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Khan S, Anwer A, Sevak JK, Trehanpati N, Kazim SN. Cytokines Expression Compared to the Determinants of Cellular Apoptosis Prominently Attributes to the Deleterious Effects of 'A' Determinant Surface Gene Mutations in HBV Transfected Hepatoma Cell Line. Immunol Invest 2024; 53:224-240. [PMID: 38095846 DOI: 10.1080/08820139.2023.2288841] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/23/2024]
Abstract
BACKGROUND Previous studies have explored the role of AKT protein in anti-apoptotic/proliferative activities. However, there has been a lack of information regarding the role of Akt in association with cytokines expression in HBV-related (wild type HBV and HBV with mutations of 'a' determinant region) studies either in the case of HBV infection or in transfected hepatoma cells. The present study tries to determine the role of Akt and cytokines expression in the presence of small surface gene mutants in the hepatoma cell line. METHODS Mutations of 'a' determinant region, viz. sA128V and sG145R, were created in wild-type pHBV1.3 by site-directed mutagenesis and transfected in hepatoma cell line. Secretory levels of HBsAg in the wild type as well as in both the mutants were analyzed by ELISA. Apoptotic analysis of transfected cells was studied by flow cytometry. Expression analysis of Akt and cytokines (TNF-alpha, IL-6, and IFN-gamma) was done by qPCR. RESULTS The presence of significantly more alive cells in sG145R than sA128V transfected cells may be due to the up-regulation of the Akt gene expression. Cytokines expression was nearly similar between sA128V and wild-type pHBV1.3 transfected cells. Presence of sG145R showed dramatically high cytokines expression than sA128V and wild-type pHBV1.3. CONCLUSION Cytokines expression predominantly contributes to the detrimental effects associated with the 'a' determinant region mutations particularly sG145R mutant. It may also be inferred that mechanisms associated with cellular apoptosis apparently do not play any major role to assign the 'a' determinant small surface gene mutation(s) for their pathological outcome.
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Affiliation(s)
- Saniya Khan
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India
| | - Ayesha Anwer
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India
| | - Jayesh Kumar Sevak
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi, India
| | - Nirupama Trehanpati
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi, India
| | - Syed Naqui Kazim
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India
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2
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Liu WC, Wu IC, Lee YC, Lin CP, Cheng JH, Lin YJ, Yen CJ, Cheng PN, Li PF, Cheng YT, Cheng PW, Sun KT, Yan SL, Lin JJ, Yang JC, Chang KC, Ho CH, Tseng VS, Chang BCH, Wu JC, Chang TT. Hepatocellular carcinoma-associated single-nucleotide variants and deletions identified by the use of genome-wide high-throughput analysis of hepatitis B virus. J Pathol 2017; 243:176-192. [PMID: 28696069 DOI: 10.1002/path.4938] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2016] [Revised: 05/31/2017] [Accepted: 07/04/2017] [Indexed: 12/26/2022]
Abstract
This study investigated hepatitis B virus (HBV) single-nucleotide variants (SNVs) and deletion mutations linked with hepatocellular carcinoma (HCC). Ninety-three HCC patients and 108 non-HCC patients were enrolled for HBV genome-wide next-generation sequencing (NGS) analysis. A systematic literature review and a meta-analysis were performed to validate NGS-defined HCC-associated SNVs and deletions. The experimental results identified 60 NGS-defined HCC-associated SNVs, including 41 novel SNVs, and their pathogenic frequencies. Each SNV was specific for either genotype B (n = 24) or genotype C (n = 34), except for nt53C, which was present in both genotypes. The pathogenic frequencies of these HCC-associated SNVs showed a distinct U-shaped distribution pattern. According to the meta-analysis and literature review, 167 HBV variants from 109 publications were categorized into four levels (A-D) of supporting evidence that they are associated with HCC. The proportion of NGS-defined HCC-associated SNVs among these HBV variants declined significantly from 75% of 12 HCC-associated variants by meta-analysis (Level A) to 0% of 10 HCC-unassociated variants by meta-analysis (Level D) (P < 0.0001). PreS deletions were significantly associated with HCC, in terms of deletion index, for both genotypes B (P = 0.030) and C (P = 0.049). For genotype C, preS deletions involving a specific fragment (nt2977-3013) were significantly associated with HCC (HCC versus non-HCC, 6/34 versus 0/32, P = 0.025). Meta-analysis of preS deletions showed significant association with HCC (summary odds ratio 3.0; 95% confidence interval 2.3-3.9). Transfection of Huh7 cells showed that all of the five novel NGS-defined HCC-associated SNVs in the small surface region influenced hepatocarcinogenesis pathways, including endoplasmic reticulum-stress and DNA repair systems, as shown by microarray, real-time polymerase chain reaction and western blot analysis. Their carcinogenic mechanisms are worthy of further research. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Affiliation(s)
- Wen-Chun Liu
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC.,Infectious Disease and Signalling Research Centre, National Cheng Kung University, Tainan, Taiwan, ROC
| | - I-Chin Wu
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC.,Infectious Disease and Signalling Research Centre, National Cheng Kung University, Tainan, Taiwan, ROC
| | - Yen-Chien Lee
- Department of Oncology, Tainan Hospital, Ministry of Health and Welfare, Tainan, Taiwan, ROC.,Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC
| | | | - Ji-Hong Cheng
- Department of Computer Science and Information Engineering, National Cheng Kung University, Tainan, Taiwan, ROC
| | - Yih-Jyh Lin
- Department of Surgery, National Cheng Kung University College of Medicine and Hospital, Tainan, Taiwan, ROC
| | - Chia-Jui Yen
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC.,Infectious Disease and Signalling Research Centre, National Cheng Kung University, Tainan, Taiwan, ROC
| | - Pin-Nan Cheng
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC.,Infectious Disease and Signalling Research Centre, National Cheng Kung University, Tainan, Taiwan, ROC
| | - Pei-Fu Li
- Institute of Medical Informatics, National Cheng Kung University, Tainan, Taiwan, ROC
| | - Yi-Ting Cheng
- Institute of Medical Informatics, National Cheng Kung University, Tainan, Taiwan, ROC
| | - Pei-Wen Cheng
- Department of Information and Learning Technology, Science and Engineering College, National University of Tainan, Tainan, Taiwan, ROC
| | - Koun-Tem Sun
- Department of Information and Learning Technology, Science and Engineering College, National University of Tainan, Tainan, Taiwan, ROC
| | - Shu-Ling Yan
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC
| | - Jia-Jhen Lin
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC
| | - Jui-Chu Yang
- Human Biobank, Research Centre of Clinical Medicine, National Cheng Kung University Hospital, Tainan, Taiwan, ROC
| | - Kung-Chao Chang
- Human Biobank, Research Centre of Clinical Medicine, National Cheng Kung University Hospital, Tainan, Taiwan, ROC
| | - Cheng-Hsun Ho
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC.,Infectious Disease and Signalling Research Centre, National Cheng Kung University, Tainan, Taiwan, ROC
| | - Vincent S Tseng
- Department of Computer Science, National Chiao Tung University, Hsinchu, Taiwan, ROC
| | | | - Jaw-Ching Wu
- Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan, ROC.,Translational Research Division, Medical Research Department, Taipei Veterans General Hospital, Taipei, Taiwan, ROC
| | - Ting-Tsung Chang
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC.,Infectious Disease and Signalling Research Centre, National Cheng Kung University, Tainan, Taiwan, ROC
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3
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Liu P, Zhang H, Liang X, Ma H, Luan F, Wang B, Bai F, Gao L, Ma C. HBV preS2 promotes the expression of TAZ via miRNA-338-3p to enhance the tumorigenesis of hepatocellular carcinoma. Oncotarget 2016; 6:29048-59. [PMID: 26315112 PMCID: PMC4745710 DOI: 10.18632/oncotarget.4804] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2015] [Accepted: 07/24/2015] [Indexed: 02/07/2023] Open
Abstract
Transactivators encoded by HBV, including HBx and preS2, play critical role in hepatocellular carcinoma (HCC). YAP, a downstream effector of the Hippo pathway, is involved in hepatocarcinogenesis mediated by HBx. Here, we investigated whether preS2, another transactivator encoded by HBV, regulates the Hippo pathway to promote HCC. We found that preS2 overexpression upregulated TAZ, a downstream effector of the Hippo pathway, at protein level but not at mRNA level. preS2 suppressed miRNA-338-3p expression in HCC cell lines. miRNA-338-3p mimics downregulated TAZ, while miRNA-338-3p inhibitor restored the expression of TAZ, suggesting that TAZ is a direct target of miRNA-338-3p. TAZ overexpression stimulated growth of HCC cell lines. Knockdown of TAZ dampened preS2-promoted HCC proliferation and migration. Thus, preS2 upregulates TAZ expression by repressing miRNA-338-3p. TAZ is necessary for preS2-promoted HCC proliferation and migration
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Affiliation(s)
- Peng Liu
- Key Laboratory for Experimental Teratology of Ministry of Education and Department of Immunology, Shandong University School of Medicine, Jinan, Shandong, 250012, P.R. China
| | - Hualin Zhang
- Key Laboratory for Experimental Teratology of Ministry of Education and Department of Immunology, Shandong University School of Medicine, Jinan, Shandong, 250012, P.R. China
| | - Xiaohong Liang
- Key Laboratory for Experimental Teratology of Ministry of Education and Department of Immunology, Shandong University School of Medicine, Jinan, Shandong, 250012, P.R. China
| | - Hongxin Ma
- Key Laboratory for Experimental Teratology of Ministry of Education and Department of Immunology, Shandong University School of Medicine, Jinan, Shandong, 250012, P.R. China
| | - Fang Luan
- Department of Clinical Laboratory, Shandong Provincial Hospital affiliated to Shandong University, Jinan, Shandong, 250021, P.R. China
| | - Bo Wang
- Key Laboratory for Experimental Teratology of Ministry of Education and Department of Immunology, Shandong University School of Medicine, Jinan, Shandong, 250012, P.R. China
| | - Fuxiang Bai
- Key Laboratory for Experimental Teratology of Ministry of Education and Department of Immunology, Shandong University School of Medicine, Jinan, Shandong, 250012, P.R. China
| | - Lifen Gao
- Key Laboratory for Experimental Teratology of Ministry of Education and Department of Immunology, Shandong University School of Medicine, Jinan, Shandong, 250012, P.R. China
| | - Chunhong Ma
- Key Laboratory for Experimental Teratology of Ministry of Education and Department of Immunology, Shandong University School of Medicine, Jinan, Shandong, 250012, P.R. China
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4
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Levrero M, Zucman-Rossi J. Mechanisms of HBV-induced hepatocellular carcinoma. J Hepatol 2016; 64:S84-S101. [PMID: 27084040 DOI: 10.1016/j.jhep.2016.02.021] [Citation(s) in RCA: 676] [Impact Index Per Article: 75.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/16/2016] [Revised: 02/17/2016] [Accepted: 02/17/2016] [Indexed: 02/06/2023]
Abstract
Hepatitis B virus (HBV) contributes to hepatocellular carcinoma (HCC) development through direct and indirect mechanisms. HBV DNA integration into the host genome occurs at early steps of clonal tumor expansion and induces both genomic instability and direct insertional mutagenesis of diverse cancer-related genes. Prolonged expression of the viral regulatory protein HBx and/or altered versions of the preS/S envelope proteins dysregulates cell transcription and proliferation control and sensitizes liver cells to carcinogenic factors. Accumulation of preS1 large envelope proteins and/or preS2/S mutant proteins activates the unfold proteins response, that can contribute to hepatocyte transformation. Epigenetic changes targeting the expression of tumor suppressor genes occur early in the development of HCC. A major role is played by the HBV protein, HBx, which is recruited on cellular chromatin and modulates chromatin dynamics at specific gene loci. Compared with tumors associated with other risk factors, HBV-related tumors have a higher rate of chromosomal alterations, p53 inactivation by mutations and overexpression of fetal liver/hepatic progenitor cells genes. The WNT/β-catenin pathway is also often activated but HBV-related tumors display a low rate of activating β-catenin mutations. HBV-related HCCs may arise on non-cirrhotic livers, further supporting the notion that HBV plays a direct role in liver transformation by triggering both common and etiology specific oncogenic pathways in addition to stimulating the host immune response and driving liver chronic necro-inflammation.
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Affiliation(s)
- Massimo Levrero
- Cancer Research Center of Lyon (CRCL) - INSERM U1052, Lyon, France; IIT Centre for Life Nanoscience (CLNS), Rome, Italy; Dept of Internal Medicine (DMISM), Sapienza University, Rome, Italy.
| | - Jessica Zucman-Rossi
- Inserm, UMR-1162, Génomique Fonctionnelle des Tumeurs Solides, Equipe Labellisée Ligue Contre le Cancer, Institut Universitaire d'Hematologie, Paris, France; Université Paris Descartes, Labex Immuno-Oncology, Sorbonne Paris Cité, Faculté de Médecine, Paris, France; Université Paris 13, Sorbonne Paris Cité, Unité de Formation et de Recherche Santé, Médecine, Biologie Humaine, Bobigny, France; Université Paris Diderot, Paris, France.
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5
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Guerrieri F, Belloni L, Pediconi N, Levrero M. Pathobiology of Hepatitis B Virus-Induced Carcinogenesis. ACTA ACUST UNITED AC 2016. [DOI: 10.1007/978-3-319-22330-8_5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
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Zhang X, Gao L, Liang X, Guo M, Wang R, Pan Y, Liu P, Zhang F, Guo C, Zhu F, Qu C, Ma C. HBV preS2 transactivates FOXP3 expression in malignant hepatocytes. Liver Int 2015; 35:1087-94. [PMID: 25047684 DOI: 10.1111/liv.12642] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/07/2013] [Accepted: 07/11/2014] [Indexed: 02/06/2023]
Abstract
BACKGROUND & AIMS Recent data reported the increased expression of forkhead box protein 3 (FOXP3), the well known master regulator of CD4(+) C25(+) regulatory T cells, in hepatocellular carcinoma (HCC) cells. However, the mechanisms remain unknown. We previously showed that preS2, one of important regulatory proteins encoded by HBV, triggers transactivation of hTERT in malignant hepatocytes. Here, we aimed to explore the role of preS2 in regulating FOXP3 expression in HCC. METHODS FOXP3 expression was detected by RT-PCR, Western blot and immunohistochemical staining. Cotransfection and siRNA knockdown were involved to study the regulation effects of preS2 on FOXP3 expression in cultured HCC cell lines. Luciferase reporter assay and EMSA assay were performed to explore the mechanism of preS2-mediated FOXP3 upregulation. RESULTS Immunohistochemical staining detected significant increased FOXP3 expression in malignant hepatocytes from sections of HCC patients. The total FOXP3 expression in hepatocytes from patients with HBsAg-positive HCC was significantly increased compared to that of HBV-negative HCCs (P = 0.002). In accordance, preS2 overexpression enhanced FOXP3 expression in HCC cell lines, while preS2 knockdown significantly reduced FOXP3 expression in HBV-integrated HepG2.2.15 cells. Results of cotransfection and luciferase report assay showed that preS2 transactivated FOXP3 promoter in a dose-dependent manner. Further study identified the AP-1 binding site at 20 bp region from -465 bp to -445 bp of FOXP3 promoter was responsible for preS2-induced FOXP3 transcriptional activation. CONCLUSIONS Our data here, for the first time, provided direct evidence to demonstrate that preS2 oncoprotein encoded by HBV transactivated FOXP3 transcription in HCC cells.
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Affiliation(s)
- Xiaoning Zhang
- Key Laboratory for Experimental Teratology of Ministry of Education and Department of Immunology, Shandong Provincial Key Laboratory of Infection & Immunity, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong, 250012, China
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7
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Hepatitis B virus PreS/S gene variants: pathobiology and clinical implications. J Hepatol 2014; 61:408-17. [PMID: 24801416 DOI: 10.1016/j.jhep.2014.04.041] [Citation(s) in RCA: 188] [Impact Index Per Article: 17.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/02/2014] [Revised: 04/21/2014] [Accepted: 04/24/2014] [Indexed: 12/16/2022]
Abstract
The emergence and takeover of hepatitis B virus (HBV) variants carrying mutation(s) in the preS/S genomic region is a fairly frequent event that may occur spontaneously or may be the consequence of immunoprophylaxis or antiviral treatments. Selection of preS/S mutants may have relevant pathobiological and clinical implications. Both experimental data and studies in humans show that several specific mutations in the preS/S gene may induce an imbalance in the synthesis of the surface proteins and their consequent retention within the endoplasmic reticulum (ER) of the hepatocytes. The accumulation of mutated surface proteins may cause ER stress with the consequent induction of oxidative DNA damage and genomic instability. Viral mutants with antigenically modified surface antigen may be potentially infectious to immune-prophylaxed patients and may account for cases of occult HBV infection. In addition, preS/S variants were reported to be associated with cases of fulminant hepatitis as well as of fibrosing cholestatic hepatitis, and they are associated with cirrhosis and hepatocellular carcinoma development.
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8
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Qu LS, Liu JX, Liu TT, Shen XZ, Chen TY, Ni ZP, Lu CH. Association of hepatitis B virus pre-S deletions with the development of hepatocellular carcinoma in Qidong, China. PLoS One 2014; 9:e98257. [PMID: 24849936 PMCID: PMC4029943 DOI: 10.1371/journal.pone.0098257] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2014] [Accepted: 04/30/2014] [Indexed: 01/05/2023] Open
Abstract
Background/Aim To investigate the roles of mutations in pre-S and S regions of hepatitis B virus (HBV) on the progression of hepatocellular carcinoma (HCC) in Qidong, China. Methods We conducted an age matched case-control study within a cohort of 2387 male HBV carriers who were recruited from August, 1996. The HBV DNA sequence in pre-S/S regions was successfully determined in 96 HCC cases and 97 control subjects. In addition, a consecutive series of samples from 11 HCC cases were employed to evaluate the pre-S deletion patterns before and after the occurrence of HCC. Results After adjustment for age, history of cigarette smoking and alcohol consumption, HBeAg positivity, pre-S deletions, pre-S2 start codon mutations, and T53C mutation were significantly associated with HCC, showing adjusted odds ratios (ORs) from 1.914 to 3.199. HCC patients also had a lower frequency of T31C mutation in pre-S2 gene, compared with control subjects (0.524; 95% CI 0.280-0.982). HBV pre-S deletions were clustered mainly in the 5′ end of pre-S2 region. Multivariate analysis showed that pre-S deletions and pre-S2 start codon mutations were independent risk factors for HCC. The OR (95% CI) were 2.434 (1.063–5.573) and 3.065 (1.099–8.547), respectively. The longitudinal observation indicated that the pre-S deletion mutations were not acquired at the beginning of HBV infection, but that the mutations occurred during the long course of liver disease. Conclusion Pre-S deletions and pre-S2 start codon mutations were independently associated with the development of HCC. The results also provided direct evidence that pre-S deletion mutations were not acquired from the beginning of infection but arose de novo during the progression of liver disease.
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Affiliation(s)
- Li-Shuai Qu
- Department of Gastroenterology, Affiliated Hospital of Nantong University, Jiangsu Province, China
| | - Jin-Xia Liu
- Department of Gastroenterology, Affiliated Hospital of Nantong University, Jiangsu Province, China
| | - Tao-Tao Liu
- Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai, China
| | - Xi-Zhong Shen
- Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai, China
| | - Tao-Yang Chen
- Qidong Liver Cancer Institute, Qidong, Jiangsu Province, China
| | - Zheng-Pin Ni
- Qidong Liver Cancer Institute, Qidong, Jiangsu Province, China
| | - Cui-Hua Lu
- Department of Gastroenterology, Affiliated Hospital of Nantong University, Jiangsu Province, China
- * E-mail:
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Occult hepatitis B in blood donors: a description of two cases. BLOOD TRANSFUSION = TRASFUSIONE DEL SANGUE 2010; 8:297-302. [PMID: 20967173 DOI: 10.2450/2010.0119-09] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Key Words] [Subscribe] [Scholar Register] [Received: 07/08/2009] [Accepted: 04/15/2010] [Indexed: 12/13/2022]
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10
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Chen CH, Changchien CS, Lee CM, Hung CH, Hu TH, Wang JH, Wang JC, Lu SN. Combined mutations in pre-s/surface and core promoter/precore regions of hepatitis B virus increase the risk of hepatocellular carcinoma: a case-control study. J Infect Dis 2008; 198:1634-42. [PMID: 18939932 DOI: 10.1086/592990] [Citation(s) in RCA: 91] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
BACKGROUND We sought to investigate the role of sequence variations in pre-S/surface and basal core promoter (BCP)/precore regions of the hepatitis B virus (HBV) in hepatocellular carcinoma (HCC). METHODS The direct sequencing in pre-S/surface and BCP/precore regions of HBV was determined for 80 patients with HCC and 160 control patients with HBV infection. RESULTS Compared with control patients, patients with HCC had higher frequencies of pre-S deletions and amino acid substitutions at codon 4, 7, and 81 in pre-S1 genes; at the start codon in pre-S2 genes; and at codon 68 in surface genes. Patients also had a lower frequency of amino acid substitution at codon 2 in pre-S2 genes, compared with control patients. In BCP/precore regions, patients with HCC had higher frequencies of C or G1753, A1762/T1764, T1846, and A1899. Multivariate analysis showed that pre-S deletions, I68T surface gene, T1762/A1764, and A1899 were independent factors associated with the development of HCC. The HBV strain with a complex mutation pattern rather than a single mutation was associated with HCC, and the HCC risks increased for patients having these factors in combination. CONCLUSIONS Pre-S deletions, I68T in surface gene, T1762/A1764, and A1899 were independent risk factors for HCC. Combination of these viral mutations appeared to increase the risk of HCC.
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Affiliation(s)
- Chien-Hung Chen
- Division of Hepatogastroenterology, Department of Internal Medicine, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, 123 Ta Pei Road, Kaohsiung, Taiwan
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11
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Zhou F, Ren JL, Lu YP, Chen MY, Xu HZ, Pan JS, Cai JY, Dong J. Interaction between fibrinogen alpha chain and the whole S protein of hepatitis B virus. Shijie Huaren Xiaohua Zazhi 2008; 16:2581-2586. [DOI: 10.11569/wcjd.v16.i23.2581] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To screen the proteins interacting with the whole S protein of hepatitis B virus (HBV) from hepatocyte cDNA library by yeast two-hybrid system, and to validate interacting behavior between fibrinogen alpha chain and the whole S protein by reverse yeast two-hybrid.
METHODS: The whole S gene of HBV was cloned into yeast expression vector pDEST32 to construct a bait plasmid, which was verified by Western blot. The bait plasmid and prey plasmids inserted liver cDNA fragments were cotransformed into yeast cells MaV203 by Liac-mediated transformation. Diploid yeasts were plated on synthetic dropout nutrient medium to screen positive colonies. After extracting and sequencing of prey plasmids from positive colonies, the inserted sequences were bioinformatically analyzed. For reverse yeast two-hybrid, the bait plasmid expressing partial fibrinogen alpha chain and four prey plasmids expressing the whole S protein mutants were re-combined. The reconstituted bait plasmid was cotransformed into yeast cells MaV203 with the four prey plasmids, respectively. Diploid yeasts were plated on synthetic dropout nutrient medium and X-gal assay was performed to validate the interacting behavior.
RESULTS: By yeast two-hybrid technique, prey plasmids that were inserted partial gene coding 266-644 amino acid of fibrinogen alpha chain had a positive reaction with bait plasmid coding the whole S protein of HBV. By reverse yeast two-hybrid, fibrinogen alpha chain protein interacted with the four whole S protein mutants. The protein binding domain of the whole S protein might be the leading 268 amino acids.
CONCLUSION: Fibrinogen alpha chain protein may bind the whole S protein of HBV. The interacting domain is in the 266-644 amino acids of fibrinogen alpha chain and the frontal 268 amino acids of the whole S protein, respectively.
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McLaughlin-Drubin ME, Munger K. Viruses associated with human cancer. BIOCHIMICA ET BIOPHYSICA ACTA 2008; 1782:127-50. [PMID: 18201576 PMCID: PMC2267909 DOI: 10.1016/j.bbadis.2007.12.005] [Citation(s) in RCA: 238] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/05/2007] [Revised: 12/13/2007] [Accepted: 12/18/2007] [Indexed: 02/07/2023]
Abstract
It is estimated that viral infections contribute to 15-20% of all human cancers. As obligatory intracellular parasites, viruses encode proteins that reprogram host cellular signaling pathways that control proliferation, differentiation, cell death, genomic integrity, and recognition by the immune system. These cellular processes are governed by complex and redundant regulatory networks and are surveyed by sentinel mechanisms that ensure that aberrant cells are removed from the proliferative pool. Given that the genome size of a virus is highly restricted to ensure packaging within an infectious structure, viruses must target cellular regulatory nodes with limited redundancy and need to inactivate surveillance mechanisms that would normally recognize and extinguish such abnormal cells. In many cases, key proteins in these same regulatory networks are subject to mutation in non-virally associated diseases and cancers. Oncogenic viruses have thus served as important experimental models to identify and molecularly investigate such cellular networks. These include the discovery of oncogenes and tumor suppressors, identification of regulatory networks that are critical for maintenance of genomic integrity, and processes that govern immune surveillance.
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Affiliation(s)
- Margaret E McLaughlin-Drubin
- The Channing Laboratory, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School, 8th Floor, 181 Longwood Avenue, Boston, MA 02115, USA.
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Liu H, Luan F, Ju Y, Shen H, Gao L, Wang X, Liu S, Zhang L, Sun W, Ma C. In vitro transfection of the hepatitis B virus PreS2 gene into the human hepatocarcinoma cell line HepG2 induces upregulation of human telomerase reverse transcriptase. Biochem Biophys Res Commun 2007; 355:379-84. [PMID: 17307151 DOI: 10.1016/j.bbrc.2007.01.160] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2007] [Accepted: 01/30/2007] [Indexed: 12/12/2022]
Abstract
The preS2 domain is the minimal functional unit of transcription activators that is encoded by the Hepatitis B virus (HBV) surface (S) gene. It is present in more than one-third of the HBV-integrates in HBV induced hepatocarcinoma (HCC). To further understand the functional role of PreS2 in hepatocytes, a PreS2 expression plasmid, pcS2, was constructed and stably transfected into HepG2 cells. We conducted growth curve and colony-forming assays to study the impact of PreS2 expression on cell proliferation. Cells transfected with PreS2 proliferated more rapidly and formed colonies in soft agar. PreS2 expressing cells also induced upregulation of human telomerase reverse transcriptase (hTERT) and telomerase activation by RT-PCR and the modified TRAP assay. Blocking expression of hTERT with antisense oligonuleotide reversed the growth rate in cells stably transfected with PreS2. Our data suggest that PreS2 may increase the malignant transformation of human HCC cell line HepG2 by upregulating hTERT and inducing telomerase activation.
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Affiliation(s)
- Hua Liu
- Institute of Immunology, Shandong University School of Medicine, #44 Wenhua Xi Road, Jinan 250012, PR China
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Mateos ML, Tato M, Casado J, Moreira V. Hepatitis crónica B por mutante G145R del virus de la hepatitis B. Med Clin (Barc) 2006; 127:596-7. [PMID: 17145020 DOI: 10.1157/13094005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
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Irshad M, Joshi YK, Sharma Y, Dhar I. Transfusion transmitted virus: A review on its molecular characteristics and role in medicine. World J Gastroenterol 2006; 12:5122-34. [PMID: 16937521 PMCID: PMC4088008 DOI: 10.3748/wjg.v12.i32.5122] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The present review gives an updated overview of transfusion transmitted virus (TTV), a novel agent, in relation to its molecular characteristics, epidemiological features, modes of transmission, tissue tropism, pathogenesis, role in various diseases and its eradication from the body. TTV, a DNA virus, is a single stranded, non-enveloped, 3.8 kb long DNA virus with a small and covalently closed circular genome comprising 3852 bases. It was tentatively designated Circinoviridae virus. TTV genome sequence is heterogeneous and reveals the existence of six different genotypes and several subtypes. TTV has been reported to transmit not only via parenteral routes, but also via alternate routes. This virus has been detected in different non-human primates as well. At present, TTV is detected by polymerase chain reaction (PCR) with no other available diagnostic assays. It shows its presence globally and was detected in high percent populations of healthy persons as well as in various disease groups. Initially it was supposed to have strong association with liver disease; however, there is little evidence to show its liver tropism and contribution in causing liver diseases. It shows high prevalence in hemodialysis patients, pointing towards its significance in renal diseases. In addition, TTV is associated with several infectious and non-infectious diseases. Although its exact pathogenesis is not yet clear, TTV virus possibly resides and multiplies in bone marrow cells and peripheral blood mononuclear cells (PBMCs). Recently, attempts have been made to eradicate this virus with interferon treatment. More information is still needed to extricate various mysteries related to TTV.
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Affiliation(s)
- M Irshad
- Clinical Biochemistry Division, Department of Laboratory Medicine, PO Box -4938, A I I M S, New Delhi-110029, India.
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Ji D, Cheng J, Chen GF, Liu Y, Wang L, Guo J. Study of transactivating effect of pre-S2 protein of hepatitis B virus and cloning of genes transactivated by pre-S2 protein with suppression subtractive hybridization. World J Gastroenterol 2005; 11:5438-43. [PMID: 16222733 PMCID: PMC4320350 DOI: 10.3748/wjg.v11.i35.5438] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the transactivating effect of pre-S2 protein of hepatitis B virus (HBV) and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization (SSH) technique, and to pave the way for elucidating the pathogenesis of HBV infection.
METHODS: pcDNA3.1(-)-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1(-)-pre-S2/pSV-lacZ and empty pcDNA3.1(-)/pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase (β-gal). SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1(-)-pre-S2 and pcDNA3.1(-) empty vector was isolated, respectively, cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR.
RESULTS: The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1(-)-pre-S2 plasmid. The activity of β-gal in HepG2 cells transfected with pcDNA3.1(-)-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid (P<0.01). The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positive clones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences were obtained, these cDNA sequences might be the target genes transactivated by pre-S2 protein.
CONCLUSION: The pre-S2 protein of HBV has transactivating effect on SV40 early promoter. The obtained sequences may be target genes transactivated by pre-S2 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity, signal transduction and cell apoptosis.This finding brings some new clues for studying the biological functions of pre-S2 protein and further understanding of HBV hepatocarcinogesis.
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Affiliation(s)
- Dong Ji
- The 7th Department of Infectious Diseases, The 302 Hospital of PLA, Beijing 100039, China.
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Thakur V, Kazim SN, Guptan RC, Hasnain SE, Bartholomeusz A, Malhotra V, Sarin SK. Transmission of G145R mutant of HBV to an unrelated contact. J Med Virol 2005; 76:40-6. [PMID: 15778957 DOI: 10.1002/jmv.20321] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Household contacts of HBV-related chronic liver disease patients constitute a high-risk group for acquisition of HBV infection. Some of the HBsAg mutants are associated with liver disease and some are reported to be transmitted vertically. There is limited information on the horizontal transmission of Gly 145 Arg (G145R) mutant to related contacts. Its possible transmission to an unrelated third degree contact is reported in the present study. An HBV related chronic liver disease patient; the index patient, and his 11 household contacts were studied. This included four 1 degrees, three 2 degrees, one 3 degrees, and a sexual contact. Surface gene sequencing including the "a" determinant region was carried out in HBV DNA+ve subjects. The sequences were aligned and compared for the homology. HBV DNA was found to be positive in one 1 degrees, three 2 degrees, and one 3 degrees contact, besides the index patient. Histopathological studies revealed evidence of chronic hepatitis in all these contacts. Mutation T118V was present in all the six subjects. Mutant G145R along with T118V and T143M was identified in three subjects who included one 1 degrees, one 2 degrees, and one 3 degrees contact. Presence of T118V and T143M mutations along with G145R mutation in these subjects provides an indirect evidence for the possible horizontal transmission of G145R HBV variant to a 3 degrees unrelated contact. Of these three contacts with G145R mutation, only one 1 degrees contact was found to be HBsAg-ve. The data also reaffirms the earlier finding of HBsAg positivity in presence of G145R mutation of the S-gene. HBV exists as quasi-species and mixed population in subjects with chronic HBV infection.
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Affiliation(s)
- Varsha Thakur
- Department of Gastroenterology, G.B. Pant Hospital, New Delhi, India
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Liu K, Lei XZ, Zhao LS, Tang H, Liu L, Feng P, Lei BJ. Tissue microarray for high-throughput analysis of gene expression profiles in hepatocellular carcinoma. World J Gastroenterol 2005; 11:1369-72. [PMID: 15761978 PMCID: PMC4250687 DOI: 10.3748/wjg.v11.i9.1369] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the expression profiles of HBsAg, HBcAg, p21WAF1/CIP1 (p21), Rb genes in hepatocellular carcinoma (HCC) and to investigate their roles in the hepatocar-cinogenesis.
METHODS: HCC tissue microarray containing 120-min tissues of 40 HCC cases was constructed. HBsAg, HBcAg, p21 and Rb proteins were immunohistochemically stained by streptavidin-peroxidase conjugated method (S-P). The expression loss of these genes in cancerous, para-cancerous tissues and adjacent normal liver tissues of 40 HCCs were comparatively examined.
RESULTS: The positive rate of HBsAg expression in cancerous tissues of 40 HCCs was 7.5%, which was lower than that in para-cancerous and adjacent normal liver tissues (χ2 =12.774, P<0.01; χ2 = 18.442, P<0.01). The positive rate of HBcAg expression in cancerous tissues of 40 HCCs was 20.0%, which was also lower than that in para-cancerous and adjacent normal liver tissues (χ2 = 9.482, P<0.01; χ2 = 14.645, P<0.01). p21 protein deletion rate in cancerous tissues of 40 HCCs was 27.5%, which was higher than that in para-cancerous and adjacent normal liver tissues (χ2 = 7.439, P<0.01; χ2 = 11.174, P<0.01). p21 protein deletion correlated remarkably with the pathological grade of HCC (χ2 = 0.072, P<0.05). Rb protein deletion rate in cancerous tissues of 40 HCCs was 42.5%, which was also higher than that in para-cancerous and adjacent normal liver tissues (χ2 = 10.551, P<0.01; χ2 = 18.353, P<0.01). Rb protein deletion rate did not correlate remarkably with tumor size or pathological grade of HCC (χ2 = 0.014, P>0.05; χ2 = 0.017, P>0.05).
CONCLUSION: Expression deletion of HBsAg, HBcAg, p21 and Rb proteins in HCCs may play important roles in the carcinogenesis of HCC. Tissue microarray is an effective high-throughput technique platform for cancer research.
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Affiliation(s)
- Kai Liu
- Division of Molecular Biology of Infectious Diseases, Key Laboratory of Biotherapy of Human Disease, Ministry of Education, West China Hospital of Sichuan University, Chengdu 610041, Sichuan Province, China
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Ma CL, Fang DX, Yao K, Li FQ, Jin HY, Li SQ, Tan WG. Incidence of HBV variants with a mutation at nt551 among hepatitis B patients in Nanjing and its neighbourhood. World J Gastroenterol 2005; 11:299-302. [PMID: 15633237 PMCID: PMC4205423 DOI: 10.3748/wjg.v11.i2.299] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the epidemiology of hepatitis B virus (HBV) strains with a mutation at nt551 in surface gene among hepatitis B patients in Nanjing and its neighbourhood.
METHODS: By using mutation-specific polymerase chain reaction (msPCR) established by our laboratory for amplifying HBV DNAs with a mutation at nt551, 117 serum samples taken from hepatitis B patients were detected.
RESULTS: The results showed that 112 samples were positive for nt551A, 4 samples were positive for nt551G. One sample was positive for nt551T. No nt551C of HBV DNA was found. The incidence of HBsAg mutants with G, C, T, A at nt551 among 117 samples was 3.42%, 0%, 0.85%, 95.73%, respectively.
CONCLUSION: In Nanjing and its neighbourhood, hepatitis B patients are mainly infected with wild genotype HBV. The incidence of mutants with a mutation at nt551 in HBV genome is significantly lower than that in wild genotype HBV DNA (P<0.01). The necessity of adding components of HBsAg mutants to HBV vaccine needs further investigation.
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Affiliation(s)
- Chun-Ling Ma
- Huadong Research Institute for Medical Biotechnics, Nanjing 210029, Jiangsu Province, China.
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Wai CT, Fontana RJ. Clinical significance of hepatitis B virus genotypes, variants, and mutants. Clin Liver Dis 2004; 8:321-52, vi. [PMID: 15481343 DOI: 10.1016/j.cld.2004.02.006] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Emerging evidence suggests that hepatitis B virus (HBV) genotypes may influence the rate of spontaneous and interferon-induced hepatitis B e antigen (HBeAg) seroconversion as well as the natural history of liver disease. In contrast, the dinical significance of precore and core promoter variants associated with HBeAg negative liver disease is less certain in light of the many competing host and virologic factors noted in reported studies. HBV surface mutants are primarily associated with prior vaccine or hepatitis B immune globulin exposure and do not appear to have untoward virulence or association with occult HBV infection. Polymerase mutants with reduced drug sensitivity and phenotypic resistance are commonly detected in patients receiving prolonged antiviral therapy and have a variable impact on disease outcomes. The introduction of additional nucleoside/nucleotide analog agents will likely lead to the development of further unique polymerase mutants with varying pathogenicity and cross-resistance to existing drugs.
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Affiliation(s)
- Chun-Tao Wai
- Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, 3912 Taubman Center, Ann Arbor, MI 48109-0362, USA
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Ma CL, Fang DX, Chen HB, Li FQ, Jin HY, Li SQ, Tan WG. A mutation specific polymerase chain reaction for detecting hepatitis B virus genome mutations at nt551. World J Gastroenterol 2003; 9:509-12. [PMID: 12632507 PMCID: PMC4621571 DOI: 10.3748/wjg.v9.i3.509] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: The hepatitis B surface antigen (HBsAg) is considered to be one of the best markers for the diagnosis of acute and chronic HBV infection. But in some patients, this antigen cannot be detected by routine serological assays despite the presence of virus. One of the most important explanations for the lack of detectable HBsAg is that mutations which occur within the “a” determinant of HBV S gene can alter expression of HBsAg and lead to changes of antigenicity and immunogenicity of HBsAg accordingly. As a result, these mutants cannot be detected by diagnosis assays. Thus, it is essential to find out specific and sensitive methods to test the new mutants and further investigate their distribution. This study is to establish a method to investigate the distribution of the HBsAg mutant at nt551.
METHODS: A mutation specific polymerase chain reaction (msPCR) was established for amplifying HBV DNA with a mutation at nt551. Four sets of primer pairs, P551A-PPS, P551G-PPS, P551C-PPS and P551T-PPS, with the same sequences except for one base at 3’ terminus were designed and synthesized according to the known HBV genome sequences and the popular HBV subtypes, adr and adw, in China. At the basis of regular PCR method, we explored the specific conditions for amplifying HBV DNAs with a mutation at nt551 by regulating annealing temperature and the concentration of these primers. 126 serum samples from patients of hepatitis B were collected, among which 16 were positive for HBV S DNA in the nested PCR amplification. These 16 HBV S DNAs were detected by using the msPCR method.
RESULTS: When the annealing temperature was raised to 71 °C, nt551A and nt551G were amplified specifically by P551A-PPS and P551G-PPS; At 72 °C and 5 pmole of the primers (each) in reaction of 25 μl volume, nt551C and nt551T were amplified specifically by P551C-PPS and P551T-PPS. 16 of HBV S gene fragments were characterized by using this method. 14 of them were positive for nt551A, one was positive for nt551G, and the other one was positive for nt551T. The results were confirmed by nucleotide sequencing.
CONCLUSION: The mutation specific polymerase chain reaction is a specific and sensitive method for detecting the mutations of HBV genome at nt551.
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Affiliation(s)
- Chun-Ling Ma
- Huadong Research Institute for Medical Biotechnics, Nanjing 210002, China.
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Affiliation(s)
- Wei Ning Chen
- Department of Clinical Research, Singapore General Hospital, Singapore
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Chakravarty R, Neogi M, Roychowdhury S, Panda CK. Presence of hepatitis B surface antigen mutant G145R DNA in the peripheral blood leukocytes of the family members of an asymptomatic carrier and evidence of its horizontal transmission. Virus Res 2002; 90:133-41. [PMID: 12457969 DOI: 10.1016/s0168-1702(02)00147-8] [Citation(s) in RCA: 39] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
An asymptomatic carrier and all six of his family members were detected positive for HBV DNA in their peripheral blood leukocytes (PBL), by polymerase chain reaction. Direct sequencing of the amplified DNA revealed that the HBV DNA from the carrier and his wife was of subtype ayw. Interestingly, the amplified HBV DNA from the five other members of the family was found to be not only of subtype adw but also contained G to A mutation at nucleotide position 587. This indicates the presence of established vaccine escape mutant of the virus (G145R) and suggests two different sources of infection within the family. Southern blot hybridization of EcoR1 digested DNA from PBL indicated presence of HBV DNA, integrated into cellular DNA and also in the form of free viral DNA. The study not only establishes the persistence of surface mutant G145R HBV DNA, within the PBL of HBsAg negative individuals from the non-vaccinated random population, but also suggests possible horizontal transmission of the mutant among the family members although none of the family members has received immunoprophylaxis against HBV or had clinically apparent disease or any other known risk factors of HBV infection. As all of them were seronegative for HBsAg/antiHBc, the presence of G145R mutant in the PBL signaled possibility of spread of the vaccine escape mutant virus by blood transfusion, unsafe injection practices or through sexual root.
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Affiliation(s)
- Runu Chakravarty
- ICMR Virus Unit, I D & B G Hospital Campus, GB-41st Floor, 57 Beliaghata Main Road, 700 010, Kolkata, India.
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Oon CJ, Chen WN, Goh KT, Mesenas S, Ng HS, Chiang G, Tan C, Koh S, Teng SW, Toh I, Moh MC, Goo KS, Tan K, Leong AL, Tan GS. Molecular characterization of hepatitis B virus surface antigen mutants in Singapore patients with hepatocellular carcinoma and hepatitis B virus carriers negative for HBsAg but positive for anti-HBs and anti-HBc. J Gastroenterol Hepatol 2002; 17 Suppl:S491-6. [PMID: 12534784 DOI: 10.1046/j.1440-1746.17.s4.16.x] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
BACKGROUND AND AIMS Mutations on the a-determinant of hepatitis B virus surface antigen (HBsAg), capable of escaping detection and vaccination, are identified in HBsAg-positive/anti-HBs-positive vaccinated infants. We studied the prevalence of these mutants in HBsAg-negative/anti-HBc-positive chronic HBV carriers and patients with hepatocellular carcinoma (HCC). METHODS DNA sequence coding for the antigenic a-determinant of HBsAg was amplified from either HCC genomic DNA or serum samples of the selected patients and sequenced. The replicative mutant genomes were reconstituted in vitro and their reactivity to commercial kits measured. RESULTS Mutations within and/or outside the a-determinant were identified in patients seronegative for HBsAg. They were then reconstituted in vitro and transiently transfected into HepG2 cells. Culture medium containing secreted HBV viral particles was collected and assayed for their binding to commercial kits. Drastic decrease of reactivity to these kits was seen with most of the identified mutations, including those located outside the a-determinant. CONCLUSION The existence of a more complex antigenic structure of HBsAg is indicated by the decreased reactivity to detection of mutations, some of which are outside the a-determinant, escape vaccination and may persist in seronegative patients. The high proportion of HBsAg mutants that are integrated in HCC genomes suggests a role of these mutants in hepatocarcinogenesis, possibly leading to mutant HBV-related HCC.
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Affiliation(s)
- Chong Jin Oon
- Department of Clinical Research, Singapore General Hospital, Ministry of the Environment, Singapore
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Zhong S, Yeo W, Tang M, Liu C, Lin XR, Ho WM, Hui P, Johnson PJ. Frequent detection of the replicative form of TT virus DNA in peripheral blood mononuclear cells and bone marrow cells in cancer patients. J Med Virol 2002; 66:428-34. [PMID: 11793398 DOI: 10.1002/jmv.2163] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The TT virus (TTV), a member of a family of human viruses related to the circoviridae viruses, was associated initially with acute and chronic liver diseases. TTV consists of a single-stranded, circular DNA genome of 3.8 kilobases (kb) and at least three open reading frames (ORFs). The objective of the present study was to determine whether or not TTV replicated in peripheral blood mononuclear cells (PBMCs) and bone marrow cells (BMCs). DNA was extracted from the PBMCs or BMCs of 153 cancer patients and from the PBMCs of 50 healthy blood donors (the controls). By using a single round of polymerase chain reaction (PCR), TTV was detected in 98.6% (141 of 143) of the PBMCs and in 90% (9 of 10) of the BMCs from cancer patients. TTV DNA was detected in significantly fewer control subjects at 86% (43 of 50; P < 0.05). Strand-specific PCR (SSPCR) targeting the ORF2 of the common genotypes of TTV was developed specifically to detect TTV positive or negative strand DNA and to examine TTV replication. TTV positive strand DNA, which may be an intermediate of viral replication, was detected in 55.3% (78 of 141) of the TTV-infected PBMCs of the cancer patients and in 7% (3 of 43) of the controls (P < 0.001). The replicative form of TTV was also detectable in 55.6% (5 of 9) of the TTV-infected BMCs. The existence of double-strand (positive and negative strands) TTV DNA in PBMCs and BMCs of the cancer patients was also supported by the finding that TTV DNA extracted from these cells was resistant to S1 nuclease. Using in situ hybridization, TTV DNA was also demonstrated to be present in the nucleus of PBMCs. It is concluded that replicative intermediate forms of TTV DNA are present in both PBMCs and BMCs, indicating that blood cells may be a site of TTV replication.
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Affiliation(s)
- Sheng Zhong
- Department of Clinical Oncology, Sir Y.K. Pao Centre for Cancer, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kong
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Hildt E, Munz B, Saher G, Reifenberg K, Hofschneider PH. The PreS2 activator MHBs(t) of hepatitis B virus activates c-raf-1/Erk2 signaling in transgenic mice. EMBO J 2002; 21:525-35. [PMID: 11847101 PMCID: PMC125852 DOI: 10.1093/emboj/21.4.525] [Citation(s) in RCA: 132] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
The large hepatitis B virus (HBV) surface protein (LHBs) and C-terminally truncated middle size surface proteins (MHBs(t)) form the family of the PreS2 activator proteins of HBV. Their transcriptional activator function is based on the cytoplasmic orientation of the PreS2 domain. MHBs(t) activators are paradigmatic for this class of activators. Here we report that MHBs(t) is protein kinase C (PKC)-dependently phosphorylated at Ser28. The integrity of the phosphorylation site is essential for the activator function. MHBs(t) triggers PKC-dependent activation of c-Raf-1/Erk2 signaling that is a prerequisite for MHBs(t)-dependent activation of AP-1 and NF-kappaB. To analyze the pathophysiological relevance of these data in vivo, transgenic mice were established that produce the PreS2 activator MHBs(t) specifically in the liver. In these mice, a permanent PreS2-dependent specific activation of c-Raf-1/Erk2 signaling was observed, resulting in an increased hepatocyte proliferation rate. In transgenics older than 15 months, an increased incidence of liver tumors occurs. These data suggest that PreS2 activators LHBs and MHBs(t) exert a tumor promoter-like function by activation of key enzymes of proliferation control.
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Affiliation(s)
- Eberhard Hildt
- Department of Virus Research, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Robert Koch-Institut, Am Nordufer 20, D-13353 Berlin and Animal Research Unit, University of Ulm, D-89081 Ulm, Germany Corresponding author e-mail:
| | - Barbara Munz
- Department of Virus Research, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Robert Koch-Institut, Am Nordufer 20, D-13353 Berlin and Animal Research Unit, University of Ulm, D-89081 Ulm, Germany Corresponding author e-mail:
| | - Gesine Saher
- Department of Virus Research, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Robert Koch-Institut, Am Nordufer 20, D-13353 Berlin and Animal Research Unit, University of Ulm, D-89081 Ulm, Germany Corresponding author e-mail:
| | - Kurt Reifenberg
- Department of Virus Research, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Robert Koch-Institut, Am Nordufer 20, D-13353 Berlin and Animal Research Unit, University of Ulm, D-89081 Ulm, Germany Corresponding author e-mail:
| | - Peter Hans Hofschneider
- Department of Virus Research, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Robert Koch-Institut, Am Nordufer 20, D-13353 Berlin and Animal Research Unit, University of Ulm, D-89081 Ulm, Germany Corresponding author e-mail:
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Kew MC. Hepatitis B virus in the etiology of hepatocellular carcinoma. VIRUSES AND LIVER CANCER 2002. [DOI: 10.1016/s0168-7069(02)06063-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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Abstract
Hepatitis B virus (HBV) is at the origin of severe liver diseases like chronic active hepatitis, liver cirrhosis and hepatocellular carcinoma. There are some groups of patients with high risk of generation of HBV mutants: infected infants, immunosuppressed individuals (including hemodialysis patients), patients treated with interferon and lamivudine for chronic HBV infection. These groups are the target for molecular investigations reviewed in this paper. The emergence of lamivudine- or other antiviral-resistant variants, rises concern regarding long term use of these drugs. Infection or immunization with one HBV subtype confers immunity to all subtypes. However, reinfection or reactivation of latent HBV infection with HBV mutants have been reported in patients undergoing transplant and those infected with HIV. Mutations of the viral genome which are not replicative incompetent can be selected in further course of infection or under prolonged antiviral treatment and might maintain the liver disease. Four open reading frames (ORF) which are called S-gene, C-gene, X-gene and P-gene were identified within the HBV genome. Mutations may affect each of the ORFs. Mutated S-genes were described to be responsible for HBV-infections in successfully vaccinated persons, mutated C-genes were found to provoke severe chronic liver diseases, mutated X-genes could cause serious medical problems in blood donors by escaping the conventional test systems and mutated P-genes were considered to be the reason for chemotherapeutic drug resistance. This paper reviews molecular, immunological and clinical aspects of the HBV mutants.
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Affiliation(s)
- C Kreutz
- International Technology for Evaluation of Clinical Pharmacology, Paris, France.
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29
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Zhong S, Yeo W, Tang MW, Lin XR, Mo F, Ho WM, Hui P, Johnson PJ. Gross elevation of TT virus genome load in the peripheral blood mononuclear cells of cancer patients. Ann N Y Acad Sci 2001; 945:84-92. [PMID: 11708500 DOI: 10.1111/j.1749-6632.2001.tb03868.x] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
TT virus (TTV) is a recently described circular DNA virus of about 3.8 kb, which is related to the circoviridae viruses. It is commonly detected in healthy subjects and no association with any specific disease has been established. TTV was initially thought to be hepatotropic, but subsequent reports have shown that it is detectable in other tissues, including kidney, prostate, mammary gland, brain, bone marrow, and peripheral blood mononuclear cells. Plasma samples from cancer patients and healthy subjects were tested for the presence or absence of TTV by heminested polymerase chain reaction (PCR). We also developed a quantitative competitive PCR (QC-PCR) assay for TTV that permits accurate measurement of TTV DNA load. Using this assay, the TTV genome load in peripheral blood mononuclear cells (PBMCs) of healthy control subjects (n = 50) and patients with various types of cancer (n = 148), including breast cancer, non-Hodgkin's lymphoma, colon cancer, hepatocellular carcinoma, nasopharyngeal carcinoma, and other cancers, was measured. TTV DNA was detected in 69 of 100 plasma samples (69%) of cancer patients tested and in 39 of 100 plasma samples (39%) randomly selected from 1000 plasma samples of blood donors (p < 0.05). TTV DNA was detectable in the PBMCs of 99% of the cancer patients and 86% of the controls. However, the median virus load was more than 100-fold higher in the cancer patients (3599 copies/100,000 cells) than among the controls (30 copies/100,000 cells; p < 0.0001). There was no significant difference in TTV load among the different cancer types. Using a cutoff value of >250 copies per 100,000 PBMCs, 93.2% of cancer patients were "positive" compared to only 4% of healthy control subjects. Almost all the cancer patients have TTV infection and their TTV genome load in PBMCs is significantly higher than that in control subjects. It remains to be elucidated whether such findings are specific to cancer patients or occur in all seriously ill subjects.
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Affiliation(s)
- S Zhong
- Department of Clinical Oncology, Institute of Molecular Oncology at the Sir Y. K. Pao Centre for Cancer, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories
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30
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Abstract
Chronic hepatitis B virus (HBV) infection is a major global cause of hepatocellular carcinoma (HCC). Individuals who are chronic carriers have a greater than 100-fold increased relative risk of developing the tumour. Several mechanisms of HBV-induced HCC have been proposed. Integration of HBV DNA into the genome of hepatocytes occurs commonly, although integration at cellular sites that are important for regulation of hepatocyte proliferation appears to be a rare event. Functions of the HBx protein are also potentially oncogenic. These include transcriptional activation of cellular growth regulatory genes, modulation of apoptosis and inhibition of nucleotide excision repair of damaged cellular DNA. The effects of HBx are mediated by interaction with cellular proteins and activation of cell signalling pathways. Variations in HBV genome sequences may be important in hepatocarcinogenesis, although their significance has not yet been completely elucidated. Necroinflammatory hepatic disease, which often accompanies chronic HBV infection, may contribute indirectly to hepatocyte transformation in a number of ways, including by facilitating HBV DNA integration, predisposing to the acquisition of cellular mutations and generating mutagenic oxygen reactive species. Although HCC is a malignancy with a poor prognosis, the availability of an effective vaccine against HBV infection, and its inclusion in the Expanded Programme of Immunization of many countries, augurs well for the eventual elimination of HBV-associated HCC.
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Affiliation(s)
- P Arbuthnot
- Department of Molecular Medicine and Haematology and Molecular Hepatology Research Unit, Department of Medicine, University of the Witwatersrand Medical School, 7 York Road, Parktown 2193, South Africa
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31
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Zhong S, Yeo W, Lin CK, Lin XR, Tang MW, Johnson PJ. Quantitative and genotypic analysis of TT virus infection in Chinese blood donors. Transfusion 2001; 41:1001-7. [PMID: 11493731 DOI: 10.1046/j.1537-2995.2001.41081001.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
BACKGROUND The TT virus (TTV) is a member of a newly described family of human viruses related to the C ircoviridae viruses. Its association with specific diseases has not been established, and screening of blood donors has not been implemented. To date, 16 genotypes have been identified. STUDY DESIGN AND METHODS Sera from 471 healthy blood donors (aged 11-58 years) were randomly selected and tested for TTV by the use of two sets of primers: NG59d/NG61d/NG63d primers and T801/T935 primers. Quantitative competitive PCR (QC-PCR) was developed to measure the TTV DNA concentration among the blood donors. Sequencing of a part of the genome was performed to identify the various genotypes. Several samples showed a mixed genotype infection. RESULTS TTV was detected in 251 (53.3%) of 471 healthy Hong Kong blood donors by the use of NG59d/NG61d/NG63d primers. The prevalence of the virus increased steadily with age (p = 0.03). TTV DNA was detected in 90 percent (90 of a randomly selected 100) of samples by the use of T801/T935 primers. TTV DNA concentration was also measured by QC-PCR in the blood donors who were positive for TTV DNA in the first round of the heminested PCR. TTV titers ranged from 4.8 x 10(2) copies per mL to 6 x 10(4) copies per mL, with a median value of 1.2 x 10(4) copies per mL. Sequencing and phylogenetic analysis of a 223-bp fragment from open reading frame 1 showed three main genotypes (G1 [60.7%], G2 [24.3%], and G3 [14%]) and a new genotype 17 (G17), with the latter bearing 60-percent nucleotide homology with other genotypes deposited at GenBank. In addition, a new TTV subtype, G2f, was found. CONCLUSION The prevalence of TTV is high in healthy Chinese blood donors. Three main genotypes (G1, G2, and G3) were detected. In addition, a new TTV genotype, tentatively designated as G17, and a new subtype, G2f, were identified.
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Affiliation(s)
- S Zhong
- Department of Clinical Oncology, Sir Y.K. Pao Centre for Cancer, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, China
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32
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Abstract
Chronic hepatitis B virus (HBV) infection is a major global cause of hepatocellular carcinoma (HCC). Individuals who are chronic carriers have a greater than 100-fold increased relative risk of developing the tumour. Several mechanisms of HBV-induced HCC have been proposed. Integration of HBV DNA into the genome of hepatocytes occurs commonly, although integration at cellular sites that are important for regulation of hepatocyte proliferation appears to be a rare event. Functions of the HBx protein are also potentially oncogenic. These include transcriptional activation of cellular growth regulatory genes, modulation of apoptosis and inhibition of nucleotide excision repair of damaged cellular DNA. The effects of HBx are mediated by interaction with cellular proteins and activation of cell signalling pathways. Variations in HBV genome sequences may be important in hepatocarcinogenesis, although their significance has not yet been completely elucidated. Necroinflammatory hepatic disease, which often accompanies chronic HBV infection, may contribute indirectly to hepatocyte transformation in a number of ways, including by facilitating HBV DNA integration, predisposing to the acquisition of cellular mutations and generating mutagenic oxygen reactive species. Although HCC is a malignancy with a poor prognosis, the availability of an effective vaccine against HBV infection, and its inclusion in the Expanded Programme of Immunization of many countries, augurs well for the eventual elimination of HBV-associated HCC.
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Affiliation(s)
- Patrick Arbuthnot
- Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School7 York Road, Parktown 2193, South Africa
- Molecular Hepatology Research Unit, Department of Medicine, University of the Witwatersrand Medical School7 York Road, Parktown 2193, South Africa
| | - Michael Kew
- Molecular Hepatology Research Unit, Department of Medicine, University of the Witwatersrand Medical School7 York Road, Parktown 2193, South Africa
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33
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Chen WN, Oon CJ, Moh MC. Detection of hepatitis B virus surface antigen mutants in paraffin-embedded hepatocellular carcinoma tissues. Virus Genes 2001; 20:263-7. [PMID: 10949955 DOI: 10.1023/a:1008100930584] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
The antigenic "a" determinant of the human hepatitis B virus surface antigen (HBsAg) is highly conserved and involved in inducing neutralizing antibody. Mutations on this determinant have been associated with the hepatitis B virus (HBV) escape from vaccination but are also found in chronic HBV carriers, but their involvement in liver diseases including hepatocellular carcinoma (HCC) is unclear. To investigate the possible liver disease-associated role of HBsAg mutants, their incidence was analyzed in 11 paraffin embedded HCC tissues using an improved DNA extraction method. Mutations on the "a" determinant (Thr126Ser, Gly145Arg, a double mutant Thr126Ser/Gln129Asn, Met 133Leu and Thr140Ile) were identified in 5 samples while the wild type sequence was found in 2 others. Future characterization of these HCC-associated HBsAg mutants should provide new insights on their role in the pathogenesis of HCC.
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Affiliation(s)
- W N Chen
- Department of Clinical Research, Singapore General Hospital, Republic of Singapore.
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34
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Oon CJ, Chen WN, Goo KS, Goh KT. Intra-familial evidence of horizontal transmission of hepatitis B virus surface antigen mutant G145R. J Infect 2000; 41:260-4. [PMID: 11120616 DOI: 10.1053/jinf.2000.0751] [Citation(s) in RCA: 43] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
OBJECTIVES To provide intra-familial evidence on the horizontal transmission of hepatitis B virus surface antigen (HBsAg) mutant G145R. METHODS Serum samples from family members of 10 vaccinated infants who carried this G145R mutant were collected. The presence of the mutant was analysed by polymerase chain reaction (PCR) and sequencing. RESULTS The G145R mutant was identified in family members of three of the 10 infants. In family 1, the mutant found initially in child 1 was identified in another child and the father. In families 2 and 3, the G145R mutant detected previously in child 1 was detected in the father. Additional mutations in HBsAg were identified in at least two members in family 1 and 2, suggesting horizontal transmission of the mutant among them. The G145R mutant was found in samples with high levels of neutralizing antibody against HBV (anti-HBs). In addition, liver damage was seen in one G145R carrier infant. CONCLUSIONS The G145R mutant could be transmitted horizontally among family members, and this could occur in the presence of high levels of anti-HBs. Improvement of detection system for the G145R and other HBsAg mutant will be needed for their effective control.
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Affiliation(s)
- C J Oon
- Ransome Research Laboratory, Singapore General Hospital, Republic of Singapore
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35
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Ireland JH, O'Donnell B, Basuni AA, Kean JD, Wallace LA, Lau GK, Carman WF. Reactivity of 13 in vitro expressed hepatitis B surface antigen variants in 7 commercial diagnostic assays. Hepatology 2000; 31:1176-82. [PMID: 10796895 DOI: 10.1053/he.2000.6407] [Citation(s) in RCA: 100] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
The primary marker of current hepatitis B infection is the surface antigen (HBsAg), however HBsAg negativity does not exclude hepatitis B viremia. HBsAg variants can be responsible for such diagnostic failures. Here 13 different HBsAg variants were cloned, variant protein produced in a mammalian expression system, and tested using 7 commercial HBsAg diagnostic assays. Of 12 variants analyzed, 6 samples displayed similar reactivity to the positive control (containing standard HBsAg sequence) in most of the assays, but 6 samples, containing various mutations throughout the entire major hydrophilic region (MHR), showed reduced reactivity. It was found that the loss of cysteine at amino acid (aa) 124 in 1 sample affected the secretion as well as the reactivity of HBsAg in the expression system. Thus, not all assays are equally able to detect HBsAg variants, implying that, to attain an acceptable level of sensitivity, the antibody repertoire of the current assays should be extended.
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Affiliation(s)
- J H Ireland
- Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland
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