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Shih C, Wu SY, Chou SF, Yuan TTT. Virion Secretion of Hepatitis B Virus Naturally Occurring Core Antigen Variants. Cells 2020; 10:cells10010043. [PMID: 33396864 PMCID: PMC7823318 DOI: 10.3390/cells10010043] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2020] [Revised: 12/21/2020] [Accepted: 12/28/2020] [Indexed: 02/07/2023] Open
Abstract
In natural infection, hepatitis B virus (HBV) core protein (HBc) accumulates frequent mutations. The most frequent HBc variant in chronic hepatitis B patients is mutant 97L, changing from an isoleucine or phenylalanine to a leucine (L) at HBc amino acid 97. One dogma in the HBV research field is that wild type HBV secretes predominantly virions containing mature double-stranded DNA genomes. Immature genomes, containing single-stranded RNA or DNA, do not get efficiently secreted until reaching genome maturity. Interestingly, HBc variant 97L does not follow this dogma in virion secretion. Instead, it exhibits an immature secretion phenotype, which preferentially secretes virions containing immature genomes. Other aberrant behaviors in virion secretion were also observed in different naturally occurring HBc variants. A hydrophobic pocket around amino acid 97 was identified by bioinformatics, genetic analysis, and cryo-EM. We postulated that this hydrophobic pocket could mediate the transduction of the genome maturation signal for envelopment from the capsid interior to its surface. Virion morphogenesis must involve interactions between HBc, envelope proteins (HBsAg) and host factors, such as components of ESCRT (endosomal sorting complex required for transport). Immature secretion can be offset by compensatory mutations, occurring at other positions in HBc or HBsAg. Recently, we demonstrated in mice that the persistence of intrahepatic HBV DNA is related to virion secretion regulated by HBV genome maturity. HBV virion secretion could be an antiviral drug target.
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Affiliation(s)
- Chiaho Shih
- Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
- Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
- Correspondence: (C.S.); (T.-T.T.Y.)
| | - Szu-Yao Wu
- Chimera Bioscience Inc., No. 18 Siyuan St., Zhongzheng Dist., Taipei 10087, Taiwan;
| | - Shu-Fan Chou
- Department of Microbiology, Harvard Medical School, Boston, MA 02115, USA;
| | - Ta-Tung Thomas Yuan
- TFBS Bioscience, Inc. 3F, No. 103, Ln 169, Kangning St., Xizhi Dist., New Taipei City 221, Taiwan
- Correspondence: (C.S.); (T.-T.T.Y.)
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Persistence of Hepatitis B Virus DNA and the Tempos between Virion Secretion and Genome Maturation in a Mouse Model. J Virol 2019; 93:JVI.01001-19. [PMID: 31462567 PMCID: PMC6819939 DOI: 10.1128/jvi.01001-19] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2019] [Accepted: 08/23/2019] [Indexed: 12/19/2022] Open
Abstract
Chronic infection with human hepatitis B virus (HBV) could lead to cirrhosis and hepatoma. At present, there is no effective treatment to eradicate the virus from patients. HBV in chronic carriers does not exist as a single homogeneous population. The most frequent naturally occurring mutation in HBV core protein occurs at amino acid 97, changing an isoleucine to leucine (I97L). One dogma in the field is that only virions containing a mature genome are preferentially secreted into the medium. Here, we demonstrated that mutant I97L can secrete immature genome in mice. Although viral DNA of mutant I97L with immature genome is less persistent than wild-type HBV in time course experiments, viral DNA of mutant P130T with genome hypermaturation, surprisingly, is more persistent. Therefore, virion secretion regulated by genome maturity could influence viral persistence. It remains an open issue whether virion secretion could be a drug target for HBV therapy. Hepatitis B virus (HBV) core protein (HBc) accumulates frequent mutations in natural infection. Wild-type HBV is known to secrete predominantly virions containing mature DNA genome. However, a frequent naturally occurring HBc variant, I97L, changing from an isoleucine to a leucine at amino acid 97, exhibited an immature secretion phenotype in culture, which preferentially secretes virions containing immature genomes. In contrast, mutant P130T, changing from a proline to a threonine at amino acid 130, exhibited a hypermaturation phenotype by accumulating an excessive amount of intracellular fully mature DNA genome. Using a hydrodynamic delivery mouse model, we studied the in vivo behaviors of these two mutants, I97L and P130T. We detected no naked core particles in all hydrodynamically injected mice. Mutant I97L in mice exhibited pleiotropic phenotypes: (i) excessive numbers of serum HBV virions containing immature genomes, (ii) significantly reduced numbers of intracellular relaxed-circle and single-stranded DNAs, and (iii) less persistent intrahepatic and secreted HBV DNAs than wild-type HBV. These pleiotropic phenotypes were observed in both immunocompetent and immunodeficient mice. Although mutant P130T also displayed a hypermaturation phenotype in vivo, it cannot efficiently rescue the immature virion secretion of mutant I97L. Unexpectedly, the single mutant P130T exhibited in vivo a novel phenotype in prolonging the persistence of HBV genome in hepatocytes. Taken together, our studies provide a plausible rationale for HBV to regulate envelopment morphogenesis and virion secretion via genome maturity, which is likely to play an important role in the persistence of viral DNA in this mouse model. IMPORTANCE Chronic infection with human hepatitis B virus (HBV) could lead to cirrhosis and hepatoma. At present, there is no effective treatment to eradicate the virus from patients. HBV in chronic carriers does not exist as a single homogeneous population. The most frequent naturally occurring mutation in HBV core protein occurs at amino acid 97, changing an isoleucine to leucine (I97L). One dogma in the field is that only virions containing a mature genome are preferentially secreted into the medium. Here, we demonstrated that mutant I97L can secrete immature genome in mice. Although viral DNA of mutant I97L with immature genome is less persistent than wild-type HBV in time course experiments, viral DNA of mutant P130T with genome hypermaturation, surprisingly, is more persistent. Therefore, virion secretion regulated by genome maturity could influence viral persistence. It remains an open issue whether virion secretion could be a drug target for HBV therapy.
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Choe WH, Kim H, Lee SY, Choi YM, Kwon SY, Moon HW, Hur M, Kim BJ. Three types of preS1 start codon deletion variants in the natural course of chronic hepatitis B infection. J Gastroenterol Hepatol 2018; 33:1370-1378. [PMID: 29232004 DOI: 10.1111/jgh.14069] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/12/2017] [Revised: 11/04/2017] [Accepted: 12/04/2017] [Indexed: 12/18/2022]
Abstract
BACKGROUND AND AIM Naturally occurring hepatitis B virus variants carrying a deletion in the preS1 start codon region may evolve during long-lasting virus-host interactions in chronic hepatitis B (CHB). The aim of this study was to determine the immune phase-specific prevalent patterns of preS1 start codon deletion variants and related factors during the natural course of CHB. METHODS A total of 399 CHB patients were enrolled. Genotypic analysis of three different preS1 start codon deletion variants (classified by deletion size: 15-base pair [bp], 18-bp, and 21-bp deletion variants) was performed. RESULTS PreS1 start codon deletion variants were detected in 155 of 399 patients (38.8%). The predominant variant was a 15-bp deletion in the immune-tolerance phase (18/50, 36%) and an 18-bp deletion in the immune-clearance phase (69/183, 37.7%). A 21-bp deletion was the predominant variant in the low replicative phase (3/25, 12.0%) and reactivated hepatitis Be antigen (HBeAg)-negative phase (22/141, 15.6%). The 15-bp and 18-bp deletion variants were more frequently found in HBeAg-positive patients (P < 0.010 and P < 0.001, respectively), whereas the 21-bp deletion variant was more frequently found in HBeAg-negative patients (P < 0.001). On multiple logistic regression analyses, the 21-bp deletion variant was independently associated with liver cirrhosis (P = 0.006), and the 15-bp deletion variant was significantly related to an incomplete response to antiviral agents (P = 0.012). CONCLUSIONS The predominant type of preS1 start codon deletion variants changes according to the immune phases of CHB infection, and each variant type is associated with different clinical parameters. PreS1 start codon deletion variants might interact with the host immune response differently according to their variant types.
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Affiliation(s)
- Won Hyeok Choe
- Department of Internal Medicine, Konkuk University School of Medicine, Seoul, Korea
| | - Hong Kim
- Department of Biomedical Sciences, Microbiology and Immunology, Liver Research Institute, Cancer Research Institute and SNUMRC, College of Medicine, Seoul National University, Seoul, Korea
| | - So-Young Lee
- Department of Biomedical Sciences, Microbiology and Immunology, Liver Research Institute, Cancer Research Institute and SNUMRC, College of Medicine, Seoul National University, Seoul, Korea
| | - Yu-Min Choi
- Department of Biomedical Sciences, Microbiology and Immunology, Liver Research Institute, Cancer Research Institute and SNUMRC, College of Medicine, Seoul National University, Seoul, Korea
| | - So Young Kwon
- Department of Internal Medicine, Konkuk University School of Medicine, Seoul, Korea
| | - Hee Won Moon
- Department of Laboratory Medicine, Konkuk University School of Medicine, Seoul, Korea
| | - Mina Hur
- Department of Laboratory Medicine, Konkuk University School of Medicine, Seoul, Korea
| | - Bum-Joon Kim
- Department of Biomedical Sciences, Microbiology and Immunology, Liver Research Institute, Cancer Research Institute and SNUMRC, College of Medicine, Seoul National University, Seoul, Korea
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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: 27] [Impact Index Per Article: 3.4] [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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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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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: 207] [Impact Index Per Article: 18.8] [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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Brichler S, Lagathu G, Chekaraou MA, Le Gal F, Edouard A, Dény P, Césaire R, Gordien E. African, Amerindian and European hepatitis B virus strains circulate on the Caribbean Island of Martinique. J Gen Virol 2013; 94:2318-2329. [PMID: 23884366 DOI: 10.1099/vir.0.055459-0] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Ten Hepatitis B virus (HBV) genotypes, as well as numerous subgenotypes, have been described in well-characterized ethnogeographical populations. Martinique has been at a crossroads between Africa, Europe, India and the Americas because of the slave trade (17th-19th centuries), followed by an important immigration of Indian and West African workers. In this work, we aimed to study the molecular epidemiology of HBV infection in Martinique according to this unique settlement pattern. To that end, blood samples from 86 consecutive HBV-infected patients from the main hospitals of the island, were retrospectively analysed. Direct sequencing of the pre-S1 or pre-C-C region or complete genome sequencing, followed by phylogenetic analyses were performed. HBV genotypes were: HBV/A1 (68.6 %), HBV/A2 (10.5 %), HBV/D, mainly HBV/D3 and HBV/D4 (8.1 %), HBV/F (3.5 %), and also HBV/E (2.3 %), two strains isolated from two West-African patients. Moreover, 74 % of the HBeAg-negative strains harboured classical pre-C-C mutations, and most HBV/A1 strains also containing specific mutations. Finally, various patterns of deletion mutants in pre-S and pre-C-C regions were found. In conclusion, our findings point to historical and migration-related issues in HBV-genotype distribution suggesting that HBV/A1, but not HBV/E, was imported from Africa during the slave trade, and further supporting the hypothesis that HBV/E has emerged recently in West Africa (<150 years). Potential origins of 'European' HBV/A2 and HBV/D3, 'Amerindian' HBV/F, and HBV/D4 strains are also discussed. Such HBV genetic diversity, beyond its epidemiological interest, may have a clinical impact on the natural history of HBV infection in Martinique.
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Affiliation(s)
- Ségolène Brichler
- Service de Bactériologie, Virologie-Hygiène, Assistance Publique-Hôpitaux de Paris; Hôpitaux Universitaires de Paris Seine Saint-Denis, Site Avicenne, Bobigny; Laboratoire Associé au Centre National de Référence des Hépatites B, C et Delta, Université Paris Nord, Sorbonne Paris Cité, France
| | - Gisèle Lagathu
- Service de Bactériologie, Virologie du Centre Hospitalier Régional et Universitaire de Rennes, Pontchaillou, France
| | - Mariama Abdou Chekaraou
- Service de Bactériologie, Virologie-Hygiène, Assistance Publique-Hôpitaux de Paris; Hôpitaux Universitaires de Paris Seine Saint-Denis, Site Avicenne, Bobigny; Laboratoire Associé au Centre National de Référence des Hépatites B, C et Delta, Université Paris Nord, Sorbonne Paris Cité, France
| | - Frédéric Le Gal
- Service de Bactériologie, Virologie-Hygiène, Assistance Publique-Hôpitaux de Paris; Hôpitaux Universitaires de Paris Seine Saint-Denis, Site Avicenne, Bobigny; Laboratoire Associé au Centre National de Référence des Hépatites B, C et Delta, Université Paris Nord, Sorbonne Paris Cité, France
| | - André Edouard
- Service d'Hépato-Gastro-Entérologie, Centre Hospitalier Universitaire de Fort-de-France, Martinique, France
| | - Paul Dény
- Centre de Recherche sur le Cancer, Équipe 16, INSERM U1052, CNRS UMR 5286, Lyon, France.,Service de Bactériologie, Virologie-Hygiène, Assistance Publique-Hôpitaux de Paris; Hôpitaux Universitaires de Paris Seine Saint-Denis, Site Avicenne, Bobigny; Laboratoire Associé au Centre National de Référence des Hépatites B, C et Delta, Université Paris Nord, Sorbonne Paris Cité, France
| | - Raymond Césaire
- Laboratoire de Virologie, Centre Hospitalier Universitaire de Fort-de-France, Martinique; EA 4537, Université Antilles-Guyane, France
| | - Emmanuel Gordien
- Service de Bactériologie, Virologie-Hygiène, Assistance Publique-Hôpitaux de Paris; Hôpitaux Universitaires de Paris Seine Saint-Denis, Site Avicenne, Bobigny; Laboratoire Associé au Centre National de Référence des Hépatites B, C et Delta, Université Paris Nord, Sorbonne Paris Cité, France
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Pollicino T, Amaddeo G, Restuccia A, Raffa G, Alibrandi A, Cutroneo G, Favaloro A, Maimone S, Squadrito G, Raimondo G. Impact of hepatitis B virus (HBV) preS/S genomic variability on HBV surface antigen and HBV DNA serum levels. Hepatology 2012; 56:434-443. [PMID: 22271491 DOI: 10.1002/hep.25592] [Citation(s) in RCA: 116] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/20/2011] [Accepted: 12/30/2011] [Indexed: 12/16/2022]
Abstract
UNLABELLED To evaluate whether hepatitis B virus (HBV) preS/S gene variability has any impact on serum hepatitis B surface antigen (HBsAg) levels and to analyze the replication capacity of naturally occurring preS/S variants, sera from 40 untreated patients with HBV-related chronic liver disease (hepatitis B e antigen [HBeAg]-positive, n = 11; HBeAg-negative, n = 29) were virologically characterized. Additionally, phenotypic analysis of three different preS/S variant isolates (carrying a 183-nucleotide deletion within the preS1 region, the deletion of preS2 start codon, and a stop signal at codon 182 within the S gene, respectively) was performed. HBV infecting 14 (35%) patients had single or multiple preS/S genomic mutations (i.e., preS1 and/or preS2 deletions, preS2 start codon mutations, C-terminally truncated and/or "a" determinant mutated S protein). Presence of preS/S variants negatively correlated with HBsAg titers (r = -0.431; P = 0.005) and its prevalence did not significantly differ between HBeAg-positive and HBeAg-negative patients. No correlation was found between HBsAg and HBV DNA levels in patients infected with preS/S mutants, whereas a significant correlation was found between HBsAg and viremia levels (r = 0.607; P = 0.001) in patients infected with wild-type HBV strains. HepG2 cells replicating the above-mentioned three preS/S variants showed significant reduction of HBsAg secretion, retention of envelope proteins in the endoplasmic reticulum, less efficient virion secretion and nuclear accumulation of significantly higher amounts of covalently closed circular DNA compared with wild-type HBV replicating cells. CONCLUSION In patients infected with preS/S variants, HBV DNA replication and HBsAg synthesis/secretion appear to be dissociated. Therefore, the use of HBsAg titer as diagnostic/prognostic tool has to take into account the frequent emergence of preS/S variants in chronic HBV infection.
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Affiliation(s)
- Teresa Pollicino
- Unit of Clinical and Molecular Hepatology, Department of Internal Medicine, University of Messina, Messina, Italy.
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9
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Locarnini S, Bowden S. Hepatitis B surface antigen quantification: not what it seems on the surface. Hepatology 2012; 56:411-4. [PMID: 22454331 DOI: 10.1002/hep.25732] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 03/11/2012] [Indexed: 01/04/2023]
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10
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The pre-s2 domain of the hepatitis B virus is dispensable for infectivity but serves a spacer function for L-protein-connected virus assembly. J Virol 2010; 84:3879-88. [PMID: 20130049 DOI: 10.1128/jvi.02528-09] [Citation(s) in RCA: 80] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
The envelope of the human hepatitis B virus (HBV) contains three membrane proteins (L, M, and S). They accomplish different functions in HBV infectivity and nucleocapsid envelopment. Infectivity determinants have been assigned to the N-terminal part of the pre-S1 domain of the L protein and the antigenic loop of the S domain in the L and/or S protein. Nucleocapsid envelopment requires a C-terminal sequence within pre-S1, including the five N-terminal amino acids of pre-S2 as part of the L protein. However, the role of the M protein and the pre-S2 domain of the L protein are not entirely understood. We addressed this question and analyzed assembly competence and infectivity of viruses that lack the M protein and, at the same time, carry alterations in the pre-S2 domain of L. These include deletions, in part frameshift mutations and a randomization of virtually the entire pre-S2 sequence. We found that the M protein is dispensable for HBV in vitro infectivity. Viruses that lack the M protein and contain a mostly randomized pre-S2 sequence assemble properly and are infectious in HepaRG cells and primary human hepatocytes. While deletions of 20 amino acids in the pre-S2 domain of L protein allowed the production of infectious virions, more extended deletions interfered with assembly. This indicates that the pre-S2 domain of the L protein serves an important role for virus assembly, presumably as a spacer that supports conformational changes of L protein but does not participate as part of the M protein or as a subdomain of the L protein in virus entry.
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Zhang KY, Imazeki F, Fukai K, Arai M, Kanda T, Mikata R, Yokosuka O. Analysis of the complete hepatitis B virus genome in patients with genotype C chronic hepatitis and hepatocellular carcinoma. Cancer Sci 2007; 98:1921-9. [PMID: 17888035 PMCID: PMC11158307 DOI: 10.1111/j.1349-7006.2007.00609.x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2007] [Revised: 07/27/2007] [Accepted: 08/05/2007] [Indexed: 12/20/2022] Open
Abstract
Hepatitis B virus (HBV) genotype C and the basic core promoter (BCP) mutations were reported to be associated with the development of hepatocellular carcinoma (HCC). In this study the full sequences of HBV genomes were analyzed in order to find the other predictors of HCC development. We determined the full sequences of HBV genomes in 24 genotype C carriers who developed HCC (HCC group) at the beginning of follow-up and at the time of HCC diagnosis, and 20 patients who did not develop HCC (non-HCC group) served as a control. The number of nucleotide and amino acid substitutions in most regions was higher in the HCC group than in the non-HCC group, and the following substitutions and deletions were found more frequently in the HCC group than in the non-HCC group: G1317A and T1341C/A/G in the X promoter region were detected in 13 and six of the HCC cases, four and none of the non-HCC cases, respectively; and pre-S2 deletion was detected in eight HCC and none of the non-HCC cases. Compared with the wild type X promoter, the mutant type X promoters, M1 (G1317A), M2 (T1341C), and M4 (T1341G) showed increases in activity of 2.3, 3.8, and 1.4 times, respectively, in HepG2 cells. Substitutions and deletion of nucleotides of the HBV genome, especially the pre-S2 deletion and G1317A and T1341C/A/G mutations may be useful markers for predicting the development of HCC.
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MESH Headings
- Adult
- Amino Acid Substitution
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/virology
- Cell Line, Tumor
- DNA, Viral/blood
- DNA, Viral/genetics
- Female
- Genes, Reporter
- Genome, Viral
- Genotype
- Hepatitis B virus/genetics
- Hepatitis C, Chronic/genetics
- Hepatitis C, Chronic/virology
- Humans
- Liver Neoplasms/genetics
- Liver Neoplasms/virology
- Male
- Middle Aged
- Polymorphism, Single Nucleotide
- RNA, Viral/blood
- RNA, Viral/genetics
- Transfection
- Viral Proteins/chemistry
- Viral Proteins/genetics
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Affiliation(s)
- Kai Yu Zhang
- Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-Ward, Chiba City, Chiba 260-8670, Japan
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12
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Gao ZY, Li T, Wang J, Du JM, Li YJ, Li J, Lu FM, Zhuang H. Mutations in preS genes of genotype C hepatitis B virus in patients with chronic hepatitis B and hepatocellular carcinoma. J Gastroenterol 2007; 42:761-8. [PMID: 17876546 DOI: 10.1007/s00535-007-2085-1] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/26/2006] [Accepted: 06/06/2007] [Indexed: 02/07/2023]
Abstract
BACKGROUND Hepatitis B virus (HBV) preS mutations are frequently isolated from patients with severe forms of liver disease. Meanwhile, genotype C has been shown to cause more serious liver disease than genotype B. This study assesses the frequency of preS mutation in Chinese patients with genotype C chronic HBV infection and its relation to liver damage. METHODS Seventy-nine persistently infected patients (25 asymptomatic carriers, 28 with chronic hepatitis, and 26 with hepatocellular carcinoma) with genotype C HBV were analyzed. Levels of HBV DNA, hepatitis B e antigen (HBeAg), alanine aminotransferase, and aspartate transaminase and mutations in the preS region were determined. RESULTS The correlations of preS deletion with disease progression were distinct: preS deletion mutations were more commonly found in the hepatocellular carcinoma (HCC) group than in the chronic hepatitis B (CHB) or asymptomatic carrier (ASC) groups, with the frequencies of 38.46% (10/26) in the HCC, 7.14% (2/28) in the CHB, and 4.00% (1/25) in the ASC (P = 0.001) groups. The HBeAg-positive rate and HBV DNA levels were comparable between patients with the preS mutation and those without. CONCLUSIONS PreS deletion mutations of genotype C HBV might play a role in HBV-related hepatocarcinogenesis.
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Affiliation(s)
- Zhi Yong Gao
- Department of Microbiology, Peking University Health Science Center, 38 Xueyuan Road, 100083, Beijing, China
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13
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Choi MS, Kim DY, Lee DH, Lee JH, Koh KC, Paik SW, Rhee JC, Yoo BC. Clinical significance of pre-S mutations in patients with genotype C hepatitis B virus infection. J Viral Hepat 2007; 14:161-8. [PMID: 17305881 DOI: 10.1111/j.1365-2893.2006.00784.x] [Citation(s) in RCA: 73] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
We investigated the overall and site-specific prevalence of pre-S mutations and its clinical significance in patients with genotype C hepatitis B virus (HBV) infection. Three hundred subjects were included: 50 asymptomatic carriers (AC), 87 chronic hepatitis (CH), 91 liver cirrhosis (LC) and 72 hepatocellular carcinoma (HCC). Pre-S mutations were determined by nucleotide sequence analysis. Possible correlations between pre-S mutations and clinical/virological parameters were examined. Pre-S mutations were detected in 82 cases (27.3%); it was more frequently found in HCC (43.1%) and LC (35.2%) group than in the CH (20.7%) and AC (2.0%) group. Pre-S2 deletion was the most commonly found mutation (10.7%), followed by pre-S2 start codon mutation (9.7%), pre-S1-S2 deletion (3.0%) and both pre-S2 deletion and start codon mutation (2.7%). Pre-S2 deletion and pre-S2 start codon mutation were more frequently detected in advanced diseases (LC and HCC). Pre-S mutations were associated with older age and higher rates of positive HBV DNA (>/=0.5 pg/mL). Advanced disease and positive HBV DNA were shown to be independent predictors of pre-S mutations by logistic regression analysis. These findings suggest that pre-S mutations, especially pre-S2 deletions and pre-S2 start codon mutations, are common in patients with genotype C HBV infection and are associated with advanced liver disease and active viral replication.
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Affiliation(s)
- M S Choi
- Department of Medicine and Digestive Disease Research Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea
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14
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Zhang K, Imazeki F, Fukai K, Arai M, Kanda T, Mikata R, Yokosuka O. Analysis of the complete hepatitis B virus genome in patients with genotype C chronic hepatitis in relation to HBeAg and anti-HBe. J Med Virol 2007; 79:683-93. [PMID: 17457922 DOI: 10.1002/jmv.20849] [Citation(s) in RCA: 5] [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
To investigate the relationship between viral factors and the development of chronic hepatitis B, the entire hepatitis B virus (HBV) genome of chronic carriers at different disease stages were analyzed. Eighty genotype C HBV carriers including 12 hepatitis B e antigen (HBeAg) positive asymptomatic carriers (Group A), 49 HBeAg positive patients with chronic liver diseases (Group B) and 19 anti-HBe positive patients with chronic liver diseases (Group C) were studied. HBV nucleic acid from serum samples was sequenced directly and compared with GenBank reference sequences HBV X01587 and M12906. On phylogenetic analysis, 76 cases were genotype C2. Of the 76 genotype C2 cases, the nucleotide and amino acid substitution rates in the precore/core region were significantly higher in Groups B and C than in Group A, also in Group C than in Group B. The nucleotide substitution rates in the full genome and the core promoter region were significantly higher in Group C than in Group A, also in group C than in Group B. The nucleotide and amino acid substitution rates in the X region were significantly higher in Group C than in Group A. The amino acid substitution rate in the pre-S2 region was significantly higher in Group C than in Group B. Deletion mutations were found mainly in Groups B and C. This whole genome analysis of HBV chronic carriers suggested that the nucleotide substitutions and deletions in HBV were closely associated with the pathogenesis of chronic HBV infection.
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Affiliation(s)
- KaiYu Zhang
- Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan
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15
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Fujiwara K, Tanaka Y, Paulon E, Orito E, Sugiyama M, Ito K, Ueda R, Mizokami M, Naoumov NV. Novel type of hepatitis B virus mutation: replacement mutation involving a hepatocyte nuclear factor 1 binding site tandem repeat in chronic hepatitis B virus genotype E. J Virol 2005; 79:14404-14410. [PMID: 16254374 PMCID: PMC1280239 DOI: 10.1128/jvi.79.22.14404-14410.2005] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2005] [Accepted: 08/17/2005] [Indexed: 12/17/2022] Open
Abstract
The genetic diversity of hepatitis B virus (HBV) strains has evolved through mutations such as point mutations, deletions or insertions, and recombination. We identified and characterized a novel type of mutation which is a complex of external insertion, deletion, and internal duplication in sequences from one of six patients with chronic hepatitis B virus genotype E (HBV/E). We provisionally named this mutation a "replacement mutation"; the core promoter upstream regulatory sequence/basic core promoter was replaced with a part of the S1 promoter covering the hepatocyte nuclear factor 1 (HNF1) binding site, followed by a tandem repeat of the HNF1 site. A longitudinal analysis of the HBV population over 6 years showed the clonal change from wild-type HBV/E to replacement-mutant type, resulting in a lower hepatitis B (HB) e antigen titer, a high HBV DNA level in serum, and progression of liver fibrosis. In an in vitro study using a replication model, the replacement-mutant HBV showed higher replication levels than the wild-type HBV/E replicon, probably mediated by altered transcription factor binding. Additionally, this HNF1 site replacement mutation was associated with excessive HB nucleocapsid protein expression in hepatocytes, in both in vivo and in vitro studies. This novel mutation may be specific to HBV genotype E, and its prevalence requires further investigation.
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Affiliation(s)
- Kei Fujiwara
- Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho, Nagoya 467-8601, Japan
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16
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Di Marco V, Stefano RD, Ferraro D, Almasio PL, Bonura C, Giglio M, Parisi P, Cappello M, Alaimo G, Craxì A. HBV-DNA Suppression and Disease Course in HBV Cirrhosis Patients on Long-Term Lamivudine Therapy. Antivir Ther 2005. [DOI: 10.1177/135965350501000305] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
In hepatitis B virus (HBV) cirrhosis patients on long-term lamivudine (LAM), the relationships between HBV suppression, development of viral resistance and disease outcome are unclear. We analysed the dynamic of serum HBV-DNA and its relationship with the clinical course of 59 patients (52 males, mean age 51.4 ±8.4 years, 12 HBeAg positive and 47 HBeAg negative, and 57 genotype D and two genotype A) with cirrhosis (45 in Child-Turcotte-Pugh class A) and high levels of serum HBV-DNA (median 14.7x107 genomes/ml) treated with LAM [median (range): 44 (15–78) months]. A total of 50 patients (84.7%) achieved a virological response (serum HBV-DNA negative by PCR) during the first 6 months of therapy, and nine (13.3%) achieved a reduction in viral load of >3 log10. Mutations in the YMDD motif of HBV polymerase were documented in 26 patients [median (range) 18: (7–42) months]. At the time of the emergence of mutants, 22 patients had HBV-DNA <105 genomes/ml and normal alanine aminotransferase (ALT) levels. The appearance of virological resistance was followed by an increase of HBV-DNA to ≥105 genomes/ml and of ALT values in 19 out of 26 patients [median (range): 8 (3–19) months]. Event-free survival was significantly longer ( P=0.001) in patients who maintained virological suppression than in those who did not have a complete virological response or suffered a breakthrough. Patients with advanced cirrhosis were more likely to develop liver failure after the emergence of YMDD mutants. The risk of development of hepatocellular carcinoma in patients with compensated cirrhosis and YMDD mutations was maintained, regardless of HBV-DNA serum levels. Profound and maintained HBV-DNA suppression correlates with a better outcome. Early identification of LAM resistance mutations allows switching to other antivirals before liver decompensation or hepatocellular carcinoma development.
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Affiliation(s)
- Vito Di Marco
- Cattedra e Unità Operativa Complessa di Gastroenterologia ed Epatologia, University of Palermo, Palermo, Italy
| | - Rosa Di Stefano
- Dipartimento di Igiene e Microbiologia, University of Palermo, Palermo, Italy
| | - Donatella Ferraro
- Dipartimento di Igiene e Microbiologia, University of Palermo, Palermo, Italy
| | - Piero Luigi Almasio
- Cattedra e Unità Operativa Complessa di Gastroenterologia ed Epatologia, University of Palermo, Palermo, Italy
| | - Celestino Bonura
- Dipartimento di Igiene e Microbiologia, University of Palermo, Palermo, Italy
| | - Maria Giglio
- Dipartimento di Igiene e Microbiologia, University of Palermo, Palermo, Italy
| | - Pietro Parisi
- Cattedra e Unità Operativa Complessa di Gastroenterologia ed Epatologia, University of Palermo, Palermo, Italy
| | - Maria Cappello
- Cattedra e Unità Operativa Complessa di Gastroenterologia ed Epatologia, University of Palermo, Palermo, Italy
| | - Giuseppe Alaimo
- Cattedra e Unità Operativa Complessa di Gastroenterologia ed Epatologia, University of Palermo, Palermo, Italy
| | - Antonio Craxì
- Cattedra e Unità Operativa Complessa di Gastroenterologia ed Epatologia, University of Palermo, Palermo, Italy
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17
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Abstract
Hepatitis B Virus (HBV) genotypes have come of age. The concept that HBV genotypes may influence the course of disease and relevant biological differences has now been recognised. However, there are still major gaps in our knowledge. Most clinical data come from Asia and describe findings in patients infected with genotypes B and C. Large scale studies with genotypes A and D as found in Europe or A, D and E from Africa are urgently needed to broaden our understanding. Experimental data which explain in vivo findings in terms of differences in molecular biology in vitro are still in the beginning. The succeeding years will see many interesting studies which will aid our understanding of how variants and genotypes of HBV influence the spectrum of disease in people infected with HBV.
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Affiliation(s)
- S Schaefer
- Abteilung Virologie, Institut für Mikrobiologie, Universität Rostock, Rostock, Germany.
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18
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Raimondo G, Costantino L, Caccamo G, Pollicino T, Squadrito G, Cacciola I, Brancatelli S. Non-sequencing molecular approaches to identify preS2-defective hepatitis B virus variants proved to be associated with severe liver diseases. J Hepatol 2004; 40:515-519. [PMID: 15123368 DOI: 10.1016/j.jhep.2003.11.025] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/08/2003] [Revised: 11/17/2003] [Accepted: 11/20/2003] [Indexed: 12/04/2022]
Abstract
BACKGROUND/AIMS PreS2-defective hepatitis B virus (HBV) variants may emerge during chronic HBV infection. These variants carry mutation(s) at the ATG-start-codon and/or in-frame deletion into the preS2 genomic region and are commonly detected by sequencing analyses. We evaluated the prevalence of these variants in a large series of chronic HBV infected patients through non-sequencing molecular approaches. METHODS We examined HBV isolates from 110 HBV carriers: 15 were inactive carriers (IC); 50 had chronic hepatitis (CH); 25 were cirrhotics; 19 had hepatocellular carcinoma (HCC). The entire preS2 genomic region was amplified by PCR technique. The amplicons were processed: (A) through electrophoresis on acrylamide gel to reveal deleted genomes; (B) through electrophoresis on agarose gel after digestion by NlaIII enzyme that cuts the wild ATG-start-codon but not the mutated one. RESULTS We detected preS2 variants in 56/110 cases (51%). In particular, we found preS2-defective mutants in 2/15 IC, 25/50 CH, 13/26 cirrhotics, and 16/19 HCC. The presence of these variants was thus significantly associated with active infection and liver disease (P<0.002). Moreover, among cases with liver disease preS2-mutants were more prevalent in HCC patients (P<0.02). CONCLUSIONS Our non-sequencing molecular methods are sensitive and specific, and simplify the identification of all preS2 HBV variant forms. Infection by these variants is significantly associated with active infection and HCC.
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Affiliation(s)
- Giovanni Raimondo
- Unità di Epatologia Clinica e Biomolecolare, Dipartimento di Medicina Interna, Policlinico Universitario di Messina, 98124 Messina, Italy.
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19
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Huy TTT, Ushijima H, Win KM, Luengrojanakul P, Shrestha PK, Zhong ZH, Smirnov AV, Taltavull TC, Sata T, Abe K. High prevalence of hepatitis B virus pre-s mutant in countries where it is endemic and its relationship with genotype and chronicity. J Clin Microbiol 2004; 41:5449-55. [PMID: 14662924 PMCID: PMC308995 DOI: 10.1128/jcm.41.12.5449-5455.2003] [Citation(s) in RCA: 84] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
It has been reported that hepatitis B virus (HBV) mutants carrying mutations in the pre-S region can be found in infected patients. In this study, we investigated the prevalence of the HBV variant with the pre-S mutant in different geographic regions, including countries with low and high levels of endemic HBV infection, and analyzed the correlation with clinical findings. We examined 387 HBV DNA-positive serum samples from individuals among 12 countries, consisting of Vietnam, Myanmar, Thailand, China, Korea, Nepal, Japan, Russia, Spain, United States, Bolivia, and Ghana. HBV pre-S mutants were detected in 71 (18.3%) of 387 serum samples tested. This mutant was the most prevalent in Vietnam (36%), followed by Nepal (27.3%), Myanmar (23.3%), China (22.4%), Korea (14.3%), Thailand (10.5%), Japan (7.7%), and Ghana (4.3%). In contrast, no case with this mutation was found in Russia, Spain, United States, and Bolivia. Among the HBV deletion mutations, 15.5% (11 of 71) occurred in the pre-S1 and 46.5% (33 of 71) in the pre-S2 regions. Eight (11.3%) cases had a mutation in both the pre-S1 and pre-S2 regions. In addition, a point mutation at the pre-S2 starting codon was observed in 19 (26.7%) cases. The detection rate of the HBV mutant in patients with hepatocellular carcinoma was significantly higher than in other patients (P < 0.05). Furthermore, these mutants were found more frequently in genotype B (25%) and genotype C (24.5%) than in the other genotypes (P < 0.05). Our results indicated that there was a high prevalence of HBV pre-S mutation in regions of endemic HBV infection in Asia. Furthermore, the pre-S mutation appeared to be correlated with hepatocellular carcinoma and HBV genotypes.
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Affiliation(s)
- Tran Thien-Tuan Huy
- Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan
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20
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Bläckberg J, Kidd-Ljunggren K. Mutations within the hepatitis B virus genome among chronic hepatitis B patients with hepatocellular carcinoma. J Med Virol 2003; 71:18-23. [PMID: 12858404 DOI: 10.1002/jmv.10458] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Chronic hepatitis B infection is a major cause of hepatocellular cancer (HCC). The pathogenesis of the carcinogenesis is not fully understood. Viral proteins such as the X protein and the truncated middle S protein have been implicated to be transactivators. In order to investigate whether any mutations within relevant parts of the hepatitis B virus (HBV) genome could be associated with the development of HCC, the genomes of 16 HBV strains from chronic HBV carriers with HCC were studied. Serum samples were subjected to PCR and the HBV DNA sequenced subsequently. Genotypes A-D were represented. The sequence analysis showed that an especially high proportion, 50% (CI 95%, 25-75%), of the patients with HCC carried HBV mutants with deletions or insertions in the N-terminal half of the pre-S2 region or had a point mutation in the start codon of pre-S2 compared with controls with chronic HBV infection, 21% (CI 95%, 3-39%). A high proportion (69%) also had mutations at position 1762 (A --> T) and/or 1764 (G --> A) in the core promoter region, but the proportion of core promoter mutations was no different from what was found in a control group of HBV carriers without HCC (68%). The pre-S2 variants, which involve deletions of immunogenic regions, may have a survival advantage as they are mostly found in long-standing HBV infection. There were no other mutations found frequently within the region coding for the X protein.
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Affiliation(s)
- Jonas Bläckberg
- Department of Infectious Diseases, Lund University, Lund, Sweden.
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21
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Sugauchi F, Ohno T, Orito E, Sakugawa H, Ichida T, Komatsu M, Kuramitsu T, Ueda R, Miyakawa Y, Mizokami M. Influence of hepatitis B virus genotypes on the development of preS deletions and advanced liver disease. J Med Virol 2003; 70:537-44. [PMID: 12794715 DOI: 10.1002/jmv.10428] [Citation(s) in RCA: 94] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Hepatitis B virus (HBV) mutants with deletions in the preS region have not been evaluated for association with viral genotypes. In a case-control study, HBV DNA samples collected from 80 each of carriers infected with HBV genotype B or C were examined for preS deletions. PreS deletion mutants were found in a total of 37 of 160 (23%) HBV carriers. Carriers with preS deletion mutants were older (56.0 +/- 12.7 vs 49.3 +/- 16.9 years, P < 0.05), were infected more frequently with HBV genotype C (84% vs 40%, P < 0.05), and had more advanced disease, such as liver cirrhosis and hepatocellular carcinoma (54% vs 31%; P < 0.05), than did those without such mutants. In a multivariate analysis, genotype C (odds ratio [OR] = 9.3, P < 0.001) and advanced liver disease (OR = 3.1, P < 0.01) were the most significant variables in association with preS deletions. A direct repeat sequence (TCAGG) was found at the start or at the end of preS1 deletions in 6 of the 20 (30%) cases examined, and preS2 deletions in these cases were clustered over the 5'-terminal half of this region. These results indicate that the development of preS deletion mutants depends on HBV genotypes and that it may be associated with progressive liver disease.
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Affiliation(s)
- Fuminaka Sugauchi
- Department of Internal Medicine and Molecular Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
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22
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Kaplan DE, Reddy KR. Rising incidence of hepatocellular carcinoma: the role of hepatitis B and C; the impact on transplantation and outcomes. Clin Liver Dis 2003; 7:683-714. [PMID: 14509534 DOI: 10.1016/s1089-3261(03)00060-6] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Hepatocellular carcinoma caused by hepatitis B and hepatitis C are global scourges but are likely to peak in incidence in the next 2 decades and then decline. Universal vaccination has been effective in stemming the incidence of chronic hepatitis B and early-onset HCC in regions of high endemicity where implemented, but preventive measures in HCV are not yet available. After the attrition of older affected generations, the incidence of HCC will likely decline rapidly. While no vaccine is currently available for hepatitis C, cases are projected to peak and decline because of a marked reduction in transmission as a result of behavioral modification and safeguarding of blood supplies. Until these epidemiologic projections come to pass, management of hepatocellular carcinoma will continue to become a progressively more frequently encountered clinical challenge. Therapy for chronic hepatitis may ameliorate but will not eliminate the development of tumors. The demand for orthotopic liver transplantation will continue to climb, and palliative therapies for non-resectable cases will require studies aimed at optimization of benefit. LDLT may remain an option for high-risk patients affording tumor-free survival for some otherwise terminal patients.
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Affiliation(s)
- David E Kaplan
- Division of Gastroenterology and Hepatology, University of Pennsylvania School of Medicine, 3 Raydin, 3400 Spruce Street, Philadelphia, PA 19104, USA
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23
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Suk FM, Lin MH, Newman M, Pan S, Chen SH, Liu JD, Shih C. Replication advantage and host factor-independent phenotypes attributable to a common naturally occurring capsid mutation (I97L) in human hepatitis B virus. J Virol 2002; 76:12069-77. [PMID: 12414948 PMCID: PMC136898 DOI: 10.1128/jvi.76.23.12069-12077.2002] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2002] [Accepted: 08/26/2002] [Indexed: 12/12/2022] Open
Abstract
Mutations of human hepatitis B virus (HBV) occur frequently within the capsid (core) protein in natural infections. The most frequent mutation of the core protein in HBV from Southeast Asia occurs at amino acid 97, changing an isoleucine (I) to a leucine (L). In our systematic study of virus-host interactions, we have examined the replication efficiency of a site-directed mutant, I97L, and its parental wild-type HBV in several different hepatoma cell lines. Interestingly, we found that this capsid variant replicated in human Huh7 hepatoma cells approximately 4.8-fold better than its parental wild-type HBV. A similar phenomenon was observed in another hepatoma cell line, J3. In addition, the level of encapsidated RNA pregenome in mutant I97L was about 5.7-fold higher than that of the wild-type HBV in Huh7 cells. Unlike Huh7 cells, no significant difference in viral DNA replication between the same I97L mutant and its parental wild-type HBV was observed in HepG2, a human hepatoblastoma cell line. This finding of a profound replication advantage for mutant I97L in Huh7 and J3 cells but not in HepG2 cells may have important implications for the emergence of this mutant in chronic HBV carriers. We speculate here that the mutation confers a host factor-independent growth advantage for the survival of HBV variants in gradually dedifferentiating hepatocytes and thus helps prolong viral persistence.
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Affiliation(s)
- Fat-Moon Suk
- Center for Tropical Diseases and Sealy Center for Vaccine Development, Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0609, USA
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24
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Kajiya Y, Hamasaki K, Nakata K, Nakagawa Y, Miyazoe S, Takeda Y, Ohkubo K, Ichikawa T, Nakao K, Kato Y, Eguchi K. Full-length sequence and functional analysis of hepatitis B virus genome in a virus carrier: a case report suggesting the impact of pre-S and core promoter mutations on the progression of the disease. J Viral Hepat 2002; 9:149-56. [PMID: 11876799 DOI: 10.1046/j.1365-2893.2002.00335.x] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
In chronic hepatitis B virus (HBV) infection, the quiescent immunotolerant phase evolves into the immunoactive phase. The aim of the present study was to clarify the virological alterations relevant to progression. Serial serum samples obtained from a patient with HBV during long-term follow-up were analysed by sequencing of the full-length HBV-DNA using polymerase chain reaction (PCR). In addition, PCR products of HBV genome from each serum sample were transfected into HuH-7 human hepatoma cells for the functional analysis of the transfected viral genomes. Based on the HBV-DNA sequence analysis, the patient had the genotype C virus, and the mutant HBV with common core promoter mutations (T(1762)A(1764)) and deletion of the pre-S region responsible for large surface protein transcription emerged before the onset of hepatitis. When the vigorous host immune response developed (indicated by the flare-up of hepatitis), the mutant HBV containing common core promoter mutations and another pre-S deletion causing lack of the surface protein promoter became predominant. The HBV-DNA sequences, other than pre-S and core promoter regions were identical to the wild-type sequence throughout the study. Transfection of PCR products containing the mutant HBV sequences resulted in increased amounts of intracellular replicative intermediates but the decreased secretion of HBsAg and HBeAg into culture media, suggesting accumulation of nonenveloped viral core particles within the cells. These results indicate that pre-S deletion and core promoter mutations may participate cooperatively in progression of the disease.
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Affiliation(s)
- Y Kajiya
- The First Department of Internal Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan
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25
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Cacciola I, Cerenzia G, Pollicino T, Squadrito G, Castellaneta S, Zanetti AR, Mieli-Vergani G, Raimondo G. Genomic heterogeneity of hepatitis B virus (HBV) and outcome of perinatal HBV infection. J Hepatol 2002; 36:426-432. [PMID: 11867188 DOI: 10.1016/s0168-8278(01)00295-1] [Citation(s) in RCA: 39] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
BACKGROUND/AIMS Data regarding hepatitis B virus (HBV) genomic heterogeneity in perinatal infection are incomplete, although HBV variants might be involved in neonatal fulminant hepatitis (ALF). We investigated HBV variability in infected babies showing different clinical courses. METHODS We analyzed HBV genomes isolated from nine vertically infected babies and the mothers of four of them. Two infants born to HBe-antigen (HBeAg)-positive women developed a chronic infection; seven babies (six born to anti-HBe mothers) developed acute hepatitis that had a fulminant course in four cases and a benign course in three. Two babies developing ALF received anti-HBV immunoprophylaxis at birth. RESULTS Viruses carrying no significant mutation infected infants born to HBeAg-positive women. HBeAg-defective viruses were detected both in children with benign and fulminant hepatitis and their mothers. A double nucleotide mutation at positions 1762 and 1764 of the HBV core-promoter was found in two of the four infants with ALF, although it was not detected in isolates from the mother of one of them. No significant S gene mutation was found in HBV from any of the babies. CONCLUSIONS This study indicates that HBV genomic heterogeneity is not primarily involved either in the evolution of the infection or the failure of neonatal HBV immunoprophylaxis.
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Affiliation(s)
- Irene Cacciola
- Department of Internal Medicine, University of Messina, Messina, Italy
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Tai PC, Suk FM, Gerlich WH, Neurath AR, Shih C. Hypermodification and immune escape of an internally deleted middle-envelope (M) protein of frequent and predominant hepatitis B virus variants. Virology 2002; 292:44-58. [PMID: 11878907 DOI: 10.1006/viro.2001.1239] [Citation(s) in RCA: 82] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Naturally occurring deletions within the human hepatitis B virus (HBV) preS2 region have frequently been identified in patients with hepatocellular carcinoma (HCC), while chronic carriers without cirrhosis and HCC contain no detectable preS2 deletion variants. We have characterized two different preS2 internal deletion variants from two patients. In addition to several weak phenotypes, our study revealed three unexpected strong phenotypes: (1) a paradoxical "hypermodification" phenomenon was observed with significantly increased size heterogeneity and molecular weights of the secreted middle (M) envelope proteins containing a preS2 internal deletion. This phenomenon was observed in transient transfection with a human hepatoma Huh7 cell line as well as in stable transfection with a rodent hepatoma cell line 7777. (2) A significantly increased intracellular accumulation of all three envelope proteins (large, middle, and small) was detected by both Western blot analysis and immunofluorescence microscopy. (3) The middle envelope proteins with a preS2 internal deletion were not recognized in vitro by a putative neutralizing antiserum, suggesting that these variants can evade immune recognition in vivo. To our knowledge, this is the first identification and characterization of the M deletion variant protein in HBV natural infection.
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Affiliation(s)
- Pei-Ching Tai
- Center for Tropical Diseases, University of Texas Medical Branch, Galveston, Texas, 77555-0609, USA
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27
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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
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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Castro LD, Niel C, Gomes SA. Low frequency of mutations in the core promoter and precore regions of hepatitis B virus in anti-HBe positive Brazilian carriers. BMC Microbiol 2001; 1:10. [PMID: 11472634 PMCID: PMC35280 DOI: 10.1186/1471-2180-1-10] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2001] [Accepted: 07/06/2001] [Indexed: 01/21/2023] Open
Abstract
BACKGROUND Mutations in the core promoter and precore regions of the hepatitis B virus (HBV) genome, notably the double substitution (AGG to TGA) at nt positions 1762-1764 in the core promoter, and the precore stop codon mutation G to A at nt 1896, can often explain the anti-HBe phenotype in chronic carriers. However, the A1896 mutation is restricted to HBV isolates that have T at nt 1858. The double substitution at positions 1762-1764 has been described to occur preferentially in patients infected with strains showing C instead of T at nt 1858. RESULTS HBV DNAs from 29 anti-HBe Brazilian samples were characterized by nucleotide sequencing of PCR products from precore region. Among them, 18 isolates presented C at nt 1858 (mostly genotype A strains). The 11 remaining isolates (genotypes D and F) had T1858. The stop codon mutation at nt 1896 was found in seven isolates (24% of the total and 63% of the isolates that had T1858). The frequency of the double substitution at positions 1762-1764 was surprisingly low (20%) among C1858 isolates. An association between A1896 and TGA 1762-1764 mutations was observed among genotype D isolates: these showed either none of the two mutations or both. Furthermore, strains mutated at positions 1896 and/or 1762-1764 also presented an elevated number of other, less common substitutions in the core promoter and precore regions. CONCLUSIONS The data reported here are not in accordance with some reports from other parts of the world. In half of the isolates, none of the mutations previously described could explain the anti-HBe phenotype.
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Affiliation(s)
- Liane De Castro
- Department of Virology, Oswaldo Cruz Foundation, Av. Brasil 4365, Rio de Janeiro, BR-21045-900, Brazil
| | - Christian Niel
- Department of Virology, Oswaldo Cruz Foundation, Av. Brasil 4365, Rio de Janeiro, BR-21045-900, Brazil
| | - Selma A Gomes
- Department of Virology, Oswaldo Cruz Foundation, Av. Brasil 4365, Rio de Janeiro, BR-21045-900, Brazil
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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)
- 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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Fan YF, Lu CC, Chang YC, Chang TT, Lin PW, Lei HY, Su IJ. Identification of a pre-S2 mutant in hepatocytes expressing a novel marginal pattern of surface antigen in advanced diseases of chronic hepatitis B virus infection. J Gastroenterol Hepatol 2000; 15:519-28. [PMID: 10847439 DOI: 10.1046/j.1440-1746.2000.02187.x] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
BACKGROUND AND AIMS The expression of hepatitis B viral (HBV) antigens in liver tissue reflects the replicative status of chronic HBV infection. We have previously recognized a novel marginal pattern of hepatitis B surface antigen (HBsAg) in hepatocytes, which usually clusters in groups and emerges at the late non-replicative phase. This study was designed to investigate whether the marginal-type HBsAg represented the gene product of a specific HBV-surface mutant. METHODS Microdissection of cirrhotic nodules homogeneously expressing marginal HBsAg was performed on two of 12 resected livers from HBsAg-seropositive patients with hepatocellular carcinoma. The gene presumably encoding marginal HBsAg was polymerase chain reaction (PCR)-cloned, sequenced and analysed. In vitro transfection and expression of the cloned surface mutant plasmids were performed on the Huh7 cell line to illustrate intrahepatic HBsAg expression. RESULTS Immunohistochemical staining revealed that the marginal HBsAg was positive for pre-S1 and thus contained large surface proteins. The PCR cloning and sequencing of the genes presumably encoding marginal-type HBsAg in both cases revealed the same deletion at the 5' terminus (nt 2-55) of pre-S2. A point mutation on the small-surface (S) antigen was also found in one case. The pre-S2 deletion sequence and the mutation sites of the S gene coincide with human lymphocyte antigen-restricted T- and/or B-cell epitopes. In vitro transfection of the mutant plasmid revealed a blot-like retention or accumulation of HBsAg in the cytoplasm or at the periphery of hepatocytes, accompanied by a decreased secretion of HBsAg in the culture supernatant, mimicking intrahepatic expression. CONCLUSION A natural pre-S2 deletion mutant was identified in hepatocytes expressing a novel marginal pattern of HBsAg, which probably contains mutant, large, surface proteins. The biological significance of the pre-S2 deletion mutant should be interesting in view of the clustering proliferation of hepatocytes expressing marginal HBsAg.
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Affiliation(s)
- Y F Fan
- Institute of Basic Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan
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32
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Maeng CY, Ryu CJ, Gripon P, Guguen-Guillouzo C, Hong HJ. Fine mapping of virus-neutralizing epitopes on hepatitis B virus PreS1. Virology 2000; 270:9-16. [PMID: 10772975 DOI: 10.1006/viro.2000.0250] [Citation(s) in RCA: 65] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
We identified the epitopes on the preS1 which induce antibodies that neutralize both ad and ay subtypes of hepatitis B virus (HBV). Previously we generated murine monoclonal antibodies KR359 and KR127 that bind specifically to the preS1 of HBV. In this study we have performed fine mappings of the epitopes of the antibodies by examining their reactivity with GST fusion proteins, which contain a series of deletion mutants of the preS1. KR359 and KR127 specifically recognize aa 19-26 and 37-45 of the preS1, respectively. The antibodies neutralized both adr and ayw subtypes of the virus in an in vitro neutralization assay using in vitro infection of adult human hepatocyte primary culture by HBV. The epitopes showed little sequence divergence and the antibodies bound to the preS1 of all the HBV subtypes and variants tested.
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Affiliation(s)
- C Y Maeng
- Antibody Engineering Research Unit, Korea Research Institute of Bioscience and Biotechnology, Taejon, Yuseong, 305-600, Korea
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33
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Cacciola I, Pollicino T, Squadrito G, Cerenzia G, Villari D, de Franchis R, Santantonio T, Brancatelli S, Colucci G, Raimondo G. Quantification of intrahepatic hepatitis B virus (HBV) DNA in patients with chronic HBV infection. Hepatology 2000; 31:507-512. [PMID: 10655278 DOI: 10.1002/hep.510310235] [Citation(s) in RCA: 63] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
No data are available about the amount of hepatitis B virus (HBV) genomes in liver of patients with chronic HBV infection. The aim of this study was to quantify the intrahepatic HBV DNA in hepatitis B surface antigen (HBsAg)-positive patients with either active or suppressed viral replication and in HBsAg-negative subjects with occult HBV infection. We optimized the Roche "Amplicor HBV Monitor" kit for quantifying liver HBV DNA and analyzed hepatic DNA extracts and serum samples from 19 HBs-Ag-positive and 43 HBsAg-negative individuals. Eight of the HBsAg carriers had active HBV replication, and for 3 of them we analyzed samples obtained before and at the end of 1 year of lamivudine treatment. Five hepatitis Delta virus (HDV) coinfected patients and 6 healthy HBsAg carriers had inhibited HBV activity. Among the HBsAg-negative subjects 21 had occult HBV infection and 22 had no evidence of HBV infection. The median of HBV genomes per microgram of liver DNA milliliter of serum was 34,500 to 2,620,000 in patients with active viral replication, 20,000 to 3,900, 000 before and 10,000 to 2,800 at the end of therapy in lamivudine-treated individuals, 9,800 to 600 in HDV-infected individuals, and 7,450 to 17,400 in healthy HBsAg carriers. These data indicate that cases with suppressed HBV activity, despite the very low levels of viremia, maintain a relatively high amount of intrahepatic viral genomes. This virus reservoir is likely involved in HBV reactivation, which is usually observed after stopping lamivudine treatment. Finally, the analysis of cases with occult HBV infection showed that the assay we used was able to specifically detect and quantify as few as 100 copies of viral genomes per microgram of liver DNA.
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Affiliation(s)
- I Cacciola
- Dipartimento di Medicina Interna, Università di Messina, Messina, Italy
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Yuan TT, Tai PC, Shih C. Subtype-independent immature secretion and subtype-dependent replication deficiency of a highly frequent, naturally occurring mutation of human hepatitis B virus core antigen. J Virol 1999; 73:10122-8. [PMID: 10559327 PMCID: PMC113064 DOI: 10.1128/jvi.73.12.10122-10128.1999] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
The most frequent mutation of the human hepatitis B virus (HBV) core antigen occurs at amino acid 97. Recently, a phenylalanine (F)-to-leucine (L) mutation at this position (mutant F97L) in HBV surface antigen subtype ayw has been shown to result in an immature secretion phenotype, which is characterized by the nonselective export of an excessive amount of virions containing minus-strand, single-stranded HBV DNA. While subtype ayw mutant F97L has been found in Europe, the major reservoir of HBV resides in Asia and Africa. We report here that the immature secretion phenotype indeed can be found in an HBV strain (subtype adr) prevalent in Asia, changing from an isoleucine (I) to a leucine (mutant I97L). Despite its immature secretion phenotype, the adr variant I97L replicates as well as its parental adr wild-type I97I, supporting the conclusion that the extracellular phenotype of immature secretion is not a consequence of the intracellular HBV DNA replication defect. Further studies demonstrated that it is the acquisition of a leucine, rather than the loss of a wild-type amino acid at codon 97, that is important for immature secretion. We conclude that immature secretion is a subtype-independent phenotype and deficiency in intracellular DNA synthesis is a subtype-dependent phenotype. The former is caused by the trans-acting effect of a mutant core protein, while the latter by a cis-acting effect of a mutated nucleotide on the ayw genome. These immature secretion variants provide an important tool for studying the regulation of HBV virion assembly and secretion.
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Affiliation(s)
- T T Yuan
- Center for Tropical Diseases, Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0609, USA
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35
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Tang RX, Gao FG, Zeng LY, Wang YW, Wang YL. Detection of HBV DNA and its existence status in liver tissues and peripheral blood lymphocytes from chronic hepatitis B patients. World J Gastroenterol 1999; 5:359-361. [PMID: 11819468 PMCID: PMC4695555 DOI: 10.3748/wjg.v5.i4.359] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
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36
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Yuan TT, Sahu GK, Whitehead WE, Greenberg R, Shih C. The mechanism of an immature secretion phenotype of a highly frequent naturally occurring missense mutation at codon 97 of human hepatitis B virus core antigen. J Virol 1999; 73:5731-40. [PMID: 10364324 PMCID: PMC112633 DOI: 10.1128/jvi.73.7.5731-5740.1999] [Citation(s) in RCA: 97] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/1998] [Accepted: 03/29/1999] [Indexed: 01/12/2023] Open
Abstract
A very frequent missense mutation at codon 97 of human hepatitis B virus (HBV) core antigen (HBcAg) has been found in chronic carriers worldwide. Functional characterization of this mutant revealed one intracellular and two extracellular phenotypes in contrast to wild-type HBV: (i) a 6- to 12-fold decrease in the level of the full-length relaxed circular DNA, a 4- to 5-fold decrease in the plus-strand DNA, and an approximately 1.8-fold decrease in the minus-strand and overall DNA levels in the intracellular viral core particles; (ii) a 5.7-fold increase in the immature secretion of Dane particles, containing minus-strand, single-stranded virion DNA; and (iii) a significant reduction of nonenveloped core particles in the medium. The steady-state levels of mutant and wild-type core proteins expressed from the same vector appeared to be similar. Using a complementation assay and gradient centrifugation analysis, we demonstrated that this mutant core protein alone is necessary and sufficient for immature secretion. The decreased level of intracellular HBV DNA is caused by both the cis defect of the mutant genome and the trans defect of the mutant core protein. We have dissected further the relationship between the intracellular and extracellular phenotypes of mutant F97L. The pleiotropic effects of the HBcAg codon 97 mutation were observed consistently in several different experimental settings. The mechanism and biological significance of these findings are discussed.
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Affiliation(s)
- T T Yuan
- Departments of Pathology and of Microbiology and Immunology, Center for Tropical Diseases, University of Texas Medical Branch, Galveston, Texas 77555-0609, USA
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37
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Cacciola I, Pollicino T, Squadrito G, Cerenzia G, Orlando ME, Raimondo G. Occult hepatitis B virus infection in patients with chronic hepatitis C liver disease. N Engl J Med 1999; 341:22-26. [PMID: 10387938 DOI: 10.1056/nejm199907013410104] [Citation(s) in RCA: 474] [Impact Index Per Article: 18.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
BACKGROUND Hepatitis B virus (HBV) infections in patients who lack detectable hepatitis B surface antigen (HBsAg) are called occult infections. Although such infections have been identified in patients with chronic hepatitis C liver disease, their prevalence and clinical significance are not known. METHODS With the polymerase chain reaction, we searched for HBV DNA in liver and serum samples from 200 HBsAg-negative patients with hepatitis C virus (HCV)-related liver disease (147 with chronic hepatitis, 48 with cirrhosis, and 5 with minimal histologic changes). One hundred of the patients had detectable antibodies to the HBV core antigen (anti-HBc); 100 were negative for all HBV markers. Eighty-three were treated with interferon alfa. We also studied 50 patients with liver disease who were negative both for HBsAg and for HCV markers. In six patients found to have occult HBV infection, we evaluated possible genomic rearrangements through cloning or direct sequencing procedures. RESULTS Sixty-six of the 200 patients with chronic hepatitis C liver disease (33 percent) had HBV sequences, as did 7 of the 50 patients with liver disease unrelated to hepatitis C (14 percent, P=0.01). Among the 66 patients, 46 were anti-HBc-positive and 20 were negative for all HBV markers (P<0.001). Twenty-two of these 66 patients (33 percent) had cirrhosis, as compared with 26 of the 134 patients with hepatitis C infection but no HBV sequences (19 percent, P=0.04). HBV sequences were detected in 26 of the 55 patients in whom interferon therapy was ineffective and 7 of the 28 patients in whom interferon therapy was effective (P=0.06). None of the sequenced HBV genomes had changes known to interfere with viral activity and gene expression. CONCLUSIONS Occult hepatitis B infection occurs frequently in patients with chronic hepatitis C liver disease and may have clinical significance.
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Affiliation(s)
- I Cacciola
- Dipartimento di Medicina Interna, Università di Messina, Italy
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38
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Günther S, Fischer L, Pult I, Sterneck M, Will H. Naturally occurring variants of hepatitis B virus. Adv Virus Res 1999; 52:25-137. [PMID: 10384235 DOI: 10.1016/s0065-3527(08)60298-5] [Citation(s) in RCA: 174] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Affiliation(s)
- S Günther
- Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie, Universität Hamburg, Federal Republic of Germany.
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Kramvis A, Kew MC, Bukofzer S. Hepatitis B virus precore mutants in serum and liver of Southern African Blacks with hepatocellular carcinoma. J Hepatol 1998; 28:132-41. [PMID: 9537850 DOI: 10.1016/s0168-8278(98)80212-2] [Citation(s) in RCA: 64] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
BACKGROUND/AIM The aim of this study was to sequence the precore region of HBV isolated from serum and tumorous and non-tumorous liver tissue from patients with hepatocellular carcinoma to identify mutations that might play a role in malignant transformation. METHODS HBV DNA was extracted from 62 sera, 14 tumorous and 12 non-tumorous liver tissue samples of patients with hepatocellular carcinoma, amplified by the polymerase chain reaction and sequenced directly. RESULTS Thirty-nine patients were HBeAg-negative and 23 HBeAg-positive. Missense mutations were present predominantly in HBeAg-negative sera. The most common missense mutation, a guanine to thymine transversion, occurred at nucleotide 1862 in the bulge of the encapsidation signal; it was more prevalent in HBeAg-negative (10/39) than in HBeAg-positive patients (1/23) (p = 0.03). Mutations known to prevent HBeAg synthesis were detected in seven sera; five with an 1896 stop-codon mutation, one with an 1817 nonsense mutation, and one with a frameshift mutation caused by an insertion between 1838 and 1839. Missense mutations and deletions were present more often in tumorous tissue derived from HBsAg-negative patients. In the tumours missense mutations occurred at position 1862 and 1899, and the deletions affected direct repeat 1 and/or the encapsidation signal and included the x gene stop-codon. CONCLUSIONS The 1862 mutation, and other missense mutations and deletions detected in the precore gene, may disrupt HBV DNA replication and/or signal peptide cleavage leading to HBeAg-negativity. Disruption of viral replication may promote integration of unencapsidated replicative intermediates and hence contribute to hepatocarcinogenesis.
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Affiliation(s)
- A Kramvis
- Medical Research Council, Department of Medicine, University of the Witwatersrand, Johannesburg, South Africa
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40
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Karasawa T, Shirasawa T, Okawa Y, Kuramoto A, Shimada N, Aizawa Y, Zeniya M, Toda G. Association between frequency of amino acid changes in core region of hepatitis B virus (HBV) and the presence of precore mutation in Japanese HBV carriers. J Gastroenterol 1997; 32:611-622. [PMID: 9349986 DOI: 10.1007/bf02934110] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
To elucidate the relationship between the frequency of core mutations and precore mutation of hepatitis B virus (HBV) in Japanese HBV carriers, we investigated the nucleotide sequence of the precore/core region of HBV in 26 Japanese HBV carriers [15 who were HBe antigen-negative (HBeAg-) and 11 who were HBeAg-positive (HBeAg+)]. The number of amino acid changes (5.9 +/- 3.8) in the core region of HBV in HBeAg-carriers was significantly greater than that in the HBeAg+ carriers (1.5 +/- 1.0; P < 0.005). The precore stop codon mutation was found in 93.3% of HBeAg-negative HBV carriers, while no precore mutation was found in the HBeAg-positive HBV carriers, suggesting that the frequency of core mutations may be associated with the presence of the precore stop codon mutation. However, there was no significant difference in the frequency of amino acid changes among HBeAg-HBV carriers. The mean number of core amino acid changes of liver cirrhosis patients, chronic active hepatitis patients, chronic persistent hepatitis patients, and asymptomatic carriers were 2.7 +/- 1.5, 6.0 +/- 2.2, 4.7 +/- 1.2, and 8.4 +/- 5.3, respectively. We detected hot spots for core mutations, which showed characteristic localizations and specific substitutions: Gly-87, Leu-97, and Thr-130 were detected exclusively in patients with chronic liver disease with or without HBeAg. To address further the relationship between frequency of core mutations and the presence of the precore stop codon mutation, we investigated the precore/core nucleotide sequence serially along with seroconversion in three patients with chronic hepatitis B in whom the hepatitis either became inactive or remained active after the seroconversion. Emergence of the precore stop codon mutation and a significant increase in core amino-acid changes after seroconversion were noted in all three patients. Our results suggest a close association between the frequency of core amino acid changes and the presence of the precore stop codon mutation; some characteristic core mutations may be associated with the clinical course of chronic hepatitis B in Japanese patients.
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Affiliation(s)
- T Karasawa
- First Department of Internal Medicine, Jikei University School of Medicine, Tokyo, Japan
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41
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Schepis F, Verucchi G, Pollicino T, Attard L, Brancatelli S, Longo G, Raimondo G. Outcome of liver disease and response to interferon treatment are not influenced by hepatitis B virus core gene variability in children with chronic type B hepatitis. J Hepatol 1997; 26:765-70. [PMID: 9126787 DOI: 10.1016/s0168-8278(97)80240-1] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
BACKGROUND/AIMS Hepatitis B virus (HBV) core gene heterogeneity may influence the outcome of liver disease and the response to interferon (IFN) therapy in adult HBV carriers. The aim of this study was to evaluate the possible association between HBV core gene variability and evolution of chronic hepatitis in children. METHODS We examined serum samples from 25 children with HBV chronic hepatitis and HBe antigen (HBeAg) positivity who were followed-up for a mean of 7.4 years. Seven cases spontaneously seroconverted to anti-HBe, becoming HBV healthy carriers; nine cases were successfully treated with IFN; nine cases were non-responders to IFN therapy. HBV-DNA was extracted from one serum sample ("I") collected during the HBeAg positive phase, and from a second sample ("II") collected after the anti-HBe seroconversion or, in non-responders, after stopping therapy. The entire core gene of the HBV isolates was amplified and sequenced. RESULTS Each isolate showed single or no missense mutation independently of the clinical behavior of the patients. HBeAg-defective viruses were detected in one case in both samples and in two cases only in sample "II". CONCLUSIONS Core gene variability does not seem to be involved either in the outcome of infection or in the response to IFN treatment in children with HBV chronic hepatitis. Considering that most of the HBV carriers in our area acquire the infection in childhood, our data suggest that core gene heterogeneity is not a major cause of progression to chronicity.
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Affiliation(s)
- F Schepis
- Dipartimento di Medicina Interna, Universita di Messina, Italy
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42
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Bozkaya H, Akarca US, Ayola B, Lok AS. High degree of conservation in the hepatitis B virus core gene during the immune tolerant phase in perinatally acquired chronic hepatitis B virus infection. J Hepatol 1997; 26:508-16. [PMID: 9075657 DOI: 10.1016/s0168-8278(97)80415-1] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
BACKGROUND Mutations in the hepatitis B virus genome have been implicated in the persistence of hepatitis B virus infection and the pathogenesis of hepatitis B virus related liver disease. In view of the heterogeneity in published sequences, data from cross-sectional studies of unrelated subjects cannot differentiate true mutations from infections with variant sequences. AIMS/METHODS We compared the hepatitis B virus core gene sequences of 42 HBsAg positive subjects from 11 Chinese families with those of the index patients (maternal carriers) to determine the frequency and rate of true hepatitis B virus core gene mutations in patients with chronic hepatitis B virus infection. RESULTS Completely identical nucleotide sequences were present in all the family members and index patients in two families, suggesting that the hepatitis B virus core gene can be conserved for more than 20 years. The high degree of sequence conservation in these families is related to the young age of the subjects (mean 19.2+/-8.9 years), the fact that they were all HBeAg positive and that 75% of them had persistently normal aminotransferase levels. Longitudinal studies confirmed that mutations were rare in those who remained HBeAg positive with normal aminotransferase levels (immune tolerant phase), but significantly more common in HBeAg positive subjects who had elevated aminotransferase levels and in those who cleared HBeAg (immune clearance phase), the rates of nucleotide and amino acid changes were respectively: 0.28+/-0.12 vs 1.30+/-0.26/10(3) nt position/yr and 0.04+/-0.01 vs 0.18+/-0.5/10(2) codon/yr. CONCLUSIONS Identical nucleotide differences could be found in the sequences of all the subjects in some families. These differences were more likely to be due to intra-familial transmission of stable variants. Sequence analysis based on comparisons with published sequences would have led to over-reporting of mutations. The hepatitis B virus core gene can remain highly conserved for more than two decades during the immune tolerant phase of perinatally acquired chronic hepatitis B virus infection. However, significant changes can occur within 2-3 years during the immune clearance phase.
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Affiliation(s)
- H Bozkaya
- Section of Gastroenterology and Hepatology, Tulane University, New Orleans, LA, USA
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Okumura A, Takayanagi M, Aiyama T, Iwata K, Wakita T, Ishikawa T, Yoshioka K, Kakumu S. Serial analysis of hepatitis B virus core nucleotide sequence of patients with acute exacerbation during chronic infection. J Med Virol 1996; 49:103-109. [PMID: 8991932 DOI: 10.1002/(sici)1096-9071(199606)49:2<103::aid-jmv6>3.0.co;2-9] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
Recent studies suggest that hepatitis B virus (HBV) core region could be an immunological target and that amino acid (aa) substitutions are mostly restricted to a small segment located in the middle of the core region. We sequenced the middle portion of HBV core gene during the course of acute exacerbation of chronic hepatitis B, and compared aa variations between the region including ideal HLA-A2 binding motifs and the nonbinding region. Five HBeAg+ chronic hepatitis patients with subtype adr (three with HLA-A2 and two without HLA-A2) were selected and using polymerase chain reaction (PCR) and cloning system, the central part of core region (nt 2063 to 2365, 303 bp) was sequenced in sera from each patient at three time points; before, at the peak of, and after exacerbation of hepatitis. The second set of sera showed higher aa substitution rates in five and in three out of five patients compared with those of the first and third sera, respectively. No significant difference was found in the aa substitution rates for the region with ideal HLA-A2 binding motifs between patients with and without HLA-A2. In asymptomatic HBV carriers with persistently normal aminotransferase values, alterations of the aa sequence were not observed within the same time frame. The results suggest that aa substitutions often occur at some particular positions in the middle of HBV core region during acute exacerbation of the disease under possible host immune pressures. Furthermore, unidentified epitopes appear to exist in the central part of HBV core region and HLA-unrestricted lymphocytes may play a role in the immune response of chronic HBV carriers.
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Affiliation(s)
- A Okumura
- Third Department of Internal Medicine, Nagoya University School of Medicine, Japan
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