg and HBV DNA in all four patients receiving entecavir. Escape mutations from anti-HBs caused de novo activation of HBV under HBIG prophylaxis after liver transplantation. Early administration of entecavir was effective on de novo activation of HBV with anti-HBs escape mutations.

Translation of the first upstream ORF in the hepatitis B virus pregenomic RNA modulates translation at the core and polymerase initiation codons

PubMed Central

Chen, Augustine; Kao, Y. F.; Brown, Chris M.

2005-01-01

The human hepatitis B virus (HBV) has a compact genome encoding four major overlapping coding regions: the core, polymerase, surface and X. The polymerase initiation codon is preceded by the partially overlapping core and four or more upstream initiation codons. There is evidence that several mechanisms are used to enable the synthesis of the polymerase protein, including leaky scanning and ribosome reinitiation. We have examined the first AUG in the pregenomic RNA, it precedes that of the core. It initiates an uncharacterized short upstream open reading frame (uORF), highly conserved in all HBV subtypes, we designated the C0 ORF. This arrangement suggested that expression of the core and polymerase may be affected by this uORF. Initiation at the C0 ORF was confirmed in reporter constructs in transfected cells. The C0 ORF had an inhibitory role in downstream expression from the core initiation site in HepG2 cells and in vitro, but also stimulated reinitiation at the polymerase start when in an optimal context. Our results indicate that the C0 ORF is a determinant in balancing the synthesis of the core and polymerase proteins. PMID:15731337

[The true story and advantages of the famous Hepatitis B virus core particles: Outlook 2016].

PubMed

Pumpens, P; Grens, E

2016-01-01

This review article is a continuation of the paper "Hepatitis B core particles as a universal display model: a structure-function basis for development" written by Pumpens P. and Grens E., ordered by Professor Lev Kisselev and published in FEBS Letters, 1999, 442, 1-6. The past 17 years have strengthened the paper's finding that the human hepatitis B virus core protein, along with other Hepadnaviridae family member core proteins, is a mysterious, multifunctional protein. The core gene of the Hepadnaviridae genome encodes five partially collinear proteins. The most important of these is the HBV core protein p21, or HBc. It can self-assemble by forming viral HBc particles, but also plays a crucial role in the regulation of viral replication. Since 1986, the HBc protein has been one of the first and the most successful tools of the virus-like particle (VLP) technology. Later, the woodchuck hepatitis virus core protein (WHc) was also used as a VLP carrier. The Hepadnaviridae core proteins remain favourite VLP candidates for the knowledge-based design of future vaccines, gene therapy vectors, specifically targeted nanocontainers, and other modern nanotechnological tools for prospective medical use.

Suppression of HBV replication by the expression of nickase- and nuclease dead-Cas9.

PubMed

Kurihara, Takeshi; Fukuhara, Takasuke; Ono, Chikako; Yamamoto, Satomi; Uemura, Kentaro; Okamoto, Toru; Sugiyama, Masaya; Motooka, Daisuke; Nakamura, Shota; Ikawa, Masato; Mizokami, Masashi; Maehara, Yoshihiko; Matsuura, Yoshiharu

2017-07-21

Complete removal of hepatitis B virus (HBV) DNA from nuclei is difficult by the current therapies. Recent reports have shown that a novel genome-editing tool using Cas9 with a single-guide RNA (sgRNA) system can cleave the HBV genome in vitro and in vivo. However, induction of a double-strand break (DSB) on the targeted genome by Cas9 risks undesirable off-target cleavage on the host genome. Nickase-Cas9 cleaves a single strand of DNA, and thereby two sgRNAs are required for inducing DSBs. To avoid Cas9-induced off-target mutagenesis, we examined the effects of the expressions of nickase-Cas9 and nuclease dead Cas9 (d-Cas9) with sgRNAs on HBV replication. The expression of nickase-Cas9 with a pair of sgRNAs cleaved the target HBV genome and suppressed the viral-protein expression and HBV replication in vitro. Moreover, nickase-Cas9 with the sgRNA pair cleaved the targeted HBV genome in mouse liver. Interestingly, d-Cas9 expression with the sgRNAs also suppressed HBV replication in vitro without cleaving the HBV genome. These results suggest the possible use of nickase-Cas9 and d-Cas9 with a pair of sgRNAs for eliminating HBV DNA from the livers of chronic hepatitis B patients with low risk of undesirable off-target mutation on the host genome.

Update Treatment for HBV Infection and Persistent Risk for Hepatocellular Carcinoma: Prospect for an HBV Cure.

PubMed

Yoo, Joseph; Hann, Hie-Won; Coben, Robert; Conn, Mitchell; DiMarino, Anthony J

2018-04-20

Since the discovery of the hepatitis B virus (HBV) by Blumberg et al. in 1965, its genome, sequence, epidemiology, and hepatocarcinogenesis have been elucidated. Globally, hepatitis B virus (HBV) is still responsible for the majority of hepatocellular carcinoma (HCC). HCC is the sixth-most common cancer in the world and the second-most common cancer death. The ultimate goal of treating HBV infection is the prevention of HCC. Fortunately, anti-HBV treatment with nucleos(t)ide analogues (NAs), which began with lamivudine in 1998, has resulted in remarkable improvements in the survival of patients with chronic hepatitis B and a reduced incidence of HCC. These results were documented with lamivudine, entecavir, and tenofovir. Nonetheless, as the duration of antiviral treatment increases, the risk for HCC still remains despite undetectable HBV DNA in serum, as reported by different investigators with observation up to 4⁻5 years. In our own experience, we are witnessing the development of HCC in patients who have received antiviral treatment. Some have enjoyed negative serum HBV DNA for over 12 years before developing HCC. Current treatment with NAs can effectively suppress the replication of the virus but cannot eradicate the covalently closed circular DNA (cccDNA) that is within the nucleus of hepatocytes. There still remains a great need for a cure for HBV. Fortunately, several compounds have been identified that have the potential to eradicate HBV, and there are ongoing clinical trials in progress in their early stages.

NTCP-Reconstituted In Vitro HBV Infection System.

PubMed

Sun, Yinyan; Qi, Yonghe; Peng, Bo; Li, Wenhui

2017-01-01

Sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a functional receptor for hepatitis B virus (HBV). Expressing human NTCP in human hepatoma HepG2 cells (HepG2-NTCP) renders these cells susceptible for HBV infection. The HepG2-NTCP stably transfected cell line provides a much-needed and easily accessible platform for studying the virus. HepG2-NTCP cells could also be used to identify chemicals targeting key steps of the virus life cycle including HBV covalent closed circular (ccc) DNA, and enable the development of novel antivirals against the infection.Many factors may contribute to the efficiency of HBV infection on HepG2-NTCP cells, with clonal differences among cell line isolates, the source of viral inoculum, and infection medium among the most critical ones. Here, we provide detailed protocols for efficient HBV infection of HepG2-NTCP cells in culture; generation and selection of single cell clones of HepG2-NTCP; production of infectious HBV virion stock through DNA transfection of recombinant plasmid that enables studying primary clinical HBV isolates; and assessing the infection with immunostaining of HBV antigens and Southern blot analysis of HBV cccDNA.

Hepatitis B virus sequencing and liver fibrosis evaluation in HIV/HBV co-infected Nigerians.

PubMed

Grant, Jennifer; Agbaji, Oche; Kramvis, Anna; Yousif, Mukhlid; Auwal, Mu'azu; Penugonda, Sudhir; Ugoagwu, Placid; Murphy, Robert; Hawkins, Claudia

2017-06-01

Molecular characteristics of hepatitis B virus (HBV), such as genotype and genomic mutations, may contribute to liver-related morbidity and mortality. The association of these characteristics with liver fibrosis severity in sub-Saharan Africa is uncertain. We aimed to characterise molecular HBV features in human immunodeficiency virus (HIV)/HBV co-infected Nigerians and evaluate associations between these characteristics and liver fibrosis severity before and after antiretroviral therapy (ART) initiation. HIV/HBV co-infected Nigerians underwent liver fibrosis estimation by transient elastography (TE) prior to and 36 months after ART initiation. Basal core promoter/precore (BCP/PC) and preS1/preS2/S regions of HBV were sequenced from baseline plasma samples. We evaluated associations between HBV mutations and liver fibrosis severity by univariate and multivariable regression. At baseline, 94 patients underwent TE with median liver stiffness of 6.4 (IQR 4.7-8.7) kPa. Patients were predominantly infected with HBV genotype E (45/46) and HBe-antigen negative (75/94, 79.8%). We identified BCP A1762T/G1764A in 15/35 (43%), PC G1896A in 20/35 (57%), 'a' determinant mutations in 12/45 (26.7%) and preS2 deletions in 6/16 (37.5%). PreS2 mutations were associated with advanced fibrosis in multivariable analysis. At follow-up, median liver stiffness was 5.2 (IQR 4.1-6.6) kPa. No HBV molecular characteristics were associated with lack of fibrosis regression, although HIV virologic control, body mass index (BMI) and baseline CD4+ T-cell count were associated with a decline in fibrosis stage. Frequent BCP/PC and preS1/preS2/S mutations were found in ART-naïve HIV/HBV co-infected Nigerians. Median liver stiffness declined after initiation of ART, regardless of pre-ART HBV mutational pattern or virologic characteristics. © 2017 John Wiley & Sons Ltd.

Identification of Factors Promoting HBV Capsid Self-Assembly by Assembly-Promoting Antivirals.

PubMed

Rath, Soumya Lipsa; Liu, Huihui; Okazaki, Susumu; Shinoda, Wataru

2018-02-26

Around 270 million individuals currently live with hepatitis B virus (HBV) infection. Heteroaryldihydropyrimidines (HAPs) are a family of antivirals that target the HBV capsid protein and induce aberrant self-assembly. The capsids formed resemble the native capsid structure but are unable to propagate the virus progeny because of a lack of RNA/DNA. Under normal conditions, self-assembly is initiated by the viral genome. The mode of action of HAPs, however, remains largely unknown. In this work, using molecular dynamics simulations, we attempted to understand the action of HAP by comparing the dynamics of capsid proteins with and without HAPs. We found that the inhibitor is more stable in higher oligomers. It retains its stability in the hexamer throughout 1 μs of simulation. Our results also show that the inhibitor might help in stabilizing the C-terminus, the HBc 149-183 arginine-rich domain of the capsid protein. The C-termini of dimers interact with each other, assisted by the HAP inhibitor. During capsid assembly, the termini are supposed to directly interact with the viral genome, thereby suggesting that the viral genome might work in a similar way to stabilize the capsid protein. Our results may help in understanding the underlying molecular mechanism of HBV capsid self-assembly, which should be crucial for exploring new drug targets and structure-based drug design.

Quantitative hepatitis B core antibody levels in the natural history of hepatitis B virus infection.

PubMed

Song, L-W; Liu, P-G; Liu, C-J; Zhang, T-Y; Cheng, X-D; Wu, H-L; Yang, H-C; Hao, X-K; Yuan, Q; Zhang, J; Kao, J-H; Chen, D-S; Chen, P-J; Xia, N-S

2015-02-01

We previously demonstrated that pretreatment quantitative anti-hepatitis B core protein (qAnti-HBc) levels can predict the treatment response for both interferon and nucleoside analogue therapy, but the characteristics of qAnti-HBc during chronic hepatitis B virus (HBV) infection remain poorly understood. To understand this issue, the qAnti-HBc levels were evaluated in individuals with past HBV infection, occult HBV infection and chronic HBV infection in the immune tolerance phase, immune clearance phase, low-replicative phase and hepatitis B e antigen (HBeAg)-negative hepatitis phase. Individuals with hepatitis B surface antigen (n = 598, 3.74 ± 0.90 log10 IU/mL) had significantly higher (p < 0.001, approximately 1000-fold) serum qAnti-HBc levels than those who had occult HBV, and serum qAnti-HBc levels were significantly higher in the occult HBV group than in the past HBV infection group (p < 0.001). qAnti-HBc levels were positively correlated with alanine aminotransferase levels (R = 0.663, p < 0.001), and subjects with an abnormal alanine aminotransferase level had a higher qAnti-HBc level (p < 0.001). Serum qAnti-HBc level varied in different phases of HBV infection, as determined by host immune status. Serum qAnti-HBc level is strongly associated with hepatitis activity in subjects with chronic HBV infection. Copyright © 2014 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

HBX Protein-Induced Downregulation of microRNA-18a is Responsible for Upregulation of Connective Tissue Growth Factor in HBV Infection-Associated Hepatocarcinoma.

PubMed

Liu, Xiaomin; Zhang, Yingjian; Wang, Ping; Wang, Hongyun; Su, Huanhuan; Zhou, Xin; Zhang, Lamei

2016-07-16

BACKGROUND This study was designed to improve our understanding of the role of miR-18a and its target (connective tissue growth factor (CTGF), which are mediators in HBX-induced hepatocellular carcinoma (HCC). MATERIAL AND METHODS We first investigated the expression of several candidate microRNAs (miRNAs) reported to have been aberrantly expressed between HepG2 and HepG2.2.15, which is characterized by stable HBV infection, while the CTGF is identified as a target of miR-18a. Furthermore, the expression of CTGF evaluated in HepG2 was transfected with HBX, while the HepG2.2.15 was transfected with miR-18a and CTGF siRNA. We examined the cell cycle at the same time. RESULTS We found that the expression of miR-18a was abnormally reduced in the HBV-positive HCC tissue samples compared with HBV-negative HCC samples. Through the use of a luciferase reporter system, we also identified CTGF 3'UTR (1046-1052 bp) as the exact binding site for miR-18a. We also observed a clear increase in CTGF mRNA and protein expression levels in HBV-positive HCC human tissue samples in comparison with the HBV-negative controls, indicating a possible negatively associated relationship between miR-18a and CTGF. Furthermore, we investigated the effect of HBX overexpression on miR-18a and CTGF, as well as the viability and cell cycle status of HepG2 cells. In addition, we found that HBX introduction downregulated miR-18a, upregulated CTGF, elevated the viability, and promoted cell cycle progression. We transfected HepG2.2.15 with miR-18a mimics and CTGF siRNA, finding that upregulated miR-18a and downregulated CTGF suppress the viability and cause cell cycle arrest. CONCLUSIONS Our study shows the role of the CTGF gene as a target of miR-18a, and identifies the function of HBV/HBX/miR-18a/CTGF as a key signaling pathway mediating HBV infection-induced HCC.

Clinical outcomes of liver transplantation for HBV-related hepatocellular carcinoma: data from the NIH HBV OLT study.

PubMed

Han, Steven-Huy; Reddy, K Rajender; Keeffe, Emmet B; Soldevila-Pico, Consuelo; Gish, Robert; Chung, Raymond T; Degertekin, Bulent; Lok, Anna

2011-01-01

Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is an indication for orthotopic liver transplantation (OLT) in patients with tumor stage within the United Network for Organ Sharing criteria. The number of patients listed for HBV-related HCC is increasing, while the number of patients listed for HBV-related cirrhosis is declining presumptively because of the availability of more effective oral nucleos(t)ide analogues. This study presents the final, long-term outcome of patients transplanted for HBV-related HCC in the National Institutes of Health (NIH) HBV OLT Study Group. Ninety-eight patients (52.4%) in the NIH HBV OLT cohort underwent OLT for HBV-related HCC. With a mean follow-up of 36.5 months post-OLT, 12 (12.2%) patients developed recurrence of HCC. Multivariate analysis did not find a statistically significant role of gender, tumor stage at OLT, pre-OLT HCC treatment, recurrence of HBV, or duration of HCC diagnosis pre-OLT in predicting HCC recurrence. Serum alpha-fetoprotein (AFP) level >200 ng/mL at transplant was found to be statistically significant in predicting HCC recurrence (p=0.003). HCC recurrence was significantly associated with decreased post-OLT survival. HCC is the most common indication for OLT in patients with chronic hepatitis B in the era of more effective oral antivirals. Serum AFP at the time of OLT is significantly associated with HCC recurrence. © 2010 John Wiley & Sons A/S.

Live Cell Imaging Confocal Microscopy Analysis of HBV Myr-PreS1 Peptide Binding and Uptake in NTCP-GFP Expressing HepG2 Cells.

PubMed

König, Alexander; Glebe, Dieter

2017-01-01

To obtain basic knowledge about specific molecular mechanisms involved in the entry of pathogens into cells is the basis for establishing pharmacologic substances blocking initial viral binding, infection, and subsequent viral spread. Lack of information about key cellular factors involved in the initial steps of HBV infection has hampered the characterization of HBV binding and entry for decades. However, recently, the liver-specific sodium-dependent taurocholate cotransporting polypeptide (NTCP) has been discovered as a functional receptor for HBV and HDV, thus opening the field for new concepts of basic binding and entry of HBV and HDV. Here, we describe practical issues of a basic in vitro assay system to examine kinetics and mechanisms of receptor-dependent HBV binding, uptake, and intracellular trafficking by live-cell imaging confocal microscopy. The assay system is comprised of HepG2 cells expressing a NTCP-GFP fusion-protein and chemically synthesized, fluorophore-labeled part of HBV surface protein, spanning the first N-terminal 48 amino acids of preS1 of the large hepatitis B virus surface protein.

Prognostic significance of catalase expression and its regulatory effects on hepatitis B virus X protein (HBx) in HBV-related advanced hepatocellular carcinomas

PubMed Central

Cho, Mi-Young; Cheong, Jae Youn; Lim, Wonchung; Jo, Sujin; Lee, Youngsoo; Wang, Hee-Jung; Han, Kyou-Hoon; Cho, Hyeseong

2014-01-01

Hepatitis B virus X protein (HBx) plays a role in liver cancer development. We previously showed that ROS increased HBx levels and here, we investigated the role of antioxidants in the regulation of HBx expression and their clinical relevance. We found that overexpression of catalase induced a significant loss in HBx levels. The cysteine null mutant of HBx (Cys−) showed a dramatic reduction in its protein stability. In clonogenic proliferation assays, Huh7-X cells produced a significant number of colonies whereas Huh7-Cys− cells failed to generate them. The Cys at position 69 of HBx was crucial to maintain its protein stability and transactivation function in response to ROS. Among 50 HBV-related hepatocellular carcinoma (HCC) specimens, 72% of HCCs showed lower catalase levels than those of surrounding non-tumor tissues. In advanced stage IV, catalase levels in non-tumor tissues were increased whereas those in tumors were further reduced. Accordingly, patients with a high T/N ratio for catalase showed significantly longer survival than those with a low T/N ratio. Together, catalase expression in HCC patients can be clinically useful for prediction of patient survival, and restoration of catalase expression in HCCs could be an important strategy for intervention in HBV-induced liver diseases. PMID:25361011

Spinoculation Enhances HBV Infection in NTCP-Reconstituted Hepatocytes.

PubMed

Yan, Ran; Zhang, Yongmei; Cai, Dawei; Liu, Yuanjie; Cuconati, Andrea; Guo, Haitao

2015-01-01

Hepatitis B virus (HBV) infection and its sequelae remain a major public health burden, but both HBV basic research and the development of antiviral therapeutics have been hindered by the lack of an efficient in vitro infection system. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as the HBV receptor. We herein report that we established a NTCP-complemented HepG2 cell line (HepG2-NTCP12) that supports HBV infection, albeit at a low infectivity level following the reported infection procedures. In our attempts to optimize the infection conditions, we found that the centrifugation of HepG2-NTCP12 cells during HBV inoculation (termed "spinoculation") significantly enhanced the virus infectivity. Moreover, the infection level gradually increased with accelerated speed of spinoculation up to 1,000g tested. However, the enhancement of HBV infection was not significantly dependent upon the duration of centrifugation. Furthermore, covalently closed circular (ccc) DNA was detected in infected cells under optimized infection condition by conventional Southern blot, suggesting a successful establishment of HBV infection after spinoculation. Finally, the parental HepG2 cells remained uninfected under HBV spinoculation, and HBV entry inhibitors targeting NTCP blocked HBV infection when cells were spinoculated, suggesting the authentic virus entry mechanism is unaltered under centrifugal inoculation. Our data suggest that spinoculation could serve as a standard protocol for enhancing the efficiency of HBV infection in vitro.

Spinoculation Enhances HBV Infection in NTCP-Reconstituted Hepatocytes

PubMed Central

Yan, Ran; Zhang, Yongmei; Cai, Dawei; Liu, Yuanjie; Cuconati, Andrea; Guo, Haitao

2015-01-01

Hepatitis B virus (HBV) infection and its sequelae remain a major public health burden, but both HBV basic research and the development of antiviral therapeutics have been hindered by the lack of an efficient in vitro infection system. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as the HBV receptor. We herein report that we established a NTCP-complemented HepG2 cell line (HepG2-NTCP12) that supports HBV infection, albeit at a low infectivity level following the reported infection procedures. In our attempts to optimize the infection conditions, we found that the centrifugation of HepG2-NTCP12 cells during HBV inoculation (termed “spinoculation”) significantly enhanced the virus infectivity. Moreover, the infection level gradually increased with accelerated speed of spinoculation up to 1,000g tested. However, the enhancement of HBV infection was not significantly dependent upon the duration of centrifugation. Furthermore, covalently closed circular (ccc) DNA was detected in infected cells under optimized infection condition by conventional Southern blot, suggesting a successful establishment of HBV infection after spinoculation. Finally, the parental HepG2 cells remained uninfected under HBV spinoculation, and HBV entry inhibitors targeting NTCP blocked HBV infection when cells were spinoculated, suggesting the authentic virus entry mechanism is unaltered under centrifugal inoculation. Our data suggest that spinoculation could serve as a standard protocol for enhancing the efficiency of HBV infection in vitro. PMID:26070202

Successful treatment of HCV/HBV/HDV-coinfection with pegylated interferon and ribavirin

PubMed Central

Hartl, Janine; Ott, Claudia; Kirchner, Gabriele; Salzberger, Bernd; Wiest, Reiner

2012-01-01

Dual and triple infections with hepatitis virus C (HCV), B (HBV) and D (HDV) frequently lead to severe liver damage. Hereby we describe a 38-year-old Caucasian male coinfected with HCV (genotype 3a), HBV [positive hepatitis B surface antigen (HbsAg) and antibody to hepatitis B core antigen; negative hepatitis B e antigen (HbeAg) and antibody to hepatitis B e antigen (anti-HBe)] and HDV. Laboratory diagnostics revealed increased liver enzymes and histological examination of the liver showed signs of fibrosis with moderate inflammation. On therapy with pegIFN-α2b and ribavirin HCV-RNA was undetectable at week 8. After week 24 the antiviral therapy was stopped because of a HBs-seroconversion, the loss of HbeAg and the detection of anti-HBe. Furthermore the HCV-RNA was negative. Six months after successful treatment of the triple-infection, HCV- and HDV-RNA and HbsAg remained negative and the liver enzymes had been completely normalized. In conclusion, pegylated-interferon plus ribavirin may be an effective therapy for HCV, HBV and HDV-coinfected patients. PMID:24765463

Random close packing in protein cores

NASA Astrophysics Data System (ADS)

Gaines, Jennifer C.; Smith, W. Wendell; Regan, Lynne; O'Hern, Corey S.

2016-03-01

Shortly after the determination of the first protein x-ray crystal structures, researchers analyzed their cores and reported packing fractions ϕ ≈0.75 , a value that is similar to close packing of equal-sized spheres. A limitation of these analyses was the use of extended atom models, rather than the more physically accurate explicit hydrogen model. The validity of the explicit hydrogen model was proved in our previous studies by its ability to predict the side chain dihedral angle distributions observed in proteins. In contrast, the extended atom model is not able to recapitulate the side chain dihedral angle distributions, and gives rise to large atomic clashes at side chain dihedral angle combinations that are highly probable in protein crystal structures. Here, we employ the explicit hydrogen model to calculate the packing fraction of the cores of over 200 high-resolution protein structures. We find that these protein cores have ϕ ≈0.56 , which is similar to results obtained from simulations of random packings of individual amino acids. This result provides a deeper understanding of the physical basis of protein structure that will enable predictions of the effects of amino acid mutations to protein cores and interfaces of known structure.

Random close packing in protein cores.

PubMed

Gaines, Jennifer C; Smith, W Wendell; Regan, Lynne; O'Hern, Corey S

2016-03-01

Shortly after the determination of the first protein x-ray crystal structures, researchers analyzed their cores and reported packing fractions ϕ ≈ 0.75, a value that is similar to close packing of equal-sized spheres. A limitation of these analyses was the use of extended atom models, rather than the more physically accurate explicit hydrogen model. The validity of the explicit hydrogen model was proved in our previous studies by its ability to predict the side chain dihedral angle distributions observed in proteins. In contrast, the extended atom model is not able to recapitulate the side chain dihedral angle distributions, and gives rise to large atomic clashes at side chain dihedral angle combinations that are highly probable in protein crystal structures. Here, we employ the explicit hydrogen model to calculate the packing fraction of the cores of over 200 high-resolution protein structures. We find that these protein cores have ϕ ≈ 0.56, which is similar to results obtained from simulations of random packings of individual amino acids. This result provides a deeper understanding of the physical basis of protein structure that will enable predictions of the effects of amino acid mutations to protein cores and interfaces of known structure.

The preS deletion of hepatitis B virus (HBV) is associated with liver fibrosis progression in patients with chronic HBV infection.

PubMed

Li, Fan; Li, Xiaodong; Yan, Tao; Liu, Yan; Cheng, Yongqian; Xu, Zhihui; Shao, Qing; Liao, Hao; Huang, Pengyu; Li, Jin; Chen, Guo-Feng; Xu, Dongping

2018-03-01

Limited data are available regarding the association of hepatitis B virus (HBV) mutations with liver fibrosis in HBV infection. The study aimed to clarify whether HBV preS deletion mutation is associated with liver fibrosis progression. A total of 469 patients were enrolled, including 324 with chronic hepatitis B (CHB), 28 with HBV-related compensated liver cirrhosis (LC), and 117 with HBV-related decompensated LC. All CHB and compensated LC patients received liver biopsy. Fibrosis grade was assessed using METAVIR score. HBV preS deletion was determined by direct sequencing and verified by clonal sequencing. Overall preS deletion was detected in 12.6% (59/469) patients, specifically, in 7.51% (13/173), 10.60% (16/151), and 20.69% (30/145) of patients with no-to-mild liver fibrosis (F0-1), moderate-to-severe liver fibrosis (F2-3), and cirrhosis (F4), respectively (p < 0.01). Patients with preS-deleted HBV had lower serum HBV DNA and albumin levels compared to patients with wild-type HBV. The median length of preS deletion was 39-base pairs (bp) (3-204 bp) and the deletion most frequently emerged in preS2 initial region. Multivariate analysis identified the preS2 deletion rather than preS1 deletion to be an independent risk factor of significant fibrosis, i.e., METAVIR F ≥ 2 (p = 0.007). In addition, preS-deleted viral sequences were detected in the pool of intrahepatic HBV covalently closed circular DNA. HBV preS deletion is positively associated with liver fibrosis progression in chronic HBV-infected patients. HBV preS2 deletion may serve as a warning indicator for liver fibrosis progression.

Association of preS/S Mutations with Occult Hepatitis B Virus (HBV) Infection in South Korea: Transmission Potential of Distinct Occult HBV Variants

PubMed Central

Kim, Hong; Kim, Bum-Joon

2015-01-01

Occult hepatitis B virus infection (HBV) is characterized by HBV DNA positivity but HBV surface antigen (HBsAg) negativity. Occult HBV infection is associated with a risk of HBV transmission through blood transfusion, hemodialysis, and liver transplantation. Furthermore, occult HBV infection contributes to the development of cirrhosis and hepatocellular carcinoma. We recently reported the characteristic molecular features of mutations in the preS/S regions among Korean individuals with occult infections caused by HBV genotype C2; the variants of preS and S related to severe liver diseases among chronically infected patients were also responsible for the majority of HBV occult infections. We also reported that HBsAg variants from occult-infected Korean individuals exhibit lower HBsAg secretion capacity but not reduced HBV DNA levels. In addition, these variants exhibit increased ROS-inducing capacity compared with the wild-type strain, linking HBV occult infections to liver cell damage. Taken together, our previous reports suggest the transmission potential of distinct HBV occult infection-related variants in South Korea. PMID:26084041

Association of preS/S Mutations with Occult Hepatitis B Virus (HBV) Infection in South Korea: Transmission Potential of Distinct Occult HBV Variants.

PubMed

Kim, Hong; Kim, Bum-Joon

2015-06-15

Occult hepatitis B virus infection (HBV) is characterized by HBV DNA positivity but HBV surface antigen (HBsAg) negativity. Occult HBV infection is associated with a risk of HBV transmission through blood transfusion, hemodialysis, and liver transplantation. Furthermore, occult HBV infection contributes to the development of cirrhosis and hepatocellular carcinoma. We recently reported the characteristic molecular features of mutations in the preS/S regions among Korean individuals with occult infections caused by HBV genotype C2; the variants of preS and S related to severe liver diseases among chronically infected patients were also responsible for the majority of HBV occult infections. We also reported that HBsAg variants from occult-infected Korean individuals exhibit lower HBsAg secretion capacity but not reduced HBV DNA levels. In addition, these variants exhibit increased ROS-inducing capacity compared with the wild-type strain, linking HBV occult infections to liver cell damage. Taken together, our previous reports suggest the transmission potential of distinct HBV occult infection-related variants in South Korea.

Performance evaluation of four dominant anti-hepatitis B core antigen (HBcAb) kits in Japan for preventing de novo hepatitis B virus (HBV) infection.

PubMed

Kobayashi, Eiji; Deguchi, Matsuo; Kagita, Masanori; Yoshioka, Nori; Kita, Mifumi; Asari, Seishi; Suehisa, Etsuji; Hidaka, Yoh; Iwatani, Yoshinori

2015-01-01

The determination of antibody to hepatitis B core antigen (HBcAb) has become an important means of evaluating the risk factors of de novo hepatitis B virus (HBV) infection before starting intensive immunosuppressive drug therapies. Four dominant HBcAb determination reagents used in Japan were evaluated with HBcIgM, HBsAg, HBsAb, HBeAb, and HBV DNA reagents in order to study their clinical utility. Four kinds of HBcAb reagent kits (HBcAb Total and HBcAb-IgG reagent) were evaluated with 526 clinical specimens, including 344 negative specimens, at Osaka University Hospital. The dynamic range of each kit was evaluated by testing serially diluted serum from pooled sera with high HBcAb concentration. The reagent that showed the largest dynamic range was the Lumipulse HBcAb-N (HBcAb-IgG reagent). Regarding clinical sensitivity and specificity, Centaur HBcAb (HBcAb Total reagent) gave several "doubtful negative" results and ARCHITECT HBcII (HBcAb Total reagent) had the most discrepant positive results. By comparing the cut-off-index distribution of negative specimens using a parameter of "distance from the mean to the cut-off divided by the SD", Centaur was determined to be the best (distance/SD = 12.65), with Lumipulse and Elecsys Anti-HBc (HBcAb Total reagent) in the second group (8.13 and 7.00, respectively), and ARCHITECT rated as the worst (3.25). In this evaluation, Elecsys and Lumipulse HBcAb kits showed good clinical sensitivity and specificity and were considered to be suitable for evaluating the risk factors of de novo HBV infection.

The detection of (total and ccc) HBV DNA in liver transplant recipients with hepatitis B vaccine against HBV reinfection.

PubMed

Duan, Bin-Wei; Lu, Shi-Chun; Lai, Wei; Liu, Xue-En; Liu, Yuan

2015-01-01

To investigate the levels of hepatitis B virus total DNA (HBV DNA) and covalently closed circular (ccc) DNA in liver transplant recipients who received hepatitis B vaccination, including responders and non-responders, following liver transplantation due to hepatitis B-related diseases and to investigate the efficacy of hepatitis B immune reconstitution against HBV reinfection. Twenty responders and 34 non-responders were enrolled in the present study. The levels of HBV total DNA and ccc DNA in peripheral blood mononuclear cells (PBMCs) and the liver and plasma were detected by real-time polymerase chain reaction (PCR). Fifty-three blood samples and 38 liver allograft tissues were acquired. For the responders, the mean serum titer for anti-HBs (antibodies against hepatitis B surface antigen) was 289 (46.64-1000) IU/ml. Also for the responders, HBV total DNA was detected in PBMCs for one recipient and in the liver for another recipient, but ccc DNA was not detected in either of those 2 recipients. For the non-responders, HBV total DNA was detected in PBMCS for 2 recipients, neither of whom had ccc DNA. Also for the non-responders, HBV total DNA was detected in the livers of 3 recipients, 2 of whom also had ccc DNA. All responders had discontinued hepatitis B immunoglobulin (HBIG), and 13 responders had discontinued antiviral agents. One responder experienced HBV recurrence during the follow-up period. For the majority of liver transplant recipients, no HBV total DNA or ccc DNA was detected in the blood or liver. The lack of HBV total DNA and ccc DNA both in PBMCs and the liver in liver transplant recipients who received hepatitis B vaccination to prevent HBV reinfection should be a prerequisite for the withdrawal of HBIG and/or antiviral agents.

Combined branched-DNA and conventional HBV PCR assays for detection of serum HBV-DNA in hepatitis B e antigen-positive chronic hepatitis B patients.

PubMed

Ozdarendeli, Aykut; Toroman, Zulal Asci; Bulut, Yasemin; Demirbag, Kutbeddin; Kalkan, Ahmet; Ozden, Mehmet; Kilic, Suleyman Sirri

2006-01-01

Monitoring of HBV replication level is very useful for the management of patients with chronic HBV. However, the use of the correct tools to quantify HBV-DNA levels in serum and monitor the replication of HBV is of paramount importance in terms of diagnosis, and antiviral treatment of patients with chronic HBV infection. The aim of this study was to combine the bDNA assay and HBV PCR to improve detection of viremia the patients with HBeAg-positive chronic hepatitis B infection. In this study, 67 HBeAg-positive chronic hepatitis B patients were analyzed to determine viremia level using bDNA and HBV PCR assays. Sixty-four patients with HBeAg-positive chronic hepatitis B showed positivity by conventional HBV PCR, whereas 56 subjects with HBeAg-positive chronic hepatitis B showed HBV-DNA levels by bDNA. The results indicated that it is reasonable to use the bDNA assay to determine HBV replicative activity first, and use conventional HBV-PCR for HBeAg-positive chronic hepatitis B patient samples that are negative in bDNA assay.

A mouse model with age-dependent immune response and immune-tolerance for HBV infection.

PubMed

Yi, Xuerui; Yuan, Youcheng; Li, Na; Yi, Lu; Wang, Cuiling; Qi, Ying; Gong, Liang; Liu, Guangze; Kong, Xiangping

2018-02-01

Viral clearance of human HBV infection largely depends on the age of exposure. Thus, a mouse model with age-dependent immune response and immune-tolerance for HBV infection was established. HBVRag1 mice were generated by crossing Rag1 -/- mice with HBV-Tg mice. Following adoptive transfer of splenocytes adult (8-9 weeks old) and young (3 weeks old) HBVRag1 mice were named as HBVRag-ReA and HBVRag-ReY mice respectively. The biochemical parameters that were associated with viral load and immune function, as well as the histological evaluation of the liver tissues between the two mouse models were detected. The immune tolerance of HBVRag-ReY mice that were reconstituted at the early stages of life was evaluated by quantitative hepatitis B core antibody assay, adoptive transfer, and modulation of gut microbiota with the addition of antibiotics. HBVRag-ReA mice indicated apparent hepatocytes damage, clearance of HBsAg and production of HBsAb and HBcAb. HBVRag-ReY mice did not develop ALT elevation, and produced HBcAb and HBsAg. A higher number of hepatic CD8 + T and B cells promoted clearance of HBsAg in HBVRag-ReA mice following 30 days of lymphocyte transfer. In contrast to HBVRag-ReA mice, HBVRag-ReY mice exhibited higher levels of Th1/Th2 cytokines. HBVRag-ReY mice exhibited significantly higher (P < .01, approximately 10-fold) serum quantitative anti-HBc levels than HBV-Tg mice, which might be similar to the phase of immune clearance and immune tolerance in human HBV infection. Furthermore, the age-related tolerance in HBVRag-ReY mice that were sensitive to antibiotic treatment was different from that noted in HBV-Tg mice. GS-9620 could inhibit the production of HBsAg, whereas HBV vaccination could induce sustained seroconversion in HBVRag-ReY mice with low levels of HBsAg. The present study described a mouse model with age-dependent immunity and immune-tolerance for HBV infection in vivo, which may mimic chronic HBV infection in humans. Copyright © 2017

Distribution of HBV genotypes in Poland.

PubMed

Świderska, Magdalena; Pawłowska, Małgorzata; Mazur, Włodzimierz; Tomasiewicz, Krzysztof; Simon, Krzysztof; Piekarska, Anna; Wawrzynowicz-Syczewska, Marta; Jaroszewicz, Jerzy; Rajewski, Paweł; Zasik, Ewelina; Murias-Bryłowska, Elżbieta; Pniewska, Anna; Halota, Waldemar; Flisiak, Robert

2015-05-01

To identify distribution of HBV genotypes in particular regions of Poland. The study included 270 treatment-naïve, HBV-infected individuals, enrolled in 7 centers of Poland. HBV genotyping was performed in 243 of them with the INNO-LiPA HBV Genotyping assay (Innogenetics). Genotype A present in 2/3 patients was demonstrated as the most predominant in Poland. It was followed by D (20%), H (5%) and mixed A + D (5%). Remaining patients were infected with genotype F, mixed D + G, A + C or D + F. Analysis of distribution demonstrated regional differences, with a higher rate of genotype D prevalence (about 30%) in the eastern (Białystok and Lublin) and south-western (Wrocław) parts compared to other regions, where the prevalence rate was below 15%. The highest prevalence of genotype A (exceeding 80%) was observed in central Poland (Bydgoszcz, Łódź). The presented data reveal the current distribution of HBV genotypes across Poland, which is the first and the largest such epidemiological analysis.

Progress and Prospects of Anti-HBV Gene Therapy Development

PubMed Central

Maepa, Mohube B.; Roelofse, Ilke; Ely, Abdullah; Arbuthnot, Patrick

2015-01-01

Despite the availability of an effective vaccine against hepatitis B virus (HBV), chronic infection with the virus remains a major global health concern. Current drugs against HBV infection are limited by emergence of resistance and rarely achieve complete viral clearance. This has prompted vigorous research on developing better drugs against chronic HBV infection. Advances in understanding the life cycle of HBV and improvements in gene-disabling technologies have been impressive. This has led to development of better HBV infection models and discovery of new drug candidates. Ideally, a regimen against chronic HBV infection should completely eliminate all viral replicative intermediates, especially covalently closed circular DNA (cccDNA). For the past few decades, nucleic acid-based therapy has emerged as an attractive alternative that may result in complete clearance of HBV in infected patients. Several genetic anti-HBV strategies have been developed. The most studied approaches include the use of antisense oligonucleotides, ribozymes, RNA interference effectors and gene editing tools. This review will summarize recent developments and progress made in the use of gene therapy against HBV. PMID:26263978

Static structures and dynamics of hemoglobin vesicle (HBV) developed as a transfusion alternative.

PubMed

Sato, Takaaki; Sakai, Hiromi; Sou, Keitaro; Medebach, Martin; Glatter, Otto; Tsuchida, Eishun

2009-06-18

Hemoglobin vesicle (HbV) is an artificial oxygen carrier that encapsulates solution of purified and highly concentrated (ca. 38 g dL(-1)) human hemoglobin. Its exceptionally high concentration as a liposomal product (ca. 40% volume fraction) achieves an oxygen-carrying capacity comparable to that of blood. We use small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) to investigate the hierarchical structures and dynamics of HbVs in concentrated suspensions. SAXS data revealed unilamellar shell structure and internal density profile of the artificial cell membrane for Hb encapsulation. The SAXS intensity of HbV at scattering vector q > 0.5 nm(-1) manifests dissolution states of the encapsulated Hbs in the inner aqueous phase of the vesicle having ca. 240 nm diameter. The peak position as well as the height and width of static structure factor of Hb before and after encapsulation are almost identical, demonstrating the preserved protein-protein interactions in the confined space. To overcome multiple scattering from turbid samples, we employed thin layer-cell DLS combined with the so-called bruteforce and echo techniques, which allows us to observe collective diffusion dynamics of HbVs without dilution. A pronounced slowdown of the HbV diffusion and eventual emergence of dynamically arrested state in the presence of high-concentration plasma substitutes (water-soluble polymers), such as dextran, modified fluid gelatin, and hydroxylethyl starch, can be explained by depletion interaction. A significantly weaker effect of recombinant human serum albumin on HbV flocculation and viscosity enhancement than those induced by other polymers is clearly attributed to the specificity as a protein; its compact structure efficiently reduces the reservoir polymer volume fraction that determines the depth of the attractive potential between HbVs. These phenomena are technically essential for controlling the suspension rheology, which is advantageous for versatile

[A quantitative real time polymerase chain reaction for detection of HBV covalently closed circular DNA in livers of the HBV infected patients].

PubMed

Wang, Mei-Rong; Qiu, Ning; Lu, Shi-Chun; Xiu, Dian-Rong; Yu, Jian-Guo; Li, Tong; Liu, Xue-En; Zhuang, Hui

2011-05-01

To establish and optimize a sensitive and specific quantitative real-time polymerase chain reaction (PCR) method for detection of hepatitis B virus covalently closed circular DNA (HBV cccDNA) in liver tissue. Specific primers and probes were designed to detect HBV DNA (tDNA) and cccDNA. A series of plasmids (3.44 × 10(0) - 3.44 × 10(9) copies/µl) containing a full double-stranded copies of HBV genome (genotype C) were used to establish the standard curve of real-time PCR. Liver samples of 33 patients with HBV related hepatocellular carcinoma (HCC), 13 Chronic hepatitis B patients (CHB) and 10 non-HBV patients were collected to verify the sensitivity and specificity of the assay. A fraction of extracted DNA was digested with a Plasmid-Safe ATP-dependent Dnase (PSAD) for HBV cccDNA detection and the remaining was used for tDNA and β-globin detection. The amount (copies/cell) of HBV cccDNA and tDNA were measured by a real-time PCR, using β-globin housekeeping gene as a quantitation standard. The standard curves of real-time PCR with a linear range of 3.44 × 10(0) to 3.44 × 10(9) copies/µl were established for detecting HBV cccDNA and tDNA, and both of the lowest detection limits of HBV cccDNA and tDNA were 3.44 × 10(0) copies/µl. The lowest quantitation levels of HBV cccDNA in liver tissues tested in 33 HBV related HCC patients and 13 CHB patients were 0.003 copies/cell and 0.031 copies/cell, respectively. HBV cccDNA and tDNA in liver tissue of 10 non-HBV patient appeared to be negative. The true positive rate was increasing through the digestion of HBV DNA by PSAD, and the analytic specificity of cccDNA detection improved by 7.24 × 10(2) times. Liver tissues of 2 patients were retested 5 times in the PCR for detecting cccDNA and the coefficient of variations on cycle threshold (Ct) were between 0.224% - 0.609%. A highly sensitive and specific quantitative real time PCR method for the detection of HBV cccDNA in liver tissue was established and could be used

Random close packing in protein cores

NASA Astrophysics Data System (ADS)

Ohern, Corey

Shortly after the determination of the first protein x-ray crystal structures, researchers analyzed their cores and reported packing fractions ϕ ~ 0 . 75 , a value that is similar to close packing equal-sized spheres. A limitation of these analyses was the use of `extended atom' models, rather than the more physically accurate `explicit hydrogen' model. The validity of using the explicit hydrogen model is proved by its ability to predict the side chain dihedral angle distributions observed in proteins. We employ the explicit hydrogen model to calculate the packing fraction of the cores of over 200 high resolution protein structures. We find that these protein cores have ϕ ~ 0 . 55 , which is comparable to random close-packing of non-spherical particles. This result provides a deeper understanding of the physical basis of protein structure that will enable predictions of the effects of amino acid mutations and design of new functional proteins. We gratefully acknowledge the support of the Raymond and Beverly Sackler Institute for Biological, Physical, and Engineering Sciences, National Library of Medicine training grant T15LM00705628 (J.C.G.), and National Science Foundation DMR-1307712 (L.R.).

Improved Analytical Sensitivity of Lateral Flow Assay using Sponge for HBV Nucleic Acid Detection.

PubMed

Tang, Ruihua; Yang, Hui; Gong, Yan; Liu, Zhi; Li, XiuJun; Wen, Ting; Qu, ZhiGuo; Zhang, Sufeng; Mei, Qibing; Xu, Feng

2017-05-02

Hepatitis B virus (HBV) infection is a serious public health problem, which can be transmitted through various routes (e.g., blood donation) and cause hepatitis, liver cirrhosis and liver cancer. Hence, it is necessary to do diagnostic screening for high-risk HBV patients in these transmission routes. Nowadays, protein-based technologies have been used for HBV testing, which however involve the issues of large sample volume, antibody instability and poor specificity. Nucleic acid hybridization-based lateral flow assay (LFA) holds great potential to address these limitations due to its low-cost, rapid, and simple features, but the poor analytical sensitivity of LFA restricts its application. In this study, we developed a low-cost, simple and easy-to-use method to improve analytical sensitivity by integrating sponge shunt into LFA to decrease the fluid flow rate. The thickness, length and hydrophobicity of the sponge shunt were sequentially optimized, and achieved 10-fold signal enhancement in nucleic acid testing of HBV as compared to the unmodified LFA. The enhancement was further confirmed by using HBV clinical samples, where we achieved the detection limit of 10 3 copies/ml as compared to 10 4 copies/ml in unmodified LFA. The improved LFA holds great potential for diseases diagnostics, food safety control and environment monitoring at point-of-care.

[A study on the relationship between point mutation in pre-core region G1896A of hepatitis B virus and safety of breast feeding].

PubMed

Lu, Yin-ping; Cao, Wei; Hong, Mei; Zhu, Jian-fang; Liu, Zhao; Yang, Dong-liang

2008-10-01

To investigate the relationship between pre-core G1896A point mutation of hepatitis B virus (HBV) and safety of breast feeding. Serum and breast milk samples were collected from 62 pregnant women of HBV DNA positive/HBeAg negative. PCR-solid phase hybridization was used to detect the point mutation in pre-core region G1896A of HBV from pregnant women, and HBV DNA loads in sera and breast milk were determined by fluorescence quantitative PCR (FQ-PCR). The prevalence of point mutation was 61.3% (38/62) in 62 pregnant women with HBsAg positive/HBeAg negative. The positive rate of HBV DNA in breast milk of group with point mutation (28.9%) was similar to that of group without mutation (29.2%, chi2=0.0003, P>0.05). However, The positive rate of HBV DNA in breast milk of group with high HBV loads (56.0%) was significantly higher than that of group with low HBV loads (10.8%, chi2=14.79, P<0.01). The point mutation in pre-core region G1896A of HBV dose not affect the positive rate of HBV DNA in breast milk and higher HBV DNA loads in serum of pregnant women might increase the risk of mother-infant transmission.

Occult HBV among Anti-HBc Alone: Mutation Analysis of an HBV Surface Gene and Pre-S Gene.

PubMed

Kim, Myeong Hee; Kang, So Young; Lee, Woo In

2017-05-01

The aim of this study is to investigate the molecular characteristics of occult hepatitis B virus (HBV) infection in 'anti-HBc alone' subjects. Twenty-four patients with 'anti-HBc alone' and 20 control patients diagnosed with HBV were analyzed regarding S and pre-S gene mutations. All specimens were analyzed for HBs Ag, anti-HBc, and anti-HBs. For specimens with an anti-HBc alone, quantitative analysis of HBV DNA, as well as sequencing and mutation analysis of S and pre-S genes, were performed. A total 24 were analyzed for the S gene, and 14 were analyzed for the pre-S gene through sequencing. A total of 20 control patients were analyzed for S and pre-S gene simultaneously. Nineteen point mutations of the major hydrophilic region were found in six of 24 patients. Among them, three mutations, S114T, P127S/T, M133T, were detected in common. Only one mutation was found in five subjects of the control group; this mutation was not found in the occult HBV infection group, however. Pre-S mutations were detected in 10 patients, and mutations of site aa58-aa100 were detected in 9 patients. A mutation on D114E was simultaneously detected. Although five mutations from the control group were found at the same location (aa58-aa100), no mutations of occult HBV infection were detected. The prevalence of occult HBV infection is not low among 'anti-HBc alone' subjects. Variable mutations in the S gene and pre-S gene were associated with the occurrence of occult HBV infection. Further larger scale studies are required to determine the significance of newly detected mutations. © Copyright: Yonsei University College of Medicine 2017

Genomic and oncogenic preference of HBV integration in hepatocellular carcinoma

PubMed Central

Zhao, Ling-Hao; Liu, Xiao; Yan, He-Xin; Li, Wei-Yang; Zeng, Xi; Yang, Yuan; Zhao, Jie; Liu, Shi-Ping; Zhuang, Xue-Han; Lin, Chuan; Qin, Chen-Jie; Zhao, Yi; Pan, Ze-Ya; Huang, Gang; Liu, Hui; Zhang, Jin; Wang, Ruo-Yu; Yang, Yun; Wen, Wen; Lv, Gui-Shuai; Zhang, Hui-Lu; Wu, Han; Huang, Shuai; Wang, Ming-Da; Tang, Liang; Cao, Hong-Zhi; Wang, Ling; Lee, Tin-Lap; Jiang, Hui; Tan, Ye-Xiong; Yuan, Sheng-Xian; Hou, Guo-Jun; Tao, Qi-Fei; Xu, Qin-Guo; Zhang, Xiu-Qing; Wu, Meng-Chao; Xu, Xun; Wang, Jun; Yang, Huan-Ming; Zhou, Wei-Ping; Wang, Hong-Yang

2016-01-01

Hepatitis B virus (HBV) can integrate into the human genome, contributing to genomic instability and hepatocarcinogenesis. Here by conducting high-throughput viral integration detection and RNA sequencing, we identify 4,225 HBV integration events in tumour and adjacent non-tumour samples from 426 patients with HCC. We show that HBV is prone to integrate into rare fragile sites and functional genomic regions including CpG islands. We observe a distinct pattern in the preferential sites of HBV integration between tumour and non-tumour tissues. HBV insertional sites are significantly enriched in the proximity of telomeres in tumours. Recurrent HBV target genes are identified with few that overlap. The overall HBV integration frequency is much higher in tumour genomes of males than in females, with a significant enrichment of integration into chromosome 17. Furthermore, a cirrhosis-dependent HBV integration pattern is observed, affecting distinct targeted genes. Our data suggest that HBV integration has a high potential to drive oncogenic transformation. PMID:27703150

Evolution of HBV S-gene in the backdrop of HDV co-infection.

PubMed

Baig, Samina; Abidi, Syed H; Azam, Zahid; Majid, Shahid; Khan, Saeed; Khanani, Muhammad R; Ali, Syed

2018-04-16

HBV-HDV co-infected people have a higher chance of developing cirrhosis, fulminant hepatitis, and hepatocellular carcinoma (HCC) compared to those infected only with HBV. The present study was conducted to investigate HBV genotypes and phylogeny among HBV mono-infected and HBV-HDV co-infected patients, as well as analyze mutations in the surface gene of HBV in mono-infected and co-infected patients. A total of 100 blood samples (50 co-infected with HBV and HDV, and 50 mono-infected with HBV only) were collected for this study. HBV DNA was extracted from patient sera and partial surface antigen gene was amplified from HBV genome using polymerase chain reaction. HBV S gene was sequenced from 49 mono-infected and 36 co-infected patients and analyzed to identify HBV genotypes and phylogenetic patterns. Subsequently, HBV S amino acid sequences were analyzed for mutational differences between sequences from mono- and co-infected patients. HBV genotype D was predominantly found in both mono-infected as well as co-infected patients. Phylogenetic analysis showed the divergence of HBV sequences, between mono- and co-infected patients, into two distinct clusters. HBV S gene mutation analysis revealed certain mutations in HBV-HDV co-infected subjects to be distinct from those found in mono-infected patients. This might indicate the evolution of HBV S gene under selection pressures generated from HDV coinfection. © 2018 Wiley Periodicals, Inc.

Tenofovir alafenamide demonstrates broad cross-genotype activity against wild-type HBV clinical isolates and maintains susceptibility to drug-resistant HBV isolates in vitro.

PubMed

Liu, Yang; Miller, Michael D; Kitrinos, Kathryn M

2017-03-01

Tenofovir alafenamide (TAF) is a novel prodrug of tenofovir (TFV). This study evaluated the antiviral activity of TAF against wild-type genotype A-H HBV clinical isolates as well as adefovir-resistant, lamivudine-resistant, and entecavir-resistant HBV isolates. Full length HBV genomes or the polymerase/reverse transcriptase (pol/RT) region from treatment-naïve patients infected with HBV genotypes A-H were amplified and cloned into an expression vector under the control of a CMV promoter. In addition, 11 drug resistant HBV constructs were created by site-directed mutagenesis of a full length genotype D construct. Activity of TAF was measured by transfection of each construct into HepG2 cells and assessment of HBV DNA levels following treatment across a range of TAF concentrations. TAF activity in vitro was similar against wild-type genotype A-H HBV clinical isolates. All lamivudine- and entecavir-resistant isolates and 4/5 adefovir-resistant isolates were found to be sensitive to inhibition by TAF in vitro as compared to the wild-type isolate. The adefovir-resistant isolate rtA181V + rtN236T exhibited low-level reduced susceptibility to TAF. TAF is similarly active in vitro against wild-type genotype A-H HBV clinical isolates. The TAF sensitivity results for all drug-resistant isolates are consistent with what has been observed with the parent drug TFV. The in vitro cell-based HBV phenotyping assay results support the use of TAF in treatment of HBV infected subjects with diverse HBV genotypes, in both treatment-naive and treatment-experienced HBV infected patients. Copyright © 2016 Elsevier B.V. All rights reserved.

Acute HBV infection in humanized chimeric mice has multiphasic viral kinetics

DOE PAGES

Ishida, Yuji; Chung, Tje Lin; Imamura, Michio; ...

2018-03-23

Background: Chimeric uPA/SCID mice reconstituted with humanized livers are useful for studying HBV infection in the absence of an adaptive immune response. However, the detailed characterization of HBV infection kinetics necessary to enable in-depth mechanistic studies in this novel in vivo HBV infection model is lacking. Methods: To characterize HBV kinetics post-inoculation (p.i.) to steady state, 42 mice were inoculated with HBV. Serum HBV DNA was frequently measured from 1 minute to 63 days p.i. Total intrahepatic HBV DNA, HBV cccDNA, and HBV RNA was measured in a subset of mice at 2, 4, 6, 10, and 13 weeks p.i.more » HBV half-life (t 1/2) was estimated using a linear mixed-effects model. Results: During the first 6 h p.i. serum HBV declined in repopulated uPA/SCID mice with a t 1/2=62 min [95%CI=59-67min]. Thereafter, viral decline slowed followed by a 2 day lower plateau. Subsequent viral amplification was multiphasic with an initial mean doubling time of t 2= 8±3 h followed by an interim plateau before prolonged amplification (t 2=2±0.5 days) to a final HBV steady state of 9.3 ± 0.3 log copies/ml. Serum HBV and intrahepatic HBV DNA were positively correlated (R2=0.98). Conclusions: HBV infection in uPA/SCID chimeric mice is highly dynamic despite the absence of an adaptive immune response. The serum HBV t 1/2 in humanized uPA/SCID mice was estimated to be ~1 h regardless of inoculum size. Finally, the HBV acute infection kinetics presented here is an important step in characterizing this experimental model system so that it can be effectively used to elucidate the dynamics of the HBV lifecycle and thus possibly reveal effective antiviral drug targets.« less

Acute HBV infection in humanized chimeric mice has multiphasic viral kinetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishida, Yuji; Chung, Tje Lin; Imamura, Michio

Background: Chimeric uPA/SCID mice reconstituted with humanized livers are useful for studying HBV infection in the absence of an adaptive immune response. However, the detailed characterization of HBV infection kinetics necessary to enable in-depth mechanistic studies in this novel in vivo HBV infection model is lacking. Methods: To characterize HBV kinetics post-inoculation (p.i.) to steady state, 42 mice were inoculated with HBV. Serum HBV DNA was frequently measured from 1 minute to 63 days p.i. Total intrahepatic HBV DNA, HBV cccDNA, and HBV RNA was measured in a subset of mice at 2, 4, 6, 10, and 13 weeks p.i.more » HBV half-life (t 1/2) was estimated using a linear mixed-effects model. Results: During the first 6 h p.i. serum HBV declined in repopulated uPA/SCID mice with a t 1/2=62 min [95%CI=59-67min]. Thereafter, viral decline slowed followed by a 2 day lower plateau. Subsequent viral amplification was multiphasic with an initial mean doubling time of t 2= 8±3 h followed by an interim plateau before prolonged amplification (t 2=2±0.5 days) to a final HBV steady state of 9.3 ± 0.3 log copies/ml. Serum HBV and intrahepatic HBV DNA were positively correlated (R2=0.98). Conclusions: HBV infection in uPA/SCID chimeric mice is highly dynamic despite the absence of an adaptive immune response. The serum HBV t 1/2 in humanized uPA/SCID mice was estimated to be ~1 h regardless of inoculum size. Finally, the HBV acute infection kinetics presented here is an important step in characterizing this experimental model system so that it can be effectively used to elucidate the dynamics of the HBV lifecycle and thus possibly reveal effective antiviral drug targets.« less

Evaluation of the Aptima HBV Quant assay vs. the COBAS TaqMan HBV test using the high pure system for the quantitation of HBV DNA in plasma and serum samples.

PubMed

Schalasta, Gunnar; Börner, Anna; Speicher, Andrea; Enders, Martin

2018-03-28

Proper management of patients with chronic hepatitis B virus (HBV) infection requires monitoring of plasma or serum HBV DNA levels using a highly sensitive nucleic acid amplification test. Because commercially available assays differ in performance, we compared herein the performance of the Hologic Aptima HBV Quant assay (Aptima) to that of the Roche Cobas TaqMan HBV test for use with the high pure system (HPS/CTM). Assay performance was assessed using HBV reference panels as well as plasma and serum samples from chronically HBV-infected patients. Method correlation, analytical sensitivity, precision/reproducibility, linearity, bias and influence of genotype were evaluated. Data analysis was performed using linear regression, Deming correlation analysis and Bland-Altman analysis. Agreement between the assays for the two reference panels was good, with a difference in assay values vs. target <0.5 log. Qualitative assay results for 159 clinical samples showed good concordance (88.1%; κ=0.75; 95% confidence interval: 0.651-0.845). For the 106 samples quantitated by both assays, viral load results were highly correlated (R=0.92) and differed on average by 0.09 log, with 95.3% of the samples being within the 95% limit of agreement of the assays. Linearity for viral loads 1-7 log was excellent for both assays (R2>0.98). The two assays had similar bias and precision across the different genotypes tested at low viral loads (25-1000 IU/mL). Aptima has a performance comparable with that of HPS/CTM, making it suitable for use for HBV infection monitoring. Aptima runs on a fully automated platform (the Panther system) and therefore offers a significantly improved workflow compared with HPS/CTM.

Evaluation of clinical sensitivity and specificity of hepatitis B virus (HBV), hepatitis C virus, and human immunodeficiency Virus-1 by cobas MPX: Detection of occult HBV infection in an HBV-endemic area.

PubMed

Ha, Jihye; Park, Younhee; Kim, Hyon-Suk

2017-11-01

Transfusion-transmitted infectious diseases remain a major concern for blood safety, particularly with hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). Nucleic acid testing (NAT) in donor screening shortens the serologically negative window period and reduces virus transmission. The cobas MPX (Roche Molecular Systems, Inc., Branchburg, New Jersey) is a recently developed multiplex qualitative PCR system that enables the simultaneous detection of HBV, HCV, and HIV with improved sensitivity and throughput using cobas 6800 and 8800 instruments. The aim of this study was to conduct an evaluation of the clinical sensitivity and specificity of cobas MPX detection of HBV, HCV, and HIV in clinical specimens. Among samples referred for HBV, HCV, and HIV-1 quantification at Severance Hospital, Yonsei University College of Medicine, positive samples were selected to evaluate sensitivity. A total of 843 samples was tested using both cobas MPX and COBAS AmpliPrep/COBAS TaqMan Tests for HBV, HCV, and HIV-1 using the cobas 8800 system and a COBAS TaqMan 96 analyzer, respectively. Samples that showed discrepancies were confirmed by nested PCR. The cobas MPX achieved excellent sensitivity and specificity for the detection of HBV, HCV, and HIV-1 in clinical samples. We found that the lower limit of detection (LOD) of blood screening by NAT actually improves clinical sensitivity, and occult HBV infection prevalence among healthy employees of the hospital was rather high. Copyright © 2017 Elsevier B.V. All rights reserved.

Routine screening of blood donations at Qingdao central blood bank, China, for hepatitis B virus (HBV) DNA with a real-time, multiplex nucleic acid test for HBV, hepatitis C virus, and human immunodeficiency virus Types 1 and 2.

PubMed

Yang, Zhongsi; Xu, Lei; Liu, Li; Feng, Qiuxia; Zhang, Longmu; Ma, Weijuan; Saldanha, John; Wang, Mingmin; Zhao, Lin

2013-10-01

The Roche cobas TaqScreen MPX test was used to evaluate the rate of hepatitis B surface antigen (HBsAg)-negative donations that were hepatitis B virus (HBV) DNA reactive from June 2010 to January 2011 in Qingdao, China. HBsAg-negative samples from 65,800 voluntary blood donors were tested with the cobas TaqScreen MPX test in pools of 6 on the Roche cobas s 201 blood screening platform. Samples positive for HBV DNA and negative for HBsAg were quantitated with the Roche COBAS AmpliPrep/COBAS TaqMan HBV test. In addition, serologic tests for HBsAg, hepatitis B surface antibody, anti-hepatitis B core antigen (anti-HBc), anti-hepatitis B e antigen (anti-HBe), and hepatitis B e antigen (HBe) were done using the Roche electrochemiluminescence immunoassay. A total of 80 nucleic acid amplification technology (NAT) test-reactive pools were identified and 59 pools (74%) resolved to a reactive sample. All samples were HBV DNA reactive and the viral load in each sample was quantitated. The viral loads of the samples ranged from less than 20 to 34,600 IU/mL; 13 samples (22%) had viral loads of more than 20 IU/mL, 27 samples (45.8%) had viral loads of less than 20 IU/mL, and 19 samples (32.2%) had undetectable viral loads. Of the 59 NAT-reactive samples, 40 (67.8%) were anti-HBc positive. Fifteen of the 59 samples could not be confirmed as NAT reactive either by an alternative NAT test or by serology. The HBV NAT yield in blood donors in Qingdao is 0.06% (38/65,800). This study confirmed the value of NAT for interdicting HBV-positive donations and preventing transfusion-transmitted HBV infections. © 2013 American Association of Blood Banks.

Common and Distinct Capsid and Surface Protein Requirements for Secretion of Complete and Genome-free Hepatitis B Virions.

PubMed

Ning, Xiaojun; Luckenbaugh, Laurie; Liu, Kuancheng; Bruss, Volker; Sureau, Camille; Hu, Jianming

2018-05-09

During the morphogenesis of hepatitis B virus (HBV), an enveloped virus, two types of virions are secreted: (1) a minor population of complete virions containing a mature nucleocapsid with the characteristic, partially double-stranded, relaxed circular DNA genome and (2) a major population containing an empty capsid with no DNA or RNA (empty virions). Secretion of both types of virions requires interactions between the HBV capsid or core protein (HBc) and the viral surface or envelope proteins. We have studied the requirements from both HBc and envelope proteins for empty virion secretion, in comparison with those for secretion of complete virions. Substitutions within the N-terminal domain of HBc that block secretion of DNA-containing virions reduced but did not prevent secretion of empty virions. The HBc C-terminal domain was not essential for empty virion secretion. Among the three viral envelope proteins, the smallest, S, alone was sufficient for empty virion secretion at a basal level. The largest protein, L, essential for complete virion secretion, was not required for, but could stimulate empty virion secretion. Also, substitutions in L that eliminate secretion of complete virions reduced but did not eliminate empty virion secretion. S mutations that block secretion of the hepatitis D virus (HDV), an HBV satellite, did not block secretion of either empty or complete HBV virions. Together, these results indicate that both common and distinct signals on empty capsids vs. mature nucleocapsids interact with the S and L proteins during the formation of complete vs. empty virions. IMPORTANCE Hepatitis B virus (HBV) is a major cause of severe liver diseases including cirrhosis and cancer. In addition to the complete infectious virion particle, which contains an outer envelope layer and an interior capsid that, in turn, encloses a DNA genome, HBV infected cells also secrete non-infectious, incomplete viral particles in large excess over the complete virions. In

Inhibition of interferon-inducible MxA protein expression by hepatitis B virus capsid protein.

PubMed

Rosmorduc, O; Sirma, H; Soussan, P; Gordien, E; Lebon, P; Horisberger, M; Bréchot, C; Kremsdorf, D

1999-05-01

Chronic hepatitis B treatment has been significantly improved by interferon (IFN) treatment. However, some studies have suggested that hepatitis B virus (HBV) might have a direct effect on the resistance to IFN. Defective particles, generated by spliced HBV RNA and associated with chronic hepatitis B, have been previously characterized; expression of these particles leads to cytoplasmic accumulation of the capsid protein. The aim of this study was to investigate the role of these defective genomes in IFN resistance. The global antiviral activity of IFN was studied by virus yield reduction assays, the expression of three IFN-induced antiviral proteins was analysed by Western blotting and confocal microscopy, and the regulation of MxA gene expression was studied by Northern blotting and the luciferase assay, in Huh7 cells transfected with a complete or the defective HBV genome. Results showed that the expression of the defective genome reduces the antiviral activity of IFN and that this modulation involves a selective inhibition of MxA protein induction by the HBV capsid protein. Our results also show the trans-suppressive effect of the HBV capsid on the MxA promoter, which might participate in this phenomenon. In conclusion, this study shows a direct interplay between the IFN-sensitive pathway and the capsid protein and might implicate this defective HBV genome in virus persistence.

Prevalence and presentation of hepatitis B and C virus (HBV and HCV) infection in Vietnamese Americans via serial community serologic testing.

PubMed

Nguyen, Kelvin; Van Nguyen, Thai; Shen, Duke; Xia, Victor; Tran, Diep; Banh, Khanh; Ruan, Victor; Hu, Ke-Qin

2015-02-01

The prevalence of hepatitis B virus (HBV) infection is reportedly high in Vietnamese Americans (VAs), but most previous studies did not assess full HBV serology, and not the prevalence of HBV and hepatitis C virus (HCV) infection simultaneously. The aim of the study is to assess the prevalence of different HBV serologies and HCV infection in VAs. This study was based on the data collected by testing for Hepatitis B surface antigen (HBsAg), anti-hepatitis B core antibody (HBcAb IgG), anti-HBs antibody (HBsAb), and anti-HCV antibody (anti-HCV) in a series of community screening in VAs in Orange County, California. In 1,405 VA participants, the mean age was 51 (17-87) years, 45.1% were males; 68.2%, married; 97.2%, born in Vietnam. Most of the participants were non-US born with their primary language being non-English and with limited access to health care. Of the 1,405 cases, 124 (8.8%) were confirmed HBV infection by HBsAg+; 81 (5.8%), HCV infection by anti-HCV+; including four (0.3%) with HBV/HCV coinfection. Twelve percent of the participants with confirmed HBV infection thought they were previously tested negative, while 29.7% of the participants with confirmed HCV infection thought they were previously tested negative. In this cohort, 15.4% were HBsAg-/HBsAb-/HBcAb IgG-, i.e. being susceptible to HBV infection. In HCV infected participants, 65.4% were born between 1945 and 1965. This large serial survey and screening in the Vietnamese American community confirmed the rates of HBV and HCV infection to be as high as 8.8% and 5.8%, respectively. We have also identified factors related to HBV and HCV infection in this high-risk population.

SIRT3 restricts HBV transcription and replication via epigenetic regulation of cccDNA involving SUV39H1 and SETD1A histone methyltransferases.

PubMed

Ren, Ji-Hua; Hu, Jie-Li; Cheng, Sheng-Tao; Yu, Hai-Bo; Wong, Vincent Kam Wai; Law, Betty Yuen Kwan; Yang, Yong-Feng; Huang, Ying; Liu, Yi; Chen, Wei-Xian; Cai, Xue-Fei; Tang, Hua; Hu, Yuan; Zhang, Wen-Lu; Liu, Xiang; Long, Quan-Xin; Zhou, Li; Tao, Na-Na; Zhou, Hong-Zhong; Yang, Qiu-Xia; Ren, Fang; He, Lin; Gong, Rui; Huang, Ai-Long; Chen, Juan

2018-04-06

Hepatitis B virus (HBV) infection remains a major health problem worldwide. Maintenance of the covalently closed circular DNA (cccDNA) which serves as a template for HBV RNA transcription is responsible for the failure of eradicating chronic HBV during current antiviral therapy. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications. In this study, we identified SIRT3 as a host factor restricting HBV transcription and replication by screening seven members of Sirtuin family which is the class III histone deacetylase. Ectopic SIRT3 expression significantly reduced total HBV RNAs, 3.5-kb RNA as well as replicative intermediate DNA in HBV-infected HepG2-NTCP cells and PHH. In contrast, gene silencing of SIRT3 promoted HBV transcription and replication. Mechanistic study found nuclear SIRT3 was recruited to the HBV cccDNA, where it deacetylated histone 3 lysine 9 (H3K9). Importantly, occupancy of SIRT3 onto cccDNA could increase the recruitment of histone methyltransferase SUV39H1 to cccDNA and decrease recruitment of SETD1A, leading to a marked increase of H3K9me3 and a decrease of H3K4me3 on cccDNA. Moreover, SIRT3-mediated HBV cccDNA transcriptional repression involved decreased binding of host RNA polymerase II and transcription factor YY1 to cccDNA. Finally, viral protein HBx could relieve SIRT3-mediated cccDNA transcriptional repression by inhibiting both SIRT3 expression and its recruitment to cccDNA. SIRT3 is a novel host factor epigenetically restricting HBV cccDNA transcription by acting cooperatively with histone methyltransferase. These data provided a rational for the use of SIRT3 activators in the prevention or treatment of HBV infection. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

The Heteroaryldihydropyrimidine Bay 38-7690 Induces Hepatitis B Virus Core Protein Aggregates Associated with Promyelocytic Leukemia Nuclear Bodies in Infected Cells.

PubMed

Huber, Andrew D; Wolf, Jennifer J; Liu, Dandan; Gres, Anna T; Tang, Jing; Boschert, Kelsey N; Puray-Chavez, Maritza N; Pineda, Dallas L; Laughlin, Thomas G; Coonrod, Emily M; Yang, Qiongying; Ji, Juan; Kirby, Karen A; Wang, Zhengqiang; Sarafianos, Stefan G

2018-04-25

Heteroaryldihydropyrimidines (HAPs) are compounds that inhibit hepatitis B virus (HBV) replication by modulating viral capsid assembly. While their biophysical effects on capsid assembly in vitro have been previously studied, the effect of HAP treatment on capsid protein (Cp) in individual HBV-infected cells remains unknown. We report here that the HAP Bay 38-7690 promotes aggregation of recombinant Cp in vitro and causes a time- and dose-dependent decrease of Cp in infected cells, consistent with previously studied HAPs. Interestingly, immunofluorescence analysis showed Cp aggregating in nuclear foci of Bay 38-7690-treated infected cells in a time- and dose-dependent manner. We found these foci to be associated with promyelocytic leukemia (PML) nuclear bodies (NBs), which are structures that affect many cellular functions, including DNA damage response, transcription, apoptosis, and antiviral responses. Cp aggregation is not an artifact of the cell system used, as it is observed in HBV-expressing HepAD38 cells, in HepG2 cells transfected with an HBV-expressing plasmid, and in HepG2-NTCP cells infected with HBV. Use of a Cp overexpression vector without HBV sequences shows that aggregation is independent of viral replication, and use of an HBV-expressing plasmid harboring a HAP resistance mutation in Cp abrogated the aggregation, demonstrating that the effect is due to direct compound-Cp interactions. These studies provide novel insight into the effects of HAP-based treatment at a single-cell level. IMPORTANCE Despite the availability of effective vaccines and treatments, HBV remains a significant global health concern, with more than 240 million individuals chronically infected. Current treatments are highly effective at controlling viral replication and disease progression but rarely cure infections. Therefore, much emphasis is being placed on finding therapeutics with new drug targets, such as viral gene expression, covalently closed circular DNA formation and

Prevalence of HBV and HBV vaccination coverage in health care workers of tertiary hospitals of Peshawar, Pakistan

PubMed Central

2011-01-01

Background Hepatitis B Virus (HBV) may progress to serious consequences and increase dramatically beyond endemic dimensions that transmits to or from health care workers (HCWs) during routine investigation in their work places. Basic aim of this study was to canvass the safety of HCWs and determine the prevalence of HBV and its possible association with occupational and non-occupational risk factors. Hepatitis B vaccination coverage level and main barriers to vaccination were also taken in account. Results A total of 824 health care workers were randomly selected from three major hospitals of Peshawar, Khyber Pakhtunkhwa. Blood samples were analyzed in Department of Zoology, Kohat University of Science and Technology Kohat, and relevant information was obtained by means of preset questionnaire. HCWs in the studied hospitals showed 2.18% prevalence of positive HBV. Nurses and technicians were more prone to occupational exposure and to HBV infection. There was significant difference between vaccinated and non-vaccinated HCWs as well as between the doctors and all other categories. Barriers to complete vaccination, in spite of good knowledge of subjects in this regard were work pressure (39.8%), negligence (38.8%) un-affordability (20.9%), and unavailability (0.5%). Conclusions Special preventive measures (universal precaution and vaccination), which are fundamental way to protect HCW against HBV infection should be adopted. PMID:21645287

[Risk Management of HBV Reactivation: Construction of Check System].

PubMed

Tanaka, Yasuhito

2015-09-01

In recent years, reactivation of HBV in patients receiving cancer chemotherapy or immunosuppressive therapy has been a problem. Generally, HBV-DNA levels are elevated prior to HBsAg concentration, and then hepatic dysfunction is observed in the process of hepatitis by HBV reactivation. Therefore, the monitoring of HBV-DNA is useful for the prediction of hepatic dysfunction, and nucleoside/nucleoside analogue (NA) administration is able to prevent this HBV reactivation. According to these facts, "Guidelines for the Prevention of HBV Reactivation in Patients Receiving Immunosuppressive Therapy or Chemotherapy", 2009 (revised as "JSH Guidelines for the Management of Hepatitis B Virus Infection", 2013) is established, and the diagnostic algorithm of HBsAg, anti-HBc, anti-HBs, and HBV-DNA has relevant descriptions. Combination therapy with rituximab and steroid for malignant lymphoma has a high risk of leading to fulminant hepatitis and, consequently, the guidelines are widely followed in such cases. We introduced the improvement of electronic medical recording and ordering systems in collaboration with hepatologists, and such a system has been widely used. Although the monitoring of HBV-DNA levels is required every 1-3 months, the guidelines are not followed strictly in cases such as rheumatoid disease and solid tumors only with chemotherapy or steroid treatment. Since a DNA assay is complicated and expensive, cost-effective, time-saving, and highly sensitive/specific measurements are required as well. Therefore, Lumipulse HBsAg-HQ (CLIA method) with high sensitivity is expected to be used for the monitoring of HBV reactivation.

An unusual renal manifestation of chronic HBV infection.

PubMed

Aravindan, Ananthakrishnapuram; Yong, Jim; Killingsworth, Murray; Strasser, Simone; Suranyi, Michael

2010-08-01

Hepatitis B viral infection is usually a self-limiting disease in immunocompetent individuals. Chronic infection can be seen in up to 5% of infected patients. Renal manifestations of chronic HBV infection are usually glomerular. We describe here an uncommon presentation of a patient with chronic HBV infection with very high viral load and rapidly progressive renal failure. Renal biopsy showed features of tubulointerstitial nephritis and tubular epithelial inclusion bodies suggestive of HBV infection. Entecavir treatment slowed down the progression of his renal disease. Tubulointerstitial nephritis should be considered as a part of the differential diagnosis in patients with HBV infection. Early antiviral treatment may halt the progression of renal disease.

[The prevalence and clinical significance of precore and core promoter mutations in Korean patients with chronic hepatitis B virus infection].

PubMed

Kim, Hyung Joon; Yoo, Byung Chul

2002-06-01

Precore and core promoter mutations of hepatitis B virus (HBV) have been reported in Korea but their prevalence and clinical significance have not been determined. The aims of this study were to determine the prevalence of precore and core promoter mutations and their relationships to hepatitis B e antigen (HBeAg) status, viral replication level, and severity of liver disease in Korea. Among the patients who visited the Liver Diseases Clinics (Chung Ang University Hospital) between December 1998 and August 1999, 150 patients were randomly selected: 50 HBeAg-positive HBV-DNA positive patients by a branched DNA (bDNA) assay, 50 HBeAg-negative bDNA-positive patients, and 50 HBeAg-negative bDNA-negative patients. Serum HBV-DNA was amplified by a polymerase chain reaction (PCR) in these patients and the core promoter/precore HBV sequence was determined in 135 of the patients whose sera were positive for HBV-DNA by PCR. All of the 135 determined HBV-DNA sequences had HBV genotype with T at nucleotide 1858. Precore mutation (A1896) was detected in 95.7% of HBeAg-negative bDNA-positive patients and 94.9% of HBeAg-negative bDNA-negative patients. In HBeAg-positive patients 88% had wild type and 12% had mixture of wild type and A1896 mutant. Core promoter TA mutation (T1762/A1764) was detected in 93.5% of HBeAg-negative bDNA-positive patients, 94.9% of HBeAg-negative bDNA-negative patients and 74% of HBeAg-positive patients. No correlation was found between the presence of precore/core promoter mutations and liver disease severity or HBV-DNA levels. Precore stop codon mutation occurred almost invariably, along with HBeAg seroconversion, irrespective of subsequent viral replication levels or disease severity. Core promoter TA mutation was frequent both in the HBeAg-positive patients and HBeAg-negative patients irrespective of viral replication levels or disease severity.

Hepatitis B core-related antigen (HBcrAg) levels in the natural history of hepatitis B virus infection in a large European cohort predominantly infected with genotypes A and D.

PubMed

Maasoumy, B; Wiegand, S B; Jaroszewicz, J; Bremer, B; Lehmann, P; Deterding, K; Taranta, A; Manns, M P; Wedemeyer, H; Glebe, D; Cornberg, M

2015-06-01

Hepatitis B core-related antigen (HBcrAg) has been suggested as an additional marker of hepatitis B virus (HBV) infection. HBcrAg combines the antigenic reactivity resulting from denatured hepatitis B e antigen (HBeAg), HBV core antigen and an artificial core-related protein (p22cr). In Asian patients, high levels of HBcrAg have been suggested to be an independent risk factor for hepatocellular carcinoma, while low levels could guide safe cessation of treatment with nucleos(t)ide analogues. We here studied HBcrAg levels in different phases of HBV infection in a large European cohort predominantly infected with genotypes A and D: HBeAg-positive immune tolerance (n = 30), HBeAg-positive immune clearance (IC) (n = 60), HBeAg-negative hepatitis (ENH) (n = 50), HBeAg-negative inactive/quiescent carrier phase (c) (n = 109) and acute hepatitis B (n = 8). Median HBcrAg levels were high in the immune tolerance and immune clearance phases (8.41 and 8.11 log U/mL, respectively), lower in ENH subjects (4.82 log U/mL) but only 2.00 log U/mL in ENQ subjects. Correlation between HBcrAg and HBV DNA varied among the different phases of HBV infection, while HBcrAg moderately correlated with hepatitis B surface antigen in all phases. ENQ patients had HBcrAg levels <3 log U/mL in 79%, in contrast to only 12% in the ENH group. HBcrAg levels vary significantly during the different phases of HBV infection. HBcrAg may serve as valuable marker for virus replication and reflect the transcriptional activity of intrahepatic cccDNA. In HBeAg-negative patients, HBcrAg may help to distinguish between inactive carriers (ENQ) and those with active disease (ENH). Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Hepatitis B virus (HBV)-specific short hairpin RNA is capable of reducing the formation of HBV covalently closed circular (CCC) DNA but has no effect on established CCC DNA in vitro.

PubMed

Starkey, Jason L; Chiari, Estelle F; Isom, Harriet C

2009-01-01

Hepatitis B virus (HBV) covalently closed circular (CCC) DNA is the source of HBV transcripts and persistence in chronically infected patients. The novel aspect of this study was to determine the effect of RNA interference (RNAi) on HBV CCC DNA when administered prior to establishment of HBV replication or during chronic HBV infection. HBV replication was initiated in HepG2 cells by transduction with HBV baculovirus. Subculture of HBV-expressing HepG2 cells at 10 days post-transduction generates a system in which HBV replication is ongoing and HBV is expressed largely from CCC DNA, thus simulating chronic HBV infection. HepG2 cells were transduced with short hairpin RNA (shRNA)-expressing baculovirus prior to initiation of HBV replication or during chronic HBV replication, and the levels of HBV RNA, HBV surface antigens (HBsAg) and replicative intermediates (RI), extracellular (EC) and CCC DNA species were measured. HBsAg, HBV RNA and DNA levels were markedly reduced until day 8 whether cells were transduced with shRNA prior to or during a chronic infection; however, the CCC DNA species were only affected when shRNA was administered prior to initiation of infection. We conclude that RNAi may have a therapeutic value for controlling HBV replication at the level of RI and EC DNA and for reducing establishment of CCC DNA during HBV infection. Our data support previous findings demonstrating the stability of HBV CCC DNA following antiviral therapy. This study also reports the development of a novel HBV baculovirus subculture system that can be used to evaluate antiviral effects on chronic HBV replication.

Comparison of clinical application of the Abbott HBV PCR kit and the VERSANT HBV DNA 3.0 test to measure serum hepatitis B virus DNA in Taiwanese patients.

PubMed

Yang, Jeng-Fu; Lin, Ya-Yun; Huang, Jee-Fu; Liu, Shu-Fen; Chu, Pei-Yu; Hsieh, Ming-Yen; Lin, Zu-Yau; Chen, Shinn-Cherng; Wang, Liang-Yen; Dai, Chia-Yen; Chuang, Wan-Long; Yu, Ming-Lung

2009-08-01

With an estimated 350-400 million people worldwide chronically infected with hepatitis B virus (HBV), and the subsequent serious complications caused by liver damage including cirrhosis, liver failure, and hepatocellular carcinoma, HBV infection remains a global health issue, particularly in Taiwan, an HBV-hyperendemic area. Sensitive and accurate quantification of HBV DNA is necessary to monitor patients with chronic hepatitis B who are receiving antiviral therapy to determine treatment response and adapt therapy. We evaluated and compared the clinical performance of two HBV DNA assays based on different technologies: the RealArt HBV PCR Kit (Abbott HBV DNA PCR kit, real-time polymerase chain reaction assay, detection limit: 27 IU/mL) and the VERSANT bDNA 3.0 assay (Bayer, branched DNA signal amplification assay, detection limit: 357 IU/mL). Serum levels of HBV DNA in 173 chronic HBV carriers were determined using both the RealArt HBV PCR Kit and the VERSANT bDNA 3.0 test. Of the 173 samples analyzed for baseline viral load detection, HBV DNA was quantifiable in 147 patients (82.1%) by the RealArt HBV PCR Kit, which was significantly higher than the 92 (53.2%) samples quantified by the VERSANT bDNA 3.0 assay. A total of 86 (49.7%) samples were quantifiable by both assays, whereas 25 (14.5%) were below the detection limit of both assays. The HBV DNA quantification values measured by the RealArt HBV PCR Kit and the VERSANT bDNA 3.0 assay were positively correlated (Spearman's rank correlation coefficient r = 0.932, p < 0.001). On average, the results derived from the RealArt HBV PCR Kit were 0.67 log lower than those of the VERSANT bDNA 3.0 assay. HBV DNA concentrations were significantly higher in 63 HBV e antigen (HBeAg)-seropositive patients than in 110 HBeAg-seronegative patients (5.42 +/- 2.34 logs vs. 3.21 +/- 2.27 logs, p < 0.001). The RealArt HBV PCR Kit is more sensitive and has a wider dynamic range than the VERSANT bDNA 3.0 assay in the clinical setting

The Native Form and Maturation Process of Hepatitis C Virus Core Protein

PubMed Central

Yasui, Kohichiroh; Wakita, Takaji; Tsukiyama-Kohara, Kyoko; Funahashi, Shin-Ichi; Ichikawa, Masumi; Kajita, Tadahiro; Moradpour, Darius; Wands, Jack R.; Kohara, Michinori

1998-01-01

The maturation and subcellular localization of hepatitis C virus (HCV) core protein were investigated with both a vaccinia virus expression system and CHO cell lines stably transformed with HCV cDNA. Two HCV core proteins, with molecular sizes of 21 kDa (p21) and 23 kDa (p23), were identified. The C-terminal end of p23 is amino acid 191 of the HCV polyprotein, and p21 is produced as a result of processing between amino acids 174 and 191. The subcellular localization of the HCV core protein was examined by confocal laser scanning microscopy. Although HCV core protein resided predominantly in the cytoplasm, it was also found in the nucleus and had the same molecular size as p21 in both locations, as determined by subcellular fractionation. The HCV core proteins had different immunoreactivities to a panel of monoclonal antibodies. Antibody 5E3 stained core protein in both the cytoplasm and the nucleus, C7-50 stained core protein only in the cytoplasm, and 499S stained core protein only in the nucleus. These results clearly indicate that the p23 form of HCV core protein is processed to p21 in the cytoplasm and that the core protein in the nucleus has a higher-order structure different from that of p21 in the cytoplasm. HCV core protein in sera of patients with HCV infection was analyzed in order to determine the molecular size of genuinely processed HCV core protein. HCV core protein in sera was found to have exactly the same molecular weight as the p21 protein. These results suggest that p21 core protein is a component of native viral particles. PMID:9621068

Effects of HBV Genetic Variability on RNAi Strategies

PubMed Central

Panjaworayan, Nattanan; Brown, Chris M.

2011-01-01

RNAi strategies present promising antiviral strategies against HBV. RNAi strategies require base pairing between short RNAi effectors and targets in the HBV pregenome or other RNAs. Natural variation in HBV genotypes, quasispecies variation, or mutations selected by the RNAi strategy could potentially make these strategies less effective. However, current and proposed antiviral strategies against HBV are being, or could be, designed to avoid this. This would involve simultaneous targeting of multiple regions of the genome, or regions in which variation or mutation is not tolerated. RNAi strategies against single genotypes or against variable regions of the genome would need to have significant other advantages to be part of robust therapies. PMID:21760994

[Fundamental and clinical evaluation of hepatitis B virus core-related antigen assay by LUMIPULSE f].

PubMed

Tanaka, Yasuhito; Takagi, Kazumi; Hiramatsu, Kumiko; Naganuma, Hatsue; Iida, Takayasu; Takasaka, Yoshimitsu; Mizokami, Masashi

2006-07-01

A sensitive chemiluminescence enzyme immunoassay (CLEIA) has been developed for hepatitis B virus (HBV) core-related antigens (HBcrAg) detection. The HBcrAg is designated as the precore/core gene products including HBeAg. The aim of this study is to evaluate reproducibility of HBcrAg and correlation with HBV-DNA in serum using the automatic LUMIPULSE f to estimate an assay suitable for general laboratory use. In this study, we demonstrated that HBcrAg assay had highly intra-assay reproducible [coefficients of variation(CVs); 2.8-5.2%] and inter-assay reproducible [CVs; 3.9-9.1%]. When the cutoff value was tentatively set at 1 kU/ml, all healthy controls (HBsAg/HBV-DNA negative; n=100) and anti-HCV antibody-positive (n=50) sera were identified as negative. The assay showed a detection limit of 0.5 kU/ml using four serially diluted HBV high-titer sera, indicating higher sensitivity than HBV-DNA (transcription-mediated amplification). The HBcrAg concentration correlated positively with serum HBV-DNA (n=125, r = 0.860, p < 0.0001) regardless of HBeAg, although the HBcrAg levels were higher in HBeAg-positive group than in HBeAg-negative group. In the natural course of HBV infection, the HBcrAg concentration usually changed in accordance with HBV-DNA levels, however during lamivudine therapy the change of HBcrAg was more gradual than that of HBV-DNA. In conclusion, HBcrAg concentration provides a reflection of HBV virus load equivalent to HBV-DNA level, and the assay therefore offers a simple method for monitoring hepatitis B patients.

[Performance evaluation of Abbott RealTime HBV Quantification Kit for HBV viral load by real-time PCR].

PubMed

Kim, Myeong Hee; Cha, Choong Hwan; An, Dongheui; Choi, Sung Eun; Oh, Heung Bum

2008-04-01

Hepatitis B virus (HBV) DNA quantification is necessary for starting and monitoring of antiviral therapy in patients with chronic hepatitis B. This study was intended to assess the clinical performance of Abbott RealTime HBV Quantification kit (Abbott Laboratories, USA). The performance was evaluated in terms of precision, linearity, detection sensitivity, cross-reactivity, and carry-over. A correlation with the Real-Q HBV Quantification kit (BioSewoom Inc., Korea) was also examined using serum samples from 64 patients diagnosed with chronic hepatitis B and underwent lamivudine therapy in Asan Medical Center. We verified the trueness of the system by comparing the outputs with the assigned values of the BBI panel (BBI Diagnostics, USA). Within-run and between-run coefficients of variation (CV) were 3.56-4.71% and 3.03-4.98%, respectively. Linearity was manifested ranging from 53 to 10(9)copies/mL and the detection sensitivity was verified to be 51 copies/mL. None of hepatitis C virus showed cross-reactivity. No cross-contamination occurred when negative and positive samples were alternatively placed in a row. It showed a good correlation with the Real-Q HBV (r(2)=0.9609) and the test results for the BBI panel were also well agreed to the assigned values (r(2)=0.9933). The performance of Abbott RealTime HBV Quantification kit was excellent; thus, it should be widely used in starting and monitoring of antiviral therapy in Korean patients with chronic hepatitis B.

Interleukin 6 inhibits HBV entry through NTCP down regulation.

PubMed

Bouezzedine, Fidaa; Fardel, Olivier; Gripon, Philippe

2015-07-01

Hepatitis B virus (HBV) infection is a major public health problem. Recently, the human liver bile acid transporter Na(+)/taurocholate cotransporting polypeptide (NTCP) has been identified as an HBV specific receptor. NTCP expression is known to be strongly regulated by IL-6. This study was aimed at characterizing the effect of IL-6 on HBV entry. HBV entry was inhibited by up to 90% when cells were pretreated with IL-6 as shown by a strong inhibition of long term HBsAg secretion. This effect was confirmed by showing a severe reduction of intracellular HBV cccDNA. In parallel, we observed a 98% decrease in NTCP mRNA steady state level and an 80% reduction in NTCP-mediated taurocholate uptake. IL-6-mediated inhibition of NTCP-mediated taurocholate uptake and viral entry exhibited similar dose-dependence and kinetics while restoration of NTCP expression suppressed the inhibitory effect of IL-6. NTCP-mediated HBV entry is therefore markedly inhibited by IL-6. Copyright © 2015 Elsevier Inc. All rights reserved.

Detection of HBV genome in the plasma and peripheral blood mononuclear cells of Iranian HBsAg negative patients with HIV infection: occult HBV infection.

PubMed

Tajik, Zahra; Bokharaei-Salim, Farah; Ghorbani, Saied; Keyvani, Hossein; Esghaei, Maryam; Monavari, Seyed Hamidreza; Ataei-Pirkooh, Angila; Garshasbi, Saba; Donyavi, Tahereh; Fakhim, Atousa

2018-06-01

The presence of hepatitis B virus (HBV) DNA in the absence of traceable hepatitis B surface antigen (HBsAg) in the plasma specimen of patients is defined as occult HBV infection (OBI). This study aimed to detect HBV-DNA in the plasma and peripheral blood mononuclear cells (PBMCs) of Iranian HBsAg negative patients with human immunodeficiency virus (HIV) infection. This cross-sectional study was conducted on 172 patients with HIV infection from September 2015 to August 2017. The patients were tested for serological parameters (HBsAg, HBcAb, HBeAg and HBeAb) against HBV infection. Moreover, they were tested for HBV viral load (using COBAS TaqMan 48 Kit, Roche, USA) in plasma and the presence of the HBV genome in PBMC specimens using real-time PCR. The mean age of the patients was 35.4 ± 13.4 years. Of the 172 studied patients, 109 (63.4%) were male. In this study, 151 (87.8%) patients were negative for HBsAg, 111 (64.5%) patients were negative for all HBV infection serological markers, 9 (5.2%) patients were only positive for HBsAg and 29 (16.9%) patients were only positive for HBcAb. Moreover, five (3.3%) patients with HBsAg negative had OBI (in the plasma sample of four patients and PBMC specimens of all five patients, HBV-DNA was detected). The present study revealed that 3.3% of the patients with HIV infection had occult HBV infection. Presumably, designing prospective studies to identify this infection in patients with HIV infection is informative and valuable.

Genomic Methylation Inhibits Expression of Hepatitis B Virus Envelope Protein in Transgenic Mice: A Non-Infectious Mouse Model to Study Silencing of HBV Surface Antigen Genes.

PubMed

Graumann, Franziska; Churin, Yuri; Tschuschner, Annette; Reifenberg, Kurt; Glebe, Dieter; Roderfeld, Martin; Roeb, Elke

2015-01-01

The Hepatitis B virus genome persists in the nucleus of virus infected hepatocytes where it serves as template for viral mRNA synthesis. Epigenetic modifications, including methylation of the CpG islands contribute to the regulation of viral gene expression. The present study investigates the effects of spontaneous age dependent loss of hepatitis B surface protein- (HBs) expression due to HBV-genome specific methylation as well as its proximate positive effects in HBs transgenic mice. Liver and serum of HBs transgenic mice aged 5-33 weeks were analyzed by Western blot, immunohistochemistry, serum analysis, PCR, and qRT-PCR. From the third month of age hepatic loss of HBs was observed in 20% of transgenic mice. The size of HBs-free area and the relative number of animals with these effects increased with age and struck about 55% of animals aged 33 weeks. Loss of HBs-expression was strongly correlated with amelioration of serum parameters ALT and AST. In addition lower HBs-expression went on with decreased ER-stress. The loss of surface protein expression started on transcriptional level and appeared to be regulated epigenetically by DNA methylation. The amount of the HBs-expression correlated negatively with methylation of HBV DNA in the mouse genome. Our data suggest that methylation of specific CpG sites controls gene expression even in HBs-transgenic mice with truncated HBV genome. More important, the loss of HBs expression and intracellular aggregation ameliorated cell stress and liver integrity. Thus, targeted modulation of HBs expression may offer new therapeutic approaches. Furthermore, HBs-transgenic mice depict a non-infectious mouse model to study one possible mechanism of HBs gene silencing by hypermethylation.

Evolutionary dynamics of HBV-D1 genotype epidemic in Turkey.

PubMed

Ciccozzi, Massimo; Ciccaglione, Anna Rita; Lo Presti, Alessandra; Equestre, Michele; Cella, Eleonora; Ebranati, Erika; Gabanelli, Elena; Villano, Umbertina; Bruni, Roberto; Yalcinkaya, Tulay; Tanzi, Elisabetta; Zehender, Gianguglielmo

2014-01-01

Hepatitis B virus (HBV), is the leading cause of liver diseases infecting an estimated 240 million persons worldwide. The HBV prevalence rates are variables between different countries, with an high level of endemicity in the south-eastern part of Europe. Seven main HBV-D subgenotypes have been described until now (D1-D7). Turkey, seems to have played an important role in the penetration of HBV-D1 in the Mediterranean area. The importance of Turkey in the European epidemiology of HBV is also suggested by the observation that the highest spread of HBV infection in the Continent are reported in Turkey with Romania, Bulgaria, Greece, Albania and some southern regions of Italy. In this paper the molecular epidemiology and the epidemiological history of HBV-D in Turkey was studied, by characterizing 34 new Turkish isolates and performing a phylogeographic reconstruction. By using a phylodynamic and phylogeographic Bayesian approach, the analysis suggested that HBV-D1 originated in Turkey about in the early 1940s. The large prevalence of D1 in comparison to the other subgenotypes in Turkey confirms the importance of this Country as epidemiological reservoir of HBV-D1 dispersion. The phylogeny suggests that after each initial introduction of the virus in a specific population, separate transmission clusters have been evolving along independent phylogenetic lineages. Better characterization and continuous monitoring of such groups are going to be crucial to understand in detail the epidemiology of HBV-D1 subgenotype in Turkey and to assess the efficacy of prevention, vaccination and therapy in controlling the epidemic. © 2013 Wiley Periodicals, Inc.

Roadmap to control HBV and HDV epidemics in China

DOE PAGES

Goyal, Ashish; Murray, John M.

2017-04-23

Hepatitis B virus (HBV) is endemic in China. Almost 10% of HBV infected individuals are also infected with hepatitis D virus (HDV) which has a 5–10 times higher mortality rate than HBV mono-infection. The aim of this manuscript is to devise strategies that can not only control HBV infections but also HDV infections in China under the current health care budget in an optimal manner. Furthermore, using a mathematical model, an annual budget of 10 billion dollars was optimally allocated among five interventions namely, testing and HBV adult vaccination, treatment for mono-infected and dually-infected individuals, second line treatment for HBVmore » mono-infections, and awareness programs. As a result, we determine that the optimal strategy is to test and treat both infections as early as possible while applying awareness programs at full intensity. Under this strategy, an additional 19.8 million HBV, 1.9 million HDV infections and 0.25 million lives will be saved over the next 10 years at a cost-savings of 79 billion dollars than performing no intervention. Introduction of second line treatment does not add a significant economic burden yet prevents 1.4 million new HBV infections and 15,000 new HDV infections. In conclusion, test and treatment programs are highly efficient in reducing HBV and HDV prevalence in the population. Under the current health budget in China, not only test and treat programs but awareness programs and second line treatment can also be implemented that minimizes prevalence and mortality, and maximizes economic benefits.« less

Roadmap to control HBV and HDV epidemics in China

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goyal, Ashish; Murray, John M.

Hepatitis B virus (HBV) is endemic in China. Almost 10% of HBV infected individuals are also infected with hepatitis D virus (HDV) which has a 5–10 times higher mortality rate than HBV mono-infection. The aim of this manuscript is to devise strategies that can not only control HBV infections but also HDV infections in China under the current health care budget in an optimal manner. Furthermore, using a mathematical model, an annual budget of 10 billion dollars was optimally allocated among five interventions namely, testing and HBV adult vaccination, treatment for mono-infected and dually-infected individuals, second line treatment for HBVmore » mono-infections, and awareness programs. As a result, we determine that the optimal strategy is to test and treat both infections as early as possible while applying awareness programs at full intensity. Under this strategy, an additional 19.8 million HBV, 1.9 million HDV infections and 0.25 million lives will be saved over the next 10 years at a cost-savings of 79 billion dollars than performing no intervention. Introduction of second line treatment does not add a significant economic burden yet prevents 1.4 million new HBV infections and 15,000 new HDV infections. In conclusion, test and treatment programs are highly efficient in reducing HBV and HDV prevalence in the population. Under the current health budget in China, not only test and treat programs but awareness programs and second line treatment can also be implemented that minimizes prevalence and mortality, and maximizes economic benefits.« less

Evolutionary dynamics of HBV-D7 subgenotype in Tunisia.

PubMed

Ciccozzi, Massimo; Chaouch, Houda; Lo Presti, Alessandra; Taffon, Stefania; Villano, Umbertina; Equestre, Michele; Bruni, Roberto; Marcantonio, Cinzia; Tritarelli, Elena; Cella, Eleonora; Blasi, Aletheia; Aouni, Mahjoub; Letaief, Amel; Ciccaglione, Anna Rita

2017-03-01

Hepatitis B virus (HBV) is the main cause of diseases liver related infecting more than 200 milion persons worldwide. HBV infection shows high level of prevalence in South-East Europe and in Mediterranean basin. In Tunisia, a country with an intermediate level endemicity, HbsAg prevalence ranges from 2 to 5%. Most of the HBV isolates from Tunisia were classified as subgenotype D7 whose circulation is restricted to a specific area of North Africa including Maghreb region. In this paper, the phylogeny of HBV-D7 isolated from 38 Tunisian patients was investigated by analyzing the S gene region of HBV. A Bayesian coalescent-based framework was used to estimate the origin of the HBV-D7 in the country. The Tunisian D7 isolates were found to share a common ancestor whose origin was traced back to 1958. Population dynamics indicated that HBV-D7 epidemic in Tunisia grew exponentially from 1960s to 1990s. After that, the curve reached a plateau around the years 2000 likely due to the implementation of the infant vaccination program in 1996. Epidemiological data suggested that the exponential growth phase was likely sustained by intra-familial transmission events occurring during infancy. Further characterization of HBV-D7 isolates should be performed to evaluate, in the post-vaccination era, the emergence of new transmission routes, and to monitor the efficacy of the vaccination program. J. Med. Virol. 89:469-475, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Inhibition of Hepatitis C Virus Production by Aptamers against the Core Protein

PubMed Central

Shi, Shali; Yu, Xiaoyan; Gao, Yimin; Xue, Binbin; Wu, Xinjiao; Wang, Xiaohong; Yang, Darong

2014-01-01

Hepatitis C virus (HCV) core protein is essential for virus assembly. HCV core protein was expressed and purified. Aptamers against core protein were raised through the selective evolution of ligands by the exponential enrichment approach. Detection of HCV infection by core aptamers and the antiviral activities of aptamers were characterized. The mechanism of their anti-HCV activity was determined. The data showed that selected aptamers against core specifically recognize the recombinant core protein but also can detect serum samples from hepatitis C patients. Aptamers have no effect on HCV RNA replication in the infectious cell culture system. However, the aptamers inhibit the production of infectious virus particles. Beta interferon (IFN-β) and interferon-stimulated genes (ISGs) are not induced in virally infected hepatocytes by aptamers. Domains I and II of core protein are involved in the inhibition of infectious virus production by the aptamers. V31A within core is the major resistance mutation identified. Further study shows that the aptamers disrupt the localization of core with lipid droplets and NS5A and perturb the association of core protein with viral RNA. The data suggest that aptamers against HCV core protein inhibit infectious virus production by disrupting the localization of core with lipid droplets and NS5A and preventing the association of core protein with viral RNA. The aptamers for core protein may be used to understand the mechanisms of virus assembly. Core-specific aptamers may hold promise for development as early diagnostic reagents and potential therapeutic agents for chronic hepatitis C. PMID:24307579

HBV X Protein induces overexpression of HERV-W env through NF-κB in HepG2 cells.

PubMed

Liu, Cong; Liu, Lijuan; Wang, Xiuling; Liu, Youyi; Wang, Miao; Zhu, Fan

2017-12-01

Human endogenous retrovirus W family (HERV-W) envelope (env) at chromosome 7 is highly expressed in the placenta and possesses fusogenic activity in trophoblast development. HERV-W env has been found to be overexpressed in some cancers and immune diseases. Viral transactivators can induce the overexpression of HERV-W env in human cell lines. Hepatitis B virus X protein (HBx) is believed to be a multifunctional oncogenic protein. Here, we reported that HBx could increase the promoter activity of HERV-W env and upregulate the mRNA levels of non-spliced and spliced HERV-W env and also its protein in human hepatoma HepG2 cells. Interestingly, we found that the inhibition of nuclear factor κB (NF-κB) using shRNA targeting NF-κB/p65 or PDTC (an inhibitor of NF-κB) could attenuate the upregulation of HERV-W env induced by HBx. These suggested that HBx might upregulate the expression of HERV-W env through NF-κB in HepG2 cells. This study might provide a new insight in HBV-associated liver diseases including HCC.

[Characteristics of HBV transmission in families with HBsAg-positive fathers and familial clustering of HBV infection].

PubMed

Yang, Y; Jin, L; He, Y L; Liu, J F; Wang, J; Wang, K; Ma, X H; Li, Q; Feng, Y L; Yan, Z; Yi, R T; Chen, T Y; Zhao, Y R

2016-04-01

To investigate the characteristics of hepatitis B virus (HBV) transmission among family members in families with familial clustering of HBV infection and poor outcomes, as well as the prevalence and distribution characteristics of HBsAg in offspring with different parental HBsAg status. The general information of each member in families with poor outcomes were collected from 2007 to 2010, and serological test was performed to analyze the prevalence and distribution of HBsAg in family members. The chi-square test or Fisher's exact test was used to analyze and compare the sex of offspring and the prevalence of HBsAg in them in 266 nuclear families with different paternal and maternal HBsAg status. The positive rates of HBsAg in parents, siblings, children, and spouses of the probands were 20%, 88.2%, 76.8%, and 9.5%, respectively. The nuclear families with HBsAg-positive fathers and HBsAg-negative mothers had a significantly increased proportion of male offspring (male/female ratio = 2.02) compared with those with HBsAg-positive mothers and HBsAg-negative fathers (1.22) or those with HBsAg-negative fathers and mothers (0.96). In addition, in the nuclear families with HBsAg-positive fathers and HBsAg-negative mothers, the male offspring had a significantly higher HBsAg positive rate than female offspring (37.4% vs 13.8%), while in those with HBsAg-positive mothers and HBsAg-negative fathers or those with HBsAg-negative fathers and mothers, HBsAg positive rate showed no significant difference between male and female offspring. In families with familial clustering of HBV infection and poor outcomes, mother-to-child transmission is still the major route of HBV transmission, but father-to-child transmission also plays a role in HBV transmission in this special population. Positive HBsAg in fathers is associated with the increased proportion of male offspring, and father-to-son transmission of HBV is higher than father-to-daughter transmission.

Analytical performance of the Hologic Aptima HBV Quant Assay and the COBAS Ampliprep/COBAS TaqMan HBV test v2.0 for the quantification of HBV DNA in plasma samples.

PubMed

Schønning, Kristian; Johansen, Kim; Nielsen, Lone Gilmor; Weis, Nina; Westh, Henrik

2018-07-01

Quantification of HBV DNA is used for initiating and monitoring antiviral treatment. Analytical test performance consequently impacts treatment decisions. To compare the analytical performance of the Aptima HBV Quant Assay (Aptima) and the COBAS Ampliprep/COBAS TaqMan HBV Test v2.0 (CAPCTMv2) for the quantification of HBV DNA in plasma samples. The performance of the two tests was compared on 129 prospective plasma samples, and on 63 archived plasma samples of which 53 were genotyped. Linearity of the two assays was assessed on dilutions series of three clinical samples (Genotype B, C, and D). Bland-Altman analysis of 120 clinical samples, which quantified in both tests, showed an average quantification bias (Aptima - CAPCTMv2) of -0.19 Log IU/mL (SD: 0.33 Log IU/mL). A single sample quantified more than three standard deviations higher in Aptima than in CAPCTMv2. Only minor differences were observed between genotype A (N = 4; average difference -0.01 Log IU/mL), B (N = 8; -0.13 Log IU/mL), C (N = 8; -0.31 Log IU/mL), D (N = 25; -0.22 Log IU/mL), and E (N = 7; -0.03 Log IU/mL). Deming regression showed that the two tests were excellently correlated (slope of the regression line 1.03; 95% CI: 0.998-1.068). Linearity of the tests was evaluated on dilution series and showed an excellent correlation of the two tests. Both tests were precise with %CV less than 3% for HBV DNA ≥3 Log IU/mL. The Aptima and CAPCTMv2 tests are highly correlated, and both tests are useful for monitoring patients chronically infected with HBV. Copyright © 2018 Elsevier B.V. All rights reserved.

Presence of anti-HBc is associated to high rates of HBV resolved infection and low threshold for Occult HBV Infection in HIV patients with negative HBsAg in Chile.

PubMed

Vargas, Jose Ignacio; Jensen, Daniela; Sarmiento, Valeska; Peirano, Felipe; Acuña, Pedro; Fuster, Felipe; Soto, Sabrina; Ahumada, Rodrigo; Huilcaman, Marco; Bruna, Mario; Jensen, Werner; Fuster, Francisco

2016-04-01

HBV-HIV coinfection is prevalent. Frequently, anti-HBc is the only serological marker of HBV, which can be indicative of HBV resolved infection, when found together with anti-HBs reactivity; or present as "isolated anti-HBc," related to HBV occult infection with presence of detectable DNA HBV, more prevalent in HIV-positive individuals. Regional data about this condition are scarce. Anti-HBc rapid test has been used as screening, but its performance has not been described in HIV-positive patients. The aim of this study was determine prevalence of anti-HBc in HIV-positive patients, serological pattern of HBV resolved infection and isolated anti-HBc, evaluating presence of HBV occult infection. Assess anti-HBc rapid test compared to ECLIA. Methods included measurement of anti-HBc and anti-HBs in HIV-positive patients with negative HBsAg. Serum HBV DNA quantification and HBV booster vaccination to "isolated anti-HBc" individuals. Detection of anti-HBc by rapid test and ECLIA. In 192 patients, prevalence of anti-HBc was 42.7% (82/192); associated to male gender, drug use, men-sex-men, positive-VDRL, and longer time HIV diagnosis. 34.4% (66/192) had presence of anti-HBs, mean titers of 637 ui/ml. Isolated anti-HBc in 8.3% (16/192), associated to detectable HIV viral load and no-use of HAART; in them, HBV DNA was undetectable, and 60% responded to HBV vaccination booster. Anti-HBc rapid test showed low sensibility (32.9%) compared to ECLIA. These results show that prevalence of anti-HBc in HIV-positive individuals is high, in most cases accompanied with anti-HBs as HBV resolved infection. Low prevalence of "isolated anti-HBc," with undetectable HBV DNA, and most had anamnestic response to HBV vaccination; suggest low possibility of occult HBV infection. Anti-HBc rapid test cannot be recommended as screening method for anti-HBc. © 2015 Wiley Periodicals, Inc.

RNA chaperoning and intrinsic disorder in the core proteins of Flaviviridae.

PubMed

Ivanyi-Nagy, Roland; Lavergne, Jean-Pierre; Gabus, Caroline; Ficheux, Damien; Darlix, Jean-Luc

2008-02-01

RNA chaperone proteins are essential partners of RNA in living organisms and viruses. They are thought to assist in the correct folding and structural rearrangements of RNA molecules by resolving misfolded RNA species in an ATP-independent manner. RNA chaperoning is probably an entropy-driven process, mediated by the coupled binding and folding of intrinsically disordered protein regions and the kinetically trapped RNA. Previously, we have shown that the core protein of hepatitis C virus (HCV) is a potent RNA chaperone that can drive profound structural modifications of HCV RNA in vitro. We now examined the RNA chaperone activity and the disordered nature of core proteins from different Flaviviridae genera, namely that of HCV, GBV-B (GB virus B), WNV (West Nile virus) and BVDV (bovine viral diarrhoea virus). Despite low-sequence similarities, all four proteins demonstrated general nucleic acid annealing and RNA chaperone activities. Furthermore, heat resistance of core proteins, as well as far-UV circular dichroism spectroscopy suggested that a well-defined 3D protein structure is not necessary for core-induced RNA structural rearrangements. These data provide evidence that RNA chaperoning-possibly mediated by intrinsically disordered protein segments-is conserved in Flaviviridae core proteins. Thus, besides nucleocapsid formation, core proteins may function in RNA structural rearrangements taking place during virus replication.

Humic acid inhibits HBV-induced autophagosome formation and induces apoptosis in HBV-transfected Hep G2 cells

PubMed Central

Pant, Kishor; Yadav, Ajay K.; Gupta, Parul; Rathore, Abhishek Singh; Nayak, Baibaswata; Venugopal, Senthil K.

2016-01-01

Hepatitis B Virus (HBV) utilizes several mechanisms to survive in the host cells and one of the main pathways being autophagosome formation. Humic acid (HA), one of the major components of Mineral pitch, is an Ayurvedic medicinal food, commonly used by the people of the Himalayan regions of Nepal and India for various body ailments. We hypothesized that HA could induce cell death and inhibit HBV-induced autophagy in hepatic cells. Incubation of Hep G2.2.1.5 cells (HepG2 cells stably expressing HBV) with HA (100 μM) inhibited both cell proliferation and autophagosome formation significantly, while apoptosis induction was enhanced. Western blot results showed that HA incubation resulted in decreased levels of beclin-1, SIRT-1 and c-myc, while caspase-3 and β-catenin expression were up-regulated. Western blot results showed that HA significantly inhibited the expression of HBx (3-fold with 50 μM and 5-fold with 100 μM) compared to control cells. When HA was incubated with HBx-transfected Hep G2 cells, HBx-induced autophagosome formation and beclin-1 levels were decreased. These data showed that HA induced apoptosis and inhibited HBV-induced autophagosome formation and proliferation in hepatoma cells. PMID:27708347

Trafficking of Hepatitis C Virus Core Protein during Virus Particle Assembly

PubMed Central

Counihan, Natalie A.; Rawlinson, Stephen M.; Lindenbach, Brett D.

2011-01-01

Hepatitis C virus (HCV) core protein is directed to the surface of lipid droplets (LD), a step that is essential for infectious virus production. However, the process by which core is recruited from LD into nascent virus particles is not well understood. To investigate the kinetics of core trafficking, we developed methods to image functional core protein in live, virus-producing cells. During the peak of virus assembly, core formed polarized caps on large, immotile LDs, adjacent to putative sites of assembly. In addition, LD-independent, motile puncta of core were found to traffic along microtubules. Importantly, core was recruited from LDs into these puncta, and interaction between the viral NS2 and NS3-4A proteins was essential for this recruitment process. These data reveal new aspects of core trafficking and identify a novel role for viral nonstructural proteins in virus particle assembly. PMID:22028650

Clinical evaluation of the COBAS Amplicor HBV monitor test for measuring serum HBV DNA and comparison with the Quantiplex branched DNA signal amplification assay in Taiwan.

PubMed

Dai, C-Y; Yu, M-L; Chen, S-C; Lin, Z-Y; Hsieh, M-Y; Wang, L-Y; Tsai, J-F; Chuang, W-L; Chang, W-Y

2004-02-01

To evaluate the performance characteristics and clinical usefulness of the COBAS Amplicor HBV monitor (COBAS-AM) test in Taiwan and to examine its correlation with the Quantiplex branched DNA signal amplification (bDNA) assay for measuring serum hepatitis B virus (HBV) DNA concentrations. HBV DNA was measured by the COBAS-AM test in 149 sera from chronic HBV infected patients that had previously been analysed by the bDNA assay. The COBAS-AM test showed good reproducibility, with acceptable intra-assay and interassay coefficients of variation (1.6% and 0.9%, respectively) and good linearity (r2=0.98). The overall sensitivity of the COBAS-AM test was significantly higher than that of the bDNA assay (95.3% v 83.2%): 69.6% of samples with HBV DNA below the detection limit of the bDNA assay could be measured by the COBAS-AM test. There was a significant correlation between the results of the two assays (r=0.901; p<0.0001). On average, the results derived from the COBAS-AM test were 0.55 log lower than those of the bDNA assay. HBV DNA concentrations were significantly higher among HBV e antigen (HBeAg) positive patients than negative ones, and higher among patients with abnormal alanine aminotransferase (ALT) concentrations than those with normal ALT concentrations (p=0.0003). The COBAS-AM assay, more sensitive in HBeAg negative samples than the bDNA assay, can effectively measure HBV DNA concentrations in Taiwanese patients. HBV DNA values measured by the COBAS-AM test and bDNA assay correlate significantly.

Genotyping of acute HBV isolates from England, 1997-2001.

PubMed

Sloan, Richard D; Strang, Angela L; Ramsay, Mary E; Teo, Chong-Gee

2009-02-01

Increasing data shows the relevance of HBV genotypes in the outcome of infection. Most studies investigating the relationship between the genotypic characteristics of hepatitis B virus (HBV) and the clinical or epidemiological aspects of HBV infection originate from studies of patients with chronic rather than acute hepatitis B. To study a convenience sample representing ca. 5% of reported acute hepatitis B in England between 1997 and 2001 to investigate the distribution of HBV genotypes and specific HBV variants with epidemiological risk factors, thereby providing baseline data for ongoing surveillance. From 160 serum samples, PCR was carried out to amplify the first 600 bases of the HBV S gene. Amplicons were sequenced and subjected to phylogenetic analysis and risk factor analysis. Fifty-seven percent of the study samples carried HBV belonging to subtype A2, 13% to subtype D2, and the rest to genotype E (8%) and subtypes C2 and D3 (each 6%), D1 and D4 (each 3%) and B4 (1%). One particular A2 isolate was dominant, accounting for 23% of the total sample set. Drug use and homosexual transmission were equally implicated as risks within genotype A2. No mutations associated with vaccine escape or resistance to antiviral therapy were identified. Immigration and travel likely shape the observed genotype distribution and consequent prevalence of genotypes other than A2 or D in this population. Data suggests no genetic separation of parenteral and sexually transmitted virus. These data demonstrate the value in pursuing more extensive and recent surveillance.

RNA chaperoning and intrinsic disorder in the core proteins of Flaviviridae

PubMed Central

Ivanyi-Nagy, Roland; Lavergne, Jean-Pierre; Gabus, Caroline; Ficheux, Damien; Darlix, Jean-Luc

2008-01-01

RNA chaperone proteins are essential partners of RNA in living organisms and viruses. They are thought to assist in the correct folding and structural rearrangements of RNA molecules by resolving misfolded RNA species in an ATP-independent manner. RNA chaperoning is probably an entropy-driven process, mediated by the coupled binding and folding of intrinsically disordered protein regions and the kinetically trapped RNA. Previously, we have shown that the core protein of hepatitis C virus (HCV) is a potent RNA chaperone that can drive profound structural modifications of HCV RNA in vitro. We now examined the RNA chaperone activity and the disordered nature of core proteins from different Flaviviridae genera, namely that of HCV, GBV-B (GB virus B), WNV (West Nile virus) and BVDV (bovine viral diarrhoea virus). Despite low-sequence similarities, all four proteins demonstrated general nucleic acid annealing and RNA chaperone activities. Furthermore, heat resistance of core proteins, as well as far-UV circular dichroism spectroscopy suggested that a well-defined 3D protein structure is not necessary for core-induced RNA structural rearrangements. These data provide evidence that RNA chaperoning—possibly mediated by intrinsically disordered protein segments—is conserved in Flaviviridae core proteins. Thus, besides nucleocapsid formation, core proteins may function in RNA structural rearrangements taking place during virus replication. PMID:18033802

Association of single nucleotide polymorphisms in VDR and DBP genes with HBV-related hepatocellular carcinoma risk in a Chinese population.

PubMed

Peng, Qiliu; Yang, Shi; Lao, Xianjun; Li, Ruolin; Chen, Zhiping; Wang, Jian; Qin, Xue; Li, Shan

2014-01-01

Polymorphisms of genes encoding components of the vitamin D pathway including vitamin D receptor (VDR) and vitamin D binding protein (DBP) have been widely investigated because of the complex role played by vitamin D in cancer tumorogenesis. In this study, we investigated the association between VDR and DBP gene polymorphisms and HBV-related HCC risk in a Chinese population. Study subjects were divided into three groups: 184 HBV patients with HCC, 296 HBV patients without HCC, and 180 healthy controls. The VDR rs2228570, and rs3782905 and the DBP rs7041 polymorphisms were genotyped using PCR-RFLP and the VDR rs11568820 polymorphism was genotyped by PCR-SSP, respectively. DNA sequencing was performed to validate the genotype results. We found that there were significant differences in the genotype and allele frequencies of the VDR rs2228570 and DBP rs7041 polymorphisms between HBV patients with HCC and healthy controls. The rs2228570 T allele was associated with a significant increased HBV-related HCC risk as compared with the C allele. The rs2228570 TT and TT/TC genotypes were correlated with a significant increased HBV-related HCC risk when compared with the wild-type CC homozygote. Similarly, the rs7041 G allele was associated with a significant increased HBV-related HCC risk as compared with the T allele. The rs7041 GG and GG/TG genotypes were correlated with a significant increased HBV-related HCC risk when compared with the wild-type TT homozygote. However, we did not observe any significant effect of VDR rs11568820, and rs3782905 polymorphisms on HBV-related HCC risk in this population. In haplotype analysis, we also did not find any significant differences in haplotype frequencies of the VDR gene between HBV patients with HCC and the healthy controls. We conclude that the VDR rs2228570 and DBP rs7041 polymorphisms may contribute to increased susceptibility to HBV-related HCC in the Chinese population. Due to the marginal significance, further large and well

Characterization of Hepatitis C Virus Core Protein Multimerization and Membrane Envelopment: Revelation of a Cascade of Core-Membrane Interactions ▿

PubMed Central

Ai, Li-Shuang; Lee, Yu-Wen; Chen, Steve S.-L.

2009-01-01

The molecular basis underlying hepatitis C virus (HCV) core protein maturation and morphogenesis remains elusive. We characterized the concerted events associated with core protein multimerization and interaction with membranes. Analyses of core proteins expressed from a subgenomic system showed that the signal sequence located between the core and envelope glycoprotein E1 is critical for core association with endoplasmic reticula (ER)/late endosomes and the core's envelopment by membranes, which was judged by the core's acquisition of resistance to proteinase K digestion. Despite exerting an inhibitory effect on the core's association with membranes, (Z-LL)2-ketone, a specific inhibitor of signal peptide peptidase (SPP), did not affect core multimeric complex formation, suggesting that oligomeric core complex formation proceeds prior to or upon core attachment to membranes. Protease-resistant core complexes that contained both innate and processed proteins were detected in the presence of (Z-LL)2-ketone, implying that core envelopment occurs after intramembrane cleavage. Mutations of the core that prevent signal peptide cleavage or coexpression with an SPP loss-of-function D219A mutant decreased the core's envelopment, demonstrating that SPP-mediated cleavage is required for core envelopment. Analyses of core mutants with a deletion in domain I revealed that this domain contains sequences crucial for core envelopment. The core proteins expressed by infectious JFH1 and Jc1 RNAs in Huh7 cells also assembled into a multimeric complex, associated with ER/late-endosomal membranes, and were enveloped by membranes. Treatment with (Z-LL)2-ketone or coexpression with D219A mutant SPP interfered with both core envelopment and infectious HCV production, indicating a critical role of core envelopment in HCV morphogenesis. The results provide mechanistic insights into the sequential and coordinated processes during the association of the HCV core protein with membranes in the

Protein complex prediction for large protein protein interaction networks with the Core&Peel method.

PubMed

Pellegrini, Marco; Baglioni, Miriam; Geraci, Filippo

2016-11-08

Biological networks play an increasingly important role in the exploration of functional modularity and cellular organization at a systemic level. Quite often the first tools used to analyze these networks are clustering algorithms. We concentrate here on the specific task of predicting protein complexes (PC) in large protein-protein interaction networks (PPIN). Currently, many state-of-the-art algorithms work well for networks of small or moderate size. However, their performance on much larger networks, which are becoming increasingly common in modern proteome-wise studies, needs to be re-assessed. We present a new fast algorithm for clustering large sparse networks: Core&Peel, which runs essentially in time and storage O(a(G)m+n) for a network G of n nodes and m arcs, where a(G) is the arboricity of G (which is roughly proportional to the maximum average degree of any induced subgraph in G). We evaluated Core&Peel on five PPI networks of large size and one of medium size from both yeast and homo sapiens, comparing its performance against those of ten state-of-the-art methods. We demonstrate that Core&Peel consistently outperforms the ten competitors in its ability to identify known protein complexes and in the functional coherence of its predictions. Our method is remarkably robust, being quite insensible to the injection of random interactions. Core&Peel is also empirically efficient attaining the second best running time over large networks among the tested algorithms. Our algorithm Core&Peel pushes forward the state-of the-art in PPIN clustering providing an algorithmic solution with polynomial running time that attains experimentally demonstrable good output quality and speed on challenging large real networks.

Expression and Purification of a Novel Computationally Designed Antigen for Simultaneously Detection of HTLV-1 and HBV Antibodies.

PubMed

Heydari Zarnagh, Hafez; Ravanshad, Mehrdad; Pourfatollah, Ali Akbar; Rasaee, Mohammad Javad

2015-04-01

Computational tools are reliable alternatives to laborious work in chimeric protein design. In this study, a chimeric antigen was designed using computational techniques for simultaneous detection of anti-HTLV-I and anti-HBV in infected sera. Databases were searched for amino acid sequences of HBV/HLV-I diagnostic antigens. The immunodominant fragments were selected based on propensity scales. The diagnostic antigen was designed using these fragments. Secondary and tertiary structures were predicted and the B-cell epitopes were mapped on the surface of built model. The synthetic DNA coding antigen was sub-cloned into pGS21a expression vector. SDS-PAGE analysis showed that glutathione fused antigen was highly expressed in E. coli BL21 (DE3) cells. The recombinant antigen was purified by nickel affinity chromatography. ELISA results showed that soluble antigen could specifically react with the HTLV-I and HBV infected sera. This specific antigen could be used as suitable agent for antibody-antigen based screening tests and can help clinicians in order to perform quick and precise screening of the HBV and HTLV-I infections.

HBV-Associated Acute Liver Failure After Immunosuppression and Risk of Death.

PubMed

Karvellas, Constantine J; Cardoso, Filipe S; Gottfried, Michelle; Reddy, K Rajender; Hanje, A James; Ganger, Daniel; Lee, William M

2017-01-01

Acute liver failure (ALF) caused by hepatitis B virus (HBV) infection can occur after immunosuppressive treatment and be fatal, although it might be preventable. We aimed to characterize the causes, clinical course, and short-term outcomes of HBV-associated ALF after immune-suppressive therapy, compared with patients with HBV-associated ALF without immunosuppression (control subjects). We performed a retrospective multicenter study of 156 consecutive patients diagnosed with HBV-associated ALF (22 with a solid or blood malignancy) enrolled in the Acute Liver Failure Study Group registry from January 1998 through April 2015. We collected data on results of serologic and hepatic biochemistry analyses, grade of hepatic encephalopathy, Model for End-Stage Liver Disease score, and King's College criteria. We also collected data on clinical features, medical therapies, and complications in the first 7 days following study enrollment. Logistic regression was used to identify factors associated with transplant-free survival at 21 days in HBV-associated ALF (the primary outcome). Among patients with HBV-associated ALF, 28 cases (18%) occurred after immunosuppressive therapy (15 patients received systemic corticosteroids and 21 received chemotherapy); and 128 cases did not (control subjects, 82%). Significantly greater proportions of patients with HBV-associated ALF after immunosuppression were nonwhite persons, and had anemia or thrombocytopenia than controls (P < .02 for all). The serologic profile of HBV infection, severity of liver failure (based on MELD score), and complications (hepatic encephalopathy or need for mechanical ventilation, vasopressors, or renal replacement therapy) were similar between the groups (P > .17 for all). Factors associated with 21 day transplant-free survival were increased MELD score (odds ratio ∼OR, 0.894 (95% confidence interval 0.842-0.949 per increment), requirement for mechanical ventilation (OR 0.111(0.041-0.300), and immunosuppressive

In silico analysis and experimental validation of azelastine hydrochloride (N4) targeting sodium taurocholate co-transporting polypeptide (NTCP) in HBV therapy.

PubMed

Fu, L-L; Liu, J; Chen, Y; Wang, F-T; Wen, X; Liu, H-Q; Wang, M-Y; Ouyang, L; Huang, J; Bao, J-K; Wei, Y-Q

2014-08-01

The aim of this study was to explore sodium taurocholate co-transporting polypeptide (NTCP) exerting its function with hepatitis B virus (HBV) and its targeted candidate compounds, in HBV therapy. Identification of NTCP as a novel HBV target for screening candidate small molecules, was used by phylogenetic analysis, network construction, molecular modelling, molecular docking and molecular dynamics (MD) simulation. In vitro virological examination, q-PCR, western blotting and cytotoxicity studies were used for validating efficacy of the candidate compound. We used the phylogenetic analysis of NTCP and constructed its protein-protein network. Also, we screened compounds from Drugbank and ZINC, among which five were validated for their authentication in HepG 2.2.15 cells. Then, we selected compound N4 (azelastine hydrochloride) as the most potent of them. This showed good inhibitory activity against HBsAg (IC50 = 7.5 μm) and HBeAg (IC50 = 3.7 μm), as well as high SI value (SI = 4.68). Further MD simulation results supported good interaction between compound N4 and NTCP. In silico analysis and experimental validation together demonstrated that compound N4 can target NTCP in HepG2.2.15 cells, which may shed light on exploring it as a potential anti-HBV drug. © 2014 John Wiley & Sons Ltd.

Steric interactions determine side-chain conformations in protein cores.

PubMed

Caballero, D; Virrueta, A; O'Hern, C S; Regan, L

2016-09-01

We investigate the role of steric interactions in defining side-chain conformations in protein cores. Previously, we explored the strengths and limitations of hard-sphere dipeptide models in defining sterically allowed side-chain conformations and recapitulating key features of the side-chain dihedral angle distributions observed in high-resolution protein structures. Here, we show that modeling residues in the context of a particular protein environment, with both intra- and inter-residue steric interactions, is sufficient to specify which of the allowed side-chain conformations is adopted. This model predicts 97% of the side-chain conformations of Leu, Ile, Val, Phe, Tyr, Trp and Thr core residues to within 20°. Although the hard-sphere dipeptide model predicts the observed side-chain dihedral angle distributions for both Thr and Ser, the model including the protein environment predicts side-chain conformations to within 20° for only 60% of core Ser residues. Thus, this approach can identify the amino acids for which hard-sphere interactions alone are sufficient and those for which additional interactions are necessary to accurately predict side-chain conformations in protein cores. We also show that our approach can predict alternate side-chain conformations of core residues, which are supported by the observed electron density. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Associated factors for recommending HBV vaccination to children among Georgian health care workers.

PubMed

Butsashvili, Maia; Kamkamidze, George; Topuridze, Marina; Morse, Dale; Triner, Wayne; DeHovitz, Jack; Nelson, Kenrad; McNutt, Louise-Anne

2012-12-20

Most cases of hepatitis B virus (HBV) infection and subsequent liver diseases can be prevented with universal newborn HBV vaccination. The attitudes of health care workers about HBV vaccination and their willingness to recommend vaccine have been shown to impact HBV vaccination coverage and the prevention of vertical transmission of HBV. The purpose of this study was to ascertain the factors associated with health care worker recommendations regarding newborn HBV vaccination. A cross-sectional study of prevalence and awareness of hepatitis B and hepatitis B vaccine was conducted among randomly selected physicians and nurses employed in seven hospitals in Georgia in 2006 and 2007. Self-administered questionnaires included a module on recommendations for HBV, HCV and HIV. Of the 1328 participants included in this analysis, 36% reported recommending against hepatitis B vaccination for children, including 33% of paediatricians. Among the 70.6% who provided a reason for not recommending HBV vaccine, the most common concern was an adverse vaccine event. Unvaccinated physicians and nurses were more likely to recommend against HBV vaccine (40.4% vs 11.4%, PR 3.54; 95% CI: 2.38, 5.29). Additionally, health care worker age was inversely correlated with recommendations for HBV vaccine with older workers less likely to recommend it. Vaccinating health care workers against HBV may provide a dual benefit by boosting occupational safety as well as strengthening universal coverage programs for newborns.

Interaction of structural core protein of Classical Swine Fever Virus with endoplasmic reticulum-associated degradation pathway protein OS9

USDA-ARS?s Scientific Manuscript database

Classical Swine Fever Virus (CSFV) Core protein is involved in virus RNA protection, transcription regulation and virus virulence. To discover additional Core protein functions a yeast two-hybrid system was used to identify host proteins that interact with Core. Among the identified host proteins, t...

Precore and core promoter mutations of hepatitis B virus and hepatitis B e antigen-negative chronic hepatitis B in Korea.

PubMed

Yoo, Byung Chul; Park, Joong-Won; Kim, Hyung Joon; Lee, Dong Ho; Cha, Young Ju; Park, Sill Moo

2003-01-01

The aims of this study were to determine the frequency of precore/core promoter mutations and hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (e-CHB) in Korea. Patients with chronic hepatitis B virus (HBV) infection were tested for HBeAg, anti-HBe, liver profile and HBV-DNA by a branched DNA (bDNA) assay. Serum HBV-DNA was amplified by a polymerase chain reaction and the precore/core promoter sequence was determined. Among the 413 consecutive HBeAg-negative patients, 19.6% were bDNA-positive. Evidence of liver disease was found in 90.1% of bDNA-positive and 41.7% of bDNA-negative patients. Overall, 17.7% of HBeAg-negative patients had e-CHB. Precore mutation (A1896) was detected in 93.7% of HBeAg-negative bDNA-positive and 93.9% of HBeAg-negative bDNA-negative patients. In 59 HBeAg-positive patients, 78% had wild-type and 22% had a mixture of wild-type and A1896 mutant. Core promoter TA mutation was detected in 89.9% of HBeAg-negative bDNA-positive patients, 89.8% of HBeAg-negative bDNA-negative patients, and 74.6% of HBeAg-positive patients. No correlation was found between the presence of precore/core promoter mutations and HBV-DNA levels or disease severity. In Korean patients infected with HBV genotype C, precore mutation occurred almost invariably along with HBeAg seroconversion and core promoter TA mutation was frequent irrespective of viral replication levels or disease severity.

Hepatitis C virus core protein potentiates proangiogenic activity of hepatocellular carcinoma cells.

PubMed

Shao, Yu-Yun; Hsieh, Min-Shu; Wang, Han-Yu; Li, Yong-Shi; Lin, Hang; Hsu, Hung-Wei; Huang, Chung-Yi; Hsu, Chih-Hung; Cheng, Ann-Lii

2017-10-17

Increased angiogenic activity has been demonstrated in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), but the mechanism was unclear. To study the role of HCV core protein, we used tube formation and Matrigel plug assays to assess the proangiogenic activity of an HCC cell line, HuH7, and 2 of its stable clones-HuH7-core-high and HuH7-core-low, with high and low HCV core protein expression, respectively. In both assays, HuH7-core-high and HuH7-core-low cells dose-dependently induced stronger angiogenesis than control cells. HuH7 cells with HCV core protein expression showed increased mRNA and protein expression of vascular endothelial growth factor (VEGF). VEGF inhibition by bevacizumab reduced the proangiogenic activity of HuH7-core-high cells. The promotor region of VEGF contains the binding site of activator protein-1 (AP-1). Compared with controls, HuH7-core-high cells had an increased AP-1 activity and nuclear localization of phospho-c-jun. AP-1 inhibition using either RNA knockdown or AP-1 inhibitors reduced the VEGF mRNA expression and the proangiogenic activity of HuH7-core-high cells. Among 131 tissue samples from HCC patients, HCV-related HCC revealed stronger VEGF expression than did hepatitis B virus-related HCC. In conclusion, increased VEGF expression through AP-1 activation is a crucial mechanism underlying the proangiogenic activity of the HCV core protein in HCC cells.

Hepatitis B Virus (HBV) Load Response to 2 Antiviral Regimens, Tenofovir/Lamivudine and Lamivudine, in HIV/ HBV-Coinfected Pregnant Women in Guangxi, China: The Tenofovir in Pregnancy (TiP) Study.

PubMed

Wang, Liming; Wiener, Jeffrey; Bulterys, Marc; Wei, Xiaoyu; Chen, Lili; Liu, Wei; Liang, Shujia; Shepard, Colin; Wang, Linhong; Wang, Ailing; Zhang, Fujie; Kourtis, Athena P

2016-12-01

 There is limited information on antiviral therapy for hepatitis B virus (HBV) infection among pregnant women coinfected with human immunodeficiency virus (HIV) and HBV.  A phase 2 randomized, controlled trial of a regimen containing tenofovir (TDF)/lamivudine (3TC) and a regimen containing 3TC in HIV/HBV-coinfected pregnant women in China. The HBV virological response was compared in study arms.  The median decline in the HBV DNA level was 2.60 log 10 copies/mL in the TDF/3TC arm and 2.24 log 10 copies/mL in the 3TC arm (P = .41). All women achieved HBV DNA levels of <6 log 10 copies/mL at delivery.  Initiation of either regimen led to achievement of HBV DNA levels below the threshold associated with perinatal HBV transmission.  NCT01125696. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

HCV core protein induces hepatic lipid accumulation by activating SREBP1 and PPAR{gamma}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Kook Hwan; Hong, Sung Pyo; Kim, KyeongJin

2007-04-20

Hepatic steatosis is a common feature in patients with chronic hepatitis C virus (HCV) infection. HCV core protein plays an important role in the development of hepatic steatosis in HCV infection. Because SREBP1 (sterol regulatory element binding protein 1) and PPAR{gamma} (peroxisome proliferators-activated receptor {gamma}) are involved in the regulation of lipid metabolism of hepatocyte, we sought to determine whether HCV core protein may impair the expression and activity of SREBP1 and PPAR{gamma}. In this study, it was demonstrated that HCV core protein increases the gene expression of SREBP1 not only in Chang liver, Huh7, and HepG2 cells transiently transfectedmore » with HCV core protein expression plasmid, but also in Chang liver-core stable cells. Furthermore, HCV core protein enhanced the transcriptional activity of SREBP1. In addition, HCV core protein elevated PPAR{gamma} transcriptional activity. However, HCV core protein had no effect on PPAR{gamma} gene expression. Finally, we showed that HCV core protein stimulates the genes expression of lipogenic enzyme and fatty acid uptake associated protein. Therefore, our finding provides a new insight into the mechanism of hepatic steatosis by HCV infection.« less

Viruslike Nanoparticles with Maghemite Cores Allow for Enhanced MRI Contrast Agents

DOE PAGES

Malyutin, Andrey G.; Easterday, Rosemary; Lozovyy, Yaroslav; ...

2014-12-15

Here, for the first time, we demonstrate formation of virus-like nanoparticles (VNPs) utilizing gold-coated iron oxide nanoparticles as cores and capsidprotein of brome mosaic virus (BMV) or hepatitis B virus (HBV) as shells. Further, utilizing cryo-electron microscopy and single particle methods, we are able to show that the BMV coat on VNPs assembles into a structure very close to that of a native virion. This is a consequence of an optimal iron oxide NP size (~11 nm) fitting the virus cavity and an ultrathin gold layer on the maghemite cores, which allows for utilization of SH-(CH 2) 11-(CH 2-CH 2-O)more » 4-OCH 2-COOH as capping molecules to provide sufficient stability, charge density, and small form factor. MRI studies show unique relaxivity ratios that diminish only slightly with gold coating. In conclusion, a virus protein coating of a magnetic core mimicking the wild-type virus makes these VNPs a versatile platform for biomedical applications.« less

Hepatitis C virus core protein potentiates proangiogenic activity of hepatocellular carcinoma cells

PubMed Central

Shao, Yu-Yun; Hsieh, Min-Shu; Wang, Han-Yu; Li, Yong-Shi; Lin, Hang; Hsu, Hung-Wei; Huang, Chung-Yi; Hsu, Chih-Hung; Cheng, Ann-Lii

2017-01-01

Increased angiogenic activity has been demonstrated in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), but the mechanism was unclear. To study the role of HCV core protein, we used tube formation and Matrigel plug assays to assess the proangiogenic activity of an HCC cell line, HuH7, and 2 of its stable clones—HuH7-core-high and HuH7-core-low, with high and low HCV core protein expression, respectively. In both assays, HuH7-core-high and HuH7-core-low cells dose-dependently induced stronger angiogenesis than control cells. HuH7 cells with HCV core protein expression showed increased mRNA and protein expression of vascular endothelial growth factor (VEGF). VEGF inhibition by bevacizumab reduced the proangiogenic activity of HuH7-core-high cells. The promotor region of VEGF contains the binding site of activator protein-1 (AP-1). Compared with controls, HuH7-core-high cells had an increased AP-1 activity and nuclear localization of phospho-c-jun. AP-1 inhibition using either RNA knockdown or AP-1 inhibitors reduced the VEGF mRNA expression and the proangiogenic activity of HuH7-core-high cells. Among 131 tissue samples from HCC patients, HCV-related HCC revealed stronger VEGF expression than did hepatitis B virus-related HCC. In conclusion, increased VEGF expression through AP-1 activation is a crucial mechanism underlying the proangiogenic activity of the HCV core protein in HCC cells. PMID:29156827

Recognition of Core- and Polymerase-derived immunogenic peptides included in novel therapeutic vaccine by T cells from Chinese chronic hepatitis B patients.

PubMed

Huang, D; Sansas, B; Jiang, J H; Gong, Q M; Jin, G D; Calais, V; Yu, D M; Zhu, M Y; Wei, D; Zhang, D H; Inchauspé, G; Zhang, X X; Zhu, R

2017-11-01

Chronic hepatitis B (CHB) is one of the major public health challenges in the world. Due to a strong interplay between specific T-cell immunity and elimination of hepatitis B virus (HBV), efforts to develop novel immunotherapeutics are gaining attention. TG1050, a novel immunotherapy, has shown efficacy in an animal study. To support the clinical development of TG1050 in China, specific immunity to the fusion antigens of TG1050 was assessed in Chinese patients. One hundred and thirty subjects were divided into three groups as CHB patients, HBV spontaneous resolvers, and CHB patients with HBsAg loss after antiviral treatment. HBV-specific T-cell responses to pools of HBV Core or Polymerase genotype D peptides included in TG1050 were evaluated. HBV Core- or Polymerase-specific cells were detected in peripheral blood mononuclear cells (PBMCs) from the different cohorts. The frequencies and intensities of HBV Core-specific immune responses were significantly lower in CHB patients than in HBsAg loss subjects. In CHB patients, a dominant pool derived from Polymerase (Pol1) was the most immunogenic. CHB patients with low viral loads (<10 6 IU/mL) were more likely to have a positive response specific to the Core peptide pool. Overall, genotype D-derived peptides included in TG1050 could raise broad and functional T-cell responses in PBMCs from Chinese CHB patients infected with genotype B/C isolates. Core-specific immunogenic domains appeared as "hot spots" with the capacity to differentiate between CHB vs HBsAg loss subjects. These observations support the extended application and associated immune monitoring of TG1050 in China. © 2017 The Authors. Journal of Viral Hepatitis Published by John Wiley & Sons Ltd.

Antiviral prophylaxis during chemotherapy or immunosuppressive drug therapy to prevent HBV reactivation in patients with resolved HBV infection: a systematic review and meta-analysis.

PubMed

Su, Yi-Chia; Lin, Pei-Chin; Yu, Hsien-Chung; Wu, Chih-Chien

2018-05-29

Until recently, the role of antiviral prophylaxis in preventing hepatitis B virus (HBV) reactivation during immunosuppressive therapy or chemotherapy in patients with resolved HBV infection was unclear. The aim of the study reported here was to compare the efficacy of antiviral prophylaxis versus that of non-prophylaxis in resolved HBV-infected patients undergoing chemotherapy or immunosuppressive therapy. PubMed, the Cochrane library, and the ClinicalTrials.gov website were searched from inception until December 2017. Studies comparing reactivation in prophylaxis versus non-prophylaxis in patients undergoing immunosuppressive therapy or chemotherapy were included. The meta-analysis was performed to calculate the relative risk (RR) and the pooled estimates. A meta-analysis was conducted of 13 studies (2 randomized controlled trials [RCTs] and 11 cohort studies). The summary RR for HBV reactivation was 0.47 (95% confidence interval [CI] 0.13-1.69) for antiviral prophylaxis versus non-prophylaxis. Both of the RCTs included in the meta-analysis enrolled patients treated with rituximab. Subgroup analyses showed that the two RCTs ± high-quality cohort studies showed a decreased risk of HBV reactivation among the antiviral prophylaxis groups (RCT 1: RR 0.13, 95% CI 0.02-0.70; P = 0.02; RCT 2: 0.28, 95% CI 0.08-0.98; P = 0.05). Subgroup analyses further showed that the cohort studies did not support an association between the antiviral prophylaxis groups and HBV reactivation (RR 0.62, 95% CI 0.14-2.83; P = 0.54); adjusting for confounding factors, such as detectable anti-HBs antibodies, failed to produce a significant association (RR,0.29, 95% CI 0.07-1.28; P = 0.10). Our meta-analyses did not show an association between antiviral prophylaxis use and risk of HBV reactivation. As using only the RCTs ± high-quality cohort studies data rendered this association significant, clinicians can consider providing antiviral prophylaxis to patients with resolved

Comparison of the effects of formaldehyde and gaseous ozone on HBV-contaminated hospital quilts

PubMed Central

Guo, Dan; Li, Ziqiong; Jia, Bei; Che, Xiaoqiong; Song, Tianshuang; Huang, Wenxiang

2015-01-01

Background: Besides being highly infectious, Hepatitis B virus (HBV) is a major cause of liver disease worldwide. In hospital settings, it is easy for the environment and quilts to be contaminated by HBV patient blood and body fluids. Therefore, HBV can be transmitted to other patients via contaminated environmental surfaces or quilts, resulting in an HBV nosocomial infection. Formaldehyde and ozone are commonly used disinfectants that may influence this infectious situation. Objective: To investigate the clinical effectiveness of formaldehyde and gaseous ozone for the terminal cleaning of hospital quilts contaminated by HBV. Methods: Thin cloth and thick cotton soaked with the serum from high HBV copy number patients were prepared and disinfected using formaldehyde fumigation and gaseous ozone at different times. The copy numbers of HBV DNA in the HBV-contaminated cloth and cotton samples were measured quantitatively with fluorescent quantitative polymerase chain reaction (PCR). Results: When gaseous ozone was used to disinfect HBV-contaminated quilts for 23 minutes (min), 36 min, 49 min, and 90 min, the HBV DNA copy number displayed no significant decrease compared with the copy number before disinfection (P > 0.05). In comparison, the copy number of the HBV DNA in the cloth group decreased significantly (P < 0.05) after formaldehyde fumigation disinfection for 1 hour (h), and there was no difference when longer times and increased concentrations were used. In the thick cotton group, there was also a significant decrease (P < 0.05) of the HBV DNA copy numbers, but the decrease was not as dramatic. In addition, in this group, the disinfection effect observed at 4 h was the strongest. Conclusions: The application of ozone to disinfect HBV-contaminated hospital quilts possibly has no effect, whereas, formaldehyde oxide fumigation effectively reduced HBV copy numbers. PMID:26770591

Trained immunity in newborn infants of HBV-infected mothers

PubMed Central

Hong, Michelle; Sandalova, Elena; Low, Diana; Gehring, Adam J.; Fieni, Stefania; Amadei, Barbara; Urbani, Simonetta; Chong, Yap-Seng; Guccione, Ernesto; Bertoletti, Antonio

2015-01-01

The newborn immune system is characterized by an impaired Th1-associated immune response. Hepatitis B virus (HBV) transmitted from infected mothers to newborns is thought to exploit the newborns’ immune system immaturity by inducing a state of immune tolerance that facilitates HBV persistence. Contrary to this hypothesis, we demonstrate here that HBV exposure in utero triggers a state of trained immunity, characterized by innate immune cell maturation and Th1 development, which in turn enhances the ability of cord blood immune cells to respond to bacterial infection in vitro. These training effects are associated with an alteration of the cytokine environment characterized by low IL-10 and, in most cases, high IL-12p40 and IFN-α2. Our data uncover a potentially symbiotic relationship between HBV and its natural host, and highlight the plasticity of the fetal immune system following viral exposure in utero. PMID:25807344

Structural characterization of Mumps virus fusion protein core

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Yueyong; Xu Yanhui; Lou Zhiyong

2006-09-29

The fusion proteins of enveloped viruses mediating the fusion between the viral and cellular membranes comprise two discontinuous heptad repeat (HR) domains located at the ectodomain of the enveloped glycoproteins. The crystal structure of the fusion protein core of Mumps virus (MuV) was determined at 2.2 A resolution. The complex is a six-helix bundle in which three HR1 peptides form a central highly hydrophobic coiled-coil and three HR2 peptides pack against the hydrophobic grooves on the surface of central coiled-coil in an oblique antiparallel manner. Fusion core of MuV, like those of simian virus 5 and human respiratory syncytium virus,more » forms typical 3-4-4-4-3 spacing. The similar charecterization in HR1 regions, as well as the existence of O-X-O motif in extended regions of HR2 helix, suggests a basic rule for the formation of the fusion core of viral fusion proteins.« less

Hepatitis B Virus X Protein Induces Hepatic Steatosis by Enhancing the Expression of Liver Fatty Acid Binding Protein.

PubMed

Wu, Yun-Li; Peng, Xian-E; Zhu, Yi-Bing; Yan, Xiao-Li; Chen, Wan-Nan; Lin, Xu

2016-02-15

Hepatitis B virus (HBV) has been implicated as a potential trigger of hepatic steatosis although molecular mechanisms involved in the pathogenesis of HBV-associated hepatic steatosis still remain elusive. Our prior work has revealed that the expression level of liver fatty acid binding protein 1 (FABP1), a key regulator of hepatic lipid metabolism, was elevated in HBV-producing hepatoma cells. In this study, the effects of HBV X protein (HBx) mediated FABP1 regulation on hepatic steatosis and the underlying mechanism were determined. mRNA and protein levels of FABP1 were measured by quantitative RT-PCR (qPCR) and Western blotting. HBx-mediated FABP1 regulation was evaluated by luciferase assay, coimmunoprecipitation, and chromatin immunoprecipitation. Hepatic lipid accumulation was measured by using Oil-Red-O staining and the triglyceride level. It was found that expression of FABP1 was increased in HBV-producing hepatoma cells, the sera of HBV-infected patients, and the sera and liver tissues of HBV-transgenic mice. Ectopic overexpression of HBx resulted in upregulation of FABP1 in HBx-expressing hepatoma cells, whereas HBx abolishment reduced FABP1 expression. Mechanistically, HBx activated the FABP1 promoter in an HNF3β-, C/EBPα-, and PPARα-dependent manner, in which HBx increased the gene expression of HNF3β and physically interacted with C/EBPα and PPARα. On the other hand, knockdown of FABP1 remarkably blocked lipid accumulation both in long-chain free fatty acids treated HBx-expressing HepG2 cells and in a high-fat diet-fed HBx-transgenic mice. Therefore, FABP1 is a key driver gene in HBx-induced hepatic lipid accumulation via regulation of HNF3β, C/EBPα, and PPARα. FABP1 may represent a novel target for treatment of HBV-associated hepatic steatosis. Accumulating evidence from epidemiological and experimental studies has indicated that chronic HBV infection is associated with hepatic steatosis. However, the molecular mechanism underlying HBV

Hepatitis B virus core antigen determines viral persistence in a C57BL/6 mouse model.

PubMed

Lin, Yi-Jiun; Huang, Li-Rung; Yang, Hung-Chih; Tzeng, Horng-Tay; Hsu, Ping-Ning; Wu, Hui-Lin; Chen, Pei-Jer; Chen, Ding-Shinn

2010-05-18

We recently developed a mouse model of hepatitis B virus (HBV) persistence, in which a single i.v. hydrodynamic injection of HBV DNA to C57BL/6 mice allows HBV replication and induces a partial immune response, so that about 20-30% of the mice carry HBV for more than 6 months. The model was used to identify the viral antigen crucial for HBV persistence. We knocked out individual HBV genes by introducing a premature termination codon to the HBV core, HBeAg, HBx, and polymerase ORFs. The specific-gene-deficient HBV mutants were hydrodynamically injected into mice and the HBV profiles of the mice were monitored. About 90% of the mice that received the HBcAg-mutated HBV plasmid exhibited high levels of hepatitis B surface antigenemia and maintained HBsAg expression for more than 6 months after injection. To map the region of HBcAg essential for viral clearance, we constructed a set of serial HBcAg deletion mutants for hydrodynamic injection. We localized the essential region of HBcAg to the carboxyl terminus, specifically to the 10 terminal amino acids (HBcAg176-185). The majority of mice receiving this HBV mutant DNA did not elicit a proper HBcAg-specific IFN-gamma response and expressed HBV virions for 6 months. These results indicate that the immune response triggered in mice by HBcAg during exposure to HBV is important in determining HBV persistence.

Simulating the minimum core for hydrophobic collapse in globular proteins.

PubMed Central

Tsai, J.; Gerstein, M.; Levitt, M.

1997-01-01

To investigate the nature of hydrophobic collapse considered to be the driving force in protein folding, we have simulated aqueous solutions of two model hydrophobic solutes, methane and isobutylene. Using a novel methodology for determining contacts, we can precisely follow hydrophobic aggregation as it proceeds through three stages: dispersed, transition, and collapsed. Theoretical modeling of the cluster formation observed by simulation indicates that this aggregation is cooperative and that the simulations favor the formation of a single cluster midway through the transition stage. This defines a minimum solute hydrophobic core volume. We compare this with protein hydrophobic core volumes determined from solved crystal structures. Our analysis shows that the solute core volume roughly estimates the minimum core size required for independent hydrophobic stabilization of a protein and defines a limiting concentration of nonpolar residues that can cause hydrophobic collapse. These results suggest that the physical forces driving aggregation of hydrophobic molecules in water is indeed responsible for protein folding. PMID:9416609

DHX9 regulates production of hepatitis B virus-derived circular RNA and viral protein levels

PubMed Central

Sekiba, Kazuma; Otsuka, Motoyuki; Ohno, Motoko; Kishikawa, Takahiro; Yamagami, Mari; Suzuki, Tatsunori; Ishibashi, Rei; Seimiya, Takahiro; Tanaka, Eri; Koike, Kazuhiko

2018-01-01

Hepatitis B virus (HBV) infection, which is a major health concern worldwide, can lead to liver cirrhosis and hepatocellular carcinoma. Although current nucleos(t)ide analogs efficiently inhibit viral reverse transcription and viral DNA load clinically, episomal viral covalently closed circular DNA (cccDNA) minichromosomes and transcripts from cccDNA continue to be expressed over the long term. We hypothesized that, under these conditions, viral transcripts may have biological functions involved in pathogenesis. Here, we show that the host protein DExH-box helicase 9 (DXH9) is associated with viral RNAs. We also show that viral-derived circular RNA is produced during HBV replication, and the amount is increased by knockdown of the DHX9 protein, which, in turn, results in decreased viral protein levels but does not affect the levels of HBV DNA. These phenomena were observed in the HBV-producing cell culture model and HBV mini-circle model mimicking HBV cccDNA, as well as in human primary hepatocytes infected with HBV. Based on these results, we conclude that, in HBV infection, the RNA binding factor DHX9 is a novel regulator of viral circular RNA and viral protein levels. PMID:29765512

Genotype-dependent activation or repression of HBV enhancer II by transcription factor COUP-TF1

PubMed Central

Fischer, Silke F; Schmidt, Katja; Fiedler, Nicola; Glebe, Dieter; Schüttler, Christian; Sun, Jianguang; Gerlich, Wolfram H; Repp, Reinald; Schaefer, Stephan

2006-01-01

AIM: To study the expression of HBV enhancer II by transcription factor COUP-TF1. METHODS: In order to study the regulation of HBV variants in the vicinity of the NRRE we cloned luciferase constructs containing the HBV enhancer II from variants and from HBV genotypes A and D and cotransfected them together with expression vectors for COUP-TF1 into HepG2 cells. RESULTS: Our findings show that enhancer II of HBV genotype A is also repressed by COUP-TF1. In contrast, two different enhancer II constructs of HBV genotype D were activated by COUP-TF1. The activation was independent of the NRRE because a natural variant with a deletion of nt 1763-1770 was still activated by COUP-TF1. CONCLUSION: Regulation of transcription of the HBV genome seems to differ among HBV genomes derived from different genotypes. These differences in transcriptional control among HBV genotypes may be the molecular basis for differences in the clinical course among HBV genotypes. PMID:17009409

Genotype-dependent activation or repression of HBV enhancer II by transcription factor COUP-TF1.

PubMed

Fischer, Silke F; Schmidt, Katja; Fiedler, Nicola; Glebe, Dieter; Schüttler, Christian; Sun, Jianguang; Gerlich, Wolfram H; Repp, Reinald; Schaefer, Stephan

2006-10-07

To study the expression of HBV enhancer II by transcription factor COUP-TF1. In order to study the regulation of HBV variants in the vicinity of the NRRE we cloned luciferase constructs containing the HBV enhancer II from variants and from HBV genotypes A and D and cotransfected them together with expression vectors for COUP-TF1 into HepG2 cells. Our findings show that enhancer II of HBV genotype A is also repressed by COUP-TF1. In contrast, two different enhancer II constructs of HBV genotype D were activated by COUP-TF1. The activation was independent of the NRRE because a natural variant with a deletion of nt 1763-1770 was still activated by COUP-TF1. Regulation of transcription of the HBV genome seems to differ among HBV genomes derived from different genotypes. These differences in transcriptional control among HBV genotypes may be the molecular basis for differences in the clinical course among HBV genotypes.

Identification of cyclin B1 and Sec62 as biomarkers for recurrence in patients with HBV-related hepatocellular carcinoma after surgical resection.

PubMed

Weng, Li; Du, Juan; Zhou, Qinghui; Cheng, Binbin; Li, Jun; Zhang, Denghai; Ling, Changquan

2012-06-08

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Frequent tumor recurrence after surgery is related to its poor prognosis. Although gene expression signatures have been associated with outcome, the molecular basis of HCC recurrence is not fully understood, and there is no method to predict recurrence using peripheral blood mononuclear cells (PBMCs), which can be easily obtained for recurrence prediction in the clinical setting. According to the microarray analysis results, we constructed a co-expression network using the k-core algorithm to determine which genes play pivotal roles in the recurrence of HCC associated with the hepatitis B virus (HBV) infection. Furthermore, we evaluated the mRNA and protein expressions in the PBMCs from 80 patients with or without recurrence and 30 healthy subjects. The stability of the signatures was determined in HCC tissues from the same 80 patients. Data analysis included ROC analysis, correlation analysis, log-lank tests, and Cox modeling to identify independent predictors of tumor recurrence. The tumor-associated proteins cyclin B1, Sec62, and Birc3 were highly expressed in a subset of samples of recurrent HCC; cyclin B1, Sec62, and Birc3 positivity was observed in 80%, 65.7%, and 54.2% of the samples, respectively. The Kaplan-Meier analysis revealed that high expression levels of these proteins was associated with significantly reduced recurrence-free survival. Cox proportional hazards model analysis revealed that cyclin B1 (hazard ratio [HR], 4.762; p = 0.002) and Sec62 (HR, 2.674; p = 0.018) were independent predictors of HCC recurrence. These results revealed that cyclin B1 and Sec62 may be candidate biomarkers and potential therapeutic targets for HBV-related HCC recurrence after surgery.

Non-invasive optical detection of HBV based on serum surface-enhanced Raman spectroscopy

NASA Astrophysics Data System (ADS)

Zheng, Zuci; Wang, Qiwen; Weng, Cuncheng; Lin, Xueliang; Lin, Yao; Feng, Shangyuan

2016-10-01

An optical method of surface-enhanced Raman spectroscopy (SERS) was developed for non-invasive detection of hepatitis B surface virus (HBV). Hepatitis B virus surface antigen (HBsAg) is an established serological marker that is routinely used for the diagnosis of acute or chronic hepatitis B virus(HBV) infection. Utilizing SERS to analyze blood serum for detecting HBV has not been reported in previous literature. SERS measurements were performed on two groups of serum samples: one group for 50 HBV patients and the other group for 50 healthy volunteers. Blood serum samples are collected from healthy control subjects and patients diagnosed with HBV. Furthermore, principal components analysis (PCA) combined with linear discriminant analysis (LDA) were employed to differentiate HBV patients from healthy volunteer and achieved sensitivity of 80.0% and specificity of 74.0%. This exploratory work demonstrates that SERS serum analysis combined with PCA-LDA has tremendous potential for the non-invasive detection of HBV.

Differences in antiproliferative effect of STAT3 inhibition in HCC cells with versus without HBV expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong, Yun; Zhou, Lin; Xie, Haiyang

2015-06-05

Chronic infection with hepatitis B virus (HBV) plays an important role in the etiology of hepatocellular carcinoma (HCC). Signal transducer and activator of transcription 3 (STAT3) inactivation could inhibit the tumor growth of HCC. In this study, differential antiproliferative effect of STAT3 inhibition was observed with HBV-related HCC cells being more resistant than non-HBV-related HCC cells. Resistance of HBV-related HCC cells to STAT3 inhibition was positively correlated to the expression of HBV. Enhanced ERK activation after STAT3 blockade was detected in HBV-related HCC cells but not in non-HBV-related HCC cells. Combined ERK and STAT3 inhibition eliminates the discrepancy between themore » two types of HCC cells. Moderate reduced HBV expression was found after STAT3 inhibition. These findings disclose a discrepancy in cellular response to STAT3 inhibition between non-HBV-related and HBV-related HCC cells and underscore the complexity of antiproliferative effect of STAT3 inactivation in HBV-related HCC cells. - Highlights: • HBV endows HCC cells with resistance to STAT3 inactivation on proliferation. • Abnormal ERK activation after STAT3 inhibition in HBV-related HCC cells. • Combined ERK and STAT3 inhibition eliminates the discrepancy. • STAT3 inhibition moderately reduces HBV expression.« less

Increased ERp57 Expression in HBV-Related Hepatocellular Carcinoma: Possible Correlation and Prognosis.

PubMed

Liu, Miao; Du, Lingyao; He, Zhiliang; Yan, Libo; Shi, Ying; Shang, Jin; Tang, Hong

2017-01-01

Aim. ERp57 is involved in virus induced endoplasmic reticulum stress (ERS) and plays an important role in tumorigenesis. This study aimed to find whether HBV infection altered ERp57 expression and whether ERp57 regulation was involved in hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) genesis. Materials and Methods. HBV-HCC tissues, chronic hepatitis B (CHB) liver tissues, and normal liver tissues were acquired. ERp57 expressions in these tissues were detected through immunohistochemistry (IHC). And ERp57 expression in liver cell line L02, HBV replicative liver cell line L02-pHBV4.1, and HCC cell lines were detected through western blot for verification. Then medical data on patients providing HCC tissues were collected and analyzed along with ERp57 expression. Results. Higher ERp57 expression was found in HCC and CHB tissues ( p < 0.001). And HCC cell lines and L02-pHBV4.1 presented higher ERp57 expression as well. In patients, ERp57 expression showed significant differences between death and survival groups ( p = 0.037). And cumulative survival in patients with higher ERp57 (score ⩾ 8.75) is significantly lower ( p = 0.009). Conclusion. Our study found increased expression of ERp57 in HBV-HCC. Such altered expression could be related to HBV infection and high ERp57 expression may lead to poor prognosis of HBV-HCC patients.

Hepatitis B Virus Core Gene Mutations Which Block Nucleocapsid Envelopment

PubMed Central

Koschel, Matthias; Oed, Daniela; Gerelsaikhan, Tudevdagwa; Thomssen, Reiner; Bruss, Volker

2000-01-01

Recently we generated a panel of hepatitis B virus core gene mutants carrying single insertions or deletions which allowed efficient expression of the core protein in bacteria and self-assembly of capsids. Eleven of these mutations were introduced into a eukaryotic core gene expression vector and characterized by trans complementation of a core-negative HBV genome in cotransfected human hepatoma HuH7 cells. Surprisingly, four mutants (two insertions [EFGA downstream of A11 and LDTASALYR downstream of R39] and two deletions [Y38-R39-E40 and L42]) produced no detectable capsids. The other seven mutants supported capsid formation and pregenome packaging/viral minus- and plus-strand-DNA synthesis but to different levels. Four of these seven mutants (two insertions [GA downstream of A11 and EHCSP downstream of P50] and two deletions [S44 and A80]) allowed virion morphogenesis and secretion. The mutant carrying a deletion of A80 at the tip of the spike protruding from the capsid was hepatitis B virus core antigen negative but wild type with respect to virion formation, indicating that this site might not be crucial for capsid-surface protein interactions during morphogenesis. The other three nucleocapsid-forming mutants (one insertion [LS downstream of S141] and two deletions [T12 and P134]) were strongly blocked in virion formation. The corresponding sites are located in the part of the protein forming the body of the capsid and not in the spike. These mutations may alter sites on the particle which contact surface proteins during envelopment, or they may block the appearance of a signal for the transport or the maturation of the capsid which is linked to viral DNA synthesis and required for envelopment. PMID:10590084

Pleiotrophin, a target of miR-384, promotes proliferation, metastasis and lipogenesis in HBV-related hepatocellular carcinoma.

PubMed

Bai, Pei-Song; Xia, Nan; Sun, Hong; Kong, Ying

2017-11-01

Hepatitis B virus (HBV) infection plays a crucial role and is a major cause of hepatocellular carcinoma (HCC) in China. microRNAs (miRNAs) have emerged as key players in hepatic steatosis and carcinogenesis. We found that down-regulation of miR-384 expression was a common event in HCC, especially HBV-related HCC. However, the possible function of miR-384 in HBV-related HCC remains unclear. The oncogene pleiotrophin (PTN) was a target of miR-384. HBx inhibited miR-384, increasing PTN expression. The PTN receptor N-syndecan was highly expressed in HCC. PTN induced by HBx acted as a growth factor via N-syndecan on hepatocytes and further promoted cell proliferation, metastasis and lipogenesis. PTN up-regulated sterol regulatory element-binding protein 1c (SREBP-1c) through the N-syndecan/PI3K/Akt/mTORC1 pathway and the expression of lipogenic genes, including fatty acid synthesis (FAS). PTN-mediated de novo lipid synthesis played an important role in HCC proliferation and metastasis. PI3K/AKT and an mTORC1 inhibitor diminished PTN-induced proliferation, metastasis and lipogenesis. Taken together, these data strongly suggest that the dysregulation of miR-384 could play a crucial role in HBV related to HCC, and the target gene of miR-384, PTN, represents a new potential therapeutic target for the prevention of hepatic steatosis and further progression to HCC after chronic HBV infection. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Virus-producing cells determine the host protein profiles of HIV-1 virion cores

PubMed Central

2012-01-01

Background Upon HIV entry into target cells, viral cores are released and rearranged into reverse transcription complexes (RTCs), which support reverse transcription and also protect and transport viral cDNA to the site of integration. RTCs are composed of viral and cellular proteins that originate from both target and producer cells, the latter entering the target cell within the viral core. However, the proteome of HIV-1 viral cores in the context of the type of producer cells has not yet been characterized. Results We examined the proteomic profiles of the cores purified from HIV-1 NL4-3 virions assembled in Sup-T1 cells (T lymphocytes), PMA and vitamin D3 activated THP1 (model of macrophages, mMΦ), and non-activated THP1 cells (model of monocytes, mMN) and assessed potential involvement of identified proteins in the early stages of infection using gene ontology information and data from genome-wide screens on proteins important for HIV-1 replication. We identified 202 cellular proteins incorporated in the viral cores (T cells: 125, mMΦ: 110, mMN: 90) with the overlap between these sets limited to 42 proteins. The groups of RNA binding (29), DNA binding (17), cytoskeleton (15), cytoskeleton regulation (21), chaperone (18), vesicular trafficking-associated (12) and ubiquitin-proteasome pathway-associated proteins (9) were most numerous. Cores of the virions from SupT1 cells contained twice as many RNA binding proteins as cores of THP1-derived virus, whereas cores of virions from mMΦ and mMN were enriched in components of cytoskeleton and vesicular transport machinery, most probably due to differences in virion assembly pathways between these cells. Spectra of chaperones, cytoskeletal proteins and ubiquitin-proteasome pathway components were similar between viral cores from different cell types, whereas DNA-binding and especially RNA-binding proteins were highly diverse. Western blot analysis showed that within the group of overlapping proteins, the level of

Social Norm, Family Communication, and HBV Screening among Asian Americans.

PubMed

Juon, Hee-Soon; Rimal, Rajiv N; Klassen, Ann; Lee, Sunmin

2017-12-01

Individuals' behaviors are influenced by those of others in their social environment (i.e., descriptive norms), as well as by how individuals perceive they should behave in that environment (e.g., injunctive norms). Although social norms are thought to play an important role in hepatitis B virus (HBV) screening, limited theoretical or empirical guidance exists on how the underlying process works. In addition, norms are social phenomena that are spread through family discussion about the importance of getting HBV screening. Using the theory of normative social behavior (TNSB), this study examined the roles of injunctive norms (IN), descriptive norms (DN), and family discussion in HBV screening behavior among Asian Americans. Data from a survey of Asian Americans in the Baltimore Washington metropolitan area (N = 877) were used to test underlying theoretical propositions. DN and family discussion emerged as key factors in HBV screening behavior among all Asian Americans. IN were associated with HBV screening among Chinese and Korean Americans, but not for Vietnamese Americans. Family discussion moderated the influence of DN on behavior among Chinese and Vietnamese Americans. However, the main effect of DN on screening behavior was not modified by IN (no interactions between DN and IN). The results indicate that family discussion and social norms are integral in enabling Asian Americans to undergo HBV screening and warrant sensitivity in the design and implementation of a liver cancer prevention program in this high-risk group of Asian Americans.

Discovery and Pre-Clinical Characterization of Third-Generation 4-H Heteroaryldihydropyrimidine (HAP) Analogues as Hepatitis B Virus (HBV) Capsid Inhibitors.

PubMed

Qiu, Zongxing; Lin, Xianfeng; Zhang, Weixing; Zhou, Mingwei; Guo, Lei; Kocer, Buelent; Wu, Guolong; Zhang, Zhisen; Liu, Haixia; Shi, Houguang; Kou, Buyu; Hu, Taishan; Hu, Yimin; Huang, Mengwei; Yan, S Frank; Xu, Zhiheng; Zhou, Zheng; Qin, Ning; Wang, Yue Fen; Ren, Shuang; Qiu, Hongxia; Zhang, Yuxia; Zhang, Yi; Wu, Xiaoyue; Sun, Kai; Zhong, Sheng; Xie, Jianxun; Ottaviani, Giorgio; Zhou, Yuan; Zhu, Lina; Tian, Xiaojun; Shi, Liping; Shen, Fang; Mao, Yi; Zhou, Xue; Gao, Lu; Young, John A T; Wu, Jim Zhen; Yang, Guang; Mayweg, Alexander V; Shen, Hong C; Tang, Guozhi; Zhu, Wei

2017-04-27

Described herein are the discovery and structure-activity relationship (SAR) studies of the third-generation 4-H heteroaryldihydropyrimidines (4-H HAPs) featuring the introduction of a C6 carboxyl group as novel HBV capsid inhibitors. This new series of 4-H HAPs showed improved anti-HBV activity and better drug-like properties compared to the first- and second-generation 4-H HAPs. X-ray crystallographic study of analogue 12 (HAP_R01) with Cp149 Y132A mutant hexamer clearly elucidated the role of C6 carboxyl group played for the increased binding affinity, which formed strong hydrogen bonding interactions with capsid protein and coordinated waters. The representative analogue 10 (HAP_R10) was extensively characterized in vitro (ADMET) and in vivo (mouse PK and PD) and subsequently selected for further development as oral anti-HBV infection agent.

The timing of hepatitis B virus (HBV) immunization relative to human immunodeficiency virus (HIV) diagnosis and the risk of HBV infection following HIV diagnosis.

PubMed

Landrum, Michael L; Hullsiek, Katherine Huppler; Chun, Helen M; Crum-Cianflone, Nancy F; Ganesan, Anuradha; Weintrob, Amy C; Barthel, R Vincent; O'Connell, Robert J; Agan, Brian K

2011-01-01

To assess associations between the timing of hepatitis B virus (HBV) immunization relative to human immunodeficiency virus (HIV) diagnosis and vaccine effectiveness, US Military HIV Natural History Study cohort participants without HBV infection at the time of HIV diagnosis were grouped by vaccination status, retrospectively followed from HIV diagnosis for incident HBV infection, and compared using Cox proportional hazards models. A positive vaccine response was defined as hepatitis B surface antibody level ≥ 10 IU/L. Of 1,877 participants enrolled between 1989 and 2008, 441 (23%) were vaccinated prior to HIV diagnosis. Eighty percent of those who received vaccine doses only before HIV diagnosis had a positive vaccine response, compared with 66% of those who received doses both before and after HIV and 41% of those who received doses only after HIV (P < 0.01 for both compared with persons vaccinated before HIV only). Compared with the unvaccinated, persons vaccinated only before HIV had reduced risk of HBV infection after HIV diagnosis (hazard ratio = 0.38, 95% confidence interval: 0.20, 0.75). No reduction in HBV infection risk was observed for other vaccination groups. These data suggest that completion of the vaccine series prior to HIV infection may be the optimal strategy for preventing this significant comorbid infection in HIV-infected persons.

HBV Bypasses the Innate Immune Response and Does Not Protect HCV From Antiviral Activity of Interferon.

PubMed

Mutz, Pascal; Metz, Philippe; Lempp, Florian A; Bender, Silke; Qu, Bingqian; Schöneweis, Katrin; Seitz, Stefan; Tu, Thomas; Restuccia, Agnese; Frankish, Jamie; Dächert, Christopher; Schusser, Benjamin; Koschny, Ronald; Polychronidis, Georgios; Schemmer, Peter; Hoffmann, Katrin; Baumert, Thomas F; Binder, Marco; Urban, Stephan; Bartenschlager, Ralf

2018-05-01

Hepatitis C virus (HCV) infection is sensitive to interferon (IFN)-based therapy, whereas hepatitis B virus (HBV) infection is not. It is unclear whether HBV escapes detection by the IFN-mediated immune response or actively suppresses it. Moreover, little is known on how HBV and HCV influence each other in coinfected cells. We investigated interactions between HBV and the IFN-mediated immune response using HepaRG cells and primary human hepatocytes (PHHs). We analyzed the effects of HBV on HCV replication, and vice versa, at the single-cell level. PHHs were isolated from liver resection tissues from HBV-, HCV-, and human immunodeficiency virus-negative patients. Differentiated HepaRG cells overexpressing the HBV receptor sodium taurocholate cotransporting polypeptide (dHepaRGNTCP) and PHHs were infected with HBV. Huh7.5 cells were transfected with circular HBV DNA genomes resembling viral covalently closed circular DNA (cccDNA), and subsequently infected with HCV; this served as a model of HBV and HCV coinfection. Cells were incubated with IFN inducers, or IFNs, and antiviral response and viral replication were analyzed by immune fluorescence, reverse-transcription quantitative polymerase chain reaction, enzyme-linked immunosorbent assays, and flow cytometry. HBV infection of dHepaRGNTCP cells and PHHs neither activated nor inhibited signaling via pattern recognition receptors. Incubation of dHepaRGNTCP cells and PHHs with IFN had little effect on HBV replication or levels of cccDNA. HBV infection of these cells did not inhibit JAK-STAT signaling or up-regulation of IFN-stimulated genes. In coinfected cells, HBV did not prevent IFN-induced suppression of HCV replication. In dHepaRGNTCP cells and PHHs, HBV evades the induction of IFN and IFN-induced antiviral effects. HBV infection does not rescue HCV from the IFN-mediated response. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Bloodborne Pathogens: HIV and HBV Contagion Risks at Camp.

ERIC Educational Resources Information Center

Skaros, Susan

1996-01-01

AIDS and hepatitis B are diseases caused by the viruses HIV and HBV, respectively, which are spread in blood and body fluids. HBV is 100 times more contagious than HIV. Diligent implementation of universal precautions, an exposure control plan, use of personal protective equipment, a vaccination program, and ongoing staff and camper education can…

Increasing Sequence Diversity with Flexible Backbone Protein Design: The Complete Redesign of a Protein Hydrophobic Core

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murphy, Grant S.; Mills, Jeffrey L.; Miley, Michael J.

2015-10-15

Protein design tests our understanding of protein stability and structure. Successful design methods should allow the exploration of sequence space not found in nature. However, when redesigning naturally occurring protein structures, most fixed backbone design algorithms return amino acid sequences that share strong sequence identity with wild-type sequences, especially in the protein core. This behavior places a restriction on functional space that can be explored and is not consistent with observations from nature, where sequences of low identity have similar structures. Here, we allow backbone flexibility during design to mutate every position in the core (38 residues) of a four-helixmore » bundle protein. Only small perturbations to the backbone, 12 {angstrom}, were needed to entirely mutate the core. The redesigned protein, DRNN, is exceptionally stable (melting point >140C). An NMR and X-ray crystal structure show that the side chains and backbone were accurately modeled (all-atom RMSD = 1.3 {angstrom}).« less

Molecular characterization of hepatitis B virus (HBV) strains circulating in the northern coast of the Persian Gulf and its comparison with worldwide distribution of HBV subgenotype D1.

PubMed

Pourkarim, Mahmoud Reza; Vergote, Valentijn; Amini-Bavil-Olyaee, Samad; Sharifi, Zohre; Sijmons, Steven; Lemey, Philippe; Maes, Piet; Alavian, Seyed Moayed; Van Ranst, Marc

2014-05-01

Iran is a large country that covers the northern coast of the Persian Gulf. Iranian residents of this coastal region interact closely with people from neighboring countries because of historical and cultural relationships, as well as economic activities. In addition, the inhabitants of this border region have experienced several wars, which have affected public health infrastructures. This study characterized for the first time, the evolution of the full-length genome of HBV strains in asymptomatic carrier patients living in this particular region. In addition, this study was compared and complemented by a comprehensive evolutionary analysis of the worldwide geographical distribution of HBV subgenotype D1. Evolutionary analysis demonstrates that patients living in the northern coast of the Persian Gulf are mainly infected with HBV subgenotype D1, subtype ayw2. Specific mutations related to advanced liver disease were found more frequently in these strains compared to other strains isolated from asymptomatic carriers from other regions of Iran. This global comprehensive analysis showed that HBV subgenotype D1 strains have a worldwide distribution and that human mobility and immigration had a large impact on dispersal of HBV subgenotype D1, subtype ayw2 in Middle Eastern countries such as Iran, Syria, and Turkey. In addition to association of subtype ayw2 with subgenotype D1, it was demonstrated that other HBV subtypes like adw2, ayw1, and ayw3 are associated with HBV subgenotype D1 in different regions of the world. This study also revealed a remarkable distribution of subgenotype D1, subtype ayw4 although this particular subtype is associated with subgenotype D4 of HBV in European countries. © 2014 Wiley Periodicals, Inc.

Hepatitis B Virus Capsid Assembly Modulators, but Not Nucleoside Analogs, Inhibit the Production of Extracellular Pregenomic RNA and Spliced RNA Variants

PubMed Central

Ren, Suping; Espiritu, Christine; Kelly, Mollie; Lau, Vincent; Zheng, Lingjie; Hartman, George D.; Flores, Osvaldo A.; Klumpp, Klaus

2017-01-01

ABSTRACT The hepatitis B virus (HBV) core protein serves multiple essential functions in the viral life cycle, and antiviral agents that target the core protein are being developed. Capsid assembly modulators (CAMs) are compounds that target core and misdirect capsid assembly, resulting in the suppression of HBV replication and virion production. Besides HBV DNA, circulating HBV RNA has been detected in patient serum and can be associated with the treatment response. Here we studied the effect of HBV CAMs on the production of extracellular HBV RNA using infected HepaRG cells and primary human hepatocytes. Representative compounds from the sulfonamide carboxamide and heteroaryldihydropyrimidine series of CAMs were evaluated and compared to nucleos(t)ide analogs as inhibitors of the viral polymerase. The results showed that CAMs blocked extracellular HBV RNA with efficiencies similar to those with which they blocked pregenomic RNA (pgRNA) encapsidation, HBV DNA replication, and Dane particle production. Nucleos(t)ide analogs inhibited viral replication and virion production but not encapsidation or production of extracellular HBV RNA. Profiling of HBV RNA from both culture supernatants and patient serum showed that extracellular viral RNA consisted of pgRNA and spliced pgRNA variants with an internal deletion(s) but still retained the sequences at both the 5′ and 3′ ends. Similar variants were detected in the supernatants of infected cells with and without nucleos(t)ide analog treatment. Overall, our data demonstrate that HBV CAMs represent direct antiviral agents with a profile differentiated from that of nucleos(t)ide analogs, including the inhibition of extracellular pgRNA and spliced pgRNA. PMID:28559265

The paradox of HBV evolution as revealed from a 16th century mummy

PubMed Central

Duggan, Ana T.; Poinar, Debi; Poinar, Hendrik N.

2018-01-01

Hepatitis B virus (HBV) is a ubiquitous viral pathogen associated with large-scale morbidity and mortality in humans. However, there is considerable uncertainty over the time-scale of its origin and evolution. Initial shotgun data from a mid-16th century Italian child mummy, that was previously paleopathologically identified as having been infected with Variola virus (VARV, the agent of smallpox), showed no DNA reads for VARV yet did for hepatitis B virus (HBV). Previously, electron microscopy provided evidence for the presence of VARV in this sample, although similar analyses conducted here did not reveal any VARV particles. We attempted to enrich and sequence for both VARV and HBV DNA. Although we did not recover any reads identified as VARV, we were successful in reconstructing an HBV genome at 163.8X coverage. Strikingly, both the HBV sequence and that of the associated host mitochondrial DNA displayed a nearly identical cytosine deamination pattern near the termini of DNA fragments, characteristic of an ancient origin. In contrast, phylogenetic analyses revealed a close relationship between the putative ancient virus and contemporary HBV strains (of genotype D), at first suggesting contamination. In addressing this paradox we demonstrate that HBV evolution is characterized by a marked lack of temporal structure. This confounds attempts to use molecular clock-based methods to date the origin of this virus over the time-frame sampled so far, and means that phylogenetic measures alone cannot yet be used to determine HBV sequence authenticity. If genuine, this phylogenetic pattern indicates that the genotypes of HBV diversified long before the 16th century, and enables comparison of potential pathogenic similarities between modern and ancient HBV. These results have important implications for our understanding of the emergence and evolution of this common viral pathogen. PMID:29300782

Clonorchis sinensis Co-infection Could Affect the Disease State and Treatment Response of HBV Patients.

PubMed

Li, Wenfang; Dong, Huimin; Huang, Yan; Chen, Tingjin; Kong, Xiangzhan; Sun, Hengchang; Yu, Xinbing; Xu, Jin

2016-06-01

Clonorchis sinensis (C. sinensis) is considered to be an important parasitic zoonosis because it infects approximately 35 million people, while approximately 15 million were distributed in China. Hepatitis B virus (HBV) infection is a major public health issue. Two types of pathogens have the potential to cause human liver disease and eventually hepatocellular carcinoma. Concurrent infection with HBV and C. sinensis is often observed in some areas where C. sinensis is endemic. However, whether C. sinensis could impact HBV infection or vice versa remains unknown. Co-infection with C. sinensis and HBV develops predominantly in males. Co-infected C. sinensis and HBV patients presented weaker liver function and higher HBV DNA titers. Combination treatment with antiviral and anti-C. sinensis drugs in co-infected patients could contribute to a reduction in viral load and help with liver function recovery. Excretory-secretory products (ESPs) may, in some ways, increase HBV viral replication in vitro. A mixture of ESP and HBV positive sera could induce peripheral blood mononuclear cells (PBMCs) to produce higher level of Th2 cytokines including IL-4, IL-6 and IL-10 compared to HBV alone, it seems that due to presence of ESP, the cytokine production shift towards Th2. C. sinensis/HBV co-infected patients showed higher serum IL-6 and IL-10 levels and lower serum IFN-γ levels. Patients with concomitant C. sinensis and HBV infection presented weaker liver function and higher HBV DNA copies. In co-infected patients, the efficacy of anti-viral treatment was better in patients who were prescribed with entecavir and praziquantel than entecavir alone. One possible reason for the weaker response to antiviral therapies in co-infected patients was the shift in cytokine production from Th1 to Th2 that may inhibit viral clearance. C. sinensis/HBV co-infection could exacerbate the imbalance of Th1/Th2 cytokine.

A European multicenter study on the analytical performance of the VERIS HBV assay.

PubMed

Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Izopet, Jacques; Lombardi, Alessandra; Mancon, Alessandro; Marcos, Maria Angeles; Sauné, Karine; O Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel

Hepatitis B viral load monitoring is an essential part of managing patients with chronic Hepatits B infection. Beckman Coulter has developed the VERIS HBV Assay for use on the fully automated Beckman Coulter DxN VERIS Molecular Diagnostics System. 1 OBJECTIVES: To evaluate the analytical performance of the VERIS HBV Assay at multiple European virology laboratories. Precision, analytical sensitivity, negative sample performance, linearity and performance with major HBV genotypes/subtypes for the VERIS HBV Assay was evaluated. Precision showed an SD of 0.15 log 10 IU/mL or less for each level tested. Analytical sensitivity determined by probit analysis was between 6.8-8.0 IU/mL. Clinical specificity on 90 unique patient samples was 100.0%. Performance with 754 negative samples demonstrated 100.0% not detected results, and a carryover study showed no cross contamination. Linearity using clinical samples was shown from 1.23-8.23 log 10 IU/mL and the assay detected and showed linearity with major HBV genotypes/subtypes. The VERIS HBV Assay demonstrated comparable analytical performance to other currently marketed assays for HBV DNA monitoring. Copyright © 2017 Elsevier B.V. All rights reserved.

JS-K, a nitric oxide prodrug, induces DNA damage and apoptosis in HBV-positive hepatocellular carcinoma HepG2.2.15 cell.

PubMed

Liu, Zhengyun; Li, Guangmin; Gou, Ying; Xiao, Dongyan; Luo, Guo; Saavedra, Joseph E; Liu, Jie; Wang, Huan

2017-08-01

Hepatocellular carcinoma (HCC) is the most important cause of cancer-related death, and 85% of HCC is caused by chronic HBV infection, the prognosis of patients and the reduction of HBV DNA levels remain unsatisfactory. JS-K, a nitric oxide-releasing diazeniumdiolates, is effective against various tumors, but little is known on its effects on HBV positive HCC. We found that JS-K reduced the expression of HBsAg and HBeAg in HBV-positive HepG2.2.15 cells. This study aimed to further examine anti-tumor effects of JS-K on HepG2.2.15 cells. The MTT assay and colony forming assay were used to study the cell growth inhibition of JS-K; scratch assay and transwell assay were performed to detect cell migration. The cell cycle was detected by flow cytometry. The immunofluorescence, flow cytometry analysis, and western blot were used to study DNA damage and cell apoptosis. JS-K inhibited HepG2.2.15 cell growth in a dose-dependent manner, suppressed cell colony formation and migration, arrested cells gather in the G2 phase. JS-K (1-20μM) increased the expression of DNA damage-associated protein phosphorylation H 2 AX (γH 2 AX), phosphorylation of checkpoint kinase 1 (p-Chk1), phosphorylation of checkpoint kinase 2 (p-Chk2), ataxia-telangiectasia mutated (ATM), phosphorylation of ataxia-telangiectasia mutated rad3-related (p-ATR) and apoptotic-associated proteins cleaved caspase-3, cleaved caspase-7, cleaved poly ADP-ribose polymerase (cleaved PARP). The study demonstrated JS-K is effective against HBV-positive HepG2.2.15 cells, the mechanisms are not only related to inhibition of HBsAg and HBeAg secretion, but also related with induction of DNA damage and apoptosis. JS-K is a promising anti-cancer candidate against HBV-positive HCC. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Hepatitis B Core Antigen in Hepatocytes of Chronic Hepatitis B: Comparison between Indirect Immunofluorescence and Immunoperoxidase Method

PubMed Central

Tabassum, Shahina; Al-Mahtab, Mamun; Nessa, Afzalun; Jahan, Munira; Shamim Kabir, Chowdhury Mohammad; Kamal, Mohammad; Cesar Aguilar, Julio

2015-01-01

Background Hepatitis B virus (HBV) infection has many faces. Precore and core promoter mutants resemble inactive carrier status. The identification of hepatitis B core antigen (HBcAg) in hepatocytes may have variable clinical significance. The present study was undertaken to detect HBcAg in chronic hepatitis B (CHB) patients and to assess the efficacy of detection system by indirect immunofluorescence (IIF) and indirect immunoperoxidase (IIP). Materials and methods The study was done in 70 chronic HBV-infected patients. Out of 70 patients, eight (11.4%) were hepatitis B e antigen (HBeAg) positive and 62 (88.57%) were HBeAg negative. Hepatitis B core antigen was detected by indirect immunofluorescence (IIF) and indirect immunoperoxidase (IIP) methods in liver tissue. Results All HBeAg positive patients expressed HBcAg by both IIF and IIP methods. Out of 62 patients with HBeAg-negative CHB, HBcAg was detected by IIF in 55 (88.7%) patients and by IIP in 51 (82.26%) patients. A positive relation among viral load and HBcAg detection was also found. This was more evident in the case of HBeAg negative patients and showed a positive relation with HBV DNA levels. Conclusion Hepatitis B core antigen can be detected using the IIF from formalin fixed paraffin block preparation and also by IIP method. This seems to reflect the magnitudes of HBV replication in CHB. How to cite this article Raihan R, Tabassum S, Al-Mahtab M, Nessa A, Jahan M, Kabir CMS, Kamal M, Aguilar JC. Hepatitis B Core Antigen in Hepatocytes of Chronic Hepatitis B: Comparison between Indirect Immunofluorescence and Immunoperoxidase Method. Euroasian J Hepato-Gastroenterol 2015;5(1):7-10. PMID:29201677

Liver-Targeted Anti-HBV Single-Stranded Oligonucleotides with Locked Nucleic Acid Potently Reduce HBV Gene Expression In Vivo.

PubMed

Javanbakht, Hassan; Mueller, Henrik; Walther, Johanna; Zhou, Xue; Lopez, Anaïs; Pattupara, Thushara; Blaising, Julie; Pedersen, Lykke; Albæk, Nanna; Jackerott, Malene; Shi, Tianlai; Ploix, Corinne; Driessen, Wouter; Persson, Robert; Ravn, Jacob; Young, John A T; Ottosen, Søren

2018-06-01

Chronic hepatitis B infection (CHB) is an area of high unmet medical need. Current standard-of-care therapies only rarely lead to a functional cure, defined as durable hepatitis B surface antigen (HBsAg) loss following treatment. The goal for next generation CHB therapies is to achieve a higher rate of functional cure with finite treatment duration. To address this urgent need, we are developing liver-targeted single-stranded oligonucleotide (SSO) therapeutics for CHB based on the locked nucleic acid (LNA) platform. These LNA-SSOs target hepatitis B virus (HBV) transcripts for RNase-H-mediated degradation. Here, we describe a HBV-specific LNA-SSO that effectively reduces intracellular viral mRNAs and viral antigens (HBsAg and HBeAg) over an extended time period in cultured human hepatoma cell lines that were infected with HBV with mean 50% effective concentration (EC 50 ) values ranging from 1.19 to 1.66 μM. To achieve liver-specific targeting and minimize kidney exposure, this LNA-SSO was conjugated to a cluster of three N-acetylgalactosamine (GalNAc) moieties that direct specific binding to the asialoglycoprotein receptor (ASGPR) expressed specifically on the surface of hepatocytes. The GalNAc-conjugated LNA-SSO showed a strikingly higher level of potency when tested in the AAV-HBV mouse model as compared with its non-conjugated counterpart. Remarkably, higher doses of GalNAc-conjugated LNA-SSO resulted in a rapid and long-lasting reduction of HBsAg to below the detection limit for quantification, i.e., by 3 log10 (p < 0.0003). This antiviral effect depended on a close match between the sequences of the LNA-SSO and its HBV target, indicating that the antiviral effect is not due to non-specific oligonucleotide-driven immune activation. These data support the development of LNA-SSO therapeutics for the treatment of CHB infection. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Alteration of Mature Nucleocapsid and Enhancement of Covalently Closed Circular DNA Formation by Hepatitis B Virus Core Mutants Defective in Complete-Virion Formation.

PubMed

Cui, Xiuji; Luckenbaugh, Laurie; Bruss, Volker; Hu, Jianming

2015-10-01

Assembly of hepatitis B virus (HBV) begins with packaging of the pregenomic RNA (pgRNA) into immature nucleocapsids (NC), which are converted to mature NCs containing the genomic relaxed circular (RC) DNA as a result of reverse transcription. Mature NCs have two alternative fates: (i) envelopment by viral envelope proteins, leading to secretion extracellularly as virions, or (ii) disassembly (uncoating) to deliver their RC DNA content into the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, the template for viral transcription. How these two alternative fates are regulated remains to be better understood. The NC shell is composed of multiple copies of a single viral protein, the HBV core (HBc) protein. HBc mutations located on the surface of NC have been identified that allow NC maturation but block its envelopment. The potential effects of some of these mutations on NC uncoating and CCC DNA formation have been analyzed by transfecting HBV replication constructs into hepatoma cells. All envelopment-defective HBc mutations tested were competent for CCC DNA formation, indicating that core functions in envelopment and uncoating/nuclear delivery of RC DNA were genetically separable. Some of the envelopment-defective HBc mutations were found to alter specifically the integrity of mature, but not immature, NCs such that RC DNA became susceptible to nuclease digestion. Furthermore, CCC DNA formation could be enhanced by NC surface mutations that did or did not significantly affect mature NC integrity, indicating that the NC surface residues may be closely involved in NC uncoating and/or nuclear delivery of RC DNA. Hepatitis B virus (HBV) infection is a major health issue worldwide. HBV assembly begins with the packaging into immature nucleocapsids (NCs) of a viral RNA pregenome, which is converted to the DNA genome in mature NCs. Mature NCs are then selected for envelopment and secretion as complete-virion particles or, alternatively, can

Efficacy of Ledipasvir and Sofosbuvir Treatment of HCV Infection in Patients Coinfected With HBV.

PubMed

Liu, Chun-Jen; Chuang, Wan-Long; Sheen, I-Shyan; Wang, Horng-Yuan; Chen, Chi-Yi; Tseng, Kuo-Chih; Chang, Ting-Tsung; Massetto, Benedetta; Yang, Jenny C; Yun, Chohee; Knox, Steven J; Osinusi, Anu; Camus, Gregory; Jiang, Deyuan; Brainard, Diana M; McHutchison, John G; Hu, Tsung-Hui; Hsu, You-Chun; Lo, Gin-Ho; Chu, Chi-Jen; Chen, Jyh-Jou; Peng, Cheng-Yuan; Chien, Ron-Nan; Chen, Pei-Jer

2018-03-01

There have been reports of reactivation of hepatitis B virus (HBV) infection during treatment of hepatitis C virus (HCV) infection with direct-acting antiviral agents. We performed a prospective study of risks and outcomes of HCV infection treatment with ledipasvir and sofosbuvir in patients with HBV infection. We performed a phase 3b, multicenter, open-label study in Taiwan of 111 patients with HCV infection (61% HCV genotype 1, 39% HCV genotype 2 infection; 62% women, 16% with compensated cirrhosis) along with HBV infection. All but 1 were positive for the hepatitis B surface antigen (HBsAg); 1 patient who was HBsAg-positive at screening was found to be HBsAg-negative at baseline. Overall, 33% of participants had received prior treatment for HCV and 5% had previously been treated for HBV; no patient was on HBV therapy at the start of the study. All patients received a fixed-dose combination of 90 mg of the HCV NS5A inhibitor ledipasvir with 400 mg of the NS5B nucleotide analogue inhibitor sofosbuvir, once daily for 12 weeks. The primary endpoint was sustained virologic response 12 weeks after the end of therapy. All 111 patients (100%) achieved a sustained virologic response. Of the 37 patients with baseline HBV DNA below 20 IU/mL, 31 (84%) had at least 1 episode of quantifiable HBV DNA through posttreatment week 12. Of the 74 patients with baseline HBV DNA levels of 20 IU/mL or more, 39 (53%) had increases of HBV DNA greater than 1 log 10 IU/mL through posttreatment week 12. Overall, 5 patients had increased levels of HBV DNA concomitant with a level of alanine aminotransferase >2 times the upper limit of normal through posttreatment week 12. Of these, 3 patients started HBV treatment. In addition, 1 patient with HBV reactivation since week 8 and concomitant alanine aminotransferase elevation >2 times upper limit of normal at posttreatment week 48 started treatment at posttreatment week 53. This patient had clinical signs and symptoms associated with HBV

The Timing of Hepatitis B Virus (HBV) Immunization Relative to Human Immunodeficiency Virus (HIV) Diagnosis and the Risk of HBV Infection Following HIV Diagnosis

PubMed Central

Landrum, Michael L.; Hullsiek, Katherine Huppler; Chun, Helen M.; Crum-Cianflone, Nancy F.; Ganesan, Anuradha; Weintrob, Amy C.; Barthel, R. Vincent; O'Connell, Robert J.; Agan, Brian K.

2011-01-01

To assess associations between the timing of hepatitis B virus (HBV) immunization relative to human immunodeficiency virus (HIV) diagnosis and vaccine effectiveness, US Military HIV Natural History Study cohort participants without HBV infection at the time of HIV diagnosis were grouped by vaccination status, retrospectively followed from HIV diagnosis for incident HBV infection, and compared using Cox proportional hazards models. A positive vaccine response was defined as hepatitis B surface antibody level ≥10 IU/L. Of 1,877 participants enrolled between 1989 and 2008, 441 (23%) were vaccinated prior to HIV diagnosis. Eighty percent of those who received vaccine doses only before HIV diagnosis had a positive vaccine response, compared with 66% of those who received doses both before and after HIV and 41% of those who received doses only after HIV (P < 0.01 for both compared with persons vaccinated before HIV only). Compared with the unvaccinated, persons vaccinated only before HIV had reduced risk of HBV infection after HIV diagnosis (hazard ratio = 0.38, 95% confidence interval: 0.20, 0.75). No reduction in HBV infection risk was observed for other vaccination groups. These data suggest that completion of the vaccine series prior to HIV infection may be the optimal strategy for preventing this significant comorbid infection in HIV-infected persons. PMID:21051446

Rapid Deterioration of Latent HBV Hepatitis during Cushing Disease and Posttraumatic Stress Disorder after Earthquake.

PubMed

Tashiro, Ryosuke; Ogawa, Yoshikazu; Tominaga, Teiji

2017-07-01

Reactivation of the hepatitis B virus (HBV) is a risk in the 350 million HBV carriers worldwide. HBV reactivation may cause hepatocellular carcinoma, cirrhosis, and fulminant hepatitis, and HBV reactivation accompanied with malignant tumor and/or chemotherapy is a critical problem for patients with chronic HBV infection. Multiple risk factors causing an immunosuppressive state can also induce HBV reactivation.We present a case of HBV reactivation during an immunosuppressive state caused by Cushing disease and physical and psychological stress after a disaster. A 47-year-old Japanese woman was an inactive HBV carrier until the Great East Japan Earthquake occurred and follow-up was discontinued. One year after the earthquake she had intractable hypertension, and her visual acuity gradually worsened. Head magnetic resonance imaging showed a sellar tumor compressing the optic chiasm, and hepatic dysfunction with HBV reactivation was identified. Endocrinologic examination established the diagnosis as Cushing disease. After normalization of hepatic dysfunction with antiviral therapy, transsphenoidal tumor removal was performed that resulted in subtotal removal except the right cavernous portion. Steroid hormone supplementation was discontinued after 3 days of administration, and gamma knife therapy was performed for the residual tumor. Eighteen months after the operation, adrenocorticotropic hormone and cortisol values returned to normal. The patient has been free from tumor regrowth and HBV reactivation throughout the postoperative course.Accomplishment of normalization with intrinsic steroid value with minimization of steroid supplementation should be established. Precise operative procedures and careful treatment planning are essential to avoid HBV reactivation in patients with this threatening condition. Georg Thieme Verlag KG Stuttgart · New York.

Type I IFN augments IL-27-dependent TRIM25 expression to inhibit HBV replication.

PubMed

Tan, Guangyun; Xiao, Qingfei; Song, Hongxiao; Ma, Feng; Xu, Fengchao; Peng, Di; Li, Na; Wang, Xiaosong; Niu, Junqi; Gao, Pujun; Qin, F Xiao-Feng; Cheng, Genhong

2018-03-01

Hepatitis B virus (HBV) can cause chronic hepatitis B, which may lead to cirrhosis and liver cancer. Type I interferon (IFN) is an approved drug for the treatment of chronic hepatitis B. However, the fundamental mechanisms of antiviral action by type I IFN and the downstream signaling pathway are unclear. TRIM25 is an IFN-stimulated gene (ISG) that has an important role in RIG-I ubiquitination and activation. Whether TRIM25 is induced in liver cells by type I IFN to mediate anti-HBV function remains unclear. Here we report that interleukin-27 (IL-27) has a critical role in IFN-induced TRIM25 upregulation. TRIM25 induction requires both STAT1 and STAT3. In TRIM25 knockout HepG2 cells, type I IFN production was consistently attenuated and HBV replication was increased, whereas overexpression of TRIM25 in HepG2 cells resulted in elevated IFN production and reduced HBV replication. More interestingly, we found that TRIM25 expression was downregulated in HBV patients and the addition of serum samples from HBV patients could inhibit TRIM25 expression in HepG2 cells, suggesting that HBV might have involved a mechanism to inhibit antiviral ISG expression and induce IFN resistance. Collectively, our results demonstrate that type I IFN -induced TRIM25 is an important factor in inhibiting HBV replication, and the IFN-IL-27-TRIM25 axis may represent a new target for treating HBV infection.

Infectivity of HBV DNA positive donations identified in look-back studies in Hyogo-Prefecture, Japan.

PubMed

Bouike, Y; Imoto, S; Mabuchi, O; Kokubunji, A; Kai, S; Okada, M; Taniguchi, R; Momose, S; Uchida, S; Nishio, H

2011-04-01

To clarify transfusion incidence of hepatitis B virus (HBV) infected blood negative for mini pool-nucleic acid amplification testing (MP-NAT). Japanese Red Cross (JRC) blood centres screen donated blood to avoid contamination with HBV. However, a low copy number of HBV may be overlooked. In Hyogo-Prefecture, JRC blood centres screened 787 695 donations for HBV from April 2005 to March 2009. Of these, 685 844 were donations from the repeat donors. To detect the donors with HBV, serological tests, MP-NAT and/or individual donation (ID)-NAT were performed. To detect the recipients with transfusion-transmitted HBV infection (TTHBI), serological analysis and/or ID-NAT were performed. In this study, 265 of the 685 844 repeat donations were serologically and/or MP-NAT positive for HBV. Their repository samples from the previous donation were examined in a look-back study; 13 of the 265 repository samples proved ID-NAT positive. Twelve recipients were transfused with HBV-infected blood components derived from 10 of the 13 HBV-infected donors. Only 1 of the 12 recipients was identified as TTHBI case. Seven of the 12 recipients escaped from our follow-up study and 4 recipients were negative for HBV during the observation period. On the basis of the look-back study among the repeat donors in Hyogo-Prefecture, Japan, donations with HBV-infected blood negative for MP-NAT occurred with a frequency of 13 in 685 844 donations (∼1/53 000 donations). However, more than half of the recipients transfused with HBV-infected blood negative for MP-NAT could not be followed up. It is necessary to establish a more cautious follow-up system. © 2010 The Authors. Transfusion Medicine © 2010 British Blood Transfusion Society.

Dynamics of an HBV Model with Drug Resistance Under Intermittent Antiviral Therapy

NASA Astrophysics Data System (ADS)

Zhang, Ben-Gong; Tanaka, Gouhei; Aihara, Kazuyuki; Honda, Masao; Kaneko, Shuichi; Chen, Luonan

2015-06-01

This paper studies the dynamics of the hepatitis B virus (HBV) model and the therapy regimens of HBV disease. First, we propose a new mathematical model of HBV with drug resistance, and then analyze its qualitative and dynamical properties. Combining the clinical data and theoretical analysis, we demonstrate that our model is biologically plausible and also computationally viable. Second, we demonstrate that the intermittent antiviral therapy regimen is one of the possible strategies to treat this kind of complex disease. There are two main advantages of this regimen, i.e. it not only may delay the development of drug resistance, but also may reduce the duration of on-treatment time compared with the long-term continuous medication. Moreover, such an intermittent antiviral therapy can reduce the adverse side effects. Our theoretical model and computational results provide qualitative insight into the progression of HBV, and also a possible new therapy for HBV disease.

New Class of Cargo Protein in Tetrahymena thermophila Dense Core Secretory Granules

PubMed Central

Haddad, Alex; Bowman, Grant R.; Turkewitz, Aaron P.

2002-01-01

Regulated exocytosis of dense core secretory granules releases biologically active proteins in a stimulus-dependent fashion. The packaging of the cargo within newly forming granules involves a transition: soluble polypeptides condense to form water-insoluble aggregates that constitute the granule cores. Following exocytosis, the cores generally disassemble to diffuse in the cell environment. The ciliates Tetrahymena thermophila and Paramecium tetraurelia have been advanced as genetically manipulatable systems for studying exocytosis via dense core granules. However, all of the known granule proteins in these organisms condense to form the architectural units of lattices that are insoluble both before and after exocytosis. Using an approach designed to detect new granule proteins, we have now identified Igr1p (induced during granule regeneration). By structural criteria, it is unrelated to the previously characterized lattice-forming proteins. It is distinct in that it is capable of dissociating from the insoluble lattice following secretion and therefore represents the first diffusible protein identified in ciliate granules. PMID:12456006

Effects of hepatitis C virus core protein and nonstructural protein 4B on the Wnt/β-catenin pathway.

PubMed

Jiang, Xiao-Hua; Xie, Yu-Tao; Cai, Ya-Ping; Ren, Jing; Ma, Tao

2017-05-25

Hepatitis C virus (HCV) core protein and nonstructural protein 4B (NS4B) are potentially oncogenic. Aberrant activation of the Wnt/β-catenin signaling pathway is closely associated with hepatocarcinogenesis. We investigated the effects of HCV type 1b core protein and NS4B on Wnt/β-catenin signaling in various liver cells, and explored the molecular mechanism underlying HCV-related hepatocarcinogenesis. Compared with the empty vector control, HCV core protein and NS4B demonstrated the following characteristics in the Huh7 cells: significantly enhanced β-catenin/Tcf-dependent transcriptional activity (F = 40.87, P < 0.01); increased nuclear translocation of β-catenin (F = 165.26, P < 0.01); upregulated nuclear β-catenin, cytoplasmic β-catenin, Wnt1, c-myc, and cyclin D1 protein expression (P < 0.01); and promoted proliferation of Huh7 cells (P < 0.01 or P < 0.05). Neither protein enhanced β-catenin/Tcf-dependent transcriptional activity in the LO2 cells (F = 0.65, P > 0.05), but they did significantly enhance Wnt3a-induced β-catenin/Tcf-dependent transcriptional activity (F = 64.25, P < 0.01), and promoted the nuclear translocation of β-catenin (F = 66.54, P < 0.01) and the Wnt3a-induced proliferation of LO2 cells (P < 0.01 or P < 0.05). Moreover, activation of the Wnt/β-catenin signaling pathway was greater with the core protein than with NS4B (P < 0.01 or P < 0.05). HCV core protein and NS4B directly activate the Wnt/β-catenin signaling pathway in Huh7 cells and LO2 cells induced by Wnt3a. These data suggest that HCV core protein and NS4B contribute to HCV-associated hepatocellular carcinogenesis.

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

PubMed Central

Sarkar, Jayeeta; Saha, Debraj; Bandyopadhyay, Bhaswati; Saha, Bibhuti; Kedia, Deepika; Guha Mazumder, D.N.; Chakravarty, Runu; Guha, Subhasish Kamal

2016-01-01

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

Clonorchis sinensis Co-infection Could Affect the Disease State and Treatment Response of HBV Patients

PubMed Central

Huang, Yan; Chen, Tingjin; Kong, Xiangzhan; Sun, Hengchang; Yu, Xinbing; Xu, Jin

2016-01-01

Background Clonorchis sinensis (C. sinensis) is considered to be an important parasitic zoonosis because it infects approximately 35 million people, while approximately 15 million were distributed in China. Hepatitis B virus (HBV) infection is a major public health issue. Two types of pathogens have the potential to cause human liver disease and eventually hepatocellular carcinoma. Concurrent infection with HBV and C. sinensis is often observed in some areas where C. sinensis is endemic. However, whether C. sinensis could impact HBV infection or vice versa remains unknown. Principal Findings Co-infection with C. sinensis and HBV develops predominantly in males. Co-infected C. sinensis and HBV patients presented weaker liver function and higher HBV DNA titers. Combination treatment with antiviral and anti-C. sinensis drugs in co-infected patients could contribute to a reduction in viral load and help with liver function recovery. Excretory-secretory products (ESPs) may, in some ways, increase HBV viral replication in vitro. A mixture of ESP and HBV positive sera could induce peripheral blood mononuclear cells (PBMCs) to produce higher level of Th2 cytokines including IL-4, IL-6 and IL-10 compared to HBV alone, it seems that due to presence of ESP, the cytokine production shift towards Th2. C. sinensis/HBV co-infected patients showed higher serum IL-6 and IL-10 levels and lower serum IFN-γ levels. Conclusions/Significance Patients with concomitant C. sinensis and HBV infection presented weaker liver function and higher HBV DNA copies. In co-infected patients, the efficacy of anti-viral treatment was better in patients who were prescribed with entecavir and praziquantel than entecavir alone. One possible reason for the weaker response to antiviral therapies in co-infected patients was the shift in cytokine production from Th1 to Th2 that may inhibit viral clearance. C. sinensis/HBV co-infection could exacerbate the imbalance of Th1/Th2 cytokine. PMID:27348302

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

PubMed

Sarkar, Jayeeta; Saha, Debraj; Bandyopadhyay, Bhaswati; Saha, Bibhuti; Kedia, Deepika; Guha Mazumder, D N; Chakravarty, Runu; Guha, Subhasish Kamal

2016-05-01

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. 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. 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. 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.

HCV core protein promotes hepatocyte proliferation and chemoresistance by inhibiting NR4A1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Yongsheng, E-mail: yongshengtanwhu@126.com; Li, Yan, E-mail: liyansd2@163.com

This study investigated the effect of HCV core protein on the proliferation of hepatocytes and hepatocellular carcinoma cells (HCC), the influence of HCV core protein on HCC apoptosis induced by the chemotherapeutic agent cisplatin, and the mechanism through which HCV core protein acts as a potential oncoprotein in HCV-related HCC by measuring the levels of NR4A1 and Runt-related transcription factor 3 (RUNX3), which are associated with tumor suppression and chemotherapy resistance. In the present study, PcDNA3.1-core and RUNX3 siRNA were transfected into LO2 and HepG2 cells using Lipofectamine 2000. LO2-core, HepG2-core, LO2-RUNX3 {sup low} and control cells were treated withmore » different concentrations of cisplatin for 72 h, and cell proliferation and apoptosis were assayed using the CellTiter 96{sup ®}Aqueous Non-Radioactive Cell Proliferation Assay Kit. Western blot and real time PCR analyses were used to detect NR4A1, RUNX3, smad7, Cyclin D1 and BAX. Confocal microscopy was used to determine the levels of NR4A1 in HepG2 and HepG2-core cells. The growth rate of HepG2-core cells was considerably greater than that of HepG2 cells. HCV core protein increased the expression of cyclin D1 and decreased the expressions of NR4A1 and RUNX3. In LO2 – RUNX3 {sup low}, the rate of cell proliferation and the level of cisplatin resistance were the same as in the LO2 -core. These results suggest that HCV core protein decreases the sensitivity of hepatocytes to cisplatin by inhibiting the expression of NR4A1 and promoting the expression of smad7, which negatively regulates the TGF-β pathway. This effect results in down regulation of RUNX3, a target of the TGF-β pathway. Taken together, these findings indicate that in hepatocytes, HCV core protein increases drug resistance and inhibits cell apoptosis by inhibiting the expressions of NR4A1 and RUNX3. - Highlights: • HCV core protein inhibits HepG2 cell sensitivity to cisplatin. • Core expression in HepG2 decreases

Combinatorial RNA Interference Therapy Prevents Selection of Pre-existing HBV Variants in Human Liver Chimeric Mice

PubMed Central

Shih, Yao-Ming; Sun, Cheng-Pu; Chou, Hui-Hsien; Wu, Tzu-Hui; Chen, Chun-Chi; Wu, Ping-Yi; Enya Chen, Yu-Chen; Bissig, Karl-Dimiter; Tao, Mi-Hua

2015-01-01

Selection of escape mutants with mutations within the target sequence could abolish the antiviral RNA interference activity. Here, we investigated the impact of a pre-existing shRNA-resistant HBV variant on the efficacy of shRNA therapy. We previously identified a highly potent shRNA, S1, which, when delivered by an adeno-associated viral vector, effectively inhibits HBV replication in HBV transgenic mice. We applied the “PICKY” software to systemically screen the HBV genome, then used hydrodynamic transfection and HBV transgenic mice to identify additional six highly potent shRNAs. Human liver chimeric mice were infected with a mixture of wild-type and T472C HBV, a S1-resistant HBV variant, and then treated with a single or combined shRNAs. The presence of T472C mutant compromised the therapeutic efficacy of S1 and resulted in replacement of serum wild-type HBV by T472C HBV. In contrast, combinatorial therapy using S1 and P28, one of six potent shRNAs, markedly reduced titers for both wild-type and T472C HBV. Interestingly, treatment with P28 alone led to the emergence of escape mutants with mutations in the P28 target region. Our results demonstrate that combinatorial RNAi therapy can minimize the escape of resistant viral mutants in chronic HBV patients. PMID:26482836

Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Wen-Ta; Li, Hui-Chun; Lee, Shen-Kao

Highlights: •HCV core and F proteins could induce hepatocyte proliferation in the transgenic mice. •β-Catenin signaling pathway was activated by core protein in the transgenic mice. •β-Catenin signaling pathway was activated by myc-F protein in the transgenic mice. •Expression of SMA protein was enhanced by core but not myc-F protein. -- Abstract: The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made itmore » more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway.« less

HBVPathDB: a database of HBV infection-related molecular interaction network.

PubMed

Zhang, Yi; Bo, Xiao-Chen; Yang, Jing; Wang, Sheng-Qi

2005-03-21

To describe molecules or genes interaction between hepatitis B viruses (HBV) and host, for understanding how virus' and host's genes and molecules are networked to form a biological system and for perceiving mechanism of HBV infection. The knowledge of HBV infection-related reactions was organized into various kinds of pathways with carefully drawn graphs in HBVPathDB. Pathway information is stored with relational database management system (DBMS), which is currently the most efficient way to manage large amounts of data and query is implemented with powerful Structured Query Language (SQL). The search engine is written using Personal Home Page (PHP) with SQL embedded and web retrieval interface is developed for searching with Hypertext Markup Language (HTML). We present the first version of HBVPathDB, which is a HBV infection-related molecular interaction network database composed of 306 pathways with 1 050 molecules involved. With carefully drawn graphs, pathway information stored in HBVPathDB can be browsed in an intuitive way. We develop an easy-to-use interface for flexible accesses to the details of database. Convenient software is implemented to query and browse the pathway information of HBVPathDB. Four search page layout options-category search, gene search, description search, unitized search-are supported by the search engine of the database. The database is freely available at http://www.bio-inf.net/HBVPathDB/HBV/. The conventional perspective HBVPathDB have already contained a considerable amount of pathway information with HBV infection related, which is suitable for in-depth analysis of molecular interaction network of virus and host. HBVPathDB integrates pathway data-sets with convenient software for query, browsing, visualization, that provides users more opportunity to identify regulatory key molecules as potential drug targets and to explore the possible mechanism of HBV infection based on gene expression datasets.

Fibrosis assessment in patients with chronic hepatitis B virus (HBV) infection

PubMed Central

Parikh, Pathik; Ryan, John D.

2017-01-01

Chronic hepatitis B virus (HBV) infection is a major cause of liver morbidity and mortality worldwide. While a proportion of the 250 million individuals chronically infected with HBV will not come to significant harm or require therapy, many others risk developing complications of the end-stage liver disease such as decompensated cirrhosis and hepatocellular carcinoma (HCC), without intervention. Due to the complex natural history of HBV infection, patients require an expert assessment to interpret biochemistry, viral serology and appropriately stage the disease, and to initiate monitoring and/or therapy where indicated. The detection and quantification of liver fibrosis is a key factor for disease management and prognostication for an individual with HBV. The reliance on invasive liver biopsy to stage disease is diminishing with the advent of robust non-invasive blood- and imaging-based algorithms which can reliably stage disease in many cases. These tests are now incorporated into International guidelines for HBV management and relied upon daily to inform clinical judgement. Both blood- and imaging-based approaches have advantages over liver biopsy, including minimal risks, lower cost, better patient acceptance and speed of results, while disadvantages include lower diagnostic accuracy in intermediate disease stages and variability with co-existing hepatic inflammation or steatosis. This review outlines the methods of fibrosis assessment in chronic HBV infection and focuses on the most commonly used blood- and imaging-based non-invasive tests, reviewing their diagnostic performance and applicability to patient care. PMID:28251119

Adolescent booster with hepatitis B virus vaccines decreases HBV infection in high-risk adults.

PubMed

Wang, Yuting; Chen, Taoyang; Lu, Ling-Ling; Wang, Minjie; Wang, Dongmei; Yao, Hongyu; Fan, Chunsun; Qi, Jun; Zhang, Yawei; Qu, Chunfeng

2017-02-15

Neutralizing antibodies (anti-HBs) after immunization with hepatitis B virus (HBV) vaccines against HBV surface antigen (HBsAg) wane after 10-15years. We analyzed the effect of an adolescent booster given to vaccination-protected children born to mothers with different HBsAg-carrying status against HBV infection in their mature adulthood. A total of 9793 individuals, who were HBsAg-negative at childhood (baseline) and donated blood samples, both during childhood and adulthood, from the vaccination group in "Qidong Hepatitis B Intervention Study", were enrolled. Among them 7414 received a one-dose, 10μg-recombinant HBV vaccine booster at 10-14years of age. At endpoint (23-28years of age), we determined the HBV serological markers and quantified their serum HBV-DNA in each of the chronic HBV-infected adults. Fifty-seven adults were identified as chronic HBV infection, indicated by HBsAg(+)&anti-HBc(+) for more than 6months. The individuals who were born to HBsAg-positive mothers (high-risk adults) had significantly increased risk of developing chronic HBV infections in adulthood compared with those who were born to HBsAg-negative mothers; the adjusted odds ratio (OR) was 12.56, 95%CI:7.14-22.08. The seronegative status of anti-HBs at 10-11years of age significantly increased the risk of HBV infections among the high-risk adults. When HBsAg(-)&anti-HBc(+) children who were born to HBsAg-positive mothers 70% of them remained as the status and 10% of them developed HBsAg(+)&anti-HBc(+). While when they were born to HBsAg-negative mothers 1.05% HBsAg(-)&anti-HBc(+) children developed HBsAg(+)&anti-HBc(+) and 24.74% of them remained as the status in 12-18years. One dose of adolescent booster showed significant protection on high-risk adults from chronic HBV infection; P for trend was 0.015. Maternal HBsAg-positive status was an independent risk factor for vaccination-protected children to develop HBV breakthrough infection in adulthood. Adolescent boosters might be

Serum Level of Antibodies Against Hepatitis B Core Protein Is Associated With Clinical Relapse After Discontinuation of Nucleos(t)ide Analogue Therapy.

PubMed

Chi, Heng; Li, Zhandong; Hansen, Bettina E; Yu, Tao; Zhang, Xiaoyong; Sun, Jian; Hou, Jinlin; Janssen, Harry L A; Peng, Jie

2018-06-11

Levels of antibodies against the hepatitis B virus (HBV) core protein (anti-HBc) have been associated with response to nucleos(t)ide analogue and (peg)interferon therapy in patients with chronic HBV infection. We performed a prospective study to determine whether the total serum level of anti-HBc level (immunoglobulins M and G) is associated with clinical relapse after discontinuation of nucleos(t)ide analogue-based therapy. We collected data from patients with chronic HBV infection who discontinued nucleos(t)ide analogue therapy according to pre-specified stopping criteria, recruited from November 2012 through July 2016 at an academic hospital in Guangzhou, China. Patients were followed through February 2017. We performed comprehensive biochemical and virologic tests at every visit, and anti-HBc was quantified for 2 years after treatment cessation (at baseline and weeks 4, 8, 12, 24, 48, and 96). The primary endpoint was clinical relapse, defined as level of HBV DNA >2000 IU/mL and level of alanine aminotransferase more than 2-fold the upper limit of normal-these were also the criteria for retreatment. We followed 100 patients (71% positive for HB e antigen [HBeAg] at the start of nucleos(t)ide analogue therapy, 43% treated with entecavir or tenofovir) for a median of 2.5 years after stopping therapy. Clinical relapse occurred in 39 patients (in 46% of patients at year 4 after discontinuation). High level of anti-HBc at the end of treatment (hazard ratio [HR], 0.31 per log IU/mL; P=.002) and low level of HB surface antigen (HBsAg) at the end of treatment (HR, 1.71 per log IU/mL; P=.032) were associated with a reduced risk of clinical relapse after adjusting for age, start of nucleos(t)ide analogue therapy HBeAg-status, and consolidation therapy duration. At year 4, 21% of patients with anti-HBc levels at the end of treatment ≥1000 IU/mL developed a clinical relapse compared to 85% of patients with levels <100 IU/mL (P<.001). An HBsAg level at the end of

Chronic hepatitis B infection and HBV DNA-containing capsids: Modeling and analysis

NASA Astrophysics Data System (ADS)

Manna, Kalyan; Chakrabarty, Siddhartha P.

2015-05-01

We analyze the dynamics of chronic HBV infection taking into account both uninfected and infected hepatocytes along with the intracellular HBV DNA-containing capsids and the virions. While previous HBV models have included either the uninfected hepatocytes or the intracellular HBV DNA-containing capsids, our model accounts for both these two populations. We prove the conditions for local and global stability of both the uninfected and infected steady states in terms of the basic reproduction number. Further, we incorporate a time lag in the model to encompass the intracellular delay in the production of the infected hepatocytes and find that this delay does not affect the overall dynamics of the system. The results for the model and the delay model are finally numerically illustrated.

Role of peripheral blood mononuclear cell transportation from mother to baby in HBV intrauterine infection.

PubMed

Shao, Qingliang; Zhao, Xiaxia; Yao Li, M D

2013-12-01

We aimed to investigate the role of peripheral blood mononuclear cell transportation from mother to baby in hepatitis B virus (HBV) intrauterine infection. Thirty HBsAg-positive pregnant women in the second trimester and their aborted fetuses were included in this study. Enzyme-linked-immunosorbent-assay was utilized to detect HBsAg in the peripheral blood of pregnant women and the femoral vein blood of their aborted fetuses. HBV-DNA in serum and peripheral blood mononuclear cells (PBMC) and GSTM1 alleles of pregnant women and their aborted fetuses were detected by nested polymerase chain reaction (PCR) and seminested PCR, respectively. We also examined the location of placenta HBsAg and HBcAb using immunohistochemical staining. The expression of placenta HBV-DNA was detected by in situ hybridization. For the 30 aborted fetuses, the HBV intrauterine infection rate was 43.33%. The HBV-positive rates of HBsAg in peripheral blood, serum, and PBMC were 10% (3/30), 23.33% (7/30), and 33.33% (10/30), respectively. Maternal-fetal PBMC transport was significantly positively correlated with fetal PBMC HBV-DNA (P = 0.004). Meanwhile, the rates of HBV infection gradually decreased from the maternal side to the fetus side of placenta (decidual cells > trophoblastic cells > villous mesenchymal cells > villous capillary endothelial cells). However, no significant correlation between placenta HBV infection and HBV intrauterine infection was observed (P = 0.410). HBV intrauterine infection was primarily due to peripheral blood mononuclear cell maternal-fetal transportation in the second trimester in pregnant women.

Glucose-regulated protein 78 is an intracellular antiviral factor against hepatitis B virus.

PubMed

Ma, Yan; Yu, Jun; Chan, Henry L Y; Chen, Yang-chao; Wang, Hua; Chen, Ying; Chan, Chu-yan; Go, Minnie Y Y; Tsai, Sau-na; Ngai, Sai-ming; To, Ka-fai; Tong, Joanna H M; He, Qing-Yu; Sung, Joseph J Y; Kung, Hsiang-fu; Cheng, Christopher H K; He, Ming-liang

2009-11-01

Hepatitis B virus (HBV) infection is a global public health problem that plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. However, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. In this study, two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics were applied to analyze the host response to HBV using an inducible HBV-producing cell line, HepAD38. Twenty-three proteins were identified as differentially expressed with glucose-regulated protein 78 (GRP78) as one of the most significantly up-regulated proteins induced by HBV replication. This induction was further confirmed in both HepAD38 and HepG2 cells transfected with HBV-producing plasmids by real time RT-PCR and Western blotting as well as in HBV-infected human liver biopsies by immunohistochemistry. Knockdown of GRP78 expression by RNA interference resulted in a significant increase of both intracellular and extracellular HBV virions in the transient HBV-producing HepG2 cells concomitant with enhanced levels of hepatitis B surface antigen and e antigen in the culture medium. Conversely overexpression of GRP78 in HepG2 cells led to HBV suppression concomitant with induction of the positive regulatory circuit of GRP78 and interferon-beta1 (IFN-beta1). In this connection, the IFN-beta1-mediated 2',5'-oligoadenylate synthetase and RNase L signaling pathway was noted to be activated in GRP78-overexpressing HepG2 cells. Moreover GRP78 was significantly down-regulated in the livers of chronic hepatitis B patients after effective anti-HBV treatment (p = 0.019) as compared with their counterpart pretreatment liver biopsies. In conclusion, the present study demonstrates for the first time that GRP78 functions as an endogenous anti-HBV factor via the IFN-beta1-2',5'-oligoadenylate synthetase-RNase L pathway in hepatocytes. Induction of hepatic GRP78 may provide a novel therapeutic

Glucose-regulated Protein 78 Is an Intracellular Antiviral Factor against Hepatitis B Virus*

PubMed Central

Ma, Yan; Yu, Jun; Chan, Henry L. Y.; Chen, Yang-chao; Wang, Hua; Chen, Ying; Chan, Chu-yan; Go, Minnie Y. Y.; Tsai, Sau-na; Ngai, Sai-ming; To, Ka-fai; Tong, Joanna H. M.; He, Qing-Yu; Sung, Joseph J. Y.; Kung, Hsiang-fu; Cheng, Christopher H. K.; He, Ming-liang

2009-01-01

Hepatitis B virus (HBV) infection is a global public health problem that plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. However, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. In this study, two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics were applied to analyze the host response to HBV using an inducible HBV-producing cell line, HepAD38. Twenty-three proteins were identified as differentially expressed with glucose-regulated protein 78 (GRP78) as one of the most significantly up-regulated proteins induced by HBV replication. This induction was further confirmed in both HepAD38 and HepG2 cells transfected with HBV-producing plasmids by real time RT-PCR and Western blotting as well as in HBV-infected human liver biopsies by immunohistochemistry. Knockdown of GRP78 expression by RNA interference resulted in a significant increase of both intracellular and extracellular HBV virions in the transient HBV-producing HepG2 cells concomitant with enhanced levels of hepatitis B surface antigen and e antigen in the culture medium. Conversely overexpression of GRP78 in HepG2 cells led to HBV suppression concomitant with induction of the positive regulatory circuit of GRP78 and interferon-β1 (IFN-β1). In this connection, the IFN-β1-mediated 2′,5′-oligoadenylate synthetase and RNase L signaling pathway was noted to be activated in GRP78-overexpressing HepG2 cells. Moreover GRP78 was significantly down-regulated in the livers of chronic hepatitis B patients after effective anti-HBV treatment (p = 0.019) as compared with their counterpart pretreatment liver biopsies. In conclusion, the present study demonstrates for the first time that GRP78 functions as an endogenous anti-HBV factor via the IFN-β1-2′,5′-oligoadenylate synthetase-RNase L pathway in hepatocytes. Induction of hepatic GRP78 may provide a novel therapeutic

Prevalence of HBV DNA among 20 million seronegative blood donations in China from 2010 to 2015.

PubMed

Liu, Chao; Chang, Le; Ji, Huimin; Guo, Fei; Zhang, Kuo; Lin, Guigao; Zhang, Rui; Li, Jinming; Wang, Lunan

2016-11-11

The prevalence of HBV DNA among seronegative blood donations in China has not been studied extensively on a nationwide scale. The aim of this study was to analyse the prevalence, trend, distributions, and serological characteristics of HBV DNA positive/seronegative blood donations. We collected HBV test data from all blood banks of China from 2010 to 2015 and performed supplemental serological tests and quantitative detection of HBV DNA of the seronegative/HBV DNA positive blood donations. We analysed the prevalence of HBV DNA among seronegative blood donations screened by varying nucleotide acid test (NAT) reagents. The analysis results showed that a total of 20,084,187 seronegative blood donations were screened by NAT from 2010 to 2015 in China. The average frequency of HBV DNA among seronegative blood donations was 1/1482, but there has been a steady increase from 1/1861 in 2011 to 1/1269 in 2015. The geographical distribution of seronegative and HBV DNA positive blood donations was roughly consistent with that of HBsAg. The most common serological pattern was HBeAb and HBcAb positive. In conclusion, our study offeres fundamental data of seronegative and HBV DNA positive blood donations throughout China.

Prevalence of HBV DNA among 20 million seronegative blood donations in China from 2010 to 2015

PubMed Central

Liu, Chao; Chang, Le; Ji, Huimin; Guo, Fei; Zhang, Kuo; Lin, Guigao; Zhang, Rui; Li, Jinming; Wang, Lunan

2016-01-01

The prevalence of HBV DNA among seronegative blood donations in China has not been studied extensively on a nationwide scale. The aim of this study was to analyse the prevalence, trend, distributions, and serological characteristics of HBV DNA positive/seronegative blood donations. We collected HBV test data from all blood banks of China from 2010 to 2015 and performed supplemental serological tests and quantitative detection of HBV DNA of the seronegative/HBV DNA positive blood donations. We analysed the prevalence of HBV DNA among seronegative blood donations screened by varying nucleotide acid test (NAT) reagents. The analysis results showed that a total of 20,084,187 seronegative blood donations were screened by NAT from 2010 to 2015 in China. The average frequency of HBV DNA among seronegative blood donations was 1/1482, but there has been a steady increase from 1/1861 in 2011 to 1/1269 in 2015. The geographical distribution of seronegative and HBV DNA positive blood donations was roughly consistent with that of HBsAg. The most common serological pattern was HBeAb and HBcAb positive. In conclusion, our study offeres fundamental data of seronegative and HBV DNA positive blood donations throughout China. PMID:27833112

CRISPR/Cas9-based tools for targeted genome editing and replication control of HBV.

PubMed

Peng, Cheng; Lu, Mengji; Yang, Dongliang

2015-10-01

Hepatitis B virus (HBV) infection remains a major global health problem because current therapies rarely eliminate HBV infections to achieve a complete cure. A different treatment paradigm to effectively clear HBV infection and eradicate latent viral reservoirs is urgently required. In recent years, the development of a new RNA-guided gene-editing tool, the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) system, has greatly facilitated site-specific mutagenesis and represents a very promising potential therapeutic tool for diseases, including for eradication of invasive pathogens such as HBV. Here, we review recent advances in the use of CRISPR/Cas9, which is designed to target HBV specific DNA sequences to inhibit HBV replication and to induce viral genome mutation, in cell lines or animal models. Advantages, limitations and possible solutions, and proposed directions for future research are discussed to highlight the opportunities and challenges of CRISPR/Cas9 as a new, potentially curative therapy for chronic hepatitis B infection.

Occult HBV infection status among chronic hepatitis C and hemodialysis patients in Northeastern Egypt: regional and national overview.

PubMed

Mandour, Mohamed; Nemr, Nader; Shehata, Atef; Kishk, Rania; Badran, Dahlia; Hawass, Nashaat

2015-01-01

Occult hepatitis B infection (OBI) is considered to be one of the major risks for patients suffering from end-stage renal disease (ESRD) on regular hemodialysis (HD) and patients with chronic hepatitis C virus (HCV) infection. This study compared the prevalence of OBI among these two high-risk groups in the Suez Canal region, Northeastern Egypt, to obtain a better national overview of the magnitude of OBI in this region. Serum samples were collected from 165 HD patients and 210 chronic HCV-infected patients. Anti-HCV antibody, hepatitis B surface antigen (HBsAg), total hepatitis B core (anti-HBc) antibody, and hepatitis B surface antibody (anti-HBs) were detected by enzyme-linked immunosorbent assay (ELISA). HCV RNA was detected using a quantitative real-time RT-PCR assay, and HBV was detected using a nested PCR. All patients were negative for HBsAg. A total of 49.1% and 25.2% of the patients in the HD and HCV groups, respectively, were anti-HBc-positive. In addition, more anti-HBs-positive patients were detected in the HD group compared to the HCV group (52.1% and 11.4%, respectively). Three cases were positive for HBV DNA in the HD group, while eighteen positive cases were detected in the HCV group. Both study groups showed significant differences in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) level as well as anti-HBc, anti-HBs and HBV-DNA positivity. OBI was more prevalent among chronic HCV patients than HD patients in the Suez Canal region, Egypt, with rates of 8.5% and 1.8%, respectively. However, more precise assessment of this infection requires regular patient follow-up using HBV DNA detection methods.

Renal health after long-term exposure to tenofovir disoproxil fumarate (TDF) in HIV/HBV positive adults in Ghana.

PubMed

Villa, G; Phillips, R O; Smith, C; Stockdale, A J; Beloukas, A; Appiah, L T; Chadwick, D; Ruggiero, A; Sarfo, F S; Post, F; Geretti, A M

2018-06-01

The study assessed markers of renal health in HIV/HBV co-infected patients receiving TDF-containing antiretroviral therapy in Ghana. Urinary protein-to-creatinine ratio (uPCR) and albumin-to-protein ratio (uAPR) were measured cross-sectionally after a median of four years of TDF. At this time, alongside extensive laboratory testing, patients underwent evaluation of liver stiffness and blood pressure. The estimated glomerular filtration rate (eGFR) was measured longitudinally before and during TDF therapy. Among 101 participants (66% women, median age 44 years, median CD4 count 572 cells/mm 3 ) 21% and 17% had detectable HIV-1 RNA and HBV DNA, respectively. Overall 35% showed hypertension, 6% diabetes, 7% liver stiffness indicative of cirrhosis, and 18% urinary excretion of Schistosoma antigen. Tubular proteinuria occurred in 16% of patients and was independently predicted by female gender and hypertension. The eGFR declined by median 1.8 ml/min/year during TDF exposure (IQR -4.4, -0.0); more pronounced declines (≥ 5 ml/min/year) occurred in 22% of patients and were associated with receiving ritonavir-boosted lopinavir rather than efavirenz. HBV DNA, HBeAg, transaminases, and liver stiffness were not predictive of renal function abnormalities. The findings mandate improved diagnosis and management of hypertension and suggest targeted laboratory monitoring of patients receiving TDF alongside a booster in sub-Saharan Africa. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

HBV DNA genome co-transfection procedure for the evaluation of relative fitness

PubMed Central

Sevic, Ina; Gonzalez Lopez Ledesma, Maria Mora; Flichman, Diego Martin

2017-01-01

Hepatitis B virus (HBV) has a high mutation rate and exists as a mixture of genetically different but closely related variants. We present a HBV DNA co-transfection fitness assay and use it to evaluate the relative fitness of different HBV variants in two scenarios: seroconversion process and occupation of an ecological niche. In the seroconversion experiment, subgenotype D1 (sgtD1) deletion (1763–1770) had significantly lower fitness comparing with both sgtD1 wild type and sgtD1mut G1896A, while, in the case of occupation of ecological niche experiment, the results showed the same relative fitness between all of the genotype combinations, except F1b-F4. In this case sgtF1b clearly overgrow sgtF4, which is in accordance with the observation that F1b is the most prevalent in the new infections in Argentina. In summary, we present a method aimed to evaluate HBV viral fitness which improve the analysis of the relative frequency of viral variants during the HBV infection process. PMID:28472081

Brief Report: HIV/HBV Coinfection is a Significant Risk Factor for Liver Fibrosis in Tanzanian HIV-Infected Adults.

PubMed

Hawkins, Claudia; Christian, Beatrice; Fabian, Emanuel; Macha, Irene; Gawile, Cecilia; Mpangala, Shida; Ulenga, Nzovu; Thio, Chloe L; Ammerman, Lauren R; Mugusi, Ferdinand; Fawzi, Wafaie; Green, Richard; Murphy, Robert

2017-11-01

In sub-Saharan Africa, the burden of liver disease associated with chronic hepatitis B virus (HBV) and HIV is unknown. We characterized liver disease using aspartate aminotransferase-to-platelet ratio index (APRI) and FIB-4 in patients with HIV, HBV, and HIV/HBV coinfection in Tanzania. Using a cross-sectional design, we compared the prevalence of liver fibrosis in treatment-naive HIV monoinfected, HBV monoinfected, and HIV/HBV-coinfected adults enrolled at Management and Development for Health (MDH)-supported HIV treatment clinics in Dar es Salaam, Tanzania. Risk factors associated with significant fibrosis (APRI >0.5 and FIB-4 >1.45) were examined. Two hundred sixty-seven HIV-infected, 165 HBV-infected, and 63 HIV/HBV-coinfected patients were analyzed [44% men, median age 37 (interquartile range 14), body mass index 23 (7)]. APRI and FIB-4 were strongly correlated (r = 0.78, P < 0.001, R = 0.61). Overall median APRI scores were low {HIV/HBV [0.36 (interquartile range 0.4)], HIV [0.23 (0.17)], HBV [0.29 (0.15)] (P < 0.01)}. In multivariate analyses, HIV/HBV coinfection was associated with APRI >0.5 [HIV/HBV vs. HIV: odds ratio (OR) 3.78 (95% confidence interval: 1.91 to 7.50)], [HIV/HBV vs. HBV: OR 2.61 (1.26 to 5.44)]. HIV RNA per 1 log10 copies/mL increase [OR 1.53 (95% confidence interval: 1.04 to 2.26)] and HBV DNA per 1 log10 copies/mL increase [OR 1.36 (1.15, 1.62)] were independently associated with APRI >0.5 in HIV-infected and HBV-infected patients, respectively. HIV/HBV coinfection is an important risk factor for significant fibrosis. Higher levels of circulating HIV and HBV virus may play a direct role in liver fibrogenesis. Prompt diagnosis and aggressive monitoring of liver disease in HIV/HBV coinfection is warranted.

The HBV DNA cutoff value for discriminating patients with HBeAgnegative chronic hepatitis B from inactive carriers.

PubMed

Kim, Eun Sun; Seo, Yeon Seok; Keum, Bora; Kim, Ji Hoon; A, Hyonggin; Yim, Hyung Joon; Kim, Yong Sik; Jeen, Yoon Tae; Lee, Hong Sik; Chun, Hoon Jai; Um, Soon Ho; Duck Kim, Chang; Ryu, Ho Sang

2011-05-01

Patients with HBeAg-negative chronic hepatitis B (CHB) has a significantly different prognosis than inactive carriers; there is however, no reliable strategy for accurately differentiating these two disease conditions. To determine a strategy for discriminating patients with HBeAg-negative CHB from inactive carriers. Consecutive inactive carriers (i.e. HBeAg-negativity, anti-HBe-positivity, normal ALT levels, and HBV DNA < 2000 IU/mL) were enrolled. HBV reactivation was defined as the elevation of the HBV DNA level to ≥ 2000 IU/mL. Patients were classified into true inactive carriers when their HBV DNA levels remained at < 2000 IU/mL or false inactive carriers when their HBV DNA levels increased to ≥ 2000 IU/mL during the first year. The Mean ± SD age of 208 inactive carriers (140 males) was 47.7 ± 12.6 years. The Mean ± SD serum ALT and HBV DNA levels were 22.8 ± 8.6 IU/L and 360 ± 482 IU/mL, respectively. HBV reactivation developed in 41 (19.7%) patients during the first year. Baseline HBV DNA and ALT levels differed significantly between true inactive and false inactive carriers. The AUROCs of the baseline ALT and HBV DNA levels for predicting a false inactive carrier were 0.609 and 0.831, respectively. HBV reactivation developed more often in patients with a baseline HBV DNA level of ≥ 200 IU/mL than in those with a baseline HBV DNA level of < 200 IU/mL during a Mean ± SD follow-up of 622 ± 199 days. The HBV DNA level was useful for discriminating patients with HBeAg-negative CHB from true inactive carriers. The follow-up strategies applied to inactive carriers need to vary with their HBV DNA levels.

Hepatitis C virus core protein induces hepatic steatosis via Sirt1-dependent pathway.

PubMed

Zhang, Chuanhai; Wang, Jingjing; Zhang, Hanlin; Liu, Shunai; Lee, Hyuek Jong; Jin, Wanzhu; Cheng, Jun

2018-05-01

Hepatic steatosis is a common feature of patients with chronic hepatitis C. Previous reports have shown that the overexpression of hepatitis C virus core-encoding sequences (hepatitis C virus genotypes 3a and 1b) significantly induces intracellular triglyceride accumulation. However, the underlying mechanism has not yet been revealed. To investigate whether Sirt1 is involved in hepatitis C virus-mediated hepatic steatosis, the overexpression of hepatitis C virus core 1b protein and Sirt1 and the knockdown of Sirt1 in HepG2 cells were performed. To confirm the results of the cellular experiment liver-specific Sirt1 KO mice with lentivirus-mediated hepatitis C virus core 1b overexpression were studied. Our results show that hepatitis C virus core 1b protein overexpression led to the accumulation of triglycerides in HepG2 cells. Notably the expression of PPARγ2 was dramatically increased at both the mRNA and protein levels by hepatitis C virus core 1b overexpression. The protein expression of Sirt1 is an upstream regulator of PPARγ2 and was also significantly increased after core 1b overexpression. In addition, the overexpression or knockdown of Sirt1 expression alone was sufficient to modulate p300-mediated PPARγ2 deacetylation. In vivo studies showed that hepatitis C virus core protein 1b-induced hepatic steatosis was attenuated in liver-specific Sirt1 KO mice by downregulation of PPARγ2 expression. Sirt1 mediates hepatitis C virus core protein 1b-induced hepatic steatosis by regulation of PPARγ2 expression. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

High prevalence of human parvovirus 4 infection in HBV and HCV infected individuals in shanghai.

PubMed

Yu, Xuelian; Zhang, Jing; Hong, Liang; Wang, Jiayu; Yuan, Zhengan; Zhang, Xi; Ghildyal, Reena

2012-01-01

Human parvovirus 4 (PARV4) has been detected in blood and diverse tissues samples from HIV/AIDS patients who are injecting drug users. Although B19 virus, the best characterized human parvovirus, has been shown to co-infect patients with hepatitis B or hepatitis C virus (HBV, HCV) infection, the association of PARV4 with HBV or HCV infections is still unknown.The aim of this study was to characterise the association of viruses belonging to PARV4 genotype 1 and 2 with chronic HBV and HCV infection in Shanghai.Serum samples of healthy controls, HCV infected subjects and HBV infected subjects were retrieved from Shanghai Center for Disease Control and Prevention (SCDC) Sample Bank. Parvovirus-specific nested-PCR was performed and results confirmed by sequencing. Sequences were compared with reference sequences obtained from Genbank to derive phylogeny trees.The frequency of parvovirus molecular detection was 16-22%, 33% and 41% in healthy controls, HCV infected and HBV infected subjects respectively, with PARV4 being the only parvovirus detected. HCV infected and HBV infected subjects had a significantly higher PARV4 prevalence than the healthy population. No statistical difference was found in PARV4 prevalence between HBV or HCV infected subjects. PARV4 sequence divergence within study groups was similar in healthy subjects, HBV or HCV infected subjects.Our data clearly demonstrate that PARV4 infection is strongly associated with HCV and HBV infection in Shanghai but may not cause increased disease severity.

TRC8-dependent degradation of hepatitis C virus immature core protein regulates viral propagation and pathogenesis

PubMed Central

Aizawa, Sayaka; Okamoto, Toru; Sugiyama, Yukari; Kouwaki, Takahisa; Ito, Ayano; Suzuki, Tatsuya; Ono, Chikako; Fukuhara, Takasuke; Yamamoto, Masahiro; Okochi, Masayasu; Hiraga, Nobuhiko; Imamura, Michio; Chayama, Kazuaki; Suzuki, Ryosuke; Shoji, Ikuo; Moriishi, Kohji; Moriya, Kyoji; Koike, Kazuhiko; Matsuura, Yoshiharu

2016-01-01

Signal-peptide peptidase (SPP) is an intramembrane protease that participates in the production of the mature core protein of hepatitis C virus (HCV). Here we show that SPP inhibition reduces the production of infectious HCV particles and pathogenesis. The immature core protein produced in SPP-knockout cells or by treatment with an SPP inhibitor is quickly degraded by the ubiquitin–proteasome pathway. Oral administration of the SPP inhibitor to transgenic mice expressing HCV core protein (CoreTg) reduces the expression of core protein and ameliorates insulin resistance and liver steatosis. Moreover, the haploinsufficiency of SPP in CoreTg has similar effects. TRC8, an E3 ubiquitin ligase, is required for the degradation of the immature core protein. The expression of the HCV core protein alters endoplasmic reticulum (ER) distribution and induces ER stress in SPP/TRC8 double-knockout cells. These data suggest that HCV utilizes SPP cleavage to circumvent the induction of ER stress in host cells. PMID:27142248

Immune-escape mutations and stop-codons in HBsAg develop in a large proportion of patients with chronic HBV infection exposed to anti-HBV drugs in Europe.

PubMed

Colagrossi, Luna; Hermans, Lucas E; Salpini, Romina; Di Carlo, Domenico; Pas, Suzan D; Alvarez, Marta; Ben-Ari, Ziv; Boland, Greet; Bruzzone, Bianca; Coppola, Nicola; Seguin-Devaux, Carole; Dyda, Tomasz; Garcia, Federico; Kaiser, Rolf; Köse, Sukran; Krarup, Henrik; Lazarevic, Ivana; Lunar, Maja M; Maylin, Sarah; Micheli, Valeria; Mor, Orna; Paraschiv, Simona; Paraskevis, Dimitros; Poljak, Mario; Puchhammer-Stöckl, Elisabeth; Simon, François; Stanojevic, Maja; Stene-Johansen, Kathrine; Tihic, Nijaz; Trimoulet, Pascale; Verheyen, Jens; Vince, Adriana; Lepej, Snjezana Zidovec; Weis, Nina; Yalcinkaya, Tülay; Boucher, Charles A B; Wensing, Annemarie M J; Perno, Carlo F; Svicher, Valentina

2018-06-01

HBsAg immune-escape mutations can favor HBV-transmission also in vaccinated individuals, promote immunosuppression-driven HBV-reactivation, and increase fitness of drug-resistant strains. Stop-codons can enhance HBV oncogenic-properties. Furthermore, as a consequence of the overlapping structure of HBV genome, some immune-escape mutations or stop-codons in HBsAg can derive from drug-resistance mutations in RT. This study is aimed at gaining insight in prevalence and characteristics of immune-associated escape mutations, and stop-codons in HBsAg in chronically HBV-infected patients experiencing nucleos(t)ide analogues (NA) in Europe. This study analyzed 828 chronically HBV-infected European patients exposed to ≥ 1 NA, with detectable HBV-DNA and with an available HBsAg-sequence. The immune-associated escape mutations and the NA-induced immune-escape mutations sI195M, sI196S, and sE164D (resulting from drug-resistance mutation rtM204 V, rtM204I, and rtV173L) were retrieved from literature and examined. Mutations were defined as an aminoacid substitution with respect to a genotype A or D reference sequence. At least one immune-associated escape mutation was detected in 22.1% of patients with rising temporal-trend. By multivariable-analysis, genotype-D correlated with higher selection of ≥ 1 immune-associated escape mutation (OR[95%CI]:2.20[1.32-3.67], P = 0.002). In genotype-D, the presence of ≥ 1 immune-associated escape mutations was significantly higher in drug-exposed patients with drug-resistant strains than with wild-type virus (29.5% vs 20.3% P = 0.012). Result confirmed by analysing drug-naïve patients (29.5% vs 21.2%, P = 0.032). Strong correlation was observed between sP120T and rtM204I/V (P < 0.001), and their co-presence determined an increased HBV-DNA. At least one NA-induced immune-escape mutation occurred in 28.6% of patients, and their selection correlated with genotype-A (OR[95%CI]:2.03[1.32-3.10],P = 0.001). Finally

Comparison of Abbott and Da-an real-time PCR for quantitating serum HBV DNA.

PubMed

Qiu, Ning; Li, Rui; Yu, Jian-Guo; Yang, Wen; Zhang, Wei; An, Yong; Li, Tong; Liu, Xue-En; Zhuang, Hui

2014-09-07

To compare the performance of the Da-an real-time hepatitis B virus (HBV) DNA assay and Abbott RealTime HBV assay. HBV DNA standards as well as a total of 180 clinical serum samples from patients with chronic hepatitis B were measured using the Abbott and Da-an real-time polymerase chain reaction (PCR) assays. Correlation and Bland-Altman plot analysis was used to compare the performance of the Abbott and Da-an assays. The HBV DNA levels were logarithmically transformed for analysis. All statistical analyses were performed using SPSS for Windows version 18.0. The correlation between the two assays was analyzed by Pearson's correlation and linear regression. The Bland-Altman plots were used for the analysis of agreement between the two assays. A P value of < 0.05 was considered statistically significant. The HBV DNA values measured by the Abbott or Da-an assay were significantly correlated with the expected values of HBV DNA standards (r = 0.999, for Abbott; r = 0.987, for Da-an, P < 0.001). A Bland-Altman plot showed good agreement between these two assays in detecting HBV DNA standards. Among the 180 clinical serum samples, 126 were quantifiable by both assays. Fifty-two samples were detectable by the Abbott assay but below the detection limit of the Da-an assay. Moreover, HBV DNA levels measured by the Abbott assay were significantly higher than those of the Da-an assay (6.23 ± 1.76 log IU/mL vs 5.46 ± 1.55 log IU/mL, P < 0.001). A positive correlation was observed between HBV DNA concentrations determined by the two assays in 126 paired samples (r = 0.648, P < 0.001). One hundred and fifteen of 126 (91.3%) specimens tested with both assays were within mean difference ± 1.96 SD of HBV DNA levels. The Da-an assay presented lower sensitivity and a narrower linear range as compared to the Abbott assay, suggesting the need to be improved.

Silencing of GSTP1 gene by CpG island DNA hypermethylation in HBV-associated hepatocellular carcinomas.

PubMed

Zhong, Sheng; Tang, Mandy W; Yeo, Winnie; Liu, Cuiling; Lo, Y M Dennis; Johnson, Philip J

2002-04-01

Glutathione S-transferases, enzymes that defend cells against damage mediated by oxidant and electrophilic carcinogens, may be critical determinants of cancer pathogenesis. In this report, we assess the role of epigenetic silencing of the GSTP1 gene, a gene encoding the pi-class glutathione S-transferase, in the pathogenesis of hepatitis B virus (HBV)-associated hepatocellular carcinomas (HCC). The cell lines Hep3B, HepG2, and a cohort of 43 HBV-associated HCC tissue specimens and corresponding nontumor tissues were subjected to analysis for GSTP1 epigenetic alteration and expression. GSTP1 "CpG" island DNA hypermethylation in the liver cell lines, and the tissue specimens were determined by methylation-specific PCR and correlated with expression of the gene using reverse-transcription PCR, immunoblotting, and immunohistochemistry. GSTP1 CpG island DNA hypermethylation was detected in 28 of 43 (65.1%) HCC tissues and 4 of 40 (10%) corresponding nontumor tissues. GSTP1 protein was absent in those cases showing hypermethylation of the gene. Similarly, DNA from Hep3B and HepG2 cell lines displayed complete GSTP1 hypermethylation in the CpG island, and they failed to express GSTP1 mRNA and the corresponding protein product. Treatment of the cell lines with the DNA methyltransferase inhibitor 5-aza-deoxycytidine reversed the hypermethylation, and restored GSTP1 mRNA and polypeptide expression. These data indicate that epigenetic silencing of GSTP1 gene expression by CpG island DNA hypermethylation is common in human HBV-associated HCC. In addition, somatic GSTP1 inactivation via CpG island hypermethylation may contribute to the pathogenesis of this malignancy.

Evaluation of vaccination efficiency against HBV among Syrian multitransfused patients.

PubMed

Yazji, Wadad; Habal, Wafaa; Menem, Fawza

2018-03-05

This cross-sectional study estimates HBV prevalence and evaluates vaccination efficiency among multitransfused patients. 159 patients with various hemoglobinopathies were tested for HBsAg, anti-HBs, and anti-HBc, using enzyme-linked immunosorbent assay (ELISA). The serological results were then compared with the relevant documentation in medical records. Seropositivity of HBV was detected in 1/8 of recruited patients. Serological immunity was found in only half of patients, while the other half were either infected or non-immune. The vaccination against HBV appeared inefficient in almost half of vaccinated patients and was not documented in the medical records of 1/6 of patients. Thus, multitransfused patients are at risk of acquiring hepatitis B infection. Applying prophylactic vaccination, documenting vaccine doses, and monitoring immune response are highly recommended.

Interplay between SIRT1 and hepatitis B virus X protein in the activation of viral transcription.

PubMed

Deng, Jian-Jun; Kong, Ka-Yiu Edwin; Gao, Wei-Wei; Tang, Hei-Man Vincent; Chaudhary, Vidyanath; Cheng, Yun; Zhou, Jie; Chan, Chi-Ping; Wong, Danny Ka-Ho; Yuen, Man-Fung; Jin, Dong-Yan

2017-04-01

Hepatitis B virus (HBV) genome is organized into a minichromosome known as covalently closed circular DNA (cccDNA), which serves as the template for all viral transcripts. SIRT1 is an NAD + -dependent protein deacetylase which activates HBV transcription by promoting the activity of cellular transcription factors and coactivators. How SIRT1 and viral transactivator X protein (HBx) might affect each other remains to be clarified. In this study we show synergy and mutual dependence between SIRT1 and HBx in the activation of HBV transcription. All human sirtuins SIRT1 through SIRT7 activated HBV gene expression. The steady-state levels of SIRT1 protein were elevated in HBV-infected liver tissues and HBV-replicating hepatoma cells. SIRT1 interacted with HBx and potentiated HBx transcriptional activity on precore promoter and covalently closed circular DNA (cccDNA) likely through a deacetylase-independent mechanism, leading to more robust production of cccDNA, pregenomic RNA and surface antigen. SIRT1 and HBx proteins were more abundant when both were expressed. SIRT1 promoted the recruitment of HBx as well as cellular transcriptional factors and coactivators such as PGC-1α and FXRα to cccDNA. Depletion of SIRT1 suppressed HBx recruitment. On the other hand, SIRT1 recruitment to cccDNA was compromised when HBx was deficient. Whereas pharmaceutical agonists of SIRT1 such as resveratrol activated HBV transcription, small-molecule inhibitors of SIRT1 including sirtinol and Ex527 exhibited anti-HBV activity. Taken together, our findings revealed not only the interplay between SIRT1 and HBx in the activation of HBV transcription but also new strategies and compounds for developing antivirals against HBV. Copyright © 2017 Elsevier B.V. All rights reserved.

HCV core protein represses the apoptosis and improves the autophagy of human hepatocytes

PubMed Central

Liu, Changhong; Qu, Aihua; Han, Xiaochun; Wang, Yiguo

2015-01-01

Objectives: This study aims to investigate the influence on human hepatocytes apoptosis and autophagy by the hepatitis C virus (HCV) core protein. Methods: QSG-7701, a human-derived non-neoplastic liver cell line, was transfected with PIRES-core vector that was a eukaryotic vector to express HCV core protein. Fluorescence microscope was used to observe the changes of nuclei in apoptosis cells by Annex in V-FITC/PI double staining. Flow cytometry was applied to detect the rate of cell apoptosis. Western blotting was used to detect the expression of HCV core protein, transcription factor nuclear factor-kappa B (NF-κB), autophagic biomarker microtubule associated protein 1 light chain 3 (LC3), and Beclin-1. Results: The apoptosis rate was significantly lower (P < 0.05) in QSG7701/core group (transfected with PIRES-core vector, (1.34±0.07)%) than in QSG7701 group (no transfection, (2.35±0.11)%) and in QSG7701 QSG7701/pcDNA3.1 group (transfected with pcDNA3.1 vector, (2.58±0.1)%). NF-κB expression was up-expressed in QSG7701/core group than in QSG7701/pcDNA3.1 group and QSG7701 group (P < 0.05). LC3-II expression and Beclin-1 expression was significant higher in QSG7701/core group than in the QSG7701/pcDNA3.1 group and QSG7701 group (P < 0.05). Conclusion: HCV core protein can repress the apoptosis and improve the autophagy of QSG7701 through up-regulating NF-κB and Beclin-1 expression. PMID:26629077

Immunological Properties of Hepatitis B Core Antigen Fusion Proteins

NASA Astrophysics Data System (ADS)

Francis, Michael J.; Hastings, Gillian Z.; Brown, Alan L.; Grace, Ken G.; Rowlands, David J.; Brown, Fred; Clarke, Berwyn E.

1990-04-01

The immunogenicity of a 19 amino acid peptide from foot-and-mouth disease virus has previously been shown to approach that of the inactivated virus from which it was derived after multimeric particulate presentation as an N-terminal fusion with hepatitis B core antigen. In this report we demonstrate that rhinovirus peptide-hepatitis B core antigen fusion proteins are 10-fold more immunogenic than peptide coupled to keyhole limpet hemocyanin and 100-fold more immunogenic than uncoupled peptide with an added helper T-cell epitope. The fusion proteins can be readily administered without adjuvant or with adjuvants acceptable for human and veterinary application and can elicit a response after nasal or oral dosing. The fusion proteins can also act as T-cell-independent antigens. These properties provide further support for their suitability as presentation systems for "foreign" epitopes in the development of vaccines.

The New Aptima HBV Quant Real-Time TMA Assay Accurately Quantifies Hepatitis B Virus DNA from Genotypes A to F

PubMed Central

Dauvillier, Claude; Dubernet, Fabienne; Poveda, Jean-Dominique; Laperche, Syria; Hézode, Christophe; Pawlotsky, Jean-Michel

2017-01-01

ABSTRACT Sensitive and accurate hepatitis B virus (HBV) DNA detection and quantification are essential to diagnose HBV infection, establish the prognosis of HBV-related liver disease, and guide the decision to treat and monitor the virological response to antiviral treatment and the emergence of resistance. Currently available HBV DNA platforms and assays are generally designed for batching multiple specimens within an individual run and require at least one full day of work to complete the analyses. The aim of this study was to evaluate the ability of the newly developed, fully automated, one-step Aptima HBV Quant assay to accurately detect and quantify HBV DNA in a large series of patients infected with different HBV genotypes. The limit of detection of the assay was estimated to be 4.5 IU/ml. The specificity of the assay was 100%. Intra-assay and interassay coefficients of variation ranged from 0.29% to 5.07% and 4.90% to 6.85%, respectively. HBV DNA levels from patients infected with HBV genotypes A to F measured with the Aptima HBV Quant assay strongly correlated with those measured by two commercial real-time PCR comparators (Cobas AmpliPrep/Cobas TaqMan HBV test, version 2.0, and Abbott RealTime HBV test). In conclusion, the Aptima HBV Quant assay is sensitive, specific, and reproducible and accurately quantifies HBV DNA in plasma samples from patients with chronic HBV infections of all genotypes, including patients on antiviral treatment with nucleoside or nucleotide analogues. The Aptima HBV Quant assay can thus confidently be used to detect and quantify HBV DNA in both clinical trials with new anti-HBV drugs and clinical practice. PMID:28202793

The New Aptima HBV Quant Real-Time TMA Assay Accurately Quantifies Hepatitis B Virus DNA from Genotypes A to F.

PubMed

Chevaliez, Stéphane; Dauvillier, Claude; Dubernet, Fabienne; Poveda, Jean-Dominique; Laperche, Syria; Hézode, Christophe; Pawlotsky, Jean-Michel

2017-04-01

Sensitive and accurate hepatitis B virus (HBV) DNA detection and quantification are essential to diagnose HBV infection, establish the prognosis of HBV-related liver disease, and guide the decision to treat and monitor the virological response to antiviral treatment and the emergence of resistance. Currently available HBV DNA platforms and assays are generally designed for batching multiple specimens within an individual run and require at least one full day of work to complete the analyses. The aim of this study was to evaluate the ability of the newly developed, fully automated, one-step Aptima HBV Quant assay to accurately detect and quantify HBV DNA in a large series of patients infected with different HBV genotypes. The limit of detection of the assay was estimated to be 4.5 IU/ml. The specificity of the assay was 100%. Intra-assay and interassay coefficients of variation ranged from 0.29% to 5.07% and 4.90% to 6.85%, respectively. HBV DNA levels from patients infected with HBV genotypes A to F measured with the Aptima HBV Quant assay strongly correlated with those measured by two commercial real-time PCR comparators (Cobas AmpliPrep/Cobas TaqMan HBV test, version 2.0, and Abbott RealTi m e HBV test). In conclusion, the Aptima HBV Quant assay is sensitive, specific, and reproducible and accurately quantifies HBV DNA in plasma samples from patients with chronic HBV infections of all genotypes, including patients on antiviral treatment with nucleoside or nucleotide analogues. The Aptima HBV Quant assay can thus confidently be used to detect and quantify HBV DNA in both clinical trials with new anti-HBV drugs and clinical practice. Copyright © 2017 American Society for Microbiology.

Genetic variations of NTCP are associated with susceptibility to HBV infection and related hepatocellular carcinoma.

PubMed

Wang, Peng; Mo, Ruidong; Lai, Rongtao; Xu, Yumin; Lu, Jie; Zhao, Gangde; Liu, Yuhan; Cao, Zhujun; Wang, Xiaolin; Li, Ziqiang; Lin, Lanyi; Zhou, Huijuan; Cai, Wei; Wang, Hui; Bao, Shisan; Xiang, Xiaogang; Xie, Qing

2017-12-01

Sodium taurocholate cotransporting polypeptide (NTCP), encoded by gene SLC10A1, is a receptor for hepatitis B virus (HBV). The aim of the current study was to investigate the role of NTCP polymorphisms in HBV susceptibility, cirrhosis and hepatocarcinogenesis. A total 1221 cases [including 866 chronic hepatitis B (CHB), 238 liver cirrhosis (LC), 117 hepatocellular carcinoma (HCC) patients] and 1232 healthy controls (HCs) were recruited, and 6 single nucleotide polymorphisms (SNPs) were genotyped. Meta-analysis was executed among 14591 CHBs and 12396 HCs to determine the association between NTCP polymorphisms and HBV infection, cirrhosis or hepatocarcinogenesis. The frequency of rs2296651-GA was inversely correlated with CHB, LC or HCC patients [adjusted OR(95%CI)=0.16(0.11-0.23), p <0.001; 0.34(0.21-0.55), p =0.001; or 0.46(0.25-0.83), p =0.008], respectively, compared with HCs. Meta-analysis also showed that NTCP rs2296651-GA was inversely associated with HBV infection [OR(95%CI)=0.532(0.287-0.986), p =0.028, codominant] or HBV-related HCC [OR(95%CI)=0.701(0.564-0.872), p =0.001, recessive]. Furthermore, the frequency of rs943277-GA was positively correlated with HBV infection [adjusted OR(95%CI)=2.42(1.05-5.54), p =0.032, codominant]. Our data suggest that NTCP mutants contribute to the susceptibility of HBV infection or HBV-related HCC.

Genetic variations of NTCP are associated with susceptibility to HBV infection and related hepatocellular carcinoma

PubMed Central

Xu, Yumin; Lu, Jie; Zhao, Gangde; Liu, Yuhan; Cao, Zhujun; Wang, Xiaolin; Li, Ziqiang; Lin, Lanyi; Zhou, Huijuan; Cai, Wei; Wang, Hui; Bao, Shisan; Xiang, Xiaogang; Xie, Qing

2017-01-01

Sodium taurocholate cotransporting polypeptide (NTCP), encoded by gene SLC10A1, is a receptor for hepatitis B virus (HBV). The aim of the current study was to investigate the role of NTCP polymorphisms in HBV susceptibility, cirrhosis and hepatocarcinogenesis. A total 1221 cases [including 866 chronic hepatitis B (CHB), 238 liver cirrhosis (LC), 117 hepatocellular carcinoma (HCC) patients] and 1232 healthy controls (HCs) were recruited, and 6 single nucleotide polymorphisms (SNPs) were genotyped. Meta-analysis was executed among 14591 CHBs and 12396 HCs to determine the association between NTCP polymorphisms and HBV infection, cirrhosis or hepatocarcinogenesis. The frequency of rs2296651-GA was inversely correlated with CHB, LC or HCC patients [adjusted OR(95%CI)=0.16(0.11-0.23), p<0.001; 0.34(0.21-0.55), p=0.001; or 0.46(0.25-0.83), p=0.008], respectively, compared with HCs. Meta-analysis also showed that NTCP rs2296651-GA was inversely associated with HBV infection [OR(95%CI)=0.532(0.287-0.986), p=0.028, codominant] or HBV-related HCC [OR(95%CI)=0.701(0.564-0.872), p=0.001, recessive]. Furthermore, the frequency of rs943277-GA was positively correlated with HBV infection [adjusted OR(95%CI)=2.42(1.05-5.54), p=0.032, codominant]. Our data suggest that NTCP mutants contribute to the susceptibility of HBV infection or HBV-related HCC. PMID:29285260

Analysis of core-periphery organization in protein contact networks reveals groups of structurally and functionally critical residues.

PubMed

Isaac, Arnold Emerson; Sinha, Sitabhra

2015-10-01

The representation of proteins as networks of interacting amino acids, referred to as protein contact networks (PCN), and their subsequent analyses using graph theoretic tools, can provide novel insights into the key functional roles of specific groups of residues. We have characterized the networks corresponding to the native states of 66 proteins (belonging to different families) in terms of their core-periphery organization. The resulting hierarchical classification of the amino acid constituents of a protein arranges the residues into successive layers - having higher core order - with increasing connection density, ranging from a sparsely linked periphery to a densely intra-connected core (distinct from the earlier concept of protein core defined in terms of the three-dimensional geometry of the native state, which has least solvent accessibility). Our results show that residues in the inner cores are more conserved than those at the periphery. Underlining the functional importance of the network core, we see that the receptor sites for known ligand molecules of most proteins occur in the innermost core. Furthermore, the association of residues with structural pockets and cavities in binding or active sites increases with the core order. From mutation sensitivity analysis, we show that the probability of deleterious or intolerant mutations also increases with the core order. We also show that stabilization centre residues are in the innermost cores, suggesting that the network core is critically important in maintaining the structural stability of the protein. A publicly available Web resource for performing core-periphery analysis of any protein whose native state is known has been made available by us at http://www.imsc.res.in/ ~sitabhra/proteinKcore/index.html.

Identification of a Functional, CRM-1-Dependent Nuclear Export Signal in Hepatitis C Virus Core Protein

PubMed Central

Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata

2011-01-01

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified. We show here that the aa(109–133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1–173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication. Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection. PMID:22039426

Hepatitis C virus core protein triggers abnormal porphyrin metabolism in human hepatocellular carcinoma cells.

PubMed

Nakano, Takafumi; Moriya, Kyoji; Koike, Kazuhiko; Horie, Toshiharu

2018-01-01

Porphyria cutanea tarda (PCT), the most common of the human porphyrias, arises from a deficiency of uroporphyrinogen decarboxylase. Studies have shown a high prevalence of hepatitis C virus (HCV) infection in patients with PCT. While these observations implicate HCV infection as a risk factor for PCT pathogenesis, the mechanism of interaction between the virus and porphyrin metabolism is unknown. This study aimed to assess the effect of HCV core protein on intracellular porphyrin metabolism to elucidate the link between HCV infection and PCT. The accumulation and excretion of porphyrins after treatment with 5-aminolevulinic acid, a porphyrin precursor, were compared between cells stably expressing HCV core protein and controls. Cells expressing HCV core protein had lower amounts of intracellular protoporphyrin IX and heme and had higher amounts of excreted coproporphyrin III, the oxidized form of coproporphyrinogen III, compared with controls. These observations suggest that HCV core protein affects porphyrin metabolism and facilitates the export of excess coproporphyrinogen III and/or coproporphyrin III, possibly via porphyrin transporters. Real-time PCR analysis revealed that the presence of HCV core protein increased the mRNA expression of porphyrin exporters ABCG2 and FLVCR1. Western blot analysis showed a higher expression level of FLVCR1, but not ABCG2, as well as a higher expression level of mature ALAS1, which is the rate-limiting enzyme in the heme synthesis pathway, in HCV core protein-expressing cells compared with controls. The data indicate that HCV core protein induced abnormal intracellular porphyrin metabolism, with an over-excretion of coproporphyrin III. These findings may partially account for the susceptibility of HCV-infected individuals to PCT development.

Identifying protein complex by integrating characteristic of core-attachment into dynamic PPI network.

PubMed

Shen, Xianjun; Yi, Li; Jiang, Xingpeng; He, Tingting; Yang, Jincai; Xie, Wei; Hu, Po; Hu, Xiaohua

2017-01-01

How to identify protein complex is an important and challenging task in proteomics. It would make great contribution to our knowledge of molecular mechanism in cell life activities. However, the inherent organization and dynamic characteristic of cell system have rarely been incorporated into the existing algorithms for detecting protein complexes because of the limitation of protein-protein interaction (PPI) data produced by high throughput techniques. The availability of time course gene expression profile enables us to uncover the dynamics of molecular networks and improve the detection of protein complexes. In order to achieve this goal, this paper proposes a novel algorithm DCA (Dynamic Core-Attachment). It detects protein-complex core comprising of continually expressed and highly connected proteins in dynamic PPI network, and then the protein complex is formed by including the attachments with high adhesion into the core. The integration of core-attachment feature into the dynamic PPI network is responsible for the superiority of our algorithm. DCA has been applied on two different yeast dynamic PPI networks and the experimental results show that it performs significantly better than the state-of-the-art techniques in terms of prediction accuracy, hF-measure and statistical significance in biology. In addition, the identified complexes with strong biological significance provide potential candidate complexes for biologists to validate.

Sequence and structural implications of a bovine corneal keratan sulfate proteoglycan core protein. Protein 37B represents bovine lumican and proteins 37A and 25 are unique

NASA Technical Reports Server (NTRS)

Funderburgh, J. L.; Funderburgh, M. L.; Brown, S. J.; Vergnes, J. P.; Hassell, J. R.; Mann, M. M.; Conrad, G. W.; Spooner, B. S. (Principal Investigator)

1993-01-01

Amino acid sequence from tryptic peptides of three different bovine corneal keratan sulfate proteoglycan (KSPG) core proteins (designated 37A, 37B, and 25) showed similarities to the sequence of a chicken KSPG core protein lumican. Bovine lumican cDNA was isolated from a bovine corneal expression library by screening with chicken lumican cDNA. The bovine cDNA codes for a 342-amino acid protein, M(r) 38,712, containing amino acid sequences identified in the 37B KSPG core protein. The bovine lumican is 68% identical to chicken lumican, with an 83% identity excluding the N-terminal 40 amino acids. Location of 6 cysteine and 4 consensus N-glycosylation sites in the bovine sequence were identical to those in chicken lumican. Bovine lumican had about 50% identity to bovine fibromodulin and 20% identity to bovine decorin and biglycan. About two-thirds of the lumican protein consists of a series of 10 amino acid leucine-rich repeats that occur in regions of calculated high beta-hydrophobic moment, suggesting that the leucine-rich repeats contribute to beta-sheet formation in these proteins. Sequences obtained from 37A and 25 core proteins were absent in bovine lumican, thus predicting a unique primary structure and separate mRNA for each of the three bovine KSPG core proteins.

Current antiviral practice and course of Hepatitis B virus infection in inflammatory arthritis: a multicentric observational study (A + HBV study).

PubMed

Kalyoncu, Umut; Emmungil, Hakan; Onat, Ahmet Mesut; Yılmaz, Sedat; Kaşifoglu, Timuçin; Akar, Servet; İnanç, Nevsun; Yıldız, Fatih; Küçükşahin, Orhan; Karadağ, Ömer; Mercan, Rıdvan; Bes, Cemal; Yazısız, Veli; Yılmazer, Barış; Özmen, Mustafa; Erten, Şükran; Şenel, Soner; Yazıcı, Ayten; Taşçılar, Koray; Kalfa, Melike; Kiraz, Sedat; Kısacık, Bünyamin; Pehlivan, Yavuz; Kılıç, Levent; Şimşek, İsmail; Çefle, Ayşe; Akkoç, Nurullah; Direskeneli, Haner; Erken, Eren; Turgay, Murat; Öztürk, Mehmet Akif; Soy, Mehmet; Aksu, Kenan; Dinç, Ayhan; Ertenli, İhsan

2015-12-01

The reactivation of hepatitis B virus (HBV) infection is a well-known event in hepatitis B surface antigen (HbsAg)-positive patients receiving immunosuppressive therapy. The objective of this study was to assess the antiviral practice and course of HBV infection in inflammatory arthritis. Nineteen rheumatology centers participated in this retrospective study. HbsAg-positive patients who were taking disease-modifying antirheumatic drugs and who were being tested for HBV viral load at a minimum of two different time points were included. The case report form (CRF) consisted of demographic data, rheumatic diseases, treatment profiles, transaminase levels, viral hepatitis serological markers, and HBV viral load. The reactivation of HBV was defined as the abrupt rise in HBV replication by an increase in serum HBV DNA levels in a patient with a previously inactive HBV infection. In total, the data of 101 (female 50.5%) patients were included (76 patients with inactive HBV carriers and 25 patients with chronic HBV infection). The mean age of patients was 44±12 years, and the mean follow-up duration was 31±22 months. Of the 101 patients, 70 (69.3%) received antiviral treatment. HBV reactivation was detected in 13 of 76 (17.1%) patients with inactive HBV carriers. HBV reactivation was observed less frequently, not although significantly, in those patients receiving antiviral prophylaxis compared with those not receiving prophylaxis [5/41 (12.2%) vs. 8/33 (24.2%), p=0.17]. Forty-two patients (31 patients had inactive HBV carriers) were using anti-tumor necrosis factor agents. HBV reactivation was detected in 6 of the 31 (19.3%) patients. Twenty-five patients had chronic hepatitis, and five (20%) of them had not received antiviral prophylaxis. HBV viral loads were persistently elevated in 7 (28%) of 25 patients (three patients under and four patients not under antiviral treatment). HBV reactivation was observed in approximately 17% of patients under immunosuppressive

Current antiviral practice and course of Hepatitis B virus infection in inflammatory arthritis: a multicentric observational study (A + HBV study)

PubMed Central

Kalyoncu, Umut; Emmungil, Hakan; Onat, Ahmet Mesut; Yılmaz, Sedat; Kaşifoglu, Timuçin; Akar, Servet; İnanç, Nevsun; Yıldız, Fatih; Küçükşahin, Orhan; Karadağ, Ömer; Mercan, Rıdvan; Bes, Cemal; Yazısız, Veli; Yılmazer, Barış; Özmen, Mustafa; Erten, Şükran; Şenel, Soner; Yazıcı, Ayten; Taşçılar, Koray; Kalfa, Melike; Kiraz, Sedat; Kısacık, Bünyamin; Pehlivan, Yavuz; Kılıç, Levent; Şimşek, İsmail; Çefle, Ayşe; Akkoç, Nurullah; Direskeneli, Haner; Erken, Eren; Turgay, Murat; Öztürk, Mehmet Akif; Soy, Mehmet; Aksu, Kenan; Dinç, Ayhan; Ertenli, İhsan

2015-01-01

Objective The reactivation of hepatitis B virus (HBV) infection is a well-known event in hepatitis B surface antigen (HbsAg)-positive patients receiving immunosuppressive therapy. The objective of this study was to assess the antiviral practice and course of HBV infection in inflammatory arthritis. Material and Methods Nineteen rheumatology centers participated in this retrospective study. HbsAg-positive patients who were taking disease-modifying antirheumatic drugs and who were being tested for HBV viral load at a minimum of two different time points were included. The case report form (CRF) consisted of demographic data, rheumatic diseases, treatment profiles, transaminase levels, viral hepatitis serological markers, and HBV viral load. The reactivation of HBV was defined as the abrupt rise in HBV replication by an increase in serum HBV DNA levels in a patient with a previously inactive HBV infection. Results In total, the data of 101 (female 50.5%) patients were included (76 patients with inactive HBV carriers and 25 patients with chronic HBV infection). The mean age of patients was 44±12 years, and the mean follow-up duration was 31±22 months. Of the 101 patients, 70 (69.3%) received antiviral treatment. HBV reactivation was detected in 13 of 76 (17.1%) patients with inactive HBV carriers. HBV reactivation was observed less frequently, not although significantly, in those patients receiving antiviral prophylaxis compared with those not receiving prophylaxis [5/41 (12.2%) vs. 8/33 (24.2%), p=0.17]. Forty-two patients (31 patients had inactive HBV carriers) were using anti-tumor necrosis factor agents. HBV reactivation was detected in 6 of the 31 (19.3%) patients. Twenty-five patients had chronic hepatitis, and five (20%) of them had not received antiviral prophylaxis. HBV viral loads were persistently elevated in 7 (28%) of 25 patients (three patients under and four patients not under antiviral treatment). Conclusion HBV reactivation was observed in

Application of k-means clustering algorithm in grouping the DNA sequences of hepatitis B virus (HBV)

NASA Astrophysics Data System (ADS)

Bustamam, A.; Tasman, H.; Yuniarti, N.; Frisca, Mursidah, I.

2017-07-01

Based on WHO data, an estimated of 15 millions people worldwide who are infected with hepatitis B (HBsAg+), which is caused by HBV virus, are also infected by hepatitis D, which is caused by HDV virus. Hepatitis D infection can occur simultaneously with hepatitis B (co infection) or after a person is exposed to chronic hepatitis B (super infection). Since HDV cannot live without HBV, HDV infection is closely related to HBV infection, hence it is very realistic that every effort of prevention against hepatitis B can indirectly prevent hepatitis D. This paper presents clustering of HBV DNA sequences by using k-means clustering algorithm and R programming. Clustering processes are started with collecting HBV DNA sequences from GenBank, then performing extraction HBV DNA sequences using n-mers frequency and furthermore the extraction results are collected as a matrix and normalized using the min-max normalization with interval [0, 1] which will later be used as an input data. The number of clusters is two and the initial centroid selected of the cluster is chosen randomly. In each iteration, the distance of every object to each centroid are calculated using the Euclidean distance and the minimum distance is selected to determine the membership in a cluster until two convergent clusters are created. As the result, the HBV viruses in the first cluster is more virulent than the HBV viruses in the second cluster, so the HBV viruses in the first cluster can potentially evolve with HDV viruses that cause hepatitis D.

HBV serum DNA and RNA levels in nucleos(t)ide analogue-treated or untreated patients during chronic and acute infection.

PubMed

Butler, Emily K; Gersch, Jeffrey; McNamara, Anne; Luk, Ka-Cheung; Holzmayer, Vera; de Medina, Maria; Schiff, Eugene; Kuhns, Mary; Cloherty, Gavin A

2018-05-07

Treatment of chronic hepatitis B (CHB) patients with nucleos(t)ide analogues (NA) suppresses HBV DNA synthesis but does not affect synthesis of HBV pregenomic RNA (pgRNA). HBV pgRNA is detectable in the serum during NA treatment and has been proposed as a marker of HBV covalently closed circular DNA (cccDNA) activity within the infected hepatocyte. We developed an automated assay for the quantification of serum HBV pgRNA using a dual-target qRT-PCR approach on the Abbott m2000sp/rt system. We demonstrate accurate detection and quantification of serum HBV RNA. HBV DNA was quantified using the Abbott RealTime HBV viral load assay. We further compared serum nucleic acid levels and kinetics in HBV-positive populations. Samples included: on-therapy CHB samples (N=16), samples (N=89) from 10 treatment naïve CHB subjects receiving 12-weeks of NA treatment with 8-week follow-up, HBsAg-positive blood donor samples (N=102), and 3 seroconversion series from plasmapheresis donors (N=79 samples). During NA treatment of CHB subjects, we observed low correlation of HBV DNA to pgRNA levels; pgRNA concentration was generally higher than HBV DNA concentrations. In contrast, when NA treatment was absent we observed serum pgRNA at concentrations that correlated to HBV DNA and were approximately 2 log lower than HBV DNA. Importantly, we observe this trend in untreated subject samples from both chronic infections and throughout seroconversion during acute infection. Results demonstrate that the presence of pgRNA in serum is part of the HBV lifecycle; constant relative detection of pgRNA and HBV DNA in the serum is suggestive of a linked mechanism for egress for HBV DNA or pgRNA containing virions. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

Lack of infectivity of HBV in feces from patients with chronic hepatitis B virus infection, and infection using chimeric mice.

PubMed

Komatsu, Haruki; Inui, Ayano; Murano, Takeyoshi; Tsunoda, Tomoyuki; Sogo, Tsuyoshi; Fujisawa, Tomoo

2015-08-20

Body fluids such as saliva and tears from patients with hepatitis B virus (HBV) infection are known as infectious agents. The infectivity of feces from patients with HBV infection has not been established. The aim of this study was to determine whether feces from HBV carriers can be a source of HBV infection. Thirty-three children and 17 adults (ages 0-49 years, median age 13 years) who were chronically infected with HBV were enrolled. The levels of HBV DNA in the feces from these patients were quantified by real-time PCR, and the levels of fecal HBsAg were measured. Isolated human hepatocytes from chimeric mice with humanized livers were co-cultured with serum, tears and feces from the HBV carriers. Four chimeric mice were inoculated intravenously with sterilized feces from HBV carriers. HBV DNA was detected in the feces of 37 (74%) of the 50 patients. The fecal HBV DNA levels ranged from 2.8 to 8.4 log copies/mL (mean ± SD  =  5.6 ± 1.2 log copies/mL). A significant correlation was observed in the levels of HBV DNA between serum and feces (r  =  0.54, p < 0.05). Of the 13 HBV carries, 7 (54%) were positive for fecal HBsAg. The fecal HBsAg levels ranged from 0.06 to 1.0 IU/mL (median 0.28 IU/mL). Immunogold electron microscopy showed Dane particles in feces. HBV DNA was detected in the human hepatocytes co-cultured with serum and tears, but not in those co-cultured with feces. HBV DNA was not detected in the serum of the chimeric mice after oral or intravenous inoculation with sterilized fecal samples, which contained 5 log copies/mL of HBV DNA levels. Although the positive rate of fecal HBV DNA was high, the fecal HBsAg levels were extremely low. The chimeric mice were not infected with HBV after oral or intravenous inoculation with sterilized fecal samples. Therefore, feces from HBV carriers seem not to serve as an infectious vehicle for the transmission of HBV.

In Vitro Assembly of Alphavirus Cores by Using Nucleocapsid Protein Expressed in Escherichia coli

PubMed Central

Tellinghuisen, Timothy L.; Hamburger, Agnes E.; Fisher, Bonnie R.; Ostendorp, Ralf; Kuhn, Richard J.

1999-01-01

The production of the alphavirus virion is a multistep event requiring the assembly of the nucleocapsid core in the cytoplasm and the maturation of the glycoproteins in the endoplasmic reticulum and the Golgi apparatus. These components associate during the budding process to produce the mature virion. The nucleocapsid proteins of Sindbis virus and Ross River virus have been produced in a T7-based Escherichia coli expression system and purified. In the presence of single-stranded but not double-stranded nucleic acid, the proteins oligomerize in vitro into core-like particles which resemble the native viral nucleocapsid cores. Despite their similarities, Sindbis virus and Ross River virus capsid proteins do not form mixed core-like particles. Truncated forms of the Sindbis capsid protein were used to establish amino acid requirements for assembly. A capsid protein starting at residue 19 [CP(19–264)] was fully competent for in vitro assembly, whereas proteins with further N-terminal truncations could not support assembly. However, a capsid protein starting at residue 32 or 81 was able to incorporate into particles in the presence of CP(19–264) or could inhibit assembly if its molar ratio relative to CP(19–264) was greater than 1:1. This system provides a basis for the molecular dissection of alphavirus core assembly. PMID:10364277

Quantitative analysis of core fucosylation of serum proteins in liver diseases by LC-MS-MRM.

PubMed

Ma, Junfeng; Sanda, Miloslav; Wei, Renhuizi; Zhang, Lihua; Goldman, Radoslav

2018-02-07

Aberrant core fucosylation of proteins has been linked to liver diseases. In this study, we carried out multiple reaction monitoring (MRM) quantification of core fucosylated N-glycopeptides of serum proteins partially deglycosylated by a combination of endoglycosidases (endoF1, endoF2, and endoF3). To minimize variability associated with the preparatory steps, the analysis was performed without enrichment of glycopeptides or fractionation of serum besides the nanoRP chromatography. Specifically, we quantified core fucosylation of 22 N-glycopeptides derived from 17 proteins together with protein abundance of these glycoproteins in a cohort of 45 participants (15 disease-free control, 15 fibrosis and 15 cirrhosis patients) using a multiplex nanoUPLC-MS-MRM workflow. We find increased core fucosylation of 5 glycopeptides at the stage of liver fibrosis (i.e., N630 of serotransferrin, N107 of alpha-1-antitrypsin, N253 of plasma protease C1 inhibitor, N397 of ceruloplasmin, and N86 of vitronectin), increase of additional 6 glycopeptides at the stage of cirrhosis (i.e., N138 and N762 of ceruloplasmin, N354 of clusterin, N187 of hemopexin, N71 of immunoglobulin J chain, and N127 of lumican), while the degree of core fucosylation of 10 glycopeptides did not change. Interestingly, although we observe an increase in the core fucosylation at N86 of vitronectin in liver fibrosis, core fucosylation decreases on the N169 glycopeptide of the same protein. Our results demonstrate that the changes in core fucosylation are protein and site specific during the progression of fibrotic liver disease and independent of the changes in the quantity of N-glycoproteins. It is expected that the fully optimized multiplex LC-MS-MRM assay of core fucosylated glycopeptides will be useful for the serologic assessment of the fibrosis of liver. We have quantified the difference in core fucosylation among three comparison groups (healthy control, fibrosis and cirrhosis patients) using a sensitive and

Novel function of the endoplasmic reticulum degradation-enhancing α-mannosidase-like proteins in the human hepatitis B virus life cycle, mediated by the middle envelope protein.

PubMed

Lazar, Catalin; Uta, Mihaela; Petrescu, Stefana Maria; Branza-Nichita, Norica

2017-02-01

Cells replicating the human hepatitis B virus (HBV) express high levels of degradation-enhancing α-mannosidase-like proteins (EDEMs), a family of proteins involved in the endoplasmic reticulum associated degradation, one of the pathways activated during the unfolded protein response. Owing to their α-1,2 mannosidase activity, the EDEM1-3 proteins are able to process the N-linked glycans of misfolded or incompletely folded proteins, providing the recognition signal for their subsequent degradation. The HBV small (S), medium (M), and large (L) surface proteins bear an N-linked glycosylation site in the common S domain that is partially occupied in all proteins. The M protein contains an additional site in its preS2 domain, which is always functional. Here, we report that these oligosaccharides are processed by EDEMs, more efficiently by EDEM3, which induces degradation of L and S proteins, accompanied by a reduction of subviral particles production. In striking contrast, M not only is spared from degradation but its trafficking is also accelerated leading to an improved secretion. This unusual behavior of the M protein requires strictly the mannose trimming of the preS2 N-linked glycan. Furthermore, we show that HBV secretion is significantly inhibited under strong endoplasmic reticulum stress conditions when M expression is prevented by mutagenesis of the viral genome. These observations unfold unique properties of the M protein in the HBV life cycle during unfolded protein response and point to alternative mechanisms employed by EDEMs to alleviate this stress in case of necessity by promoting glycoprotein trafficking rather than degradation. © 2016 John Wiley & Sons Ltd.

Active co-infection with HBV and/or HCV in South African HIV positive patients due for cancer therapy.

PubMed

Musyoki, Andrew M; Msibi, Thembeni L; Motswaledi, Mojakgomo H; Selabe, Selokela G; Monokoane, Tshweu S; Mphahlele, M Jeffrey

2015-02-01

Human immunodeficiency virus (HIV), Hepatitis B virus (HBV) and Hepatitis C virus (HCV) share routes of transmission. There is limited data on the incidence of active co-infection with HBV and/or HCV in cancer patients infected with HIV in Africa. This was a prospective study based on 34 patients with varied cancer diagnosis, infected with HIV and awaiting cancer therapy in South Africa. HIV viral load, CD4+ cell counts, Alanine-aminotransferase and aspartate aminotransferase levels were tested. Exposure to HBV and HCV was assessed serologically using commercial kits. Active HBV and/or HCV co-infection was detected using viral specific nested PCR assays. HCV 5'-UTR PCR products were sequenced to confirm active HCV infection. Active viral infection was detected in 64.7% of patients for HBV, 38.2% for HCV, and 29.4% for both HBV and HCV. Occult HBV infection was observed in 63.6% of the patients, while seronegative HCV infection was found in 30.8% of patients. In addition, CD4+ cell count < 350 cells/µl was not a risk factor for increased active HBV, HCV or both HBV and HCV co-infections. A total of 72.7%, 18.2% and 9.1% of the HCV sequences were assigned genotype 5, 1 and 4 respectively.The study revealed for the first time a high active HBV and/or HCV co-infection rate in cancer patients infected with HIV. The findings call for HBV and HCV testing in such patients, and where feasible, appropriate antiviral treatment be indicated, as chemotherapy or radiotherapy has been associated with reactivation of viral hepatitis and termination of cancer therapy. © 2014 Wiley Periodicals, Inc.

HBV and HIV co-infection: Prevalence and clinical outcomes in tertiary care hospital Malaysia.

PubMed

Akhtar, Ali; Khan, Amer Hayat; Sulaiman, Syed Azhar Syed; Soo, Chow Ting; Khan, Kashifullah

2016-03-01

According to WHO, Malaysia has been classified as a concentrated epidemic country due to progression of HIV infection in the population of injecting drug users. The main objectives of current study are to determine the prevalence of HBV among HIV-positive individuals in a tertiary care hospital of Malaysia and to assess the predictors involved in the outcomes of HIV-HBV co-infected patients. A retrospective, cross-sectional study is conducted at Hospital Palau Pinang, Malaysia. The collection of socio-demographic data as well as clinical data is done with the help of data collection form. Data were analyzed after putting the collected values of required data by using statistical software SPSS version 20.0 and P > 0.05 is considered as significant. Results show that the overall prevalence of HBV was 86 (13%) including 495 (74.5%) males and 169 (25.5%) females among a total of 664 HIV-infected patients. It was observed that there is a high prevalence of HIV-HBV co-infection in males 76 (11.4%) as compared to females 10 (1.5%) (P = 0.002). The median age of the study population was 39 years. The statistical significant risk factors involved in the outcomes of HIV-HBV co-infected patients were observed in the variables of gender, age groups, and injecting drug users. The findings of the present study shows that the prevalence of HBV infection among HIV-positive patients was 13% and the risk factors involved in the outcomes of HIV-HBV co-infected patients were gender, age, and intravenous drug users. © 2015 Wiley Periodicals, Inc.

Deep sequencing shows low-level oncogenic hepatitis B virus variants persists post-liver transplant despite potent anti-HBV prophylaxis.

PubMed

Lau, K C K; Osiowy, C; Giles, E; Lusina, B; van Marle, G; Burak, K W; Coffin, C S

2018-06-01

Recent studies suggest that withdrawal of hepatitis B immune globulin (HBIG) and nucleos(t)ide analogues (NA) prophylaxis may be considered in HBV surface antigen (HBsAg)-negative liver transplant (LT) recipients with a low risk of disease recurrence. However, the frequency of occult HBV infection (OBI) and HBV variants after LT in the current era of potent NA therapy is unknown. Twelve LT recipients on prophylaxis were tested in matched plasma and peripheral blood mononuclear cells (PBMCs) for HBV quasispecies by in-house nested PCR and next-generation sequencing of amplicons. HBV covalently closed circular DNA (cccDNA) was detected in Hirt DNA isolated from PBMCs with cccDNA-specific primers and confirmed by nucleic acid hybridization and Sanger sequencing. HBV mRNA in PBMC was detected with reverse-transcriptase nested PCR. In LT recipients on immunosuppressive therapy (10/12 male; median age 57.5 [IQR: 39.8-66.5]; median follow-up post-LT 60 months; 6 pre-LT hepatocellular carcinoma [HCC]), 9 were HBsAg-. HBV DNA was detected in all plasma and PBMC tested; cccDNA and/or mRNA was detected in the PBMC of 10/12 patients. Significant HBV quasispecies diversity (ie 143-2212 nonredundant HBV species) was noted in both sites, and single nucleotide polymorphisms associated with cirrhosis and HCC were detected at varying frequencies. In conclusion, OBI and HBV variants associated with severe liver disease persist in LT recipients on prophylaxis. Although HBV control and cccDNA transcriptional silencing may occur despite immunosuppression, complete virological eradication does not occur in LT recipients with a history of HBV-related end-stage liver disease. © 2018 John Wiley & Sons Ltd.

Economic evaluation of HBV vaccination: A systematic review of recent publications (2000-2013).

PubMed

La Torre, Giuseppe; Mannocci, Alice; Saulle, Rosella; Colamesta, Vittoria; Meggiolaro, Angela; Mipatrini, Daniele; Sinopoli, Alessandra

2016-09-01

To conduct a systematic review of the economic evaluations (EE) of HBV vaccination, taking also into account the studies published in the new millennium. An extensive scientific literature review was conducted using two electronic medical journal databases: Scopus and PubMed engines for published studies on EE of HBV vaccination. 22 articles were reviewed, 9, 5 and 8 cost-effectiveness, cost-benefit and cost-utility analysis, respectively. Studies were mainly concerning EE of universal vaccination (UV), mostly with regards to low or low-medium income countries. For high income countries, EE were focused on the possible implementation of HBV vaccination in particular settings, such as diabetic, renal and other chronic conditions care, as well as infectious diseasesUV has usually a very good cost-effectiveness ratio (80%), ranging from cost-saving (China) or few Euro per LY/QALY gained (in Thailand, and Vietnam) to 630.00$/QALY in USA (Asian and Pacific Islands) Moreover, EE of HBV vaccination are favorable in the infectious diseases field as well as for chronic conditions. In relation to diabetes the studies gave controversial results. This systematic review highlighted the importance of introducing HBV vaccination not only for infant UV program but also for other settings in which patients are people affected by communicable and non-communicable diseases.

Characterization of the fusion core in zebrafish endogenous retroviral envelope protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Jian; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071; Zhang, Huaidong

2015-05-08

Zebrafish endogenous retrovirus (ZFERV) is the unique endogenous retrovirus in zebrafish, as yet, containing intact open reading frames of its envelope protein gene in zebrafish genome. Similarly, several envelope proteins of endogenous retroviruses in human and other mammalian animal genomes (such as syncytin-1 and 2 in human, syncytin-A and B in mouse) were identified and shown to be functional in induction of cell–cell fusion involved in placental development. ZFERV envelope protein (Env) gene appears to be also functional in vivo because it is expressible. After sequence alignment, we found ZFERV Env shares similar structural profiles with syncytin and other type Imore » viral envelopes, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR) which were crucial for membrane fusion. We expressed the regions of N + C protein in the ZFERV Env (residues 459–567, including predicted NHR and CHR) to characterize the fusion core structure. We found N + C protein could form a stable coiled-coil trimer that consists of three helical NHR regions forming a central trimeric core, and three helical CHR regions packing into the grooves on the surface of the central core. The structural characterization of the fusion core revealed the possible mechanism of fusion mediated by ZFERV Env. These results gave comprehensive explanation of how the ancient virus infects the zebrafish and integrates into the genome million years ago, and showed a rational clue for discovery of physiological significance (e.g., medicate cell–cell fusion). - Highlights: • ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes. • The fusion core of ZFERV Env forms stable coiled-coil trimer including three NHRs and three CHRs. • The structural mechanism of viral entry mediated by ZFERV Env is disclosed. • The results are helpful for further discovery of physiological function of ZFERV Env in zebrafish.« less

The adenovirus major core protein VII is dispensable for virion assembly but is essential for lytic infection

PubMed Central

Suomalainen, Maarit; Zheng, Yueting; Boucke, Karin

2017-01-01

The Adenovirus (Ad) genome within the capsid is tightly associated with a virus-encoded, histone-like core protein—protein VII. Two other Ad core proteins, V and X/μ, also are located within the virion and are loosely associated with viral DNA. Core protein VII remains associated with the Ad genome during the early phase of infection. It is not known if naked Ad DNA is packaged into the capsid, as with dsDNA bacteriophage and herpesviruses, followed by the encapsidation of viral core proteins, or if a unique packaging mechanism exists with Ad where a DNA-protein complex is simultaneously packaged into the virion. The latter model would require an entirely new molecular mechanism for packaging compared to known viral packaging motors. We characterized a virus with a conditional knockout of core protein VII. Remarkably, virus particles were assembled efficiently in the absence of protein VII. No changes in protein composition were evident with VII−virus particles, including the abundance of core protein V, but changes in the proteolytic processing of some capsid proteins were evident. Virus particles that lack protein VII enter the cell, but incoming virions did not escape efficiently from endosomes. This greatly diminished all subsequent aspects of the infectious cycle. These results reveal that the Ad major core protein VII is not required to condense viral DNA within the capsid, but rather plays an unexpected role during virus maturation and the early stages of infection. These results establish a new paradigm pertaining to the Ad assembly mechanism and reveal a new and important role of protein VII in early stages of infection. PMID:28628648

Characterization of the honeybee venom proteins C1q-like protein and PVF1 and their allergenic potential.

PubMed

Russkamp, Dennis; Van Vaerenbergh, Matthias; Etzold, Stefanie; Eberlein, Bernadette; Darsow, Ulf; Schiener, Maximilian; De Smet, Lina; Absmaier, Magdalena; Biedermann, Tilo; Spillner, Edzard; Ollert, Markus; Jakob, Thilo; Schmidt-Weber, Carsten B; de Graaf, Dirk C; Blank, Simon

2018-05-26

Honeybee (Apis mellifera) venom (HBV) represents an ideal model to study the role of particular venom components in allergic reactions in sensitized individuals as well as in the eusociality of Hymenoptera species. The aim of this study was to further characterize the HBV components C1q-like protein (C1q) and PDGF/VEGF-like factor 1 (PVF1). C1q and PVF1 were produced as recombinant proteins in insect cells. Their allergenic properties were examined by determining the level of specific IgE antibodies in the sera of HBV-allergic patients (n = 26) as well as by their capacity to activate patients' basophils (n = 11). Moreover, the transcript heterogeneity of PVF1 was analyzed. It could be demonstrated that at least three PVF1 variants are present in the venom gland, which all result from alternative splicing of one transcript. Additionally, recombinant C1q and PVF1 from Spodoptera frugiperda insect cells exhibited specific IgE reactivity with approximately 38.5% of sera of HBV-allergic patients. Interestingly, both proteins were unable to activate basophils of the patients, questioning their role in the context of clinically relevant sensitization. Recombinant C1q and PVF1 can build the basis for a deeper understanding of the molecular mechanisms of Hymenoptera venoms. Moreover, the conflicting results between IgE sensitization and lack of basophil activation, might in the future contribute to the identification of factors that determine the allergenic potential of proteins. Copyright © 2018 Elsevier Ltd. All rights reserved.

HBV and HCV infection among non-European Union immigrants in North-East Italy.

PubMed Central

Chiaramonte, M.; Pupo, A.; Menegon, T.; Baldo, V.; Malatesta, R.; Trivello, R.

1998-01-01

The status of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection among non-European Union (non-EU) immigrants in North-East Italy was evaluated. Among the 1683 individuals tested the prevalence of HBsAg was 8.9% (150 subjects) and of HBV antibodies (anti-HBc with/without anti-HBs) was 38.9% (654 subjects). The distribution of HBV serological markers showed significant differences according to region of origin; the highest prevalence of infection (76.9%) and carriage (16.1%) was found in immigrants from sub-Saharan Africa. Among the 933 individuals screened for HCV infection, prevalence of antibody was much lower (0.9%) than that observed in the Italian general population (3.2-12.6%). The large number of HBV carriers among immigrants could increase the number of new adult infections due to life-style habits or professional risks in the host population. In contrast, the risk of HCV spread from non-EU immigrants is very low. PMID:9747770

The detection of HBV DNA with gold-coated iron oxide nanoparticle gene probes

NASA Astrophysics Data System (ADS)

Xi, Dong; Luo, XiaoPing; Lu, QiangHua; Yao, KaiLun; Liu, ZuLi; Ning, Qin

2008-03-01

Gold-coated iron oxide nanoparticle Hepatitis B virus (HBV) DNA probes were prepared, and their application for HBV DNA measurement was studied. Gold-coated iron oxide nanoparticles were prepared by the citrate reduction of tetra-chloroauric acid in the presence of iron oxide nanoparticles which were added as seeds. With a fluorescence-based method, the maximal surface coverage of hexaethiol 30-mer oligonucleotides and the maximal percentage of hybridization strands on gold-coated iron oxide nanoparticles were (120 ± 8) oligonucleotides per nanoparticle, and (14 ± 2%), respectively, which were comparable with those of (132 ± 10) and (22 ± 3%) in Au nanoparticle groups. Large network aggregates were formed when gold-coated iron oxide nanoparticle HBV DNA gene probe was applied to detect HBV DNA molecules as evidenced by transmission electron microscopy and the high specificity was verified by blot hybridization. Our results further suggested that detecting DNA with iron oxide nanoparticles and magnetic separator was feasible and might be an alternative effective method.

An Unusual Hydrophobic Core Confers Extreme Flexibility to HEAT Repeat Proteins

PubMed Central

Kappel, Christian; Zachariae, Ulrich; Dölker, Nicole; Grubmüller, Helmut

2010-01-01

Alpha-solenoid proteins are suggested to constitute highly flexible macromolecules, whose structural variability and large surface area is instrumental in many important protein-protein binding processes. By equilibrium and nonequilibrium molecular dynamics simulations, we show that importin-β, an archetypical α-solenoid, displays unprecedentedly large and fully reversible elasticity. Our stretching molecular dynamics simulations reveal full elasticity over up to twofold end-to-end extensions compared to its bound state. Despite the absence of any long-range intramolecular contacts, the protein can return to its equilibrium structure to within 3 Å backbone RMSD after the release of mechanical stress. We find that this extreme degree of flexibility is based on an unusually flexible hydrophobic core that differs substantially from that of structurally similar but more rigid globular proteins. In that respect, the core of importin-β resembles molten globules. The elastic behavior is dominated by nonpolar interactions between HEAT repeats, combined with conformational entropic effects. Our results suggest that α-solenoid structures such as importin-β may bridge the molecular gap between completely structured and intrinsically disordered proteins. PMID:20816072

Synthesis and Evaluation of N-phenyl-3-sulfamoyl-benzamide Derivatives as Capsid Assembly Modulators inhibiting Hepatitis B Virus (HBV).

PubMed

Vandyck, Koen; Rombouts, Geert; Stoops, Bart; Tahri, Abdellah; Vos, Ann; Verschueren, Wim; Wu, Yiming; Yang, Jingmei; Hou, Fuliang; Huang, Bing; Vergauwen, Karen; Dehertogh, Pascale; Berke, Jan-Martin; Raboisson, Pierre Jean Marie Bernard

2018-06-15

Small molecule induced Hepatitis B virus (HBV) capsid assembly modulation is considered an attractive approach for new antiviral therapies against HBV. Here we describe efforts towards the discovery of a HBV capsid assembly modulator in a hit-to-lead optimization, resulting in JNJ-632, a tool compound used to further profile the mode of action. Administration of JNJ-632 (54) in HBV genotype D infected chimeric mice, resulted in a 2.77 log reduction of the HBV DNA viral load.

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

PubMed

Sengupta, Isha; Das, Dipanwita; Singh, Shivaram Prasad; Chakravarty, Runu; Das, Chandrima

2017-12-15

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. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The Role of Infected Cell Proliferation in the Clearance of Acute HBV Infection in Humans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goyal, Ashish; Ribeiro, Ruy Miguel; Perelson, Alan S.

Around 90–95% of hepatitis B virus (HBV) infected adults do not progress to the chronic phase and, instead, recover naturally. The strengths of the cytolytic and non-cytolytic immune responses are key players that decide the fate of acute HBV infection. In addition, it has been hypothesized that proliferation of infected cells resulting in uninfected progeny and/or cytokine-mediated degradation of covalently closed circular DNA (cccDNA) leading to the cure of infected cells are two major mechanisms assisting the adaptive immune response in the clearance of acute HBV infection in humans. We employed fitting of mathematical models to human acute infection datamore » together with physiological constraints to investigate the role of these hypothesized mechanisms in the clearance of infection. Results suggest that cellular proliferation of infected cells resulting in two uninfected cells is required to minimize the destruction of the liver during the clearance of acute HBV infection. In contrast, we find that a cytokine-mediated cure of infected cells alone is insufficient to clear acute HBV infection. Lastly, our modeling indicates that HBV clearance without lethal loss of liver mass is associated with the production of two uninfected cells upon proliferation of an infected cell.« less

The Role of Infected Cell Proliferation in the Clearance of Acute HBV Infection in Humans

DOE PAGES

Goyal, Ashish; Ribeiro, Ruy Miguel; Perelson, Alan S.

2017-11-18

Around 90–95% of hepatitis B virus (HBV) infected adults do not progress to the chronic phase and, instead, recover naturally. The strengths of the cytolytic and non-cytolytic immune responses are key players that decide the fate of acute HBV infection. In addition, it has been hypothesized that proliferation of infected cells resulting in uninfected progeny and/or cytokine-mediated degradation of covalently closed circular DNA (cccDNA) leading to the cure of infected cells are two major mechanisms assisting the adaptive immune response in the clearance of acute HBV infection in humans. We employed fitting of mathematical models to human acute infection datamore » together with physiological constraints to investigate the role of these hypothesized mechanisms in the clearance of infection. Results suggest that cellular proliferation of infected cells resulting in two uninfected cells is required to minimize the destruction of the liver during the clearance of acute HBV infection. In contrast, we find that a cytokine-mediated cure of infected cells alone is insufficient to clear acute HBV infection. Lastly, our modeling indicates that HBV clearance without lethal loss of liver mass is associated with the production of two uninfected cells upon proliferation of an infected cell.« less

Association of neutrophil-lymphocyte ratio and T lymphocytes with the pathogenesis and progression of HBV-associated primary liver cancer

PubMed Central

Han, Junyan; Wang, Lijia; Li, Mengge; Jiang, Yuyong; Wang, Xianbo; Yang, Zhiyun

2017-01-01

Background The neutrophil–lymphocyte ratio (NLR) is a new prognostic predictor for patients with liver cancer. The association of NLR and T lymphocytes with the pathogenesis and progression of liver cancer is poorly understood. Methods Seventy-three patients with hepatitis B virus (HBV)-associated primary liver cancer (HBV-PLC), 50 patients with HBV-associated liver cirrhosis (HBV-LC) and 37 patients with chronic HBV infection (CHB) were prospectively enrolled from July 1, 2013 to February 28, 2014 in Beijing Ditan Hospital, Capital Medical University (Beijing, China). The NLR, proportions and concentrations of neutrophils and lymphocytes, concentration of subpopulations of lymphocytes, and the expression of CD31 (index for recent thymic output) and HLA-DR (index for activation of T lymphocytes) of T cells in the peripheral blood samples of the patients were assessed and statistically compared between different groups. Results The NLR was significantly increased from patients with CHB, those with HBV-LC to those with HBV-PLC (P<0.05), along with significant increase of neutrophils and decrease of lymphocytes in the same way (P<0.05). The concentrations of T lymphocytes, natural killer cells, B cells, CD4+ T cells and CD8+ T cells were decreased from patients with CHB, those with HBV-LC to those with HBV-PLC, and were significantly reduced in patients with HBV-PLC compared with those in patients with CHB (P<0.05). The CD31 and HLA-DR expression of naive CD4+ and CD8+ T cells was significantly decreased and increased, respectively in patients with HBV-PLC compared with that in patients with CHB. Conclusions Elevated NLR, resulted from the increase of neutrophils and decrease of lymphocytes, is positively associated with the pathogenesis and progression of HBV-PLC. The reduced thymic output and hyperactivation of T lymphocytes may contribute to the decrease of T lymphocytes, which could be also related to the pathogenesis of HBV-PLC. PMID:28231294

Cellular chromosome DNA interferes with fluorescence quantitative real-time PCR detection of HBV DNA in culture medium.

PubMed

Pan, Xiao-Ben; Wei, Lai; Han, Jin-Chao; Gao, Yan

2008-01-01

Fluorescence quantitative real-time PCR (FQ-PCR) is a recently developed technique increasingly used for clinical diagnosis by detection of hepatitis B virus (HBV) DNA in serum. FQ-PCR is also used in scientific research for detection of HBV DNA in cell culture. Understanding potential FQ-PCR interference factors can improve the accuracy of HBV DNA quantification in cell culture medium. HBV positive serum was diluted with culture medium to produce three test groups with HBV DNA levels of 5 x 10(7) copies/ml (high), 5 x 10(5) copies/ml (medium), and 5 x 10(3) copies/ml (low). Chromosome DNA was extracted from HepG2 cells and then added to high, medium, and low group samples at final concentrations of 0, 12.5, 25, 50, and 100 microg/ml. The samples were quantified by FQ-PCR and data were evaluated using statistical software. No marked changes were seen in the quantitative curves for high level HBV DNA samples when the samples were supplemented with 0-100 microg/ml of chromosome DNA. Interference was observed in medium level samples when 50 and 100 microg/ml of chromosome DNA was added. Interference was also observed in low level HBV DNA samples when the concentration of added chromosome DNA was greater than 25 microg/ml. The interference was eliminated when samples were digested by DNase I prior to PCR detection. In Conclusions, the presence of cellular chromosome DNA can interfere with the detection of HBV DNA by FQ-PCR. Removal of cellular chromosome DNA from culture media prior to FQ-PCR is necessary for reliable HBV DNA quantitative detection. (c) 2007 Wiley-Liss, Inc.

Inhibition of HBV replication in vivo using helper-dependent adenovirus vectors to deliver antiviral RNA interference expression cassettes.

PubMed

Crowther, Carol; Mowa, Mohube B; Ely, Abdullah; Arbuthnot, Patrick B

2014-01-01

HBV is hyperendemic to southern Africa and parts of Asia, but licensed antivirals have little effect on limiting life-threatening complications of the infection. Although RNA interference (RNAi)-based gene silencing has shown therapeutic potential, difficulties with delivery of anti-HBV RNAi effectors remain an obstacle to their clinical use. To address concerns about the transient nature of transgene expression and toxicity resulting from immunostimulation by recombinant adenovirus vectors (Ads), utility of RNAi-activating anti-HBV helper-dependent (HD) Ads were assessed in this study. Following intravenous administration of 5×10(9) unmodified or pegylated HD Ad infectious particles to HBV transgenic mice, HBV viral loads and serum HBV surface antigen levels were monitored for 12 weeks. Immunostimulation of HD Ads was assessed by measuring inflammatory cytokines, hepatic function and immune response to the co-delivered LacZ reporter gene. Unmodified and pegylated HD Ads transduced 80-90% of hepatocytes and expressed short hairpin RNAs (shRNAs) were processed to generate intended HBV-targeting guides. Markers of HBV replication were decreased by approximately 95% and silencing was sustained for 8 weeks. Unmodified HD Ads induced release of proinflammatory cytokines and there was evidence of an adaptive immune response to β-galactosidase. However the HD Ad-induced innate immune response was minimal in preparations that were enriched with infectious particles. HD Ads have potential utility for delivery of therapeutic HBV-silencing sequences and alterations of these vectors to attenuate their immune responses may further improve their efficacy.

A Cul-3-BTB ubiquitylation pathway regulates junctional levels and asymmetry of core planar polarity proteins

PubMed Central

Strutt, Helen; Searle, Elizabeth; Thomas-MacArthur, Victoria; Brookfield, Rosalind; Strutt, David

2013-01-01

The asymmetric localisation of core planar polarity proteins at apicolateral junctions is required to specify cell polarity in the plane of epithelia. This asymmetric distribution of the core proteins is proposed to require amplification of an initial asymmetry by feedback loops. In addition, generation of asymmetry appears to require the regulation of core protein levels, but the importance of such regulation and the underlying mechanisms is unknown. Here we show that ubiquitylation acts through more than one mechanism to control core protein levels in Drosophila, and that without this regulation cellular asymmetry is compromised. Levels of Dishevelled at junctions are regulated by a Cullin-3-Diablo/Kelch ubiquitin ligase complex, the activity of which is most likely controlled by neddylation. Furthermore, activity of the deubiquitylating enzyme Fat facets is required to maintain Flamingo levels at junctions. Notably, ubiquitylation does not alter the total cellular levels of Dishevelled or Flamingo, but only that of the junctional population. When junctional core protein levels are either increased or decreased by disruption of the ubiquitylation machinery, their asymmetric localisation is reduced and this leads to disruption of planar polarity at the tissue level. Loss of asymmetry by altered core protein levels can be explained by reference to feedback models for amplification of asymmetry. PMID:23487316

Quantitation of HBV DNA in human serum using a branched DNA (bDNA) signal amplification assay.

PubMed

Hendricks, D A; Stowe, B J; Hoo, B S; Kolberg, J; Irvine, B D; Neuwald, P D; Urdea, M S; Perrillo, R P

1995-11-01

The aim of this study was to establish the performance characteristics of a nonradioisotopic branched DNA (bDNA) signal amplification assay for quantitation of hepatitis B virus (HBV) DNA in human serum. Quantitation was determined from a standard curve and expressed as HBV DNA equivalents/mL (Eq/mL; 285,000 Eq = 1 pg of double stranded HBV DNA). The bDNA assay exhibited a nearly four log dynamic range of quantitation and an analytical detection limit of approximately 100,000 Eq/mL. To ensure a specificity of 99.7%, the quantitation limit was set at 700,000 Eq/mL. The interassay percent coefficient of variance for quantification values ranged from 10% to 15% when performed by novice users with different sets of reagents. Using the bDNA assay, HBV DNA was detected in 94% to 100% of hepatitis B e antigen-positive specimens and 27% to 31% of hepatitis B e antigen-negative specimens from chronic HBV-infected patients. The bDNA assay may be useful as a prognostic and therapy monitoring tool for the management of HBV-infected patients undergoing antiviral treatment.

Novel HBV recombinants between genotypes B and C in 3'-terminal reverse transcriptase (RT) sequences are associated with enhanced viral DNA load, higher RT point mutation rates and place of birth among Chinese patients.

PubMed

Liu, Baoming; Yang, Jing-Xian; Yan, Ling; Zhuang, Hui; Li, Tong

2018-01-01

As one of the major global public health concerns, hepatitis B virus (HBV) can be divided into at least eight genotypes, which may be related to disease severity and treatment response. We previously demonstrated that genotypes B and C HBV, with distinct geographical distribution in China, had divergent genotype-dependent amino acid polymorphisms and variations in reverse transcriptase (RT) gene region, a target of antiviral therapy using nucleos(t)ide analogues. Recently recombination between HBV genotypes B and C was reported to occur in the RT region. However, their frequency and clinical significance is poorly understood. Here full-length HBV RT sequences from 201 Chinese chronic hepatitis B (CHB) patients were amplified and sequenced, among which 31.34% (63/201) were genotype B whereas 68.66% (138/201) genotype C. Although no intergenotypic recombination was detected among C-genotype HBV, 38.10% (24/63) of B-genotype HBV had recombination with genotype C in the 3'-terminal RT sequences. The patients with B/C intergenotypic recombinants had significantly (P<0.05) higher serum HBV DNA level than the "pure" B-genotype cohort did. Moreover, the B/C intergenotypic recombinants were prone to more substitutions at several specific residues in the RT region than genotype B or C. Besides, unlike their parental genotypes, the recombinant HBV appeared to display an altered geographic distribution feature in China. Our findings provide novel insight into the virological, clinical and epidemiological features of new HBV B/C intergenotypic recombinants at the 3' end of RT sequences among Chinese CHB patients. The highly complex genetic background of the novel recombinant HBV carrying new mutations affecting RT protein may contribute to an enhanced heterogeneity in treatment response or prognosis among CHB patients. Published by Elsevier B.V.

Replication and gene expression of hepatitis B virus in a transgenic mouse that contains the complete viral genome.

PubMed Central

Farza, H; Hadchouel, M; Scotto, J; Tiollais, P; Babinet, C; Pourcel, C

1988-01-01

We have sought to address the problem of the host and tissue specificity of the hepatitis B virus (HBV) by using transgenic mice obtained after injection of head-to-tail dimers of the HBV genome. Viral DNA replication and protein synthesis were obtained in one of nine transgenic mice containing integrated HBV DNA. The RNAs encoding the HBV surface antigen and the core antigen were synthesized in the liver, the kidney, and the heart. In these organs, DNA replicative intermediates similar to those found during normal infection were associated with corelike structures. Large amounts of core polypeptides and capsids were detected in the nuclei in the absence of any pathological effect. These results show that the different steps of HBV multiplication can take place in nonliver nonhuman cells once the problem of entry into the host cell is overcome. In the absence of a small laboratory animal infectable by HBV, such transgenic mice should be helpful for the study of many aspects of viral multiplication. Images PMID:2845128

Interaction of TLR-IFN and HLA polymorphisms on susceptibility of chronic HBV infection in Southwest Han Chinese.

PubMed

He, Dengming; Tao, Shiqi; Guo, Shimin; Li, Maoshi; Wu, Junqiu; Huang, Hongfei; Guo, Xinwu; Yan, Guohua; Zhu, Peng; Wang, Yuming

2015-08-01

The toll-like receptor-interferon (TLR-IFN) signalling pathway plays a crucial role in HBV infection. Human leucocyte antigen (HLA) polymorphisms are associated with chronic HBV infection by genome wide association study (GWAS). We aimed to explore interaction between TLR-IFN and HLA gene polymorphisms in susceptibility of chronic HBV infection. In the Chinese Southwest Han population, 1191 chronic HBV infection patients and 273 HBV clearance were selected. A total of 39 single nucleotide polymorphism loci in 23 genes of the TLR-IFN pathway and four HLA polymorphism loci associated with chronic HBV infection identified by GWAS were selected for genotyping. SNPStats, QVALUE, and multifactor dimensionality reduction were used for statistical analysis. A significant association was seen in several of the TLR-IFN pathway genes, TLR9 rs352140 (OR = 0.70, P = 0.0088), IL1B rs16944 (OR = 0.67, P = 0.016), IL12B rs3212227 (OR = 1.38, P = 0.021), IFNGR1 rs3799488 (OR = 1.48, P = 0.0048), IFNGR2 rs1059293 (OR = 0.27, P = 0.011), MX1 rs467960 (OR = 0.68, P = 0.022), as well as four loci in HLA, rs3077 (OR = 0.55, P < 0.0001), rs2856718 (OR = 0.60, P = 4e-04), rs9277535 (OR = 0.54, P < 0.0001) and rs7453920 (OR = 0.43, P < 0.0001). A synergistic relationship was seen between rs9277535 and rs16944 (0.13%), rs1143623 and rs6613 (0.10%). The combination of rs9277535 in HLA and rs16944 in IL1B was the best model to predict chronic HBV infection (testing accuracy = 0.6040, P = 0.0010, cross-validation consistency = 10/10). TLR-IFN pathway gene polymorphisms are associated with chronic HBV infection. Interactions with polymorphisms in these genes may be one mechanism by which HLA polymorphisms influence susceptibility to chronic HBV infection, as specific single nucleotide polymorphism combinations are highly predictive of chronic HBV infection. © 2014 The Authors. Liver International Published by John Wiley & Sons Ltd.

Prevalence, risk factors, and impact of isolated antibody to hepatitis B core antigen and occult hepatitis B virus infection in HIV-1-infected pregnant women.

PubMed

Khamduang, Woottichai; Ngo-Giang-Huong, Nicole; Gaudy-Graffin, Catherine; Jourdain, Gonzague; Suwankornsakul, Weerapong; Jarupanich, Tapnarong; Chalermpolprapa, Veeradate; Nanta, Sirisak; Puarattana-Aroonkorn, Noossara; Tonmat, Sakchai; Lallemant, Marc; Goudeau, Alain; Sirirungsi, Wasna

2013-06-01

Prevalence and risk factors for isolated antibody to hepatitis B core antigen (anti-HBc) and occult hepatitis B virus (HBV) infection are not well known in human immunodeficiency virus type 1 (HIV-1)-infected pregnant women. It is unclear if women with occult infections are at risk of transmitting HBV to their infants. HIV-1-infected and HBV surface antigen (HBsAg)-negative pregnant women were tested for antibody to HBsAg (anti-HBs) and anti-HBc using enzyme immunoassay. Women with isolated anti-HBc were assessed for occult HBV infection, defined as HBV DNA levels >15 IU/mL, using the Abbott RealTime HBV DNA assay. Infants born to women with isolated anti-HBc and detectable HBV DNA were tested at 4 months of age for HBV DNA. Logistic regression analysis was used to identify factors associated with isolated anti-HBc and occult HBV infection. Among 1812 HIV-infected pregnant women, 1682 were HBsAg negative. Fourteen percent (95% confidence interval [CI], 12%-15%) of HBsAg-negative women had an isolated anti-HBc that was independently associated with low CD4 count, age >35 years, birth in northern Thailand, and positive anti-hepatitis C virus serology. Occult HBV infection was identified in 24% (95% CI, 18%-30%) of women with isolated anti-HBc, representing 2.6% (95% CI, 1.9%-3.5%) of HIV-1-infected pregnant women, and was inversely associated with HIV RNA levels. None of the women with isolated anti-HBc and occult HBV infection transmitted HBV to their infants. HIV-1-infected pregnant women with isolated anti-HBc and occult HBV infection have very low HBV DNA levels and are thus at very low risk to transmit HBV to their infants.

Quantitative intrahepatic HBV cccDNA correlates with histological liver inflammation in chronic hepatitis B virus infection.

PubMed

Liang, Ling-Bo; Zhu, Xia; Yan, Li-Bo; Du, Ling-Yao; Liu, Cong; Liao, Juan; Tang, Hong

2016-11-01

The aim of this study was to determine the role of baseline hepatitis B virus (HBV) forming covalently closed circular DNA (HBV cccDNA) in liver inflammation in patients infected with HBV with serum alanine aminotransferase (ALT) levels under two times the upper limit of normal (2×ULN). After liver biopsy and serum virological and biochemical marker screening, patients diagnosed with chronic HBV infection with serum ALT levels under 2×ULN and histological liver inflammation of less than grade G2 were prospectively recruited into this study. Recruitment took place between March 2009 and November 2010 at the Center of Infectious Disease, Sichuan University. Patient virological and biochemical markers, as well as markers of liver inflammation, were monitored. A total of 102 patients were recruited and 68 met the inclusion criteria; the median follow-up was 4.1 years (range 3.9-5.2 years). During follow-up, 41 patients (60.3%) exhibited signs of inflammation. Baseline HBV cccDNA >1 copy/cell (odds ratio 9.43, p=0.049) and liver inflammation grade ≥G1 (odds ratio 5.77, p=0.046) were both independent predictors of liver inflammation. A higher baseline intrahepatic HBV cccDNA level may increase the risk of liver inflammation. Further investigations will be required to validate HBV cccDNA as an intrahepatic virological marker of patients who require extended outpatient management. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Primary and booster vaccination in Latin American children with a DTPw-HBV/Hib combination: a randomized controlled trial.

PubMed

Espinoza, Felix; Tregnaghi, Miguel; Gentile, Angela; Abarca, Katia; Casellas, Javier; Collard, Alix; Lefevre, Inge; Jacquet, Jeanne-Marie

2010-10-15

Diphtheria-tetanus-whole-cell pertussis (DTPw)-based combination vaccines are an attractive option to rapidly achieve high coverage and protection against other important pathogens, such as hepatitis B virus (HBV) and Haemophilus influenzae type B (Hib). To ensure adequate antigen supply, GlaxoSmithKline Biologicals has introduced a new DTPw antigen source and developed a new DTPw-HBV/Hib combination vaccine containing a reduced amount of Hib polyribosylribitol phosphate (PRP). This study was undertaken to compare the immunogenicity and reactogenicity of this new DTPw-HBV/Hib vaccine with a licensed DTPw-HBV/Hib vaccine (Tritanrix™-HBV/Hib). This was a randomized, partially-blind, multicenter study in three countries in Latin America (Argentina, Chile and Nicaragua). Healthy children received either the new DTPw-HBV/Hib vaccine (1 of 3 lots; n = 439; double-blind) or Tritanrix™-HBV/Hib (n = 146; single-blind) co-administered with oral poliovirus vaccine (OPV) at 2, 4 and 6 months, with a booster dose at 18-24 months. One month after the end of the 3-dose primary vaccination course, the new DTPw-HBV/Hib vaccine was non-inferior to Tritanrix™-HBV/Hib in terms of seroprotection/vaccine response rates for all component antigens; ≥97.3% and ≥93.9% of subjects in the two groups, respectively, had seroprotective levels of antibodies against diphtheria, tetanus, hepatitis B and Hib and a vaccine response to the pertussis component. Persistence of antibodies against all vaccine antigens was comparable between groups, with marked increases in all antibody concentrations after booster administration in both groups. Both vaccines were generally well-tolerated as primary and booster doses. Results confirm the suitability of this new DTPw-HBV/Hib vaccine comprising antigens from a new source and a reduced PRP content for inclusion into routine childhood vaccination programs. http://www.clinicaltrials.gov NCT00332566.

Substantial variation in the hepatitis B surface antigen (HBsAg) in hepatitis B virus (HBV)-positive patients from South Africa: Reliable detection of HBV by the Elecsys HBsAg II assay.

PubMed

Gencay, Mikael; Vermeulen, Marion; Neofytos, Dionysis; Westergaard, Gaston; Pabinger, Stephan; Kriegner, Albert; Seffner, Anja; Gohl, Peter; Huebner, Kirsten; Nauck, Markus; Kaminski, Wolfgang E

2018-04-01

It is essential that hepatitis B surface antigen (HBsAg) diagnostic assays reliably detect genetic diversity in the major hydrophilic region (MHR) of HBsAg to avoid false-negative results. Mutations in this domain display marked ethno-geographic variation and may lead to failure to diagnose hepatitis B virus (HBV) infection. Evaluate diagnostic performance of the Elecsys ® HBsAg II Qualitative assay in a cohort of South African HBV-positive blood donors. A total of 179 South African HBsAg- and HBV DNA > 100 IU/mL-positive blood donor samples were included. Samples were sequenced for genetic variation in HBsAg MHR using next-generation ultra-deep sequencing. HBsAg seropositivity was determined using the Roche Elecsys HBsAg II Qualitative assay. Mutation rates were compared between the first (amino acids 124-137) and second (amino acids 139-147) loops of the immunodominant MHR 'a' determinant region. Frequency of occult HBV infection-associated Y100C mutations was also determined. We observed a total of 279 MHR mutations (117 variants) in 102 (57%) samples, of which 91 were located in the 'a' determinant region. The major vaccine-induced escape mutation G145R was observed in two samples. All occult HBV infection-associated Y100C and common diagnostic and vaccine-escape-associated P120T, G145R, K122R, M133L, M133T, Q129H, G130N, and T126S mutations were reliably detected by the assay, which consistently detected the presence of HBsAg in all 179 samples including samples with 11 novel mutations. Despite substantial variation in HBsAg MHR, the Elecsys HBsAg II Qualitative assay robustly detects HBV infection in this South African cohort. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

Eliminating hepatitis B by antagonizing cellular inhibitors of apoptosis.

PubMed

Ebert, Gregor; Allison, Cody; Preston, Simon; Cooney, James; Toe, Jesse G; Stutz, Michael D; Ojaimi, Samar; Baschuk, Nikola; Nachbur, Ueli; Torresi, Joseph; Silke, John; Begley, C Glenn; Pellegrini, Marc

2015-05-05

We have shown that cellular inhibitor of apoptosis proteins (cIAPs) impair clearance of hepatitis B virus (HBV) infection by preventing TNF-mediated killing/death of infected cells. A key question, with profound therapeutic implications, is whether this finding can be translated to the development of drugs that promote elimination of infected cells. Drug inhibitors of cIAPs were developed as cancer therapeutics to promote TNF-mediated tumor killing. These drugs are also known as Smac mimetics, because they mimic the action of the endogenous protein Smac/Diablo that antagonizes cIAP function. Here, we show using an immunocompetent mouse model of chronic HBV infection that birinapant and other Smac mimetics are able to rapidly reduce serum HBV DNA and serum HBV surface antigen, and they promote the elimination of hepatocytes containing HBV core antigen. The efficacy of Smac mimetics in treating HBV infection is dependent on their chemistry, host CD4(+) T cells, and TNF. Birinapant enhances the ability of entecavir, an antiviral nucleoside analog, to reduce viral DNA production in HBV-infected animals. These results indicate that birinapant and other Smac mimetics may have efficacy in treating HBV infection and perhaps, other intracellular infections.

HIV, HBV and HCV Coinfection Prevalence in Iran--A Systematic Review and Meta-Analysis.

PubMed

Bagheri Amiri, Fahimeh; Mostafavi, Ehsan; Mirzazadeh, Ali

2016-01-01

worldwide, hepatitis C and B virus infections (HCV and HCV), are the two most common coinfections with human immunodeficiency virus (HIV) and has become a major threat to the survival of HIV-infected persons. The review aimed to estimate the prevalence of HIV, HBV, HCV, HIV/HCV and HIV/HBV and triple coinfections in different subpopulations in Iran. Following PRISMA guidelines, we conducted a systematic review and meta-analysis of reports on prevalence of HIV, HBV, HCV and HIV coinfections in different subpopulations in Iran. We systematically reviewed the literature to identify eligible studies from January 1996 to March 2012 in English or Persian/Farsi databases. We extracted the prevalence of HIV antibodies (diagnosed by Elisa confirmed with Western Blot test), HCV antibodies and HBsAg (with confirmatory laboratory test) as the main primary outcome. We reported the prevalence of the three infections and coinfections as point and 95% confidence intervals. HIV prevalence varied from %0.00 (95% CI: 0.00-0.003) in the general population to %17.25 (95% CI: 2.94-31.57) in people who inject drugs (PWID). HBV prevalence ranged from % 0.00 (95% CI: 0.00-7.87) in health care workers to % 30.9 (95% CI: 27.88-33.92) in PWID. HCV prevalence ranged from %0.19 (95% CI: 0.00-0.66) in health care workers to %51.46 (95% CI: 34.30-68.62) in PWID. The coinfection of HIV/HBV and also HIV/HCV in the general population and in health care workers was zero, while the most common coinfections were HIV/HCV (10.95%), HIV/HBV (1.88%) and triple infections (1.25%) in PWID. We found that PWID are severely and disproportionately affected by HIV and the other two infections, HCV and HBV. Screenings of such coinfections need to be reinforced to prevent new infections and also reduce further transmission in their community and to others.

HIV, HBV and HCV Coinfection Prevalence in Iran - A Systematic Review and Meta-Analysis

PubMed Central

Bagheri Amiri, Fahimeh; Mostafavi, Ehsan; Mirzazadeh, Ali

2016-01-01

Background worldwide, hepatitis C and B virus infections (HCV and HCV), are the two most common coinfections with human immunodeficiency virus (HIV) and has become a major threat to the survival of HIV-infected persons. The review aimed to estimate the prevalence of HIV, HBV, HCV, HIV/HCV and HIV/HBV and triple coinfections in different subpopulations in Iran. Method Following PRISMA guidelines, we conducted a systematic review and meta-analysis of reports on prevalence of HIV, HBV, HCV and HIV coinfections in different subpopulations in Iran. We systematically reviewed the literature to identify eligible studies from January 1996 to March 2012 in English or Persian/Farsi databases. We extracted the prevalence of HIV antibodies (diagnosed by Elisa confirmed with Western Blot test), HCV antibodies and HBsAg (with confirmatory laboratory test) as the main primary outcome. We reported the prevalence of the three infections and coinfections as point and 95% confidence intervals. Findings HIV prevalence varied from %0.00 (95% CI: 0.00–0.003) in the general population to %17.25 (95% CI: 2.94–31.57) in people who inject drugs (PWID). HBV prevalence ranged from % 0.00 (95% CI: 0.00–7.87) in health care workers to % 30.9 (95% CI: 27.88–33.92) in PWID. HCV prevalence ranged from %0.19 (95% CI: 0.00–0.66) in health care workers to %51.46 (95% CI: 34.30–68.62) in PWID. The coinfection of HIV/HBV and also HIV/HCV in the general population and in health care workers was zero, while the most common coinfections were HIV/HCV (10.95%), HIV/HBV (1.88%) and triple infections (1.25%) in PWID. Conclusions We found that PWID are severely and disproportionately affected by HIV and the other two infections, HCV and HBV. Screenings of such coinfections need to be reinforced to prevent new infections and also reduce further transmission in their community and to others. PMID:27031352

Identifying the Genotypes of Hepatitis B Virus (HBV) with DNA Origami Label.

PubMed

Liu, Ke; Pan, Dun; Wen, Yanqin; Zhang, Honglu; Chao, Jie; Wang, Lihua; Song, Shiping; Fan, Chunhai; Shi, Yongyong

2018-02-01

The hepatitis B virus (HBV) genotyping may profoundly affect the accurate diagnosis and antiviral treatment of viral hepatitis. Existing genotyping methods such as serological, immunological, or molecular testing are still suffered from substandard specificity and low sensitivity in laboratory or clinical application. In a previous study, a set of high-efficiency hybridizable DNA origami-based shape ID probes to target the templates through which genetic variation could be determined in an ultrahigh resolution of atomic force microscopy (AFM) nanomechanical imaging are established. Here, as a further confirmatory research to explore the sensitivity and applicability of this assay, differentially predesigned DNA origami shape ID probes are also developed for precisely HBV genotyping. Through the specific identification of visualized DNA origami nanostructure with clinical HBV DNA samples, the genetic variation information of genotypes can be directly identified under AFM. As a proof-of-concept, five genotype B and six genotype C are detected in 11 HBV-infected patients' blood DNA samples of Han Chinese population in the single-blinded test. The AFM image-based DNA origami shape ID genotyping approach shows high specificity and sensitivity, which could be promising for virus infection diagnosis and precision medicine in the future. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

On the mineral core of ferritin-like proteins: structural and magnetic characterization

NASA Astrophysics Data System (ADS)

García-Prieto, A.; Alonso, J.; Muñoz, D.; Marcano, L.; Abad Díaz de Cerio, A.; Fernández de Luis, R.; Orue, I.; Mathon, O.; Muela, A.; Fdez-Gubieda, M. L.

2015-12-01

It is generally accepted that the mineral core synthesized by ferritin-like proteins consists of a ferric oxy-hydroxide mineral similar to ferrihydrite in the case of horse spleen ferritin (HoSF) and an oxy-hydroxide-phosphate phase in plant and prokaryotic ferritins. The structure reflects a dynamic process of deposition and dissolution, influenced by different biological, chemical and physical variables. In this work we shed light on this matter by combining a structural (High Resolution Transmission Electron Microscopy (HRTEM) and Fe K-edge X-ray Absorption Spectroscopy (XAS)) and a magnetic study of the mineral core biomineralized by horse spleen ferritin (HoSF) and three prokaryotic ferritin-like proteins: bacterial ferritin (FtnA) and bacterioferritin (Bfr) from Escherichia coli and archaeal ferritin (PfFtn) from Pyrococcus furiosus. The prokaryotic ferritin-like proteins have been studied under native conditions and inside the cells for the sake of preserving their natural attributes. They share with HoSF a nanocrystalline structure rather than an amorphous one as has been frequently reported. However, the presence of phosphorus changes drastically the short-range order and magnetic response of the prokaryotic cores with respect to HoSF. The superparamagnetism observed in HoSF is absent in the prokaryotic proteins, which show a pure atomic-like paramagnetic behaviour attributed to phosphorus breaking the Fe-Fe exchange interaction.It is generally accepted that the mineral core synthesized by ferritin-like proteins consists of a ferric oxy-hydroxide mineral similar to ferrihydrite in the case of horse spleen ferritin (HoSF) and an oxy-hydroxide-phosphate phase in plant and prokaryotic ferritins. The structure reflects a dynamic process of deposition and dissolution, influenced by different biological, chemical and physical variables. In this work we shed light on this matter by combining a structural (High Resolution Transmission Electron Microscopy (HRTEM

Proteoglycan biosynthesis in chondrocytes: protein A-gold localization of proteoglycan protein core and chondroitin sulfate within Golgi subcompartments

PubMed Central

1985-01-01

The intracellular pathway of cartilage proteoglycan biosynthesis was investigated in isolated chondrocytes using a protein A-gold electron microscopy immunolocalization procedure. Proteoglycans contain a protein core to which chondroitin sulfate and keratan sulfate chains and oligosaccharides are added in posttranslational processing. Specific antibodies have been used in this study to determine separately the distribution of the protein core and chondroitin sulfate components. In normal chondrocytes, proteoglycan protein core was readily localized only in smooth-membraned vesicles which co-labeled with ricin, indicating them to be galactose-rich medial/trans-Golgi cisternae, whereas there was only a low level of labeling in the rough endoplasmic reticulum. Chondroitin sulfate was also localized in medial/trans-Golgi cisternae of control chondrocytes but was not detected in other cellular compartments. In cells treated with monensin (up to 1.0 microM), which strongly inhibits proteoglycan secretion (Burditt, L.J., A. Ratcliffe, P. R. Fryer, and T. Hardingham, 1985, Biochim. Biophys. Acta., 844:247-255), there was greatly increased intracellular localization of proteoglycan protein core in both ricin- positive vesicles, and in ricin-negative vesicles (derived from cis- Golgi stacks) and in the distended rough endoplasmic reticulum. Chondroitin sulfate also increased in abundance after monensin treatment, but continued to be localized only in ricin-positive vesicles. The results suggested that the synthesis of chondroitin sulfate on proteoglycan only occurs in medial/trans-Golgi cisternae as a late event in proteoglycan biosynthesis. This also suggests that glycosaminoglycan synthesis on proteoglycans takes place in a compartment in common with events in the biosynthesis of both O-linked and N-linked oligosaccharides on other secretory glycoproteins. PMID:3934179

Soluble ST2 Plasma Concentrations Predict Mortality in HBV-Related Acute-on-Chronic Liver Failure

PubMed Central

Mo, Zhishuo; Zhu, Jianyun; Pang, Xiuqing; Wu, Zhebin; Wang, Ke; Li, Xinhua; Xie, Dongying; Gao, Zhiliang

2015-01-01

Hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) is a rapidly progressing and frequently fatal condition. The aim of this study was to determine whether interleukin- (IL-) 33 and soluble ST2 (sST2) were associated with disease severity and mortality in HBV-ACLF. We found that plasma levels of sST2 but not IL-33 were higher in HBV-ACLF patients compared with chronic hepatitis B (CHB) patients and healthy controls. However, plasma levels of IL-33, TNF-α, IFN-γ, and IL-10 did not correlate with sST2 levels. Similarly, immunohistochemistry revealed low IL-33 expression and high ST2 expression in liver sections of patients with HBV-ACLF. Evaluation of dynamic changes of sST2 in HBV-ACLF showed that plasma sST2 levels increased over time in patients who died during the 180-day follow-up but decreased in those who survived. In addition, plasma sST2 level after week 1 correlated with disease severity, as assessed by total bilirubin, prothrombin time, and model for end-stage liver disease score. Results of Kaplan-Meier survival analysis showed that higher sST2 concentration (≥87 ng/mL) at week 3 was associated with poor survival. These findings indicate the potential usefulness of sST2 as a predictor of disease severity and in making treatment decisions for patients with HBV-ACLF. PMID:25892854

Genetic polymorphism of interleukin-6 influences susceptibility to HBV-related hepatocellular carcinoma in a male Chinese Han population.

PubMed

Tang, Shengli; Yuan, Yufeng; He, Yueming; Pan, Dingyu; Zhang, Yongxi; Liu, Yuanyuan; Liu, Quanyan; Zhang, Zhonglin; Liu, Zhisu

2014-04-01

As a multifunctional cytokine, interleukin-6 (IL-6) plays a key role in chronic inflammation as well as tumor growth and progression of hepatitis B virus (HBV) infection. Recent studies have implicated that single nucleotide polymorphism (SNP) -572C>G (rs1800796) located within the promoter region of IL-6 gene was associated with susceptibility to several diseases. Here, a case-control study was undertaken to investigate the association between this polymorphism and HBV-related hepatocellular carcinoma (HCC) susceptibility in a Chinese Han population. A total of 900 patients with chronic HBV infection, including 505 HBV-related HCC patients and 395 HBV infected patients without HCC were enrolled, and rs1800796 polymorphism was genotyped by the TaqMan method and DNA sequencing technology. The results indicated no significant association between rs1800796 polymorphism and the risk of HBV-related HCC in all subjects; however, a significant difference was identified in male subjects. Under the dominant model, male subjects with the G allele (CG/GG) have higher susceptibility to HBV-related HCC than those with CC genotype after adjusting confounding factors (P=0.012, odds ratio [OR] 1.68, 95% confidence interval [95% CI] 1.15-2.42). Our results suggested that rs1800796 polymorphism of IL-6 gene was associated with susceptibility to HBV-related HCC in a male Chinese Han population. Copyright © 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

PRMT5 restricts hepatitis B virus replication through epigenetic repression of covalently closed circular DNA transcription and interference with pregenomic RNA encapsidation.

PubMed

Zhang, Wen; Chen, Jieliang; Wu, Min; Zhang, Xiaonan; Zhang, Min; Yue, Lei; Li, Yaming; Liu, Jiangxia; Li, Baocun; Shen, Fang; Wang, Yang; Bai, Lu; Protzer, Ulrike; Levrero, Massimo; Yuan, Zhenghong

2017-08-01

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. The covalently closed circular DNA (cccDNA) minichromosome, which serves as the template for the transcription of viral RNAs, plays a key role in viral persistence. While accumulating evidence suggests that cccDNA transcription is regulated by epigenetic machinery, particularly the acetylation of cccDNA-bound histone 3 (H3) and H4, the potential contributions of histone methylation and related host factors remain obscure. Here, by screening a series of methyltransferases and demethylases, we identified protein arginine methyltransferase 5 (PRMT5) as an effective restrictor of HBV transcription and replication. In cell culture-based models for HBV infection and in liver tissues of patients with chronic HBV infection, we found that symmetric dimethylation of arginine 3 on H4 on cccDNA was a repressive marker of cccDNA transcription and was regulated by PRMT5 depending on its methyltransferase domain. Moreover, PRMT5-triggered symmetric dimethylation of arginine 3 on H4 on the cccDNA minichromosome involved an interaction with the HBV core protein and the Brg1-based human SWI/SNF chromatin remodeler, which resulted in down-regulation of the binding of RNA polymerase II to cccDNA. In addition to the inhibitory effect on cccDNA transcription, PRMT5 inhibited HBV core particle DNA production independently of its methyltransferase activity. Further study revealed that PRMT5 interfered with pregenomic RNA encapsidation by preventing its interaction with viral polymerase protein through binding to the reverse transcriptase-ribonuclease H region of polymerase, which is crucial for the polymerase-pregenomic RNA interaction. PRMT5 restricts HBV replication through a two-part mechanism including epigenetic suppression of cccDNA transcription and interference with pregenomic RNA encapsidation; these findings improve the understanding of epigenetic regulation of HBV transcription and host-HBV

A new series of HAPs as anti-HBV agents targeting at capsid assembly.

PubMed

Yang, Xiu-yan; Xu, Xiao-qian; Guan, Hua; Wang, Li-li; Wu, Qin; Zhao, Guo-ming; Li, Song

2014-09-01

A series of novel Heteroaryldihydropyrimidines (HAPs) derivatives were designed and synthesized as potent inhibitors of HBV capsid assembly. These compounds were prepared from efforts to optimize an earlier series of HAPs, and compounds Mo1, Mo7, Mo8, Mo10, Mo12, and Mo13 demonstrated potent inhibition of HBV DNA replication at submicromolar range. Copyright © 2014. Published by Elsevier Ltd.

The ESFRI Instruct Core Centre Frankfurt: automated high-throughput crystallization suited for membrane proteins and more.

PubMed

Thielmann, Yvonne; Koepke, Juergen; Michel, Hartmut

2012-06-01

Structure determination of membrane proteins and membrane protein complexes is still a very challenging field. To facilitate the work on membrane proteins the Core Centre follows a strategy that comprises four labs of protein analytics and crystal handling, covering mass spectrometry, calorimetry, crystallization and X-ray diffraction. This general workflow is presented and a capacity of 20% of the operating time of all systems is provided to the European structural biology community within the ESFRI Instruct program. A description of the crystallization service offered at the Core Centre is given with detailed information on screening strategy, screens used and changes to adapt high throughput for membrane proteins. Our aim is to constantly develop the Core Centre towards the usage of more efficient methods. This strategy might also include the ability to automate all steps from crystallization trials to crystal screening; here we look ahead how this aim might be realized at the Core Centre.

Hepatitis C Virus core+1/ARF Protein Modulates the Cyclin D1/pRb Pathway and Promotes Carcinogenesis.

PubMed

Moustafa, Savvina; Karakasiliotis, Ioannis; Mavromara, Penelope

2018-05-01

Viruses often encompass overlapping reading frames and unconventional translation mechanisms in order to maximize the output from a minimum genome and to orchestrate their timely gene expression. Hepatitis C virus (HCV) possesses such an unconventional open reading frame (ORF) within the core-coding region, encoding an additional protein, initially designated ARFP, F, or core+1. Two predominant isoforms of core+1/ARFP have been reported, core+1/L, initiating from codon 26, and core+1/S, initiating from codons 85/87 of the polyprotein coding region. The biological significance of core+1/ARFP expression remains elusive. The aim of the present study was to gain insight into the functional and pathological properties of core+1/ARFP through its interaction with the host cell, combining in vitro and in vivo approaches. Our data provide strong evidence that the core+1/ARFP of HCV-1a stimulates cell proliferation in Huh7-based cell lines expressing either core+1/S or core+1/L isoforms and in transgenic liver disease mouse models expressing core+1/S protein in a liver-specific manner. Both isoforms of core+1/ARFP increase the levels of cyclin D1 and phosphorylated Rb, thus promoting the cell cycle. In addition, core+1/S was found to enhance liver regeneration and oncogenesis in transgenic mice. The induction of the cell cycle together with increased mRNA levels of cell proliferation-related oncogenes in cells expressing the core+1/ARFP proteins argue for an oncogenic potential of these proteins and an important role in HCV-associated pathogenesis. IMPORTANCE This study sheds light on the biological importance of a unique HCV protein. We show here that core+1/ARFP of HCV-1a interacts with the host machinery, leading to acceleration of the cell cycle and enhancement of liver carcinogenesis. This pathological mechanism(s) may complement the action of other viral proteins with oncogenic properties, leading to the development of hepatocellular carcinoma. In addition, given that

A Critical Role for Protein Degradation in the Nucleus Accumbens Core in Cocaine Reward Memory

PubMed Central

Ren, Zhen-Yu; Liu, Meng-Meng; Xue, Yan-Xue; Ding, Zeng-Bo; Xue, Li-Fen; Zhai, Suo-Di; Lu, Lin

2013-01-01

The intense associative memories that develop between cocaine-paired contexts and rewarding stimuli contribute to cocaine seeking and relapse. Previous studies have shown impairment in cocaine reward memories by manipulating a labile state induced by memory retrieval, but the mechanisms that underlie the destabilization of cocaine reward memory are unknown. In this study, using a Pavlovian cocaine-induced conditioned place preference (CPP) procedure in rats, we tested the contribution of ubiquitin-proteasome system-dependent protein degradation in destabilization of cocaine reward memory. First, we found that polyubiquitinated protein expression levels and polyubiquitinated N-ethylmaleimide-sensitive fusion (NSF) markedly increased 15 min after retrieval while NSF protein levels decreased 1 h after retrieval in the synaptosomal membrane fraction in the nucleus accumbens (NAc) core. We then found that infusion of the proteasome inhibitor lactacystin into the NAc core prevented the impairment of memory reconsolidation induced by the protein synthesis inhibitor anisomycin and reversed the effects of anisomycin on NSF and glutamate receptor 2 (GluR2) protein levels in the synaptosomal membrane fraction in the NAc core. We also found that lactacystin infusion into the NAc core but not into the shell immediately after extinction training sessions inhibited CPP extinction and reversed the extinction training-induced decrease in NSF and GluR2 in the synaptosomal membrane fraction in the NAc core. Finally, infusions of lactacystin by itself into the NAc core immediately after each training session or before the CPP retrieval test had no effect on the consolidation and retrieval of cocaine reward memory. These findings suggest that ubiquitin-proteasome system-dependent protein degradation is critical for retrieval-induced memory destabilization. PMID:23303053

Core protein cleavage by signal peptide peptidase is required for hepatitis C virus-like particle assembly

PubMed Central

Ait-Goughoulte, Malika; Hourioux, Christophe; Patient, Romuald; Trassard, Sylvie; Brand, Denys; Roingeard, Philippe

2006-01-01

SUMMARY Hepatitis C virus (HCV) core protein, expressed with a Semliki forest virus (SFV) replicon, self-assembles into HCV-like particles (HCV-LP) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV assembly and morphogenesis by electron microscopy. We used this model to investigate whether the processing of the HCV core protein by the signal peptide peptidase (SPP) is required for the HCV-LP assembly. We designed several mutants as there are conflicting reports concerning the cleavage of mutant proteins by SPP. Production of the only core mutant protein that escaped SPP processing led to the formation of multiple layers of electron-dense ER membrane, with no evidence of HCV-LP assembly. Our data shed light on the HCV core residues involved in SPP cleavage and suggest that this cleavage is essential for HCV assembly. PMID:16528035

Inhibition of HBV Replication in HepG2.2.15 Cells by Human Peripheral Blood Mononuclear Cell-Derived Dendritic Cells.

PubMed

Liu, Tao; Song, Hong-Li; Zheng, Wei-Ping; Shen, Zhong-Yang

2015-01-01

Anti-HBV therapy is essential for patients awaiting liver transplantation. This study aimed to explore the effects of dendritic cells (DCs) derived from the peripheral blood of hepatitis B patients on the replication of HBV in vivo and to evaluate the biosafety of DCs in clinical therapy. Peripheral blood mononuclear cells (PBMCs) were isolated from HBV-infected patients and maturation-promoting factors and both HBsAg and HBcAg were used to induce DC maturation. Mature DCs and lymphocytes were co-cultured with human hepatocyte cell HL-7702 or HBV-producing human hepatocellular carcinoma cell HepG2.2.15. We found that mature lymphocytes exposed to DCs in vitro did not influence morphology or activities of HL-7702 and HepG2.2.15 cells. Liver function indexes and endotoxin levels in the cell supernatants did not change in these co-cultures. Additionally, supernatant and intracellular HBV DNA levels were reduced when HepG2.2.15 cells were co-cultured with mature lymphocytes that had been cultured with DCs, and HBV covalently closed circular DNA (cccDNA) levels in HepG2.2.15 cells also decreased. Importantly, DC-mediated immunotherapy had no mutagenic effect on HBV genomic DNA by gene sequencing of the P, S, X, and C regions of HBV genomic DNA. We conclude that PBMC-derived DCs from HBV-infected patients act on autologous lymphocytes to suppress HBV replication and these DC clusters showed favorable biosafety. © 2015 by the Association of Clinical Scientists, Inc.

Lipid-coated mannitol core microparticles for sustained release of protein.

PubMed

Wang, Bifeng; Friess, Wolfgang

2018-07-01

Parenteral sustained release systems for proteins which provide therapeutic levels over a longer period avoiding frequent administration, which preserve protein stability during manufacturing, storage and application and which are biodegradable and highly biocompatible in the body are intensively sought after. The aim of this study was to generate and study mannitol core microparticles loaded with a monoclonal antibody IgG1 and coated with lipid either hard fat or glyceryl stearate at different coating levels. The protein was stabilized with 22.5 mg/mL sucrose, 0.1% PS 80, 10 mM methionine in 10 mM His buffer pH 7.2 during the spray loading process. 30 g protein-loaded mannitol carrier microparticles were coated with 5 g, 10 g, 20 g and 30 g of lipid, respectively. Placing more lipid onto the protein-loaded microparticles reduced both burst and release rate, and the particles maintained their geometric form during the release test. The IgG1 release from microparticles covered with a hard fat layer extended up to 6 weeks. The IgG1 was released in its monomeric form and maintained its secondary structure as shown by FTIR. Incomplete release of IgG1 from glyceryl stearate-coated microparticles was observed, which may be due to the small pore sizes of the glyceryl stearate layer or a detrimental surfactant character of glyceryl stearate to protein. Hence, these hard fat-coated mannitol core microparticles have high potential for protein delivery. Copyright © 2018 Elsevier B.V. All rights reserved.

Protective Effects of Moringa oleifera on HBV Genotypes C and H Transiently Transfected Huh7 Cells.

PubMed

Feustel, Sina; Ayón-Pérez, Fabiola; Sandoval-Rodriguez, Ana; Rodríguez-Echevarría, Roberto; Contreras-Salinas, Homero; Armendáriz-Borunda, Juan; Sánchez-Orozco, L V

2017-01-01

Chronic hepatitis B infection treatment implicates a long-lasting treatment. M. oleifera extracts contain compounds with antiviral, antioxidant, and antifibrotic properties. In this study, the effect of M. oleifera was evaluated in Huh7 cells expressing either HBV genotypes C or H for the antiviral, antifibrotic, anti-inflammatory, and antioxidative responses. Huh7 cells were treated with an aqueous extract of M. oleifera (leaves) at doses of 0, 30, 45, or 60 μ g/mL. The replicative virus and TGF-β1 , CTGF , CAT , IFN-β1 , and pgRNA expressions were measured by real time. HBsAg and IL-6 titers were determined by ELISA. CTGF , TGF-β1 , IFN-β1 , and pgRNA expressions decreased with M. oleifera treatment irrespective of the HBV genotype. HBsAg secretion in the supernatant of transfected Huh7 cells with both HBV genotypes was decreased regardless of the dose of M. oleifera . Similar effect was observed in proinflammatory cytokine IL-6, which had a tendency to decrease at 24 hours of treatment. Transfection with both HBV genotypes strongly decreased CAT expression, which is retrieved with M. oleifera treatment. M. oleifera treatment reduced fibrosis markers, IL-6, and HBsAg secretion in HBV genotypes C and H. However, at the level of replication, only HBV-DNA genotype C was slightly reduced with this treatment.

Synthesis and assembly of Hepatitis B virus envelope protein-derived particles in Escherichia coli.

PubMed

Li, Hao; Onbe, Keisuke; Liu, Qiushi; Iijima, Masumi; Tatematsu, Kenji; Seno, Masaharu; Tada, Hiroko; Kuroda, Shun' Ichi

2017-08-19

Hepatitis B virus (HBV) envelope particles have been synthesized in eukaryotic cells (e.g., mammalian cells, insect cells, and yeast cells) as an HB vaccine immunogen and drug delivery system (DDS) nanocarrier. Many researchers had made attempts to synthesize the particles in Escherichia coli for minimize the cost and time for producing HBV envelope particles, but the protein was too deleterious to be synthesized in E. coli. In this study, we generated deletion mutants of HBV envelope L protein (389 amino acid residues (aa)) containing three transmembrane domains (TM1, TM2, TM3). The ΔNC mutant spanning from TM2 to N-terminal half of TM3 (from 237 aa to 335 aa) was found as a shortest form showing spontaneous particle formation. After the N-terminal end of ΔNC mutant was optimized by the N-end rule for E. coli expression, the modified ΔNC mutant (mΔNC) was efficiently expressed as particles in E. coli. The molecular mass of mΔNC particle was approx. 670 kDa, and the diameter was 28.5 ± 6.2 nm (mean ± SD, N = 61). The particle could react with anti-HBV envelope S protein antibody, indicating the particles exhibited S antigenic domain outside as well as HBV envelope particles. Taken together, the E. coli-derived mΔNC particles could be used as a substitute of eukaryotic cell-derived HBV envelope particles for versatile applications. Copyright © 2017 Elsevier Inc. All rights reserved.

Bidirectional Lipid Droplet Velocities Are Controlled by Differential Binding Strengths of HCV Core DII Protein

PubMed Central

Lyn, Rodney K.; Hope, Graham; Sherratt, Allison R.; McLauchlan, John; Pezacki, John Paul

2013-01-01

Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein’s lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV. PMID:24223760

Functional analysis of 'a' determinant mutations associated with occult HBV in HIV-positive South Africans.

PubMed

Powell, Eleanor A; Boyce, Ceejay L; Gededzha, Maemu P; Selabe, Selokela G; Mphahlele, M Jeffrey; Blackard, Jason T

2016-07-01

Occult hepatitis B is defined by the presence of hepatitis B virus (HBV) DNA in the absence of hepatitis B surface antigen (HBsAg). Occult HBV is associated with the development of hepatocellular carcinoma, reactivation during immune suppression, and virus transmission. Viral mutations contribute significantly to the occult HBV phenotype. Mutations in the 'a' determinant of HBsAg are of particular interest, as these mutations are associated with immune escape, vaccine escape and diagnostic failure. We examined the effects of selected occult HBV-associated mutations identified in a population of HIV-positive South Africans on HBsAg production in vitro. Mutations were inserted into two different chronic HBV backbones and transfected into a hepatocyte-derived cell line. HBsAg levels were quantified by enzyme-linked immunosorbent assay (ELISA), while the detectability of mutant HBsAg was determined using an HA-tagged HBsAg expression system. Of the seven mutations analysed, four (S132P, C138Y, N146D and C147Y) resulted in decreased HBsAg expression in one viral background but not in the second viral background. One mutation (N146D) led to a decrease in HBsAg detected as compared to HA-tag, indicating that this mutation compromises the ability of the ELISA to detect HBsAg. The contribution of occult-associated mutations to the HBsAg-negative phenotype of occult HBV cannot be determined adequately by testing the effect of the mutation in a single viral background, and rigorous analysis of these mutations is required.

Hepatitis B virus X protein-induced upregulation of CAT-1 stimulates proliferation and inhibits apoptosis in hepatocellular carcinoma cells

PubMed Central

Dai, Rongjuan; Peng, Feng; Xiao, Xinqiang; Gong, Xing; Jiang, Yongfang; Zhang, Min; Tian, Yi; Xu, Yun; Ma, Jing; Li, Mingming; Luo, Yue; Gong, Guozhong

2017-01-01

The HBx protein of hepatitis B virus (HBV) is widely recognized to be a critical oncoprotein contributing to the development of HBV-related hepatocellular carcinoma (HCC). In addition, cationic amino acid transporter 1 (CAT-1) gene is a target of miR-122. In this study, we found that CAT-1 protein levels were higher in HBV-related HCC carcinomatous tissues than in para-cancerous tumor tissues, and that CAT-1 promoted HCC cell growth, proliferation, and metastasis. Moreover, HBx-induced decreases in Gld2 and miR-122 levels that contributed to the upregulation of CAT-1 in HCC. These results indicate that a Gld2/miR-122/CAT-1 pathway regulated by HBx likely participates in HBV-related hepatocellular carcinogenesis. PMID:28977838

Hepatitis B virus X protein-induced upregulation of CAT-1 stimulates proliferation and inhibits apoptosis in hepatocellular carcinoma cells.

PubMed

Dai, Rongjuan; Peng, Feng; Xiao, Xinqiang; Gong, Xing; Jiang, Yongfang; Zhang, Min; Tian, Yi; Xu, Yun; Ma, Jing; Li, Mingming; Luo, Yue; Gong, Guozhong

2017-09-22

The HBx protein of hepatitis B virus (HBV) is widely recognized to be a critical oncoprotein contributing to the development of HBV-related hepatocellular carcinoma (HCC). In addition, cationic amino acid transporter 1 (CAT-1) gene is a target of miR-122. In this study, we found that CAT-1 protein levels were higher in HBV-related HCC carcinomatous tissues than in para-cancerous tumor tissues, and that CAT-1 promoted HCC cell growth, proliferation, and metastasis. Moreover, HBx-induced decreases in Gld2 and miR-122 levels that contributed to the upregulation of CAT-1 in HCC. These results indicate that a Gld2/miR-122/CAT-1 pathway regulated by HBx likely participates in HBV-related hepatocellular carcinogenesis.

Genogeography and Immune Epitope Characteristics of Hepatitis B Virus Genotype C Reveals Two Distinct Types: Asian and Papua-Pacific

PubMed Central

Thedja, Meta Dewi; Muljono, David Handojo; Ie, Susan Irawati; Sidarta, Erick; Turyadi; Verhoef, Jan; Marzuki, Sangkot

2015-01-01

Distribution of hepatitis B virus (HBV) genotypes/subgenotypes is geographically and ethnologically specific. In the Indonesian archipelago, HBV genotype C (HBV/C) is prevalent with high genome variability, reflected by the presence of 13 of currently existing 16 subgenotypes. We investigated the association between HBV/C molecular characteristics with host ethnicity and geographical distribution by examining various subgenotypes of HBV/C isolates from the Asia and Pacific region, with further analysis on the immune epitope characteristics of the core and surface proteins. Phylogenetic tree was constructed based on complete HBV/C genome sequences from Asia and Pacific region, and genetic distance between isolates was also examined. HBV/C surface and core immune epitopes were analyzed and grouped by comparing the amino acid residue characteristics and geographical origins. Based on phylogenetic tree and geographical origins of isolates, two major groups of HBV/C isolates—East-Southeast Asia and Papua-Pacific—were identified. Analysis of core and surface immune epitopes supported these findings with several amino acid substitutions distinguishing the East-Southeast Asia isolates from the Papua-Pacific isolates. A west-to-east gradient of HBsAg subtype distribution was observed with adrq+ prominent in the East and Southeast Asia and adrq- in the Pacific, with several adrq-indeterminate subtypes observed in Papua and Papua New Guinea (PNG). This study indicates that HBV/C isolates can be classified into two types, the Asian and the Papua-Pacific, based on the virus genome diversity, immune epitope characteristics, and geographical distribution, with Papua and PNG as the molecular evolutionary admixture region in the switching from adrq+ to adrq-. PMID:26162099

Unusual presentation of hepatitis B serological markers in an Amerindian community of Venezuela with a majority of occult cases

PubMed Central

2011-01-01

Background Occult hepatitis B infection (OBI) is characterized by the presence of hepatitis B virus (HBV) DNA in the absence of HBsAg in the serum of patients. The aim of this study was to characterize HBV infection among a Piaroa community, an Amerindian group which exhibits significant evidence of exposure to HBV but relatively low presence of HBsAg, and to explore the presence of OBI in this population. Results Of 150 sera, with 17% anti-HBc and 1.3% HBsAg prevalence, 70 were tested for the presence of HBV DNA. From these, 25 (36%) were found positive for HBV DNA by PCR in the core region. Two of these 25 sera were HBsAg positive, indicating an overt infection. Of the remaining 68 sera tested, 23 exhibited OBI. Of these, 13 were HBV DNA out of 25 anti-HBc positive (52%) and 10 HBV DNA positive, out of 43 anti-HBc negative (23%), with a statistical significance of p = 0.03. Viral DNA and HBsAg were present intermittently in follow up sera of 13 individuals. Sequence analysis in the core region of the amplified DNA products showed that all the strains belonged to HBV genotype F3. The OBI isolates displayed 96-100% nucleotide identity between them. One isolate exhibited the co-circulation of a wild type variant with a variant with a premature stop codon at the core protein, and a variant exhibiting a deletion of 28 amino acids. Conclusions The frequency of OBI found in this Amerindian group warrants further studies in other communities exhibiting different degrees of HBV exposure. PMID:22152023

Prevalence of precore-defective mutant of hepatitis B virus in HBV carriers.

PubMed

Niitsuma, H; Ishii, M; Saito, Y; Miura, M; Kobayashi, K; Ohori, H; Toyota, T

1995-08-01

Two hundred and seventy-three serum specimens from hepatitis B virus (HBV) carriers were examined for the presence of a characteristic one point mutation at nucleotide (nt) 1896 from the EcoRI site of the HBV genome in the precore region (the preC mutant) using restriction fragment length polymorphism (RFLP) analysis. This assay approach could detect preC mutants or wild-type sequences when either form constituted more than 10% of the total sample. Overall, 65.5% (76/116) of HBeAg-positive carriers had only the preC wild-type. All HBeAg-positive asymptomatic carriers (n = 14) had only the preC wild-type. In patients with chronic hepatitis B and in anti-HBe-positive asymptomatic carriers, increased prevalence of the preC mutant was associated with the development of anti-HBe antibodies and normalization of the serum alanine aminotransferase concentration. Furthermore, 27 (29.0%) of 93 HBeAg-negative carriers had unexpectedly preC wild-type sequences only. Direct sequencing of the HBV precore region of HBV specimens from 24 patients revealed no mutation at nt 1896, supporting the specificity of the RFLP analysis. These results suggest that RFLP analysis was accurate for the detection of the preC mutation and that the absence of serum HBeAg cannot be explained solely by the dominance of the preC mutant.

Expression of a functional intrabody against hepatitis C virus core protein in Escherichia coli and silkworm pupae.

PubMed

Kato, Tatsuya; Hasegawa, Moeko; Yamamoto, Takeshi; Miyazaki, Takatsugu; Suzuki, Ryosuke; Wakita, Takaji; Suzuki, Tetsuro; Park, Enoch Y

2018-10-01

It has been shown that the single-domain intrabody 2H9-L against the hepatitis C virus (HCV) capsid (core) protein inhibits the viral propagation and NF-κB promoter activity induced by the HCV core. In this study, 2H9-L fused with the FLAG tag sequence was expressed in both Escherichia coli and silkworm pupae and then purified. In addition, the full-length and its C terminal deletions of the HCV core protein, i.e., 1-123 amino acid residues (C123), 1-152 amino acid residues (C152), 1-177 amino acid residues (C177) and 1-191 amino acid residues (C191), were expressed as fusion proteins with a 6 × His tag at their N-terminus in E. coli and then purified. Approximately 175 and 132 μg of the intrabody were purified from 100 ml of E. coli culture and 10 silkworm pupae, respectively, by affinity chromatography. The C123, C152, C177 and C191 HCV core protein variants were purified to approximately 152, 127, 103 and 155 μg, respectively, from 100 ml of E. coli culture. An ELISA in which the intrabodies were immobilized revealed that the intrabodies purified from both hosts were bound to all HCV core protein variants. However, their binding to the C191 appeared to be weak compared to their bindings to the other HCV core protein variants. When C152 was immobilized in the ELISA, the binding of each intrabody to the core protein was also observed. These purified intrabodies can be used in biochemical analyses of the inhibitory mechanism of HCV propagation and as protein interference reagents, thus providing a potential pathway to developing a new type of antiviral drug. Copyright © 2018 Elsevier Inc. All rights reserved.

Detection of Hepatitis B Virus DNA among Chronic and potential Occult HBV patients in resource-limited settings by Loop-Mediated Isothermal Amplification assay.

PubMed

Akram, Arifa; Islam, S M Rashedul; Munshi, Saif Ullah; Tabassum, Shahina

2018-05-16

Transmission of Hepatitis B Virus (HBV) usually occurs due to the transfusion of blood or blood products from chronic HBV (CHB) or occult HBV infected (OBI) patients. Besides serological tests e.g. HBsAg and anti-HBc (total), detection of HBV-DNA is necessary for the diagnosis of OBI patients. Different nucleic acid tests (NATs) including real-time-Polymerase Chain Reaction (qPCR) are used for the detect HBV-DNA. The NATs are expensive and require technical expertise which are barriers to introducing them in resource-limited settings. This study was undertaken to evaluate the use of Loop-Mediated Isothermal Amplification (LAMP) assay as an alternative to qPCR for the detection of HBV-DNA in CHB and potential OBI patients in resource-limited settings. Following the published protocols with some modifications, a LAMP assay was developed for detection of HBV-DNA by either using a heat block followed by detection in an agarose gel or using a qPCR thermocycler. The LAMP assay was applied to supernatant prepared from heat treated serum collected from CHB and potential OBI patients. HBV viral load in serum was measured by qPCR using a single step HBV-DNA quantification kit. Among 200 samples tested, qPCR was capable to detect HBV-DNA in 25.5% of cases, whereas LAMP assay detected HBV-DNA in 43.5% cases. The qPCR was able to detect 11 (9.16%) potential OBI cases, whereas LAMP assay identified HBV-DNA in 43 (35.83%) cases. In addition to tests for HBsAg and/or anti-HBc (total), detection of HBV-DNA by LAMP assay may aid in preventing post-transfusion HBV infection in resource-limited settings. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Hepatitis C Virus Core Protein Modulates Endoglin (CD105) Signaling Pathway for Liver Pathogenesis.

PubMed

Kwon, Young-Chan; Sasaki, Reina; Meyer, Keith; Ray, Ranjit

2017-11-01

Endoglin is part of the TGF-β receptor complex and has a crucial role in fibrogenesis and angiogenesis. It is also an important protein for tumor growth, survival, and cancer cell metastasis. In a previous study, we have shown that hepatitis C virus (HCV) infection induces epithelial-mesenchymal transition (EMT) state and cancer stem-like cell (CSC) properties in human hepatocytes. Our array data suggested that endoglin (CD105) mRNA is significantly upregulated in HCV-associated CSCs. In this study, we have observed increased endoglin expression on the cell surface of an HCV core-expressing hepatocellular carcinoma (HepG2) cell line or immortalized human hepatocytes (IHH) and activation of its downstream signaling molecules. The status of phospho-SMAD1/5 and the expression of inhibitor of DNA binding protein 1 (ID1) were upregulated in HCV-infected cells or viral core gene-transfected cells. Additionally, we observed upregulation of endoglin/ID1 mRNA expression in chronic HCV patient liver biopsy samples. CSC generation by HCV core protein was dependent on the endoglin signaling pathway using activin receptor-like kinase 1 (ALK1) Fc blocking peptide and endoglin small interfering RNA (siRNA). Further, follow-up from in vitro analysis suggested that the antiapoptosis Bcl2 protein, proliferation-related cyclin D1 protein, and CSC-associated Hes1, Notch1, Nanog, and Sox2 proteins are enhanced during infection or ectopic expression of HCV core protein. IMPORTANCE Endoglin plays a crucial role in fibrogenesis and angiogenesis and is an important protein for tumor growth, survival, and cancer cell metastasis. Endoglin enhances ALK1-SMAD1/5 signaling in different cell types, leading to increased proliferation and migration responses. We have observed endoglin expression on the HCV core-expressing cell surface of human hepatocyte origin and activation of phospho-SMAD1/5 and ID1 downstream signaling molecules. ID1 protein plays a role in CSC properties, and we found that

Glycosyltransferase Function in Core 2-Type Protein O Glycosylation▿

PubMed Central

Stone, Erica L.; Ismail, Mohd Nazri; Lee, Seung Ho; Luu, Ying; Ramirez, Kevin; Haslam, Stuart M.; Ho, Samuel B.; Dell, Anne; Fukuda, Minoru; Marth, Jamey D.

2009-01-01

Three glycosyltransferases have been identified in mammals that can initiate core 2 protein O glycosylation. Core 2 O-glycans are abundant among glycoproteins but, to date, few functions for these structures have been identified. To investigate the biological roles of core 2 O-glycans, we produced and characterized mice deficient in one or more of the three known glycosyltransferases that generate core 2 O-glycans (C2GnT1, C2GnT2, and C2GnT3). A role for C2GnT1 in selectin ligand formation has been described. We now report that C2GnT2 deficiency impaired the mucosal barrier and increased susceptibility to colitis. C2GnT2 deficiency also reduced immunoglobulin abundance and resulted in the loss of all core 4 O-glycan biosynthetic activity. In contrast, the absence of C2GnT3 altered behavior linked to reduced thyroxine levels in circulation. Remarkably, elimination of all three C2GnTs was permissive of viability and fertility. Core 2 O-glycan structures were reduced among tissues from individual C2GnT deficiencies and completely absent from triply deficient mice. C2GnT deficiency also induced alterations in I-branching, core 1 O-glycan formation, and O mannosylation. Although the absence of C2GnT and C4GnT activities is tolerable in vivo, core 2 O glycosylation exerts a significant influence on O-glycan biosynthesis and is important in multiple physiological processes. PMID:19349303

HBV or HCV Coinfection in HIV-1-Infected Pregnant Women in France: Prevalence and Pregnancy Outcomes.

PubMed

Benhammou, Valérie; Tubiana, Roland; Matheron, Sophie; Sellier, Pierre; Mandelbrot, Laurent; Chenadec, Jérôme Le; Marel, Emmanuelle; Khoshnood, Babak; Warszawski, Josiane

2018-04-15

Chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection is frequent in HIV-infected persons but their impact on pregnant HIV-infected women is understudied. We explored whether these coinfections are associated with adverse pregnancy outcomes and lower response to antiretroviral therapy (ART). Pregnancies in HIV-1-infected women included in the ANRS French Perinatal Cohort between 2005 and 2013 were analyzed if HBV and HCV infection statuses were available. Among 4236 women, the prevalence of HBV (HBs Ag+) and HCV (RNA+) were 6.2% (95% confidence interval: 5.4 to 6.8) and 1.7% (1.3 to 2.1), respectively. HCV coinfection was strongly associated with a history of drug use; HBV coinfection was 6 times more frequent in women born in Sub-Saharan Africa than in European France. Baseline HIV viral load, CD4 count, and HIV care during pregnancy were similar in coinfected and monoinfected HIV mothers, except that 90% of HBV/HIV women were receiving tenofovir and/or lamivudine or emtricitabine. HCV coinfection was significantly associated with cholestasis [adjusted odds ratio: 4.1 (1.5-10.8), P = 0.005], preterm delivery [3.0 (1.6-5.7), P < 0.001], lower CD4 [2.6 (1.0-6.4), P < 0.001], and detectable viral load [2.3 (1.0-5.5), P = 0.06] at the end of pregnancy. HBV coinfection was not associated with any of these outcomes. In HIV-infected women, chronic HBV infection, mostly treated using targeted ART, had no major impact on the course of pregnancy. By contrast, chronic HCV infection was associated with a higher risk of obstetrical complications and a poorer immune-virological response to ART. It is yet unknown whether cure of HCV infection before conception can limit these adverse outcomes.

HCV Core Protein Uses Multiple Mechanisms to Induce Oxidative Stress in Human Hepatoma Huh7 Cells

PubMed Central

Ivanov, Alexander V.; Smirnova, Olga A.; Petrushanko, Irina Y.; Ivanova, Olga N.; Karpenko, Inna L.; Alekseeva, Ekaterina; Sominskaya, Irina; Makarov, Alexander A.; Bartosch, Birke; Kochetkov, Sergey N.; Isaguliants, Maria G.

2015-01-01

Hepatitis C virus (HCV) infection is accompanied by the induction of oxidative stress, mediated by several virus proteins, the most prominent being the nucleocapsid protein (HCV core). Here, using the truncated forms of HCV core, we have delineated several mechanisms by which it induces the oxidative stress. The N-terminal 36 amino acids of HCV core induced TGFβ1-dependent expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 1 and 4, both of which independently contributed to the production of reactive oxygen species (ROS). The same fragment also induced the expression of cyclo-oxygenase 2, which, however, made no input into ROS production. Amino acids 37–191 of HCV core up-regulated the transcription of a ROS generating enzyme cytochrome P450 2E1. Furthermore, the same fragment induced the expression of endoplasmic reticulum oxidoreductin 1α. The latter triggered efflux of Ca2+ from ER to mitochondria via mitochondrial Ca2+ uniporter, leading to generation of superoxide anions, and possibly also H2O2. Suppression of any of these pathways in cells expressing the full-length core protein led to a partial inhibition of ROS production. Thus, HCV core causes oxidative stress via several independent pathways, each mediated by a distinct region of the protein. PMID:26035647

High prevalence of HBV/A1 subgenotype in native south Americans may be explained by recent economic developments in the Amazon.

PubMed

Godoy, Bibiane A; Gomes-Gouvêa, Michele S; Zagonel-Oliveira, Marcelo; Alvarado-Mora, Mónica V; Salzano, Francisco M; Pinho, João R R; Fagundes, Nelson J R

2016-09-01

Native American populations present the highest prevalence of Hepatitis B Virus (HBV) infection in the Americas, which may be associated to severe disease outcomes. Ten HBV genotypes (A–J) have been described, displaying a remarkable geographic structure, which most likely reflects historic patterns of human migrations. In this study, we characterize the HBV strains circulating in a historical sample of Native South Americans to characterize the historical viral dynamics in this population. The sample consisted of 1070 individuals belonging to 38 populations collected between 1965 and 1997. Presence of HBV DNA was checked by quantitative real-time PCR, and determination of HBV genotypes and subgenotypes was performed through sequencing and phylogenetic analysis of a fragment including part of HBsAg and Pol coding regions (S/Pol). A Bayesian Skyline Plot analysis was performed to compare the viral population dynamics of HBV/A1 strains found in Native Americans and in the general Brazilian population. A total of 109 individuals were positive for HBV DNA (~ 10%), and 70 samples were successfully sequenced and genotyped. Subgenotype A1 (HBV/A1), related to African populations and the African slave trade, was the most prevalent (66–94%). The Skyline Plot analysis showed a marked population expansion of HBV/A1 in Native Americans occurring more recently (1945–1965) than in the general Brazilian population. Our results suggest that historic processes that contributed to formation of HBV/A1 circulating in Native American are related with more recent migratory waves towards the Amazon basin, which generated a different viral dynamics in this region.

Seroepidemiology of HBV infection in South-East of iran; a population based study.

PubMed

Salehi, M; Alavian, S M; Tabatabaei, S V; Izadi, Sh; Sanei Moghaddam, E; Amini Kafi-Abad, S; Gharehbaghian, A; Khosravi, S; Abolghasemi, H

2012-05-01

Hepatitis B virus (HBV) infection is a major risk factor of cirrhosis and hepatocellular carcinoma affecting billions of people globally. Since information on its prevalence in general population is mandatory for formulating effective policies, this population based serological survey was conducted in Sistan and Baluchistan, where no previous epidemiological data were available. Using random cluster sampling 3989 healthy subjects were selected from 9 districts of Sistan and Baluchistan Province in southeastern Iran. The subjects' age ranged from 6 to 65 years old. Serum samples were tested for HBcAb, HBsAg. Screening tests were carried out by the third generation of ELISA. Various risk factors were recorded and multivariate analysis was performed. The prevalence of HBsAg and HBcAb in Sistan and Baluchistan was 3.38% (95% CI 2.85; 3.98) and 23.58% (95% CI 22.29; 24.93) respectively. We found 8 cases of positive anti-HDV antibody. Predictors of HBsAg or HBcAb in multivariate analysis were age, marital status and addiction. The rate of HBV infection in Sistan and Baluchistan was higher than other parts of Iran. Approximately 25% of general population in this province had previous exposure to HBV and 3% were HBsAg carriers. Intrafamilial and addiction were major routes of HBV transmission in this province.

Complement C4a inhibits the secretion of hepatitis B virus screened by surface-enhanced laser desorption ionization time-flight mass spectrometry-based ProteinChip analysis.

PubMed

Song, Ya-Nan; Zhang, Gui-Biao; Hu, Xue-Qing; Lu, Yi-Yu; Zhao, Yu; Yang, Yang; Yang, Yi-Fu; Zhang, Yong-Yu; Hu, Yi-Yang; Su, Shi-Bing

2015-12-01

Chronic hepatitis B (CHB) is a kind of chronic liver disease caused by persistent hepatitis B virus (HBV) infection. The study aims to seek the factors of host resistance to HBV and investigate their roles. Protein profiles of 58 healthy controls and 121 CHB patients were obtained by SELDI-TOF/MS. Predicted protein was validated by ELISA. Protein expression was evaluated by Western blot in the persistently HBV expressing cell line HepG2.2.15 and non-HBV expressing cell line HepG2. The level of HBV DNA was subsequently detected by quantitative real-time PCR in HepG2.2.15 cells with complement C4a treatment. Significantly altered protein peaks were found through statistical analysis, and m/z 4300 was predicted by databases and successfully matched with the fragment of complement C4a. According to ELISA, serum complement C4a was found to be significantly lower in CHB patients compared with healthy controls (p < 0.001) and the area under receiver operating characteristics curve is 0.78. Furthermore, complement C4a showed lower expression in HepG2.2.5 cells and the secretion of HBV DNA was inhibited by complement C4a. The present study implied the important role of complement C4a in inhibiting the HBV DNA secretion in CHB. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Occult HBV Infection: A Faceless Enemy in Liver Cancer Development

PubMed Central

Morales-Romero, Jaime; Vargas, Gustavo; García-Román, Rebeca

2014-01-01

The hepatitis B virus (HBV) represents a worldwide public health problem; the virus is present in one third of the global population. However, this rate may in fact be higher due to occult hepatitis B virus infection (OBI). This condition is characterized by the presence of the viral genome in the liver of individuals sero-negative for the virus surface antigen (HBsAg). The causes of the absence of HBsAg in serum are unknown, however, mutations have been identified that produce variants not recognized by current immunoassays. Epigenetic and immunological host mechanisms also appear to be involved in HBsAg suppression. Current evidence suggests that OBI maintains its carcinogenic potential, favoring the progression of fibrosis and cirrhosis of the liver. In common with open HBV infection, OBI can contribute to the establishment of hepatocellular carcinoma. Epidemiological data regarding the global prevalence of OBI vary due to the use of detection methods of different sensitivity and specificity. In Latin America, which is considered an area of low prevalence for HBV, diagnostic screening methods using gene amplification tests for confirmation of OBI are not conducted. This prevents determination of the actual prevalence of OBI, highlighting the need for the implementation of cutting edge technology in epidemiological surveillance systems. PMID:24717680

Acute hepatitis B virus infection with simultaneous high HBsAg and high anti-HBs signals in a previously HBV vaccinated HIV-1 positive patient.

PubMed

van Dommelen, Laura; Verbon, Annelies; van Doorn, H Rogier; Goossens, Valère J

2010-03-01

We present a case of a clinical manifest hepatitis B virus infection and a potentially misleading HBV serological profile in an HIV-1 positive patient despite previous HBV vaccination. The patient presented with an acute hepatitis B and there was no indication of chronic HBV infection or the presence of a mutation in the 'a' determinant. Remarkably, simultaneously with high HBV surface antigen and HBV viral load, high anti-HBs antibodies were present. If, due to previous HBV vaccination only anti-HBs was tested in this patient, the result of the high anti-HBs antibodies could be very misleading and offering a false sense of security. Our findings contribute to the ongoing discussion on how to assess HBV specific immunological memory and determining the role of HBV booster vaccinations in immunocompromised individuals.

Requirement of the cyclic adenosine monophosphate response element-binding protein for hepatitis B virus replication.

PubMed

Kim, Bo Kyung; Lim, Seoung Ok; Park, Yun Gyu

2008-08-01

The cyclic adenosine monophosphate-response element (CRE)-transcription factor complex participates in the regulation of viral gene expression and pathologic processes caused by various viruses. The hepatitis B virus (HBV) enhancer I directs liver-specific transcription of viral genes and contains a CRE sequence (HBV-CRE); however, whether the HBV-CRE and CRE-binding protein (CREB) are required for the HBV life cycle remains to be determined. This study was designed to investigate the role of CREB in HBV replication and gene expression. Sequence-comparison analysis of 984 HBVs reported worldwide showed that the HBV-CRE sequence is highly conserved, indicating the possibility that it plays an important role in the HBV life cycle. The binding of CREB to the HBV-CRE site was markedly inhibited by oligonucleotides containing HBV-CRE and consensus CRE sequences in vitro and in vivo. The HBV promoter activity was demonstrated to be dependent upon the transactivation activity of CREB. Treatment with CRE decoy oligonucleotides reduced HBV promoter activity, and this was reversed by CREB overexpression. The levels of viral transcripts, DNA, and antigens were remarkably decreased in response to the overexpression of CREB mutants or treatment with the CRE decoy oligonucleotides, whereas enhancing CREB activity increased the levels of viral transcripts. In addition, introduction of a three-base mutation into the HBV-CRE led to a marked reduction in HBV messenger RNA synthesis. Taken together, our results demonstrate that both replication and gene expression of HBV require a functional CREB and HBV-CRE. We have also demonstrated that CRE decoy oligonucleotides and the overexpression of CREB mutants can effectively block the HBV life cycle, suggesting that interventions against CREB activity could provide a new avenue to treat HBV infection.

Dynamic changes of HBV DNA in serum and peripheral blood mononuclear cells of chronic hepatitis patients after lamivudine treatment

PubMed Central

Ke, Chang-Zheng; Chen, Yue; Gong, Zuo-Jiong; Meng, Zhong-Ji; Liu, Li; Ren, Ze-Jiu; Zhou, Zuo-Hua

2006-01-01

AIM: To study the dynamic changes of hepatits B virus (HBV) DNA in serum and peripheral blood mononuclear cells (PBMCs) of patients after lamivudine therapy. METHODS: A total of 72 patients with chronic HBV infection were included in this study. All patients were confirmed to have the following conditions: above 16 years of age, elevated serum alanine amonotransferase (ALT), positive hepatitis B e antigen (HBeAg), positive HBV DNA in serum and PBMCs, negative antibodies against HAV, HCV, HDV, HEV. Other possible causes of chronic liver damages, such as drugs, alcohol and autoimmune diseases were excluded. Seventy-two cases were randomly divided into lamivudine treatment group (n = 42) and control group (n = 30). HBV DNA was detected both in serum and in PBMCs by fluorescence quantitative polymerase chain reaction (PCR), during and after lamivudine treatment. RESULTS: In the treatment group, HBV DNA became negative both in serum and in PBMC, of 38 and 25 out of 42 cases respectively during the 48 wk of lamivudine treatment, the negative rate was 90.5% and 59.5% respectively. In the control group, the negative rate was 23.3% and 16.7% respectively. It was statistically significant at 12, 24 and 48 wk as compared with the control group (P < 0.005). The average conversion period of HBV DNA was 6 wk (2-8 wk) in serum and 16 wk (8-24 wk) in PBMC. CONCLUSION: Lamivudine has remarkable inhibitory effects on HBV replication both in serum and in PBMCs. The inhibitory effect on HBV DNA in PBMCs is weaker than that in serum. PMID:16810760

Time trends of chronic HBV infection over prior decades - A global analysis.

PubMed

Ott, Jördis J; Horn, Johannes; Krause, Gérard; Mikolajczyk, Rafael T

2017-01-01

Information on trends in chronic hepatitis B virus (HBV) prevalence across countries is lacking. We studied changes in chronic HBV infection over previous decades by country, and assessed patterns of change between and within WHO-defined regions. Based on data from a published systematic review on chronic HBV, we applied a linear model on the logit scale to assess time trends in country-specific prevalence. Estimated HBsAg prevalence in 2000 and relative changes in prevalence over time were evaluated by country and region. Sufficient data were available for 50 countries, mostly showing reductions in prevalence over time. Various degrees of heterogeneity were observed within regions, with a relatively homogenous pattern in the Eastern Mediterranean region with strong decreases in HBsAg prevalence. Europe showed a mixed pattern: higher and stable chronic HBsAg prevalence in Eastern, and constantly low prevalence in Western Europe. In Africa, some countries demonstrated no change in prevalence; increases were seen in Uganda (odds ratio 1.05 per year; 95% confidence interval 1.04-1.06), Nigeria (1.02; 1.02-1.02), Senegal (1.01; 1.01-1.02), and South Africa (1.02; 1.01-1.02). With some exceptions, country-patterns overlapped among countries of South East Asian and Western Pacific regions, characterized by low-medium HBsAg decreases, most prominent in China and Malaysia. Most countries experienced decreases in HBsAg prevalence. Dynamics varied, even within regions; decreases occurred mostly before the direct effects of childhood vaccination may have manifested. These findings together with stable and increasing HBsAg prevalence in some countries of Africa and Eastern Europe indicate the need for further tailored country-specific prevention. This study investigated time trends in prevalence of chronic HBV infection in 50 countries worldwide over the last decade, by estimating relative changes in prevalence. Results show decreases in chronic HBV infection in most countries

Prioritization of potential drug targets against P. aeruginosa by core proteomic analysis using computational subtractive genomics and Protein-Protein interaction network.

PubMed

Uddin, Reaz; Jamil, Faiza

2018-06-01

Pseudomonas aeruginosa is an opportunistic gram-negative bacterium that has the capability to acquire resistance under hostile conditions and become a threat worldwide. It is involved in nosocomial infections. In the current study, potential novel drug targets against P. aeruginosa have been identified using core proteomic analysis and Protein-Protein Interactions (PPIs) studies. The non-redundant reference proteome of 68 strains having complete genome and latest assembly version of P. aeruginosa were downloaded from ftp NCBI RefSeq server in October 2016. The standalone CD-HIT tool was used to cluster ortholog proteins (having >=80% amino acid identity) present in all strains. The pan-proteome was clustered in 12,380 Clusters of Orthologous Proteins (COPs). By using in-house shell scripts, 3252 common COPs were extracted out and designated as clusters of core proteome. The core proteome of PAO1 strain was selected by fetching PAO1's proteome from common COPs. As a result, 1212 proteins were shortlisted that are non-homologous to the human but essential for the survival of the pathogen. Among these 1212 proteins, 321 proteins are conserved hypothetical proteins. Considering their potential as drug target, those 321 hypothetical proteins were selected and their probable functions were characterized. Based on the druggability criteria, 18 proteins were shortlisted. The interacting partners were identified by investigating the PPIs network using STRING v10 database. Subsequently, 8 proteins were shortlisted as 'hub proteins' and proposed as potential novel drug targets against P. aeruginosa. The study is interesting for the scientific community working to identify novel drug targets against MDR pathogens particularly P. aeruginosa. Copyright © 2018 Elsevier Ltd. All rights reserved.

Epidemiological profile and risk factors of HIV and HBV/HCV co-infection in Fujian Province, southeastern China.

PubMed

Wu, Shouli; Yan, Pingping; Yang, Tianfei; Wang, Zhenghua; Yan, Yansheng

2017-03-01

This study aimed to investigate the epidemiological features of HIV-infected subjects co-infected with HBV/HCV in Fujian Province, southeastern China, and identify the risk factors. Blood samples were collected from 2,028 HIV antibody-positive subjects in Fujian Province. Serum HBsAg and anti-HCV antibody were detected, and CD4 + T cell count was measured. Of the 2,028 subjects, the prevalence of HIV-HBV, HIV-HCV, and HIV-HBV-HCV co-infections was 16.22%, 3.7%, and 0.79%, respectively. Man (OR = 1.912, 95% CI: 1.371-2.667), key population (OR = 0.756, 95% CI: 0.57-0.976) and detainee (OR = 0.486, 95% CI: 0.259-0.909) were risk factors of HIV-HBV co-infection, and man (OR = 2.227, 95% CI: 1.096-4.525), minority (OR = 5.04, 95% CI: 1.696-14.98), junior high school or lower education (OR = 2.32, 95% CI: 1.071-5.025), intravenous drug use (OR = 38.46, 95% CI: 11.46-129.11) and detainee (OR = 5.687, 95% CI: 2.44-13.25) were risk factors of HIV-HCV co-infection. In addition, a lower mean CD4 + T cell count was measured in HIV/HBV and HIV/HCV co-infected subjects than in HIV-infected subjects among the untreated individuals, while in the treated populations, a higher mean CD4 + T cell count was detected in HIV/HBV and HIV/HCV co-infected subjects than in HIV-infected subjects. HIV co-infection with HBV or HCV, notably HIV-HBV co-infection, is widespread in southeastern China. Hepatitis virus screening should be included in monitoring of HIV infection, and HIV and hepatitis virus co-infection should be considered during the development of HIV antiretroviral therapy scheme. J. Med. Virol. 89:443-449, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Genetic variants in NTCP exon gene are associated with HBV infection status in a Chinese Han population.

PubMed

Wu, Wennan; Zeng, Yongbin; Lin, Jinpiao; Wu, Yingying; Chen, Tianbin; Xun, Zhen; Ou, Qishui

2018-04-01

Sodium taurocholate co-transporting polypeptide (NTCP) plays an important role in the enterohepatic circulation of bile acids. Recently, NTCP was identified as a hepatitis B virus (HBV) receptor. The aim of this study is to investigate the association of NTCP polymorphisms with HBV clinical outcomes and investigate the relationship between NTCP polymorphisms and the serum bile acid level in a Chinese Han population. The single nucleotide polymorphisms rs2296651 and rs4646285 were genotyped in 1619 Chinese Han individuals. Improved multiple ligase detection reaction was utilized to genotype. The level of bile acids was measured by the enzymatic cycling method. Quantitative polymerase chain reaction analysis was carried out to analyze the potential function. In logistic regression analysis, the frequency of rs2296651 (S267F) CT genotype was higher in HBV immune recovery and healthy control groups than in the chronic HBV infection group (P = 0.001 and P < 0.001, respectively). Patients who carried allele T showed a higher bile acid level than patients who did not carry allele T (P = 0.009). The rs4646285 AA genotype was more common in the immune recovery group than in the chronic HBV infection group (P = 0.011). No difference in serum bile acid was detected between the rs4646285 wild-type patients and mutant-type patients. Quantitative reverse transcription-polymerase chain reaction showed the NTCP mRNA levels were lower in rs4646285 variants than wild types. NTCP gene polymorphisms may be associated with the natural course of HBV infection in a Chinese Han population. The S267F variant may be a protective factor to resist chronic hepatitis B progression which showed a higher bile acid level in Chinese Han chronic HBV infection patients. The rs4646285 variants could influence the expression of NTCP at the level of transcription, and ultimately may be associated with HBV infection immune recovery. © 2017 The Japan Society of Hepatology.

Preparation and recognition of surface molecularly imprinted core-shell microbeads for protein in aqueous solutions

NASA Astrophysics Data System (ADS)

Lu, Yan; Yan, Chang-Ling; Gao, Shu-Yan

2009-04-01

In this paper, a surface molecular imprinting technique was reported for preparing core-shell microbeads of protein imprinting, and bovine hemoglobin or bovine serum albumin were used as model proteins for studying the imprinted core-shell microbeads. 3-Aminophenylboronic acid (APBA) was polymerized onto the surface of polystyrene microbead in the presence of the protein templates to create protein-imprinted core-shell microbeads. The various samples were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and Brunauer-Emmett-Teller (BET) methods. The effect of pH on rebinding of the template hemoglobin, the specific binding and selective recognition were studied for the imprinted microbeads. The results show that the bovine hemoglobin-imprinted core-shell microbeads were successfully created. The shell was a sort of imprinted thin films with porous structure and larger surface areas. The imprinted microbeads have good selectivity for templates and high stability. Due to the recognition sites locating at or closing to the surface, these imprinted microbeads have good property of mass-transport. Unfortunately, the imprint technology was not successfully applied to imprinting bovine serum albumin (BSA).

Functional analysis of ‘a’ determinant mutations associated with occult HBV in HIV-positive South Africans

PubMed Central

Powell, Eleanor A.; Boyce, Ceejay L.; Gededzha, Maemu P.; Selabe, Selokela G.; Mphahlele, M. Jeffrey

2016-01-01

Occult hepatitis B is defined by the presence of hepatitis B virus (HBV) DNA in the absence of hepatitis B surface antigen (HBsAg). Occult HBV is associated with the development of hepatocellular carcinoma, reactivation during immune suppression, and virus transmission. Viral mutations contribute significantly to the occult HBV phenotype. Mutations in the ‘a’ determinant of HBsAg are of particular interest, as these mutations are associated with immune escape, vaccine escape and diagnostic failure. We examined the effects of selected occult HBV-associated mutations identified in a population of HIV-positive South Africans on HBsAg production in vitro. Mutations were inserted into two different chronic HBV backbones and transfected into a hepatocyte-derived cell line. HBsAg levels were quantified by enzyme-linked immunosorbent assay (ELISA), while the detectability of mutant HBsAg was determined using an HA-tagged HBsAg expression system. Of the seven mutations analysed, four (S132P, C138Y, N146D and C147Y) resulted in decreased HBsAg expression in one viral background but not in the second viral background. One mutation (N146D) led to a decrease in HBsAg detected as compared to HA-tag, indicating that this mutation compromises the ability of the ELISA to detect HBsAg. The contribution of occult-associated mutations to the HBsAg-negative phenotype of occult HBV cannot be determined adequately by testing the effect of the mutation in a single viral background, and rigorous analysis of these mutations is required. PMID:27031988

HIV, HBV, and HCV molecular epidemiology among trans (transvestites, transsexuals, and transgender) sex workers in Argentina.

PubMed

Carobene, Mauricio; Bolcic, Federico; Farías, María Sol Dos Ramos; Quarleri, Jorge; Avila, María Mercedes

2014-01-01

Commercial sex work is frequent among male-to-female transvestites, transsexuals and transgenders in Argentina, leading to high susceptibility to HIV, HBV, and HCV among other sexually transmitted infections. In a global context of scarce data on the trans sex workers population, this study was aimed to study the genomic characterization of these viruses. Plasma presence of HIV, HBV, and HCV genomic material was evaluated in samples from 273 trans sex workers. Genomic sequences of HIV-gag, pol, and vif-vpu genes, HBV-S gene, and HCV-5'UT and NS5B genes were obtained. Molecular characterization involved phylogenetic analysis and several in silico tools. Resistance-associated mutations in HIV and HBV pol genes were also analyzed. The HIV genomic characterization in 62 trans sex workers samples showed that 54.8% of the isolates corresponded to BF intersubtype recombinants, and 38.7% to subtype B. The remaining were classified as subtypes C (4.8%) and A (1.6%). HBV and HCV co-infection prevalence among HIV positive trans sex workers yielded rates of 3.2% and 6.5% respectively. Drug resistance-associated mutations were found in 12/62 (19%) HIV pol sequences, but none among HBV. Based on phylogenetic relationships, HIV isolates characterized as subtypes BF and B appeared intermingled with those from other high-risk groups. Despite trans sex workers declared not to have received antiviral treatment, complex drug resistance-associated mutation patterns were found in several HIV isolates. Planned prevention, screening, and treatment are needed to reduce further transmission and morbidity. © 2013 Wiley Periodicals, Inc.

Aiming for cure in HBV and HDV infection.

PubMed

Petersen, Jörg; Thompson, Alexander J; Levrero, Massimo

2016-10-01

Chronic hepatitis B virus (HBV) infection continues to be a major health burden worldwide. Currently available antiviral treatment options for chronic hepatitis B include pegylated interferon alpha2a (PegIFN) or nucleos(t)ide analogues (NAs). The major advantages of NAs are good tolerance and potent antiviral activity associated with high rates of sustained on-treatment response to therapy. The advantages of PegIFN include a finite course of treatment, the absence of drug resistance, and an opportunity to obtain a durable post-treatment response to therapy. Furthermore, PegIFN is the only approved agent known to be active against hepatitis D virus (HDV). The use of these two antiviral agents with different mechanisms of action in combination against hepatitis B is theoretically an attractive approach for treatment. Although several studies have confirmed certain virological advantages of combination therapies, data supporting a long-term clinical benefit for patients are lacking and monotherapy with PegIFN or NAs remains the therapy of choice. Moreover, with the current treatment approaches, only a limited number of patients achieve hepatitis B surface antigen (HBsAg) loss. HBsAg loss is considered a "functional cure", but does not mean viral eradication. There is a need for novel therapeutic approaches that enable not only suppression of viral replication, but resolution of HBV infection. A key challenge is to target covalently closed circular DNA (cccDNA) in the nucleus of infected hepatocytes. The recent development and availability of innovative in vitro and in vivo systems and sensitive molecular techniques has opened new possibilities to study the complex network of interactions that HBV establishes with the host in the course of infection and to define new targets for antiviral strategies. Several new antiviral or immunomodulatory compounds have reached preclinical or clinical testing with the aim of silencing or eradicating cccDNA to achieve functional cure

Epidemiological Profile and Risk Factors for Acquiring HBV and/or HCV in HIV-Infected Population Groups in Nepal.

PubMed

Bhattarai, Manjula; Baniya, Jagat Bahadur; Aryal, Nirmal; Shrestha, Bimal; Rauniyar, Ramanuj; Adhikari, Anurag; Koirala, Pratik; Oli, Pardip Kumar; Pandit, Ram Deo; Stein, David A; Gupta, Birendra Prasad

2018-01-01

HBV and HCV infections are widespread among the HIV-infected individuals in Nepal. The goals of this study were to investigate the epidemiological profile and risk factors for acquiring HBV and/or HCV coinfection in disadvantaged HIV-positive population groups in Nepal. We conducted a retrospective study on blood samples from HIV-positive patients from the National Public Health Laboratory at Kathmandu to assay for HBsAg, HBeAg, and anti-HCV antibodies, HIV viral load, and CD4+ T cell count. Among 579 subjects, the prevalence of HIV-HBV, HIV-HCV, and HIV-HBV-HCV coinfections was 3.62%, 2.93%, and 0.34%, respectively. Multivariate regression analysis indicated that spouses of HIV-positive migrant labourers were at significant risk for coinfection with HBV infection, and an age of >40 years in HIV-infected individuals was identified as a significant risk factor for HCV coinfection. Overall our study indicates that disadvantaged population groups such as intravenous drug users, migrant workers and their spouses, female sex workers, and men who have sex with HIV-infected men are at a high and persistent risk of acquiring viral hepatitis. We conclude that Nepalese HIV patients should receive HBV and HCV diagnostic screening on a regular basis.

Detection of hepatitis B virus (HBV) genotype E carried--even in the presence of high titers of anti-HBs antibodies--by an Argentinean patient of African descent who had received vaccination against HBV.

PubMed

Mathet, Verónica L; Cuestas, María L; Ruiz, Vanesa; Minassian, María L; Rivero, Cintia; Trinks, Julieta; Daleoso, Graciela; León, Liliana M; Sala, Andrea; Libellara, Beatriz; Corach, Daniel; Oubiña, José R

2006-09-01

Genotype E hepatitis B virus (HBV) was detected in two Argentine sisters exhibiting an African mitochondrial lineage. One of them (who had been vaccinated against HBV) exhibited anti-HBs cocirculating antibodies without HBsAg escape mutants, while her unvaccinated sister showed a D144A HBsAg escape mutant without anti-HBs antibodies. Both sisters carried an unusual L209V substitution within HBsAg.

Epidemiological, virological and clinical characteristics of HBV infection in 223 HIV co-infected patients: a French multi-centre collaborative study.

PubMed

Thibault, Vincent; Gaudy-Graffin, Catherine; Colson, Philippe; Gozlan, Joël; Schnepf, Nathalie; Trimoulet, Pascale; Pallier, Coralie; Saune, Karine; Branger, Michel; Coste, Marianne; Thoraval, Francoise Roudot

2013-03-15

Chronic hepatitis B (CHB) is a clinical concern in human immunodeficiency virus (HIV)-infected individuals due to substantial prevalence, difficulties to treat, and severe liver disease outcome. A large nationwide cross-sectional multicentre analysis of HIV-HBV co-infected patients was designed to describe and identify parameters associated with virological and clinical outcome of CHB in HIV-infected individuals with detectable HBV viremia. A multicenter collaborative cross-sectional study was launched in 19 French University hospitals distributed through the country. From January to December 2007, HBV load, genotype, clinical and epidemiological characteristics of 223 HBV-HIV co-infected patients with an HBV replication over 1000 IU/mL were investigated. Patients were mostly male (82%, mean age 42 years). Genotype distribution (A 52%; E 23.3%; D 16.1%) was linked to risk factors, geographic origin, and co-infection with other hepatitis viruses. This genotypic pattern highlights divergent contamination event timelines by HIV and HBV viruses. Most patients (74.7%) under antiretroviral treatment were receiving a drug with anti-HBV activity, including 47% receiving TDF. Genotypic lamivudine-resistance detected in 26% of the patients was linked to duration of lamivudine exposure, age, CD4 count and HIV load. Resistance to adefovir (rtA181T/V) was detected in 2.7% of patients. Advanced liver lesions were observed in 54% of cases and were associated with an older age and lower CD4 counts but not with viral load or genotype. Immune escape HBsAg variants were seldom detected. Despite the detection of advanced liver lesions in most patients, few were not receiving anti-HBV drugs and for those treated with the most potent anti-HBV drugs, persistent replication suggested non-optimal adherence. Heterogeneity in HBV strains reflects epidemiological differences that may impact liver disease progression. These findings are strong arguments to further optimize clinical management

The prevalence of HBV infection in the cohort of IDPs of war against terrorism in Malakand Division of Northern Pakistan

PubMed Central

2011-01-01

Background Hepatitis B is an important public health problem in the Pakistani population and is the major cause of chronic hepatitis, cirrhosis, fibrosis and hepatocellular carcinoma. High prevalence of HBV infections has been observed especially in areas of low economic status. In spite of effective immunization programs, no significant change has been observed in the epidemiology of HBV in the rural areas of Pakistan (~67.5% of the total population) mainly due to lack of interest from government authorities and poor hygienic measures. The current study was aimed at estimating the prevalence and risk factors associated with HBV infection within internally displaced persons (IDPs) due to war against terrorism in the Malakand Division of Northern Pakistan. Methods Blood samples from 950 IDPs suspected with HBV infection (including both males and females) were collected and processed with commercial ELISA kits for HBsAg, Anti HBs, HBeAg, Anti HBe antibodies. The samples positive by ELISA were confirmed for HBV DNA by real-time PCR analysis. Results The overall prevalence of HBV observed was 21.05% of which 78.5% were males and 21.5% were females. Most confirmed HBV patients belong to the Malakand and Dir (lower) district. High-risk of infection was found in the older subjects 29.13% (46-60 years), while a lower incidence (11.97%) was observed in children aged <15 years. Lack of awareness, socioecomic conditions, sexual activities and sharing of razor blades, syringes and tattooing needles were the most common risk factors of HBV infection observed during the cohort of patients. Conclusion The present study, revealed for the first time a high degree of prevalence of HBV infection in rural areas of Northern Pakistan. The noticed prevalence is gender- and age-dependent that might be due to their high exposures to the common risk factors. To avoid the transmission of HBV infection proper awareness about the possible risk factors and extension of immunization to the rural

The impact of p53 protein core domain structural alteration on ovarian cancer survival.

PubMed

Rose, Stephen L; Robertson, Andrew D; Goodheart, Michael J; Smith, Brian J; DeYoung, Barry R; Buller, Richard E

2003-09-15

Although survival with a p53 missense mutation is highly variable, p53-null mutation is an independent adverse prognostic factor for advanced stage ovarian cancer. By evaluating ovarian cancer survival based upon a structure function analysis of the p53 protein, we tested the hypothesis that not all missense mutations are equivalent. The p53 gene was sequenced from 267 consecutive ovarian cancers. The effect of individual missense mutations on p53 structure was analyzed using the International Agency for Research on Cancer p53 Mutational Database, which specifies the effects of p53 mutations on p53 core domain structure. Mutations in the p53 core domain were classified as either explained or not explained in structural or functional terms by their predicted effects on protein folding, protein-DNA contacts, or mutation in highly conserved residues. Null mutations were classified by their mechanism of origin. Mutations were sequenced from 125 tumors. Effects of 62 of the 82 missense mutations (76%) could be explained by alterations in the p53 protein. Twenty-three (28%) of the explained mutations occurred in highly conserved regions of the p53 core protein. Twenty-two nonsense point mutations and 21 frameshift null mutations were sequenced. Survival was independent of missense mutation type and mechanism of null mutation. The hypothesis that not all missense mutations are equivalent is, therefore, rejected. Furthermore, p53 core domain structural alteration secondary to missense point mutation is not functionally equivalent to a p53-null mutation. The poor prognosis associated with p53-null mutation is independent of the mutation mechanism.

Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus.

PubMed

Sonntag, Eric; Milbradt, Jens; Svrlanska, Adriana; Strojan, Hanife; Häge, Sigrun; Kraut, Alexandra; Hesse, Anne-Marie; Amin, Bushra; Sonnewald, Uwe; Couté, Yohann; Marschall, Manfred

2017-10-01

Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.

IL-17 and IL-22 genetic polymorphisms in HBV vaccine non- and low-responders among healthcare workers

PubMed Central

Borzooy, Zohreh; Streinu-Cercel, Adrian; Mirshafiey, Abbass; Khamseh, Azam; Mahmoudie, Masoud Karkhaneh; Navabi, Shadi Sadat; Nosrati, Marjan; Najafi, Zahra; Hosseini, Mostafa; Jazayeri, Seyed Mohammad

2016-01-01

Background Healthcare workers constitute a population at high risk for HBV infection. Efficient vaccination options are available; however, the individual response to HBV vaccination may vary widely between subjects, potentially due to cytokine profiles and genetic variations. In the present study, we investigated the relationship between IL-17 and IL-22 gene polymorphisms versus non- and low-responsiveness to HBV vaccination in healthcare workers. Methods We selected the following IL-17 and IL-22 polymorphisms: rs4711998 (A/G) from IL-17 and rs2227501 (A/T), rs2227503 (A/G), rs1026786 (A/G) from IL-22 sequences genes. These were determined by polymerase chain reaction restriction fragment length polymorphisms. Results The IL-17 rs4711998 GG genotype had a significantly lower frequency in non-responders compared to low-responders (p=0.025). However, we did not identify a relationship between IL-22 rs1026780, rs2227501 and rs2227503 genotypes and the anti-HBs response following HBV vaccination. Conclusion These data suggest that genetic variation in rs4711998 polymorphisms in the IL-17 cytokine may influence vaccine-induced immune responses to HBV vaccine in healthcare workers. PMID:27019828

Genomic Diversity of Hepatitis B Virus Infection Associated With Fulminant Hepatitis B Development.

PubMed

Mina, Thomas; Amini Bavil Olyaee, Samad; Tacke, Frank; Maes, Piet; Van Ranst, Marc; Pourkarim, Mahmoud Reza

2015-06-01

After five decades of Hepatitis B Virus (HBV) vaccine discovery, HBV is still a major public health problem. Due to the high genetic diversity of HBV and selective pressure of the host immune system, intra-host evolution of this virus in different clinical manifestations is a hot topic of research. HBV infection causes a range of clinical manifestations from acute to chronic infection, cirrhosis and hepatocellular carcinoma. Among all forms of HBV infection manifestations, fulminant hepatitis B infection possesses the highest fatality rate. Almost 1% of the acutely infected patients develop fulminant hepatitis B, in which the mortality rate is around 70%. All published papers deposited in Genbank, on the topic of fulminant hepatitis were reviewed and their virological aspects were investigated. In this review, we highlight the genomic diversity of HBV reported from patients with fulminant HBV infection. The most commonly detected diversities affect regulatory motifs of HBV in the core and S region, indicating that these alterations may convert the virus to an aggressive strain. Moreover, mutations at T-cell and B-cell epitopes located in pre-S1 and pre-S2 proteins may lead to an immune evasion of the virus, likely favoring a more severe clinical course of infection. Furthermore, point and frame shift mutations in the core region increase the viral replication of HBV and help virus to evade from immune system and guarantee its persistence. Fulminant hepatitis B is associated with distinct mutational patterns of HBV, underlining that genomic diversity of the virus is an important factor determining its pathogenicity.

Viral Hepatitis: Past and Future of HBV and HDV

PubMed Central

Thomas, Emmanuel; Yoneda, Masato; Schiff, Eugene R.

2015-01-01

Viral hepatitis is a significant disease afflicting hundreds of millions of people. Hepatitis-causing viruses initiate significant morbidity and mortality by establishing both acute and chronic infections, and several of these viruses are specifically associated with the development of hepatocellular carcinoma (HCC). Consequently, intense research efforts are focused on increasing our understanding of virus biology and on improving antiviral therapy. Even though viral hepatitis can be caused by several viruses from a range of virus families, the discovery of components of the hepatitis B virus (HBV) became a catalyst for the development of diagnostic assays that differentiate between these viruses as well as strategies for novel methods of vaccine development. Improvements in both the treatment and prevention of viral hepatitis are advancing rapidly. However, HBV, along with the associated infection by the hepatitis D virus, is still among the most common pathogens afflicting humans. PMID:25646383

[Requirement of standardizing anti-HBs assay methods in Japan for HBV infection-preventing strategy--discrepancy of anti-HBs measurements among three different kits widely used in Japan].

PubMed

Ogata, Norio

2006-09-01

The strategy to eliminate hepatitis B virus (HBV) infection by administrating an HB vaccine is changing worldwide; however, this is not the case in Japan. An important concern about the HBV infection-preventing strategy in Japan may be that the assay methods for the antibody to hepatitis B surface antigen (anti-HBs) are not standardized. The minimum protective anti-HBs titer against HBV infection has been established as 10 mIU/ml by World Health Organization (WHO) -standardized assay methods worldwide, but that is still determined as a "positive" test result by the passive hemagglutination (PHA) method in Japan. We compared anti-HBs measurements in given samples among PHA(Mycell II, Institute of Immunology), chemiluminescent enzyme immunoassay (CLEIA) (Lumipulse, Fujirebio), and chemiluminescent immunoassay (CLIA) (Architect, Abbott), all of which are currently in wide use in Japan. First, anti-HBs measurements in serum from individuals who received a yeast-derived recombinant HB vaccine composed of the major surface protein of either subtype adr or subtype ayw were compared. The results clearly showed that in subtype adr-vaccinees CLIA underestimated the anti-HBs amount compared with CLEIA and PHA, but in ayw-vaccinees, the discordance in the measurements among the three kits was not prominent. Second, anti-HBs measurements in standard or calibration solutions of each assay kit were compared. Surprisingly, CLEIA showed higher measurements in all three kit-associated standard or calibration solutions than CLIA. Thus, the anti-HBs titer of 10 mIU/ml is difficult to introduce in Japan as the minimum protective level against HBV infection. Efforts to standardize anti-HBs assay methods are expected to share international evidence about the HBV infection-preventing strategy.

Mechanism of DNA binding enhancement by hepatitis B virus protein pX.

PubMed

Palmer, C R; Gegnas, L D; Schepartz, A

1997-12-09

At least three hundred million people worldwide are infected with the hepatitis B virus (HBV), and epidemiological studies show a clear correlation between chronic HBV infection and the development of hepatocellular carcinoma. HBV encodes a protein, pX, which abducts the cellular transcriptional machinery in several ways including direct interactions with bZIP transcription factors. These interactions increase the DNA affinities of target bZIP proteins in a DNA sequence-dependent manner. Here we use a series of bZIP peptide models to explore the mechanism by which pX interacts with bZIP proteins. Our results suggest that pX increases bZIP.DNA stability by increasing the stability of the bZIP dimer as well as the affinity of the dimer for DNA. Additional experiments provide evidence for a mechanism in which pX recognizes the composite structure of the peptide.DNA complex, not simply the primary peptide sequence. These experiments provide a framework for understanding how pX alters the patterns of transcription within the nucleus. The similarities between the mechanism proposed for pX and the mechanism previously proposed for the human T-cell leukemia virus protein Tax are discussed.

Human Adenovirus Core Protein V Is Targeted by the Host SUMOylation Machinery To Limit Essential Viral Functions.

PubMed

Freudenberger, Nora; Meyer, Tina; Groitl, Peter; Dobner, Thomas; Schreiner, Sabrina

2018-02-15

Human adenoviruses (HAdV) are nonenveloped viruses containing a linear, double-stranded DNA genome surrounded by an icosahedral capsid. To allow proper viral replication, the genome is imported through the nuclear pore complex associated with viral core proteins. Until now, the role of these incoming virion proteins during the early phase of infection was poorly understood. The core protein V is speculated to bridge the core and the surrounding capsid. It binds the genome in a sequence-independent manner and localizes in the nucleus of infected cells, accumulating at nucleoli. Here, we show that protein V contains conserved SUMO conjugation motifs (SCMs). Mutation of these consensus motifs resulted in reduced SUMOylation of the protein; thus, protein V represents a novel target of the host SUMOylation machinery. To understand the role of protein V SUMO posttranslational modification during productive HAdV infection, we generated a replication-competent HAdV with SCM mutations within the protein V coding sequence. Phenotypic analyses revealed that these SCM mutations are beneficial for adenoviral replication. Blocking protein V SUMOylation at specific sites shifts the onset of viral DNA replication to earlier time points during infection and promotes viral gene expression. Simultaneously, the altered kinetics within the viral life cycle are accompanied by more efficient proteasomal degradation of host determinants and increased virus progeny production than that observed during wild-type infection. Taken together, our studies show that protein V SUMOylation reduces virus growth; hence, protein V SUMOylation represents an important novel aspect of the host antiviral strategy to limit virus replication and thereby points to potential intervention strategies. IMPORTANCE Many decades of research have revealed that HAdV structural proteins promote viral entry and mainly physical stability of the viral genome in the capsid. Our work over the last years showed that this

Blocking Tim-3 or/and PD-1 reverses dysfunction of tumor-infiltrating lymphocytes in HBV-related hepatocellular carcinoma.

PubMed

Liu, Furong; Zeng, Gucheng; Zhou, Shaotang; He, Xiaoshun; Sun, Nianfeng; Zhu, Xiaofeng; Hu, Anbin

2018-05-01

The immunosuppression of tumor-infiltrating lymphocytes (TILs) is associated with rapid progression of hepatitis B virus-related hepatocellular carcinoma (HBV-HCC). T cell Ig- and mucin-domain-containing molecule-3 (Tim-3) and programmed cell death 1 (PD-1) are important inhibitory molecules expressed on the surface of T cells, but their roles in the function of TILs in HBV-HCC are poorly understood. We aimed to study the roles of these two markers in HBV-HCC. Ninety patients with pathologically confirmed HBV-associated HCC were enrolled in our study. Blood samples, paired fresh tumor tissues and adjacent tissues were collected, and isolating peripheral blood mononuclear cells, TILs and adjacent-infiltrating lymphocytes were isolated from these samples. The patients were followed-up to allow survival analysis. Tim-3 or/and PD-1 was up-regulated expressed on CD4 + and CD8 + TILs in HBV-HCC patients and a higher proportion of TILs expressed PD-1 alone. Tim-3 + and PD-1 + TILs greatly decreased secretion of IFN-? and TNF-a. Expression of Tim-3 and PD-1 on TILs negatively correlated with disease-free survival of HCC patients. Direct blockade of Tim-3 and PD-1 in vitro significantly enhanced TILs proliferation and secretion of IFN-? and TNF-a. Expression of Tim-3 and/or PD-1 on TILs impairs their function and correlates negatively with disease-free survival in HBV-HCC. Direct blockade of Tim-3 and PD-1 restores anti-tumor effects of TILs, which suggests a potential target for novel immunotherapy in HBV-HCC. Copyright © 2018 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

Stop codons in the hepatitis B surface proteins are enriched during antiviral therapy and are associated with host cell apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Colledge, Danielle; Soppe, Sally; Yuen, Lilly

Premature stop codons in the hepatitis B virus (HBV) surface protein can be associated with nucleos(t)ide analogue resistance due to overlap of the HBV surface and polymerase genes. The aim of this study was to determine the effect of the replication of three common surface stop codon variants on the hepatocyte. Cell lines were transfected with infectious HBV clones encoding surface stop codons rtM204I/sW196*, rtA181T/sW172*, rtV191I/sW182*, and a panel of substitutions in the surface proteins. HBsAg was measured by Western blotting. Proliferation and apoptosis were measured using flow cytometry. All three surface stop codon variants were defective in HBsAg secretion.more » Cells transfected with these variants were less proliferative and had higher levels of apoptosis than those transfected with variants that did not encode surface stop codons. The most cytopathic variant was rtM204I/sW196*. Replication of HBV encoding surface stop codons was toxic to the cell and promoted apoptosis, exacerbating disease progression. - Highlights: •Under normal circumstances, HBV replication is not cytopathic. •Premature stop codons in the HBV surface protein can be selected and enriched during nucleos(t)ide analogue therapy. •Replication of these variants can be cytopathic to the cell and promote apoptosis. •Inadequate antiviral therapy may actually promote disease progression.« less

Molecular characterization of occult hepatitis B virus in genotype E-infected subjects.

PubMed

Zahn, Astrid; Li, Chengyao; Danso, Kwabena; Candotti, Daniel; Owusu-Ofori, Shirley; Temple, Jillian; Allain, Jean-Pierre

2008-02-01

Occult hepatitis B virus (HBV) infection (OBI), defined as the presence of HBV DNA without detectable HBV surface antigen (HBsAg), is frequent in west Africa, where genotype E is prevalent. The prevalence of OBI in 804 blood donors and 1368 pregnant women was 1.7 and 1.5%, respectively. Nine of 32 OBI carriers were evaluated with HBV serology, viral load and complete HBV genome sequence of two to five clones. All samples except one were anti-HBV core antigen-positive and three contained antibodies against HBsAg (anti-HBs). All strains were of genotype E and formed quasispecies with 0.20-1.28% intra-sample sequence variation. Few uncommon mutations (absent in 23 genotype E reference sequences) were found across the entire genome. Two mutations in the core region encoded truncated or abnormal capsid protein, potentially affecting viral production, but were probably rescued by non-mutated variants, as found in one clone. No evidence of escape mutants was found in anti-HBs-carrying samples, as the 'a' region was consistently wild type. OBI carriers constitute approximately 10% of all HBV DNA-viraemic adult Ghanaians. OBI carriers appear as a disparate group, with a very low viral load in common, but multiple origins reflecting decades of natural evolution in an area essentially devoid of human intervention.

Clinical Significance of Quantitative HBsAg Titres and its Correlation With HBV DNA Levels in the Natural History of Hepatitis B Virus Infection.

PubMed

Karra, Vijay K; Chowdhury, Soumya J; Ruttala, Rajesh; Polipalli, Sunil K; Kar, Premashis

2016-09-01

Quantification of serum hepatitis B antigen (HBsAg) is an important test that marks active infection with hepatitis B and helps in the prediction of the clinical outcome and management of hepatitis B virus (HBV) infection. Correlation with HBV DNA quantitative levels may help in developing strategies for antiviral treatment. This study is aimed to evaluate HBsAg titres in various phase of HBV infection in HBsAg positive patients, and its correlation with HBV DNA viral load levels. 976 HBV related patients were analysed in this retrospective cross-sectional study. Patients were categorised on the basis of the phase of HBV infection: immune tolerant phase (IT, n  = 123), immune clearance phase (IC, n  = 192), low-replicative phase (LR, n  = 476), and HBeAg-negative hepatitis (ENH, n  = 185). HBsAg titres were quantified and correlated with HBV-DNA levels and clinical parameters. Median HBsAg titres were different between each phases of HBV infection ( P  < 0.001): (4.62 log10 IU/ml), IC (3.88 log10 IU/ml), LR (2.76 log10 IU/ml) and ENH (2.94 log10 IU/ml). HBsAg and HBV DNA levels showed significant correlation in the whole group ( r  = 0.694, P  < 0.001), and this was also observed in different phases of HBV infection. Strong correlation in IT phase ( r  = 0.603, P  < 0.001) and IC phase ( r  = 0.523, P  < 0.001), moderate in LR phase ( r  = 0.362, P  < 0.001) and weak in ENH ( r  = 0.110, P  = 0.04). No correlation was observed between serum HBsAg levels and biochemical parameters. The study demonstrated significant difference in the median baseline values of serum HBsAg titres in different phases of HBV infection and provides additional information in understanding the natural history of HBV-infection.

The eisosome core is composed of BAR domain proteins

PubMed Central

Olivera-Couto, Agustina; Graña, Martin; Harispe, Laura; Aguilar, Pablo S.

2011-01-01

Eisosomes define sites of plasma membrane organization. In Saccharomyces cerevisiae, eisosomes delimit furrow-like plasma membrane invaginations that concentrate sterols, transporters, and signaling molecules. Eisosomes are static macromolecular assemblies composed of cytoplasmic proteins, most of which have no known function. In this study, we used a bioinformatics approach to analyze a set of 20 eisosome proteins. We found that the core components of eisosomes, paralogue proteins Pil1 and Lsp1, are distant homologues of membrane-sculpting Bin/amphiphysin/Rvs (BAR) proteins. Consistent with this finding, purified recombinant Pil1 and Lsp1 tubulated liposomes and formed tubules when the proteins were overexpressed in mammalian cells. Structural homology modeling and site-directed mutagenesis indicate that Pil1 positively charged surface patches are needed for membrane binding and liposome tubulation. Pil1 BAR domain mutants were defective in both eisosome assembly and plasma membrane domain organization. In addition, we found that eisosome-associated proteins Slm1 and Slm2 have F-BAR domains and that these domains are needed for targeting to furrow-like plasma membrane invaginations. Our results support a model in which BAR domain protein–mediated membrane bending leads to clustering of lipids and proteins within the plasma membrane. PMID:21593205

Seroprevalence of HIV, HBV, HCV, and HTLV among Pregnant Women in Southwestern Nigeria.

PubMed

Opaleye, Oluyinka Oladele; Igboama, Magdalene C; Ojo, Johnson Adeyemi; Odewale, Gbolabo

2016-01-01

Sexually transmitted infections (STIs) are major public health challenge especially in developing countries. This study was designed to determine the prevalence of Hepatitis B virus (HBV), Hepatitis C Virus (HCV), Human immunodeficiency virus (HIV), and Human T-cell lymphotropic Virus type I (HTLV-I) among pregnant women attending antenatal clinic, in Ladoke Akintola University Teaching Hospital, Osogbo, and South-Western Nigeria. One hundred and eighty two randomly selected pregnant women were screened for HBsAg, anti-HCV, anti-HIV and HTLV-1 IgM antibodies using commercially available ELISA kit. Of the 182 blood samples of pregnant women screened whose age ranged from 15-49 years, 13 (7.1%), 5 (2.7%), 9 (4.9%), and 44 (24.2%) were positive for HBsAg, anti-HCV, anti-HIV, and HTLV-1 IgM antibodies, respectively. The co-infection rate of 0.5% was obtained for HBV/HCV, HBV/HIV, HIV/HTLV-1, and HCV/HTLV-1 while 1.1% and 0% was recorded for HBV/HTLV-1 and HCV/HIV co-infections, respectively. Expected risk factors such as history of surgery, circumcision, tattooing and incision showed no significant association with any of the viral STIs (P > 0.05). This study shows that there is the need for a comprehensive screening of all pregnant women for HBsAg, anti-HCV, anti-HIV and HTLV-1 to prevent mother to child transmission of these viral infections and its attending consequences.

Humoral and cell-mediated immune responses after a booster dose of HBV vaccine in HIV-infected children, adolescents and young adults.

PubMed

Giacomet, Vania; Masetti, Michela; Nannini, Pilar; Forlanini, Federica; Clerici, Mario; Zuccotti, Gian Vincenzo; Trabattoni, Daria

2018-01-01

HBV vaccine induces protective antibodies only in 23-56% of HIV-infected children. The aim of our study is to evaluate the immunologic effects of a booster dose of HBV vaccine in HIV-infected youth. 53 young HIV-infected patients in whom HBV vaccination did not elicit protective Ab titers were enrolled. All patients were on ART with optimal immunological and viral response. All patients received a booster dose of HBV vaccine (HBVAXPRO 10 μg i.m.). HBV-specific Ab titer, viral load and CD4+ T cells were measured at baseline (T0), T1, T6 and T12 months. In a subgroup of 16 patients HBV-specific cell mediated immune responses were evaluated at baseline, at T1 and T6. The booster dose induced seroconversion in 51% of patients at T1, 57% at T6, and49% at T12; seroconversion rate was significantly correlated with CD4+T cells at T0 and to the CD4 nadir. The booster dose induced HBV-specific cell mediated immunity at T6 mainly in Responders (Rs): Effector Memory CD8+T cells, HBV-specific TNFα-, IFNγ-, granzyme secreting CD8+ T cells and IL2-secreting CD4+ T cells were significantly increased in Rs compared to T0. In Non Responders (NRs), HBV-specific IL2-secreting CD4+ T cells, Central and Effector Memory CD8+ T cells were the only parameters modified at T6. Seroconversion induced by a booster dose of vaccine correlates with the development of T cell immunological memory in HIV-infected patients who did not respond to the standard immunization. Alternate immunization schedules need to be considered in NRs.

Effect of a hepatitis B virus inhibitor, NZ-4, on capsid formation.

PubMed

Yang, Li; Wang, Ya-Juan; Chen, Hai-Jun; Shi, Li-Ping; Tong, Xian-Kun; Zhang, Yang-Ming; Wang, Gui-Feng; Wang, Wen-Long; Feng, Chun-Lan; He, Pei-Lan; Xu, Yi-Bin; Lu, Meng-Ji; Tang, Wei; Nan, Fa-Jun; Zuo, Jian-Ping

2016-01-01

During the hepatitis B virus (HBV) life cycle, nucleocapsid assembly is essential for HBV replication. Both RNA reverse transcription and DNA replication occur within the HBV nucleocapsid. HBV nucleocapsid is consisted of core protein (HBcAg), whose carboxy-terminal domain (CTD) contains an Arg-rich domain (ARD). The ARD of HBcAg does contribute to the encapsidation of pregenomic RNA (pgRNA). Previously, we reported a small-molecule, NZ-4, which dramatically reduced the HBV DNA level in an in vitro cell setting. Here, we explore the possible mechanisms by which NZ-4 inhibits HBV function. As an HBV inhibitor, NZ-4 leads to the formation of genome-free capsids, including a new population of capsid that runs faster on agarose gels. NZ-4's activity was dependent on the presence of the ARD I, containing at least one positively charged amino acid. NZ-4 might provide a new option for further development of HBV therapeutics for the treatment of chronic hepatitis B. Copyright © 2015. Published by Elsevier B.V.

Protocol for the use of light upon extension real-time PCR for the determination of viral load in HBV infection.

PubMed

Li, Guimin; Li, Wangfeng; Liu, Lixia

2012-01-01

Real-time PCR has engendered wide acceptance for quantitation of hepatitis B virus (HBV) DNA in the blood due to its improved rapidity, sensitivity, reproducibility, and reduced contamination. Here we describe a cost-effective and highly sensitive HBV real-time quantitative assay based on the light upon extension real-time PCR platform and a simple and reliable HBV DNA preparation method using silica-coated magnetic beads.

Novel point mutations and mutational complexes in the enhancer II, core promoter and precore regions of hepatitis B virus genotype D1 associated with hepatocellular carcinoma in Saudi Arabia.

PubMed

Khan, Anis; Al Balwi, Mohammed A; Tanaka, Yasuhito; Hajeer, Ali; Sanai, Faisal M; Al Abdulkarim, Ibrahim; Al Ayyar, Latifah; Badri, Motasim; Saudi, Dib; Tamimi, Waleed; Mizokami, Masashi; Al Knawy, Bandar

2013-12-15

In this study, a cohort of 182 patients [55 hepatocellular carcinoma (HCC) and 127 non-HCC] infected with hepatitis B virus (HBV) in Saudi Arabia was investigated to study the relationship between sequence variation in the enhancer II (EnhII), basal core promoter (BCP) and precore regions of HBV genotype D (HBV/D) and the risk of HCC. HBV genotypes were determined by sequencing analysis and/or enzyme-linked immunosorbent assay. Variations in the EnhII, BCP and precore regions were compared between 107 non-HCC and 45 HCC patients infected with HBV/D, followed by age-matched analysis of 40 cases versus equal number of controls. Age and male gender were significantly associated with HCC (p = 0.0001 and p = 0.03, respectively). Serological markers such as aspartate aminotransferase, albumin and anti-HBe were significantly associated with HCC (p = 0.0001 for all), whereas HBeAg positivity was associated with non-HCC (p = 0.0001). The most prevalent HBV genotype was HBV/D (94%), followed by HBV/E (4%), HBV/A (1.6%) and HBV/C (0.5%). For HBV/D1, genomic mutations associated with HCC were T1673/G1679, G1727, C1741, C1761, A1757/T1764/G1766, T1773, T1773/G1775 and C1909. Age- and gender-adjusted stepwise logistic regression analysis indicated that mutations G1727 [odds ratio (OR) = 18.3; 95% confidence interval (CI) = 2.8-118.4; p = 0.002], A1757/T1764/G1766 (OR = 4.7; 95% CI = 1.3-17.2; p = 0.01) and T1773 (OR = 14.06; 95% CI = 2.3-84.8; p = 0.004) are independent predictors of HCC development. These results implicate novel individual and combination patterns of mutations in the X/precore region of HBV/D1 as predictors of HCC. Risk stratification based on these mutation complexes would be useful in determining high-risk patients and improving diagnostic and treatment strategies for HBV/D1. Copyright © 2013 UICC.

Cellular inhibitor of apoptosis proteins prevent clearance of hepatitis B virus.

PubMed

Ebert, Gregor; Preston, Simon; Allison, Cody; Cooney, James; Toe, Jesse G; Stutz, Michael D; Ojaimi, Samar; Scott, Hamish W; Baschuk, Nikola; Nachbur, Ueli; Torresi, Joseph; Chin, Ruth; Colledge, Danielle; Li, Xin; Warner, Nadia; Revill, Peter; Bowden, Scott; Silke, John; Begley, C Glenn; Pellegrini, Marc

2015-05-05

Hepatitis B virus (HBV) infection can result in a spectrum of outcomes from immune-mediated control to disease progression, cirrhosis, and liver cancer. The host molecular pathways that influence and contribute to these outcomes need to be defined. Using an immunocompetent mouse model of chronic HBV infection, we identified some of the host cellular and molecular factors that impact on infection outcomes. Here, we show that cellular inhibitor of apoptosis proteins (cIAPs) attenuate TNF signaling during hepatitis B infection, and they restrict the death of infected hepatocytes, thus allowing viral persistence. Animals with a liver-specific cIAP1 and total cIAP2 deficiency efficiently control HBV infection compared with WT mice. This phenotype was partly recapitulated in mice that were deficient in cIAP2 alone. These results indicate that antagonizing the function of cIAPs may promote the clearance of HBV infection.

B cell gene signature with massive intrahepatic production of antibodies to hepatitis B core antigen in hepatitis B virus-associated acute liver failure.

PubMed

Farci, Patrizia; Diaz, Giacomo; Chen, Zhaochun; Govindarajan, Sugantha; Tice, Ashley; Agulto, Liane; Pittaluga, Stefania; Boon, Denali; Yu, Claro; Engle, Ronald E; Haas, Mark; Simon, Richard; Purcell, Robert H; Zamboni, Fausto

2010-05-11

Hepatitis B virus (HBV)-associated acute liver failure (ALF) is a dramatic clinical syndrome due to a sudden loss of hepatic cells leading to multiorgan failure. The mechanisms whereby HBV induces ALF are unknown. Here, we show that liver tissue collected at the time of liver transplantation in two patients with HBV-associated ALF is characterized by an overwhelming B cell response apparently centered in the liver with massive accumulation of plasma cells secreting IgG and IgM, accompanied by complement deposition. We demonstrate that the molecular target of these antibodies is the hepatitis B core antigen (HBcAg); that these anti-bodies display a restricted variable heavy chain (V(H)) repertoire and lack somatic mutations; and that these two unrelated individuals with ALF use an identical predominant V(H) gene with unmutated variable domain (IGHV1-3) for both IgG and IgM anti-HBc antibodies, indicating that HBcAg is the target of a germline human V(H) gene. These data suggest that humoral immunity may exert a primary role in the pathogenesis of HBV-associated ALF.

First principles design of a core bioenergetic transmembrane electron-transfer protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goparaju, Geetha; Fry, Bryan A.; Chobot, Sarah E.

Here we describe the design, Escherichia coli expression and characterization of a simplified, adaptable and functionally transparent single chain 4-α-helix transmembrane protein frame that binds multiple heme and light activatable porphyrins. Such man-made cofactor-binding oxidoreductases, designed from first principles with minimal reference to natural protein sequences, are known as maquettes. This design is an adaptable frame aiming to uncover core engineering principles governing bioenergetic transmembrane electron-transfer function and recapitulate protein archetypes proposed to represent the origins of photosynthesis. This article is part of a Special Issue entitled Biodesign for Bioenergetics — the design and engineering of electronic transfer cofactors, proteinsmore » and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.« less

Hydrophobic core malleability of a de novo designed three-helix bundle protein.

PubMed

Walsh, S T; Sukharev, V I; Betz, S F; Vekshin, N L; DeGrado, W F

2001-01-12

De novo protein design provides a tool for testing the principles that stabilize the structures of proteins. Recently, we described the design and structure determination of alpha(3)D, a three-helix bundle protein with a well-packed hydrophobic core. Here, we test the malleability and adaptability of this protein's structure by mutating a small, Ala residue (A60) in its core to larger, hydrophobic side-chains, Leu and Ile. Such changes introduce strain into the structures of natural proteins, and therefore generally destabilize the native state. By contrast, these mutations were slightly stabilizing ( approximately 1.5 kcal mol(-1)) to the tertiary structure of alpha(3)D. The value of DeltaC(p) for unfolding of these mutants was not greatly affected relative to wild-type, indicating that the change in solvent accessibility for unfolding was similar. However, two-dimensional heteronuclear single quantum coherence spectra indicate that the protein adjusts to the introduction of steric bulk in different ways. A60L-alpha(3)D showed serious erosion in the dispersion of both the amide backbone as well as the side-chain methyl chemical shifts. By contrast, A60I-alpha(3)D showed excellent dispersion of the backbone resonances, and selective changes in dispersion of the aliphatic side-chains proximal to the site of mutation. Together, these data suggest that alpha(3)D, although folded into a unique three-dimensional structure, is nevertheless more malleable and flexible than most natural, native proteins. Copyright 2001 Academic Press.

The Hepatitis B Virus X Protein Elevates Cytosolic Calcium Signals by Modulating Mitochondrial Calcium Uptake

PubMed Central

Yang, Bei

2012-01-01

Chronic hepatitis B virus (HBV) infections are associated with the development of hepatocellular carcinoma (HCC). The HBV X protein (HBx) is thought to play an important role in the development of HBV-associated HCC. One fundamental HBx function is elevation of cytosolic calcium signals; this HBx activity has been linked to HBx stimulation of cell proliferation and transcription pathways, as well as HBV replication. Exactly how HBx elevates cytosolic calcium signals is not clear. The studies described here show that HBx stimulates calcium entry into cells, resulting in an increased plateau level of inositol 1,4,5-triphosphate (IP3)-linked calcium signals. This increased calcium plateau can be inhibited by blocking mitochondrial calcium uptake and store-operated calcium entry (SOCE). Blocking SOCE also reduced HBV replication. Finally, these studies also demonstrate that there is increased mitochondrial calcium uptake in HBx-expressing cells. Cumulatively, these studies suggest that HBx can increase mitochondrial calcium uptake and promote increased SOCE to sustain higher cytosolic calcium and stimulate HBV replication. PMID:22031934