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Wang T, Qin Y, Zhang J, Li X, Tong S, Zhao W, Zhang J. An antiviral drug-resistant mutant of hepatitis B virus with high replication capacity in association with a large in-frame deletion in the preS1 region of viral surface gene. Virus Genes 2020; 56:677-686. [PMID: 32840739 DOI: 10.1007/s11262-020-01787-9] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2019] [Accepted: 08/08/2020] [Indexed: 12/14/2022]
Abstract
We amplified a full-length hepatitis B virus (HBV) genome from the serum of a chronic hepatitis B patient who experienced virological breakthrough with high HBV DNA titer following adefovir (ADV) therapy. The PCR product was cloned and sequencing of the six clones revealed an isolate of C2 subgenotype. Mutation(s) in the polymerase gene responsible for ADV resistance included rtA181T (all clones) and rtN236T (four clones). The rtA181T mutation caused the W172* nonsense mutation in the overlapping S gene. In addition, all the clones harbored another nonsense mutation in the S gene (C69*) and a 207nt in-frame deletion in the preS1 region. These clones were converted to a 1.1mer construct for transient transfection of Huh7 cells. All the clones were deficient in hepatitis B surface antigen production. Three clones had similar levels of DNA replication. Comparison with a wild-type clone of the same genotype revealed a higher intracellular level of replicative DNA for clone c4, which was reduced by putting back the deleted 207nt, but not by co-transfection with an expression construct for the three surface proteins to rescue virion production. The HBcAg expression of the c4 and c4+207nt clones was mainly in the nucleus. Co-transfection with the L/M/S proteins expression construct did not alter the distribution of core. Clone c4 showed a significantly decreased susceptibility to ADV, a mild reduction in susceptibility to lamivudine and tenofovir, but remained sensitive to entecavir. In conclusion, this is an unusual ADV-resistant HBV isolate harboring two nonsense mutations in the S gene and a large in-frame deletion in the preS1 region, but still retains a high replication phenotype, which can provide a platform for recombinant vector construction.
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Affiliation(s)
- Ting Wang
- Department of Infectious Diseases, Jing'An District Centre Hospital of Shanghai (Huashan Hospital, Fudan University Jing'An Branch), Shanghai, China
- Department of Infectious Diseases, The First Affiliated Hospital of Soochow University, 188 Shizi Street, Gusu District, Soochow, 215006, Jiangsu, China
| | - Yanli Qin
- Department of Infectious Diseases, Huashan Hospital, Fudan University, 12 Middle Wulumuqi Road, Shanghai, 200040, China
| | - Jing Zhang
- Key Lab of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Xinyan Li
- Department of Hepatitis Diseases, Shanghai Public Health Clinical Center, Fudan University, Shanghai, China
| | - Shuping Tong
- Key Lab of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Weifeng Zhao
- Department of Infectious Diseases, The First Affiliated Hospital of Soochow University, 188 Shizi Street, Gusu District, Soochow, 215006, Jiangsu, China.
| | - Jiming Zhang
- Department of Infectious Diseases, Jing'An District Centre Hospital of Shanghai (Huashan Hospital, Fudan University Jing'An Branch), Shanghai, China.
- Department of Infectious Diseases, Huashan Hospital, Fudan University, 12 Middle Wulumuqi Road, Shanghai, 200040, China.
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Teppa E, Nadalin F, Combet C, Zea DJ, David L, Carbone A. Coevolution analysis of amino-acids reveals diversified drug-resistance solutions in viral sequences: a case study of hepatitis B virus. Virus Evol 2020; 6:veaa006. [PMID: 32158552 PMCID: PMC7050494 DOI: 10.1093/ve/veaa006] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
The study of mutational landscapes of viral proteins is fundamental for the understanding of the mechanisms of cross-resistance to drugs and the design of effective therapeutic strategies based on several drugs. Antiviral therapy with nucleos(t)ide analogues targeting the hepatitis B virus (HBV) polymerase protein (Pol) can inhibit disease progression by suppression of HBV replication and makes it an important case study. In HBV, treatment may fail due to the emergence of drug-resistant mutants. Primary and compensatory mutations have been associated with lamivudine resistance, whereas more complex mutational patterns are responsible for resistance to other HBV antiviral drugs. So far, all known drug-resistance mutations are located in one of the four Pol domains, called reverse transcriptase. We demonstrate that sequence covariation identifies drug-resistance mutations in viral sequences. A new algorithmic strategy, BIS2TreeAnalyzer, is designed to apply the coevolution analysis method BIS2, successfully used in the past on small sets of conserved sequences, to large sets of evolutionary related sequences. When applied to HBV, BIS2TreeAnalyzer highlights diversified viral solutions by discovering thirty-seven positions coevolving with residues known to be associated with drug resistance and located on the four Pol domains. These results suggest a sequential mechanism of emergence for some mutational patterns. They reveal complex combinations of positions involved in HBV drug resistance and contribute with new information to the landscape of HBV evolutionary solutions. The computational approach is general and can be applied to other viral sequences when compensatory mutations are presumed.
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Affiliation(s)
- Elin Teppa
- Sorbonne Université, Univ P6, CNRS, IBPS, Laboratoire de Biologie Computationnelle et Quantitative (LCQB) - UMR 7238, 4 Place Jussieu, 75005 Paris, France
- Sorbonne Université, Institut des Sciences du Calcul et des Données (ISCD), 4 Place Jussieu, 75005 Paris, France
| | - Francesca Nadalin
- Sorbonne Université, Univ P6, CNRS, IBPS, Laboratoire de Biologie Computationnelle et Quantitative (LCQB) - UMR 7238, 4 Place Jussieu, 75005 Paris, France
- Institute Curie, PSL Research University, INSERM U932, Immunity and Cancer Department, 26 rue d’Ulm, 75248 Paris, France
| | - Christophe Combet
- Univ Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Centre de recherche en cancérologie de Lyon, 151 Cours Albert Thomas, 69424 Lyon, France
| | - Diego Javier Zea
- Sorbonne Université, Univ P6, CNRS, IBPS, Laboratoire de Biologie Computationnelle et Quantitative (LCQB) - UMR 7238, 4 Place Jussieu, 75005 Paris, France
| | - Laurent David
- Sorbonne Université, Univ P6, CNRS, IBPS, Laboratoire de Biologie Computationnelle et Quantitative (LCQB) - UMR 7238, 4 Place Jussieu, 75005 Paris, France
| | - Alessandra Carbone
- Sorbonne Université, Univ P6, CNRS, IBPS, Laboratoire de Biologie Computationnelle et Quantitative (LCQB) - UMR 7238, 4 Place Jussieu, 75005 Paris, France
- Institut Universitaire de France, 1 rue Descartes, 75231 Paris, France
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Analysis of hepatitis B virus-mixed genotype infection by ultra deep pyrosequencing in Sudanese patients, 2015-2016. Infection 2019; 47:793-803. [PMID: 30963405 DOI: 10.1007/s15010-019-01306-5] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2018] [Accepted: 04/03/2019] [Indexed: 12/15/2022]
Abstract
PURPOSE The frequency of detection of HBV co-infection with multiple HBV genotypes is influenced by the detection method; usually co-infections are detected by multiplex PCR or hybridization assays, and are rarely confirmed by sequencing and conventional cloning. The objective of this study was to confirm by ultra-deep pyrosequencing (UDPS) mixed HBV infections, previously detected by multiplex-nested PCR. METHODS Sixteen samples from HBV co-infected Sudanese patients detected by multiplex-nested PCR, were amplified targeting the P/S region and sequenced by UDPS. RESULTS The only genotypes identified using UDPS were D and E, while A, B, C and F genotypes, previously detected by multiplex-nested PCR, were not detected. Specifically, 10 samples were shown to be mono-infected (D or E); in 3 out of 10 mono-infected D patients, a subtype combination was observed: D1 + D7 in 2 cases and D2 + D6 in 1 case. The remaining 6 subjects were D + E co-infected (harboring different mixtures of D subtypes). CONCLUSIONS Overall, UDPS is more effective than multiplex-nested PCR for identifying multiple HBV genotypes and subtypes infections.
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Asan A, Sayan M, Akhan S, Tekin Koruk S, Aygen B, Sirmatel F, Eraksoy H, Tuna N, Köse S, Kaya A, Eren Tulek N, Aktug Demir N, Mistik R, Ormen B, Korkmaz F, Yildirmak T, Ural O, Aydin M, Turgut H, Gunal O, Demirturk N. Molecular Characterization of Drug Resistance in Hepatitis B Viruses Isolated from Patients with Chronical Infection in Turkey. HEPATITIS MONTHLY 2018; 18. [DOI: 10.5812/hepatmon.12472] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/02/2024]
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Epidemiological, Virological and Clinical Characteristics of Hepatitis B Virus Genotypes in Chronically Infected Persons in Slovenia. HEPATITIS MONTHLY 2017. [DOI: 10.5812/hepatmon.43838] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/13/2023]
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Mulrooney-Cousins P, Michalak T. Molecular Testing in Hepatitis Virus Related Disease. DIAGNOSTIC MOLECULAR PATHOLOGY 2017:63-73. [DOI: 10.1016/b978-0-12-800886-7.00006-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
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Wang G, Liu Y, Qiu P, Zhou SF, Xu L, Wen P, Wen J, Xiao X. Cost-effectiveness analysis of lamivudine, telbivudine, and entecavir in treatment of chronic hepatitis B with adefovir dipivoxil resistance. DRUG DESIGN DEVELOPMENT AND THERAPY 2015; 9:2839-46. [PMID: 26082614 PMCID: PMC4459610 DOI: 10.2147/dddt.s73150] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
The purpose of this study was to analyze the cost-effectiveness of lamivudine (LMV), telbivudine (LdT), and entecavir (ETV) in treatment of chronic hepatitis B with adefovir dipivoxil (ADV) resistance. Two hundred and fifty-two patients were recruited and screened for resistance to ADV and randomly assigned into three groups: LMV + ADV, LdT + ADV, and ETV + ADV. The ratio of biochemical response, virological response, seroconversion of hepatitis Be antigen (HBeAg)/hepatitis Be antibody (HBeAb), viral breakthrough, and the cost and effectiveness of treatments were analyzed. A comparison of the results of the ratio of biochemical response, virological response and seroconversion of HBeAg/HBeAb, showed no statistical difference between the three groups, with the economic cost of LMV + ADV the lowest, LdT + ADV the middle, and ETV + ADV the highest. The side effects of the three plans are all rare and tolerable. LMV + ADV is the optimal rescue strategy, and LdT + ADV the alternative selection in the economically less developed regions, while ETV + ADV was used in the economically developed regions.
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Affiliation(s)
- Guiliang Wang
- Department of Digestive Internal Medicine, Gannan Medical University Pingxiang Hospital, Pingxiang, People's Republic of China ; Department of Digestive Internal Medicine, 307 Hospital of PLA, Beijing, People's Republic of China
| | - Yan Liu
- Department of Digestive Internal Medicine, 307 Hospital of PLA, Beijing, People's Republic of China
| | - Ping Qiu
- Department of Digestive Internal Medicine, Gannan Medical University Pingxiang Hospital, Pingxiang, People's Republic of China
| | - Shu-Feng Zhou
- Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA ; Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, People's Republic of China
| | - Linfang Xu
- Department of Digestive Internal Medicine, Gannan Medical University Pingxiang Hospital, Pingxiang, People's Republic of China
| | - Ping Wen
- Department of Digestive Internal Medicine, Gannan Medical University Pingxiang Hospital, Pingxiang, People's Republic of China
| | - Jianbo Wen
- Department of Digestive Internal Medicine, Gannan Medical University Pingxiang Hospital, Pingxiang, People's Republic of China
| | - Xianzhong Xiao
- Laboratory of Shock, Department of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha, People's Republic of China
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Su F, Dai J, Yang S, Jiang X, Cui X, Ning H, Li J, Huang M. Prevalence and types of drug-resistant variants in Chinese patients with acute hepatitis B. J Med Virol 2015; 87:1527-31. [PMID: 26032700 DOI: 10.1002/jmv.24006] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/03/2014] [Indexed: 12/21/2022]
Abstract
The presence of therapy-associated hepatitis B virus (HBV) variants is the main drawback of antiviral therapy for HBV infection. Moreover, drug-resistant variants are more insensitive to a second agent and more therapy-associated mutations will be present. To apply better nucleos(t)ide analogues (NA) and reduce the occurrence of resistance, the prevalence and types of drug-resistant mutations in acute hepatitis B patients were investigated in this study. One hundred three HBV DNA-positive patients with symptomatic acute hepatitis B that were observed from 2011 to 2013 were enrolled. Direct polymerase chain reaction sequencing was used firstly to screen HBV reverse-transcriptase domain to detect HBV mutants. Five lamivudine-resistant variants were identified. Clonal sequencing was performed for 5 resistance-positive samples and 10 other random samples. Interestingly, all detected samples harbored drug-resistant mutations, although with different percentage. Thirteen harbored lamivudine-related alone (five) or together with other NA related mutations (five with adefovir, one with entecavir, and one with telbivudine), and two of them harbored adefovir-related mutations. Also, mutations associated with four currently used NA were all detected, and the frequency is in accordance with the popularity of NA used in clinical practice. These data suggest that drug-resistant variants are present in patients with acute hepatitis B and NA should be applied more carefully for chronic hepatitis B patients developed from acute hepatitis B.
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Affiliation(s)
- Feifei Su
- Department of Infection Diseases, Wenzhou Central Hospital, Wenzhou, China
| | - Jianyi Dai
- Department of Infection Diseases, Wenzhou Central Hospital, Wenzhou, China
| | - Shoufeng Yang
- Department of Infection Diseases, Wenzhou Central Hospital, Wenzhou, China
| | - Xiangao Jiang
- Department of Infection Diseases, Wenzhou Central Hospital, Wenzhou, China
| | - Xiaoya Cui
- Department of Infection Diseases, Wenzhou Central Hospital, Wenzhou, China
| | - Hongye Ning
- Department of Infection Diseases, Wenzhou Central Hospital, Wenzhou, China
| | - Junhua Li
- Department of Infection Diseases, Wenzhou Central Hospital, Wenzhou, China
| | - Mohe Huang
- Department of Infection Diseases, Wenzhou Central Hospital, Wenzhou, China
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Characterization of Full-Length Genomes of Hepatitis B Virus Quasispecies in Sera of Patients at Different Phases of Infection. J Clin Microbiol 2015; 53:2203-14. [PMID: 25926495 DOI: 10.1128/jcm.00068-15] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2015] [Accepted: 04/24/2015] [Indexed: 12/17/2022] Open
Abstract
Hepatitis B virus (HBV) infection results in different clinical presentation due to different levels of immune response. Our study aimed to characterize HBV full-length genome quasispecies (QS) in patients with different phases of infection to better understand its pathogenesis. Forty treatment-naive HBV-infected patients were enrolled, including 10 cases of acute hepatitis B (AHB), 9 cases of immunotolerant (IT) HBV carriers, 11 cases of chronic hepatitis B (CHB), and 10 cases of acute-on-chronic liver failure (ACLF). The present study was conducted by clone-based sequencing. QS heterogeneity within each open reading frame was calculated. The mutation frequency index (MFI) and amino acid variations within the large HBsAg, HBcAg, and HBxAg regions were analyzed based on the different infection phases. In total, 606 HBV full-length sequences were obtained. HBV QS had higher heterogeneity in ACLF and CHB than that in IT among chronically infected individuals. AHB patients had the lower QS heterogeneity at onset than those with chronic infection. ACLF patients had the highest frequency of mutations in the core promoter and precore region. A triple mutation (A1762T/G1764A/G1896A) was observed more frequently in genotype C than in genotype B. The MFI indicated that specific peptides of the studied regions had more frequent mutations in ACLF. Furthermore, several amino acid variations, known as T- and B-cell epitopes, were potentially associated with the immunoactive phase of infection. More HBV genome mutations and deletions were observed in patients with more severe diseases, particularly in specific regions of the core and preS regions, the clinical significance and mechanism of which need to be further investigated.
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Bang KB, Kim HJ. Management of antiviral drug resistance in chronic hepatitis B. World J Gastroenterol 2014; 20:11641-11649. [PMID: 25206270 PMCID: PMC4155356 DOI: 10.3748/wjg.v20.i33.11641] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/21/2013] [Revised: 01/10/2014] [Accepted: 05/28/2014] [Indexed: 02/07/2023] Open
Abstract
Rescue antiviral treatment for patients with resistance to preexisting nucleos(t)ide analogues remains a clinical challenge. The correct choice of a first-line treatment of high potency and with a high genetic barrier to achieve sustained long-term suppression of viral replication provides the best chance of preventing treatment failure and the emergence of drug resistance. The management of treatment failure and drug resistance requires a precise and accurate clinical and virologic monitoring. Combination treatment with antiviral drugs that belong to different groups is associated with a lower chance of developing resistance to rescue drugs. To guarantee better control of viral replication in patients with drug resistance, the addition of another drug without a cross resistance profile should be given as early as possible, preferably at the time when genotypic resistance emerges. Long-term surveillance for treatment efficacy and possible emergence of drug resistance should be continued to prevent the emergence of multidrug-resistant strains.
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rtM204Q may serve as a novel lamivudine-resistance-associated mutation of hepatitis B virus. PLoS One 2014; 9:e89015. [PMID: 24586482 PMCID: PMC3933355 DOI: 10.1371/journal.pone.0089015] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2013] [Accepted: 01/14/2014] [Indexed: 02/07/2023] Open
Abstract
Background and Aims Lamivudine (LAM) is still widely used for anti-HBV therapy in China. The study aimed to clarify whether a newly-found rtM204Q mutation from patients was associated with the drug resistance. Methods HBV complete reverse-transcriptase region was screened by direct sequencing and verified by clonal sequencing. Replication-competent plasmids containing patient-derived 1.1mer mutant or wild-type viral genome were constructed and transfected into HepG2 cells. After cultured with or without serially-diluted antiviral drugs, intracellular HBV replicative intermediates were quantitated for calculating the 50% effective concentration of drug (EC50). Results A total of 12,000 serum samples of 9,830 patients with chronic HBV infection were screened. rtM204Q mutation was detected in seven LAM-refractory patients. By contrast, rtM204I/rtM204V mutations were detected in 2,502 patients' samples. The rtM204Q emerged either alone or in concomitance with rtM204I/rtM204V, and all were accompanied with virologic breakthrough in clinical course. Clonal sequencing verified that rtM204Q mutant was predominant in viral quasispecies of these samples. Phenotypic analysis showed that rtM204Q mutant had 89.9% of replication capacity and 76-fold increased LAM EC50 of the concomitant wild-type strain. By contrast, rtM204I mutant in the sample had lower replication capacity and higher LAM resistance (46.3% and 1396-fold increased LAM EC50 of the wild-type strain) compared to rtM204Q mutant. rtM204Q mutant was susceptible to adefovir dipivoxil (ADV) in vitro and ADV/ADV+LAM rescue therapy in clinic. Conclusion rtM204Q is suggested to be a novel LAM-resistance-associated mutation. It conferred a moderate resistance with higher competent natural replication capacity compared to rtM204I mutation.
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Paintsil E, Cheng YC. Antiviral Agents☆. REFERENCE MODULE IN BIOMEDICAL SCIENCES 2014. [PMCID: PMC7150273 DOI: 10.1016/b978-0-12-801238-3.02387-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/04/2022]
Abstract
Antiviral agents are drugs approved in the USA by the Food and Drug Administration (FDA) for the treatment or control of viral infections. Available antiviral agents mainly target stages in the viral life cycle. The target stages in the viral life cycle are; viral attachment to host cell, uncoating, synthesis of viral mRNA, translation of mRNA, replication of viral RNA and DNA, maturation of new viral proteins, budding, release of newly synthesized virus, and free virus in body fluids. Two important factors that can limit the utility of antiviral drugs are toxicity and the development of resistance to the antiviral agent by the virus. In addition, host phenotypic behaviors toward antiviral drugs because of either genomic or epigenetic factors could limit the efficacy of an antiviral agent in an individual. This article summarizes the most relevant pharmacologic and clinical properties of current antiviral agents, and targets for novel antiviral agents.
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Rapid detection of hepatitis B virus variants associated with lamivudine and adefovir resistance by multiplex ligation-dependent probe amplification combined with real-time PCR. J Clin Microbiol 2013; 52:460-6. [PMID: 24478474 DOI: 10.1128/jcm.02554-13] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
Drug-resistant mutations of hepatitis B virus (HBV) are the major obstacles to successful therapy for chronic hepatitis B infection. Although there are many methods for detecting the antiviral drug-resistant mutations of HBV, their applications are restricted because of their shortcomings, such as low sensitivity, the time required, and the high cost. For this study, a multiplex ligation-dependent probe real-time PCR (MLP-RT-PCR) method was developed to simultaneously detect lamivudine (LAM)- and adefovir (ADV)-resistant HBV mutants (those with the mutations rtM204V/I, rtA181V/T, and rtN236T). The new method combined the high-throughput nature of multiplex ligation-dependent probe amplification (MLPA) with the rapid and sensitive detection of real-time PCR. In this report, MLP-RT-PCR was evaluated by detecting drug-resistant mutants in 116 patients with chronic hepatitis B infection. By MLP-RT-PCR analysis, LAM-resistant mutations were detected in 41 patients (35.3%), ADV-resistant mutations were detected in 17 patients (14.7%), and LAM- and-ADV-resistant mutations were detected in 5 patients (4.3%). Based on the results of MLP-RT-PCR, the mutations rtM204V, rtM204I, rtA181T, rtA181V, and rtN236T were 95.7% (111/116 patients), 98.3% (114/116 patients), 99.1% (115/116 patients), 98.3% (114/116 patients), and 99.1% (115/116 patients) concordant, respectively, with those of direct sequencing. The MLP-RT-PCR assay was more sensitive than direct sequencing for detecting mutations with low frequencies. Four samples containing the low-frequency (<10%) mutants were identified by MLP-RT-PCR and further confirmed by clonal sequencing. MLP-RT-PCR is a rapid and sensitive method that enables the detection of multidrug-resistant HBV mutations in clinical practice.
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Devi U, Locarnini S. Hepatitis B antivirals and resistance. Curr Opin Virol 2013; 3:495-500. [PMID: 24016777 DOI: 10.1016/j.coviro.2013.08.006] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2013] [Revised: 08/04/2013] [Accepted: 08/06/2013] [Indexed: 12/19/2022]
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Afshar RM, Mollaie HR. Use of ALLGIO probe assays for detection of HBV resistance to adefovir in patients with chronic hepatitis B, Kerman, Iran. Asian Pac J Cancer Prev 2013; 13:5463-7. [PMID: 23317201 DOI: 10.7314/apjcp.2012.13.11.5463] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
Hepatitis B virus (HBV) infection is contagious with transmissiobn vertically or horizontally by blood products and body secretions. Over 50% of Iranian carriers contracted the infection prenatally, making this the most likely route of transmission of HBV in Iran. To evaluate the resistance to adefovir (ADV) therapy in patients with chronic hepatitis B infection, a study was conducted on 70 patients (63 males and 7 females), who had received in first line lamivudine and second line adefovir. All were tested for the presence of hepatitis B surface antigen (HBsAg), hepatitis B envelope antigen (HBeAg), serum alanine amino transferase (ALT) level and HBV DNA load before and after treatment with ADV. In all samples, resistance to lamivudine and ADV was tested with real time PCR. Among seventy patients with chronic hepatitis B infection, 18 (25.7%) were resistant to LAM and 8 (11.4%) were resistant to ADV. Only one patient was negative for the presence of HBS-Ag (5.6%) and two were negative for HBe-Ag (11.1%). In this study we used a new method (ALLGIO probe assay) that has high sensitivity in detection of adefovir resistance mutants, which we recommend to other researchers. Mutant strains of the YMDD motif of HBV polymerase can be found in some patients under treatment with lamivudine and ADV. ADV has been demonstrated to be efficient in patients with lamivudine resistant HBV.
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Polymerase mutations rtN238R, rtT240Y and rtN248H of hepatitis B virus decrease susceptibility to adefovir. ACTA ACUST UNITED AC 2013. [DOI: 10.1007/s11434-013-5770-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
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De Clercq E. A cutting-edge view on the current state of antiviral drug development. Med Res Rev 2013; 33:1249-77. [PMID: 23495004 DOI: 10.1002/med.21281] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Prominent in the current stage of antiviral drug development are: (i) for human immunodeficiency virus (HIV), the use of fixed-dose combinations (FDCs), the most recent example being Stribild(TM); (ii) for hepatitis C virus (HCV), the pleiade of direct-acting antivirals (DAAs) that should be formulated in the most appropriate combinations so as to obtain a cure of the infection; (iii)-(v) new strategies (i.e., AIC316, AIC246, and FV-100) for the treatment of herpesvirus infections: herpes simplex virus (HSV), cytomegalovirus (CMV), and varicella-zoster virus (VZV), respectively; (vi) the role of a new tenofovir prodrug, tenofovir alafenamide (TAF) (GS-7340) for the treatment of HIV infections; (vii) the potential use of poxvirus inhibitors (CMX001 and ST-246); (viii) the usefulness of new influenza virus inhibitors (peramivir and laninamivir octanoate); (ix) the position of the hepatitis B virus (HBV) inhibitors [lamivudine, adefovir dipivoxil, entecavir, telbivudine, and tenofovir disoproxil fumarate (TDF)]; and (x) the potential of new compounds such as FGI-103, FGI-104, FGI-106, dUY11, and LJ-001 for the treatment of filoviruses (i.e., Ebola). Whereas for HIV and HCV therapy is aimed at multiple-drug combinations, for all other viruses, HSV, CMV, VZV, pox, influenza, HBV, and filoviruses, current strategies are based on the use of single compounds.
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Affiliation(s)
- Erik De Clercq
- Rega Institute for Medical Research, KU Leuven, B-3000, Leuven, Belgium.
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Monavari SH, Keyvani H, Mollaie H, Roudsari RV. Detection of rtN236T mutation associated with adefovir dipivoxil resistance in Hepatitis B infected patients with YMDD mutations in Tehran. IRANIAN JOURNAL OF MICROBIOLOGY 2013; 5:76-80. [PMID: 23467016 PMCID: PMC3577563] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 10/29/2022]
Abstract
BACKGROUND OBJECTIVES The risk of adefovir dipivoxil resistance emergence has increased in lamivudine-resistant hepatitis B infected patients. The mutations known as causing adefovir resistance, rtN236T and rtA181V/T, are detected within the D and B functional domain of the HBV polymerase, respectively. In this study, we intended to determine the pre-existing adefovir-resistance mutations in patients infected with LAM resistant mutants prior to starting adefovir therapy. MATERIAL AND METHODS The study included 30 patients with chronic hepatitis B with lamivudine resistance mutations in the YMDD motif that experienced viral breakthrough. RESULTS After alignment of protein coding sequences, the rtN236T mutation was observed in two (6.6%) patients, while twenty-eight others had neither rtN236T, nor rtA181V/T mutation. All 30 patients were infected with genotype D of hepatitis B virus. CONCLUSIONS The early detection of LAM-resistance mutations may allow a timely chance of therapy to avoid hepatitis flare-up. This data suggests that monitoring of ADV-resistance mutations in ADV naïve patients can be considered in selecting the appropriate anti-viral regimen.
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Affiliation(s)
- Seyed Hamidreza Monavari
- Antimicrobial Resistance Research Center, Tehran University of Medical Sciences, Tehran, Iran, Corresponding author: Dr. Seyed Hamid Reza Monavari, Address: Department of Virology, Tehran University of Medical Sciences. Tel: +98-21-88602205. Fax: +98-21-88602205. E-mail:
| | - Hossein Keyvani
- Department of Virology, Tehran University of Medical Sciences, Tehran, Iran
| | - Hamidreza Mollaie
- Department of Virology, Tehran University of Medical Sciences, Tehran, Iran
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Wong GLH, Wong VWS, Chan HY, Tse PCH, Wong J, Chim AML, Yiu KKL, Chu SHT, Chan HLY. Undetectable HBV DNA at month 12 of entecavir treatment predicts maintained viral suppression and HBeAg-seroconversion in chronic hepatitis B patients at 3 years. Aliment Pharmacol Ther 2012; 35:1326-35. [PMID: 22506552 DOI: 10.1111/j.1365-2036.2012.05098.x] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/26/2012] [Revised: 02/22/2012] [Accepted: 03/22/2012] [Indexed: 02/06/2023]
Abstract
BACKGROUND On-treatment monitoring of serum hepatitis B virus (HBV) DNA to guide treatment strategy for patients on entecavir has received little attention. AIM To investigate the predictive value of on-treatment HBV DNA levels for responses to entecavir. METHODS This was a retrospective cohort study among nucleos(t)ide analogue-naïve HBV-infected patients on entecavir with a minimum follow-up of 2 years. Maintained virological suppression was defined as undetectable HBV DNA (<20 IU/mL) until the last visit. Genotypic drug resistance was screened by using the INNO-LiPA DR assay. RESULTS A total of 440 chronic hepatitis B patients (160 HBeAg-positive) followed for 34 ± 9 months were included. The cumulative probability of maintained virological suppression at year 1, 2 and 3 were 76.5%, 83.0% and 88.3% respectively. On multivariate analysis, lower baseline HBV DNA, undetectable HBV DNA at month 12 and negative HBeAg were the independent predictors of maintained virological suppression. M12 responders (who had undetectable HBV DNA at month 12) had higher probability of maintained virological suppression at 3 years (99.1%) as compared to non responders (57.5%; P < 0.001). The cumulative probability of HBeAg-seroconversion at year 1, 2 and 3 were 19.0%, 27.2% and 33.5% respectively. M12 responders had higher probability of HBeAg-seroconversion at 3 years (43.2%) than the non responders (19.0%; P = 0.003). M12 responders had lower probability of drug resistance at 3 years (0%) than the non responders (2.6%; P = 0.004). CONCLUSION Month 12 HBV DNA responses could predict the probability of maintained virological suppression, HBeAg-seroconversion and risk of drug resistance among patients on entecavir treatment at 3 years.
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Affiliation(s)
- G L-H Wong
- Institute of Digestive Disease, The Chinese University of Hong Kong, Hong Kong SAR, China
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Michailidis E, Kirby KA, Hachiya A, Yoo W, Hong SP, Kim SO, Folk WR, Sarafianos SG. Antiviral therapies: focus on hepatitis B reverse transcriptase. Int J Biochem Cell Biol 2012; 44:1060-71. [PMID: 22531713 DOI: 10.1016/j.biocel.2012.04.006] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2012] [Revised: 04/04/2012] [Accepted: 04/05/2012] [Indexed: 12/20/2022]
Abstract
Hepatitis B virus (HBV) is the etiologic agent of mankind's most serious liver disease. While the availability of a vaccine has reduced the number of new HBV infections, the vaccine does not benefit the approximately 350 million people already chronically infected by the virus. Most of the drugs approved by the FDA for the treatment of hepatitis B target the reverse transcriptase (RT or P gene product) and are nucleoside RT inhibitors (NRTIs) that suppress viral replication. However, prolonged monotherapies directed against a single target result in the emergence of viral resistance. HBV genotypic differences affect NRTI resistance, and because the reading frames of the S (surface antigen) and P genes partially overlap, genomic differences that affect the surface of the virus may also alter the viral polymerase sequence, function and drug susceptibility. The scope of this review is to assess the effects of HBV genotypic variation on the development of drug resistance to NRTIs. Some RT residues that vary among different genotypes are in the vicinity of residues that mutate and give rise to NRTI resistance. Interactions between these amino acids can help explain the effect of HBV genotype on the development of NRTI resistance during antiviral therapies, and might help in the design of improved therapeutic strategies.
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Affiliation(s)
- Eleftherios Michailidis
- Christopher S. Bond Life Sciences Center, Department of Molecular Microbiology & Immunology, University of Missouri, Columbia, MO 65211, USA
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21
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Hu Y, Zhang WL, Xie SL, Zhao Y, Hu JL, Cai XF, Lai GQ, Huang AL. An improved reverse dot hybridization for simple and rapid detection of adefovir dipivoxil-resistant hepatitis B virus. GENETICS AND MOLECULAR RESEARCH 2012; 11:53-60. [PMID: 22290465 DOI: 10.4238/2012.january.9.6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Early detection of adefovir dipivoxil-resistant mutants during long-term treatment of chronic hepatitis B virus (HBV) infection with this drug is of great clinical importance. We developed an improved reverse dot hybridization test for simple and rapid detection of the rtA181V/T and rtN236T mutations associated with adefovir dipivoxil resistance in chronic hepatitis B patients. Probes were designed for genotypes B, C, and D of this resistance characteristic; a total of 70 clinical samples were analyzed with this improved reverse dot hybridization assay. Its usefulness was validated by comparing with sequencing data. Discordant results were confirmed by subclone sequencing. This reverse dot hybridization assay was sufficiently sensitive to detect 10(3) copies/mL; it also detected adefovir dipivoxil-resistant mutant strains when they comprised more than 5% of a mixed virus population. This reverse dot hybridization array correctly identified adefovir dipivoxil-resistant mutants; it had high concordance (98.5%) with direct sequencing data. There was no clear relationship between the HBV genotype and the development of adefovir dipivoxil-resistant mutants. This reverse dot hybridization assay proved to be simple and rapid for detection of rtA181V/T and rtN236T mutations associated with resistance to adefovir dipivoxil.
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Affiliation(s)
- Y Hu
- Key Laboratory of Molecular Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China
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Li W, Han M, Li Y, Chen D, Luo X, Ning Q. Antiviral resistance mutations potentiate HBV surface antigen-induced transcription of hfgl2 prothrombinase gene. BIOCHEMISTRY (MOSCOW) 2011; 76:1043-50. [PMID: 22082274 DOI: 10.1134/s0006297911090094] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Antiviral resistance mutations in the hepatitis B virus (HBV) polymerase (pol) gene have been demonstrated to play an important role in the progression of liver disease and the development of hepatocellular carcinoma. The HBV pol gene overlaps the S gene encoding surface antigen (HBsAg). Previous studies from our laboratory have shown that HBV core protein (HBc) and X protein (HBx), but not HBV S protein (HBs), promote hfgl2 prothrombinase transcription. To investigate whether the nucleotide (nucleoside)-induced resistant mutations of HBs potentiate transcription of hfgl2 prothrombinase gene, we generated two mutant HB expression constructs harboring rtM204V/sI195M or rtM204I/sW196L mutations. Two mutant expression plasmids were co-transfected with hfgl2 promoter luciferase-reporter plasmids and β-galactosidase plasmid in CHO cells and HepG2 cells, respectively. Luciferase assay showed that the rtM204I/V mutant HBs could activate the transcription of hfgl2 promoter compared with the wild type HBs. Site-directed mutagenesis and further experiment (co-transfection) demonstrated that transcription factor Ets translocated to its cognate cis-element in the hfgl2 promoter. The results show that mutated HBs caused by antiviral drug resistance induce transcription of the hfgl2 gene dependent on the transcription factor Ets.
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Affiliation(s)
- Weina Li
- Department and Institute of Infectious Disease, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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Liu H, Mao R, Fan L, Xia J, Li Y, Yin Y, Li X, Zhao X, Guo H, Zhu H, Zhang Y, Kang Y, Zhang J. Detection of lamivudine- or adefovir-resistant hepatitis B virus mutations by a liquid array. J Virol Methods 2011; 175:1-6. [PMID: 21513743 DOI: 10.1016/j.jviromet.2011.04.005] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2010] [Revised: 03/27/2011] [Accepted: 04/05/2011] [Indexed: 01/07/2023]
Abstract
A novel polymerase chain reaction (PCR)-Luminex assay was developed for rapid, accurate, and high-throughput detection of the most important hepatitis B virus (HBV) variants, including those with reverse transcriptase (RT) domain L180M, M204I/V, A181T/V/S, I233V and N236T mutations associated with resistance to lamivudine (LAM) or adefovir (ADV). Using mixtures of mutant and wild-type HBV, this method was sufficiently sensitive for detecting 10(3)HBV ml(-1) and could detect minor mutants when they comprised 5% of the total viral population. Comparison of the PCR-Luminex assay with INNO-LiPA for detecting clinical LAM- or ADV-resistant chronic hepatitis B virus infection in 64 patients confirmed the following: the 2 methods were 97.9% (48 of 49) and 93.3% (14 of 15) concordant for detecting LAM- or ADV-resistance mutations, respectively. The agreement with direct sequencing was 70.3% (45 of 64). The PCR-Luminex assay or multi-analyte suspension array can detect simultaneously and efficiently minor populations HBV mutants early during infection in many clinical samples. It is a simple, cost-effective method for resistance surveillance or selecting appropriate antiviral agents and initiating timely rescue treatment before the development drug-resistance related virus or biochemical breakthrough.
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Affiliation(s)
- Hongyan Liu
- Department of Infectious Diseases, Huashan Hospital, and Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, 12 Middle Wulumuqi Road, Shanghai 200040, China
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Peptide nucleic acid array for detection of point mutations in hepatitis B virus associated with antiviral resistance. J Clin Microbiol 2010; 48:3127-31. [PMID: 20573874 DOI: 10.1128/jcm.02058-09] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The detection of antiviral-resistant hepatitis B virus (HBV) mutations is important for monitoring the response to treatment and for effective treatment decisions. We have developed an array using peptide nucleic acid (PNA) probes to detect point mutations in HBV associated with antiviral resistance. PNA probes were designed to detect mutations associated with resistance to lamivudine, adefovir, and entecavir. The PNA array assay was sensitive enough to detect 10(2) copies/ml. The PNA array assay was able to detect mutants present in more than 5% of the virus population when the total HBV DNA concentration was greater than 10(4) copies/ml. We analyzed a total of 68 clinical samples by this assay and validated its usefulness by comparing results to those of the sequencing method. The PNA array correctly identified viral mutants and has high concordance (98.3%) with direct sequencing in detecting antiviral-resistant mutations. Our results showed that the PNA array is a rapid, sensitive, and easily applicable assay for the detection of antiviral-resistant mutation in HBV. Thus, the PNA array is a useful and powerful diagnostic tool for the detection of point mutations or polymorphisms.
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Antiviral treatment of chronic hepatitis B virus (HBV) infections. Viruses 2010; 2:1279-1305. [PMID: 21994680 PMCID: PMC3185710 DOI: 10.3390/v2061279] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2010] [Revised: 05/18/2010] [Accepted: 05/25/2010] [Indexed: 12/21/2022] Open
Abstract
While 25 compounds have been formally licensed for the treatment of HIV infection (AIDS), only seven licensed products are currently available for the treatment of chronic hepatitis B virus (HBV) infection: interferon-α, pegylated interferon-α, lamivudine, adefovir (dipivoxil), entecavir, telbivudine and tenofovir (disoproxil fumarate). In contrast to the treatment of HIV infections where the individual drugs are routinely used in combination, for the treatment of chronic HBV infection the individual drugs are generally used in monotherapy. In principle, combination drug therapy should allow reducing the likelihood of drug-resistant development.
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Sayan M, Sentürk O, Akhan SÇ, Hülagü S, Cekmen MB. Monitoring of hepatitis B virus surface antigen escape mutations and concomitantly nucleos(t)ide analog resistance mutations in Turkish patients with chronic hepatitis B. Int J Infect Dis 2010; 14 Suppl 3:e136-41. [PMID: 20382061 DOI: 10.1016/j.ijid.2009.11.039] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2009] [Revised: 10/30/2009] [Accepted: 11/25/2009] [Indexed: 12/26/2022] Open
Abstract
BACKGROUND The hepatitis B virus (HBV) polymerase gene completely overlaps with the envelope gene. In the present study we aimed to monitor the prevalence and pattern of the typical mutations for hepatitis B surface antigen (HBsAg) escape, and concomitantly nucleos(t)ide analog (NUC) resistance mutations, in Turkish patients undergoing different antiviral therapies and in treatment-naïve patients with chronic hepatitis B (CHB). METHODS The investigation was undertaken between March 2007 and August 2009 and involved a total of 142 patients under NUC therapy (88 males; mean age 42 years (range 13-68); hepatitis B e antigen (HBeAg) negativity in 94 patients; HBV DNA median log 4.3 log(10) IU/ml (range 2.0->6.0); alanine aminotransferase (ALT) median level 76.1 IU/ml (range 12-1082)) and 185 treatment-naïve CHB patients (120 males; mean age 39 years (range 1-76 years); HBeAg negativity in 132 patients; HBV DNA median log 3.5 log(10) IU/ml (range 2.0-6.0); ALT median level 60.7 IU/l (range 8-874)). RESULTS The overall prevalence of typical HBsAg escape mutations found in the CHB patients was 8.3% (27/327). In the NUC therapy group the prevalence was 8.5% (12/142), with the following patterns: sY100C+sI110V, sL109I, sP120T, sP127T, sG130R+sG145X, sS132A+sY134N, sY134N+sG145R, sC137G, sD144E, sG145R. In the treatment-naïve group the prevalence was 8.1% (15/185), with the following patterns: sL109I, sI110V, sS117INST, sP120T, sP127T, sM133I, sC137L+sG145R, sS143L. However, NUC resistance mutations were found in 7.7% (11/142) of the patients on NUC therapy and 3.8% (7/185) of the treatment-naïve group patients. Interestingly, the treatment-naïve patients had preexisting drug resistance mutations related to lamivudine (rtL180M+rtM204I), adefovir (rtA181V, rtQ215S, rtI233V), entecavir (intermediate susceptibility with rtL180M+rtM204IHBV variant), telbivudine (rtL180M+rtM204I), and tenofovir (rtA194T). CONCLUSIONS The findings of this study show preexisting typical HBsAg escape and NUC resistance mutations are possible. The genetic arrangement of the HBV genome with polymerase and surface genes overlapping has substantial public health and diagnostic implications and relevance.
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Affiliation(s)
- M Sayan
- Clinical Laboratory, PCR Unit, Faculty of Medicine, University of Kocaeli, İzmit-Kocaeli, Turkey.
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Zhao WF, Shao YL, Chen LY, Wu JH, Zhu YL, Gan JH, Xiong H. Establishment of a new quantitative detection approach to adefovir-resistant HBV and its clinical application. World J Gastroenterol 2010; 16:1267-1273. [PMID: 20222172 PMCID: PMC2839181 DOI: 10.3748/wjg.v16.i10.1267] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/04/2009] [Revised: 03/27/2009] [Accepted: 04/03/2009] [Indexed: 02/06/2023] Open
Abstract
AIM To establish the more feasible and sensitive assessment approach to the detection of adefovir (ADV) resistance-associated hepatitis B virus (HBV) quasispecies. METHODS Based on the characteristics of rtA181V/T and rtN236T mutations, a new approach based on real-time fluorescent quantitative polymerase chain reaction (RT-PCR) was established for the detection of ADV-resistant HBV quasispecies, total HBV DNA, rtA181 and rtN236 mutations in blood samples from 32 chronic hepatitis B (CHB) patients with unsatisfactory curative effect on ADV and compared with routine HBV DNA sequencing. RESULTS Both the sensitivity and specificity of this new detection approach to ADV-resistant HBV quasispecies were 100%, which were much higher than those of direct HBV DNA sequencing. The approach was able to detect 0.1% of mutated strains in a total plasmid population. Among the 32 clinical patients, single rtA181 and rtN236T mutation and double rtA181T and rtN236T mutations were detected in 20 and 8, respectively, while ADV-resistant mutations in 6 (including, rtA181V/T mutation alone in 5 patients) and no associated mutations in 26. CONCLUSION This new approach is more feasible and efficient to detect ADV-resistant mutants of HBV and ADV-resistant mutations before and during ADV treatment with a specificity of 100% and a sensitivity of 100%.
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Validation of the INNO-LiPA HBV DR assay (version 2) in monitoring hepatitis B virus-infected patients receiving nucleoside analog treatment. Antimicrob Agents Chemother 2010; 54:1283-9. [PMID: 20065049 DOI: 10.1128/aac.00970-09] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Hepatitis B virus (HBV) antiviral drug resistance mutations prevent successful outcome of treatment and lead to worsening of liver disease. Detection of its emergence permits opportune treatment with alternative drugs. Unfortunately, the use of newly approved antivirals, including adefovir dipivoxil, emtricitabine, and telbivudine, is also associated with the development of drug resistance, albeit to a lesser extent than the use of lamivudine. The objectives of this work were to assess the performance characteristics (sensitivity and accuracy) of an updated drug resistance test, the INNO-LiPA HBV DR v2, which includes detection of mutations associated with lamivudine, adefovir, emtricitabine, and telbivudine resistance, and to compare the results with consensus sequencing of serum samples from patients treated with HBV antivirals. Diagnostic sensitivity, defined as detection of a positive amplification line on the line probe assay (LiPA) strip, was 94.8% (95% confidence interval [CI], 89.7 to 97.9) after initial testing, increasing to 96.3% (95% CI, 91.6 to 98.8) after repeat test 1 and to 100% (95% CI, 97.3 to 100.0) after repeat test 2. In diagnostic accuracy determinations, full concordance was observed between sequencing and LiPA for 77.0% of the codons tested (620/805 codons [95% CI, 74.0 to 79.9]), whereas LiPA and sequencing were partially concordant 22% of the time (177/805 codons). In 167 out of 177 cases, LiPA detected a wild-type/mutant mixture whereas sequencing detected only one of the two results. Performance testing of the new LiPA test, the INNO-LiPA HBV DR v2, showed convincing diagnostic sensitivity and accuracy. The ability of the test to detect mixed infections and minority viral populations associated with resistance to the current generation of antivirals, including adefovir, emtricitabine, and telbivudine, makes it a useful tool for HBV therapy monitoring.
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Selection and counterselection of the rtI233V adefovir resistance mutation during antiviral therapy. J Clin Microbiol 2009; 48:631-4. [PMID: 20007398 DOI: 10.1128/jcm.01073-09] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Recently, we reported on three patients with chronic hepatitis B virus (HBV) infection for whom adefovir (ADF) therapy virologically failed, most likely due to a preexisting rtI233V HBV polymerase mutation. Here, we describe two further patients with chronic HBV infection who were found to develop the rtI233V mutation after initiation of ADF therapy. These patients represent the first cases known so far in which the rtI233V ADF resistance mutation evolved under persistent HBV replication during HBV therapy with ADF. Interestingly, one of the previously described patients, who was initially successfully switched from ADF to tenofovir (TDF) and became virologically suppressed subsequently, experienced a moderate but remarkable rebound of HBV viremia after switching from TDF to entecavir, due to the emergence of renal toxicity. Thus, we provide evidence for the selection and counterselection of the rtI233V ADF resistance mutation during antiviral therapy.
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Detection of the rtA181V/T and rtN236T mutations associated with resistance to adefovir dipivoxil using a ligase detection reaction assay. Clin Chim Acta 2009; 408:70-4. [DOI: 10.1016/j.cca.2009.07.016] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2009] [Revised: 07/16/2009] [Accepted: 07/16/2009] [Indexed: 12/15/2022]
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Prevalence, viral replication efficiency and antiviral drug susceptibility of rtQ215 polymerase mutations within the hepatitis B virus genome. J Hepatol 2009; 51:647-54. [PMID: 19586679 DOI: 10.1016/j.jhep.2009.04.022] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/13/2009] [Revised: 04/22/2009] [Accepted: 04/27/2009] [Indexed: 02/06/2023]
Abstract
BACKGROUND/AIMS The rtQ215S mutation in the hepatitis B virus (HBV) polymerase has been described as a secondary mutation associated with resistance to lamivudine (LAM) and adefovir (ADV). We aimed at assessing the prevalence of substitutions at rtQ215 of the HBV polymerase and determining the molecular and functional consequences using phenotypic analyses in vitro. METHODS The polymerase region was directly sequenced in HBV isolates from a cohort of 249 HBV genotype D-infected patients from Iran (174 males/75 females, 194 treatment-nai ve/ 55 LAM-treated). Replication-competent HBV constructs containing the naturally occurring rtQ215H, rtQ215P and rtQ215S mutations were generated, and compared to wild-type, LAM- (rtM204I, rtL180M/rtM204V) and ADV-resistant (rtN236T) clones. RESULTS In an Iranian cohort of 249 HBV infected patients, 14.5% (36/249) showed mutations in the rtQ215 locus, namely 6.8% rtQ215S, 3.6% rtQ215P and 4.1% rtQ215H. The frequency of rtQ215 substitutions was higher in LAM-treated than treatment-nai ve patients (25% vs. 11%), but not associated with clinical complications. In phenotypic assays, rtQ215S, rtQ215P and rtQ215H constructs showed equivalent levels of viral replication as wild-type HBV, whereas LAM- and ADV-resistant mutants had significantly impaired replicative capacities. Furthermore, rtQ215S, rtQ215P and rtQ215H harbouring constructs remained susceptible towards treatment with LAM or ADV in vitro. CONCLUSIONS Our study reveals that rtQ215 substitutions in the HBV polymerase frequently occur in chronic hepatitis B, even without exogenous selection pressures. As these substitutions do neither impair the viral replication efficiency nor susceptibility to LAM or ADV in vitro, rtQ215 substitutions likely represent background polymorphisms rather than resistance mutations with clinical implications.
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Beale MA, Ijaz S, Tedder RS. The genetic backbone modulates the phenotype of hepatitis B surface antigen mutants. J Gen Virol 2009; 91:68-73. [PMID: 19759242 DOI: 10.1099/vir.0.013078-0] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Hepatitis B virus (HBV) vaccine and diagnostic escape mutants are a growing concern. The principle target of detection, hepatitis B surface antigen (HBsAg), encoded by S, is completely overlapped by the reverse transcriptase encoding P. With the increased incidence of nucleos(t)ide analogue resistance altering P, the concurrent impact on S must be assessed. HBV DNA from 59 HBsAg-positive plasma samples was sequenced across the polymerase/surface region and the amino acid sequence of HBsAg was inferred. ELISAs were formatted containing individually bound monoclonal antibodies directed against three discrete epitopes on HBsAg. Similar point mutations occurring in different genotypes were shown to influence epitope conformation differently, indicating that the genetic backbone is a major factor in predicting phenotype. C-terminal changes associated with antiviral resistance were found to modulate epitope profiles of HBsAg. Treatment options which may promote drug resistance should be avoided to both protect antiviral treatment and prevent facilitation of vaccine and diagnostic escape mutants.
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Affiliation(s)
- Mathew A Beale
- Blood Borne Virus Unit, Virus Reference Department, Centre for Infections, Health Protection Agency, London, UK
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Ginya H, Asahina J, Nakao R, Tamada Y, Takahashi M, Yohda M, Yatsuhashi H. Semi-quantitative discrimination of HBV mutants using allele-specific oligonucleotide hybridization with Handy Bio-Strand. J Biosci Bioeng 2009; 109:94-100. [PMID: 20129090 DOI: 10.1016/j.jbiosc.2009.06.023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2009] [Revised: 06/17/2009] [Accepted: 06/27/2009] [Indexed: 11/15/2022]
Abstract
The analysis of hepatitis B virus (HBV) mutations is important for understanding HBV progression and for deciding on appropriate clinical treatments. However, it is difficult to determine the quantitative abundance of various mutants in heterogeneous mixtures by conventional methods such as direct sequencing or the TaqMan assay. In this study, we investigated the possibility of using both allele-specific oligonucleotide hybridization (ASOH) and allele-specific oligonucleotide competitive hybridization (ASOCH) with the Handy Bio-Strand system for the quantitative identification of three well-defined HBV variants: the basal core promoter (BCP) mutations (nt1762 and nt1764), the pre-core (PC) mutation (nt1896), and variance at nt1858. Using standardized mixtures of wild-type and mutant DNA, optimal hybridization conditions for ASOH and ASOCH were determined. Next, the performance of these methods was evaluated using actual serum DNAs from HBV patients. Excellent reproducibility was obtained both in the analysis of internal positive controls and in the semi-quantitative categorization of heterogeneous viral mixtures into five abundance groups (0%, 25%, 50%, 75%, and 100% mutant virus). Combined with real-time PCR to determine the HBV viral load, this hybridization method offers a new tool with applications both in HBV clinical research and treatment.
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Affiliation(s)
- Harumi Ginya
- Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Naka-cho, Koganei, Japan.
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Tian JH, Zhao H, Cong X, Yan J, Du SC, Wei L. Evaluation of INNO-LiPA in detection of HBV drug resistance mutation. Shijie Huaren Xiaohua Zazhi 2009; 17:1366-1369. [DOI: 10.11569/wcjd.v17.i13.1366] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To assess the accuracy of INNO-LiPA HBV DR v2 for detecting hepatitis B virus (HBV) drug resistance.
METHODS: Sera from 237 chronic hepatitis B patients undergoing lamivudine or adefovir treatment were detected using INNO-LiPA and direct sequencing of the reverse transcriptase region of the HBV Polymerase gene.
RESULTS: Sequencing results were observed for 179/235 samples (76.1%) and 1293/1410 amino acid positions (91.7%). It showed high concordance with directing antiviral drug-resistance mutations, particularly for the sensitive detection of mixed drug-resistance mutations.
CONCLUSION: The INNO-LiPA HBV DR v2 appears to be useful for the rapid antiviral drug-resistance mutations assay of HBV.
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Yuen MF, Fung J, Wong DKH, Lai CL. Prevention and management of drug resistance for antihepatitis B treatment. THE LANCET. INFECTIOUS DISEASES 2009; 9:256-64. [PMID: 19324298 DOI: 10.1016/s1473-3099(09)70056-8] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Emergence of drug resistance in antiviral therapy for chronic hepatitis B negates treatment benefits. There is a lower chance for emergence of resistance for drugs with rapid and potent viral suppression and a high genetic barrier for resistant mutations. Measurement of viral load at 24 weeks' treatment to aid decision making is mandatory for patients receiving drugs that are associated with a higher resistance rate. Combination treatment with drugs that belong to different groups is associated with a lower chance of resistance. To ensure better control of viral replication in patients with drug resistance, the addition of another drug without an overlapping resistance profile should be given as early as possible, preferably at the time when genotypic resistance emerges. With such strategies, most patients can be maintained in clinical remission. However, because of the mechanism of viral persistence, research efforts should continue to anticipate and prevent the emergence of multidrug-resistant strains.
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Affiliation(s)
- Man-Fung Yuen
- Department of Medicine, University of Hong Kong, Queen Mary Hospital, Hong Kong
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36
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Jardi R, Rodriguez-Frias F, Tabernero D, Homs M, Schaper M, Esteban R, Buti M. Use of the novel INNO-LiPA line probe assay for detection of hepatitis B virus variants that confer resistance to entecavir therapy. J Clin Microbiol 2009; 47:485-488. [PMID: 19052182 PMCID: PMC2643676 DOI: 10.1128/jcm.01678-08] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2008] [Revised: 10/08/2008] [Accepted: 11/22/2008] [Indexed: 02/07/2023] Open
Abstract
A line probe assay (INNO-LiPA DR, version 3) for the detection of hepatitis B virus mutations that confer resistance to entecavir therapy was evaluated. The INNO-LiPA DR assay is a highly sensitive assay that is easily applicable for the detection and monitoring of entecavir resistance-conferring mutations and is more sensitive than sequencing for the detection of mixed sequences.
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Affiliation(s)
- Rosendo Jardi
- Departments of Biochemistry, Hospital Universitario Vall d'Hebron, Barcelona, Spain.
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37
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Osiowy C, Gordon D, Borlang J, Giles E, Villeneuve JP. Hepatitis B virus genotype G epidemiology and co-infection with genotype A in Canada. J Gen Virol 2009; 89:3009-3015. [PMID: 19008387 DOI: 10.1099/vir.0.2008/005124-0] [Citation(s) in RCA: 65] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Hepatitis B virus (HBV) genotype G (HBV/G) is an unusual variant, and little is known about its epidemiology and natural history, particularly the requirement for a co-infecting HBV genotype and their relationship during infection. This study investigated the quasispecies nature of co-infecting genotypes in 39 samples collected over a 6 year period from 13 HBV/G-infected patients. HBV/G infections were found to occur predominantly in males (92 %) and were primarily associated with male homosexual sex (67 %). All patients were infected with HBV/G and HBV/A, or a recombinant HBV/A/G strain. Co-infecting genotypic prevalence was often observed to fluctuate over time, with periods of HBV/G monoinfection in some patients. The average sequence divergence among Canadian HBV/G strains was 1.57+/-0.62 %. Thus, all HBV/G infections in Canada occur in the context of co-infection or recombination with HBV/A, and strains display increased sequence divergence compared with all known HBV/G sequences described to date.
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Affiliation(s)
- Carla Osiowy
- Section of Hepatology, Department of Internal Medicine, University of Manitoba, Winnipeg, MB, Canada.,Bloodborne Pathogens and Hepatitis, National Microbiology Laboratory, Winnipeg, MB, Canada
| | - Diane Gordon
- Bloodborne Pathogens and Hepatitis, National Microbiology Laboratory, Winnipeg, MB, Canada
| | - Jamie Borlang
- Bloodborne Pathogens and Hepatitis, National Microbiology Laboratory, Winnipeg, MB, Canada
| | - Elizabeth Giles
- Bloodborne Pathogens and Hepatitis, National Microbiology Laboratory, Winnipeg, MB, Canada
| | - Jean-Pierre Villeneuve
- Hôpital Saint Luc du Centre Hospitalier, Universitaire de Montréal, Montréal, QC, Canada
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38
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Panessa C, Hill WD, Giles E, Yu A, Harvard S, Butt G, Andonov A, Krajden M, Osiowy C. Genotype D amongst injection drug users with acute hepatitis B virus infection in British Columbia. J Viral Hepat 2009; 16:64-73. [PMID: 19192159 DOI: 10.1111/j.1365-2893.2008.01045.x] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
The eight genotypes of hepatitis B virus (HBV) exhibit distinct geographical distributions. This study identified HBV genotypes and transmission modes associated with acute infection in British Columbia (BC), Canada, from 2001 to 2005. Seventy cases of acute HBV in BC were identified from laboratory reports using a standardized case definition. Interviews for risk factors and hepatitis history were conducted for each case. HBV genotypes were determined by BLAST comparison analysis of the surface (S) or preS gene sequence. To illustrate the distribution of genotypes identified amongst acute cases in BC, an annotated map was produced showing the global occurrence of HBV genotypes. The majority of acute HBV cases occurred in Caucasian, Canadian-born males, with 30% of cases reporting injection drug use (IDU) and 21% reporting incarceration. The most common genotype observed was genotype D (62.9%), followed by genotypes A (18.6%), C (11.4%), B (4.3%), and E (1.4%). A significant association was observed between Genotype D and IDU (P = 0.0025) and previous incarceration (P = 0.0067). Phylogenetic analysis of the S gene sequence demonstrated identical or high genetic relatedness amongst genotype D viral strains (86% sub-genotype D3), thus verifying transmission clustering amongst BC injection drug users. The association between acute HBV genotype and reported transmission modes has not been previously described in North America. Tracking of genotypes can help identify disease transmission patterns and target at-risk populations for preventive immunization.
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Affiliation(s)
- C Panessa
- BC Centre for Disease Control, Vancouver, BC, Canada
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39
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Degertekin B, Hussain M, Tan J, Oberhelman K, Lok AS. Sensitivity and accuracy of an updated line probe assay (HBV DR v.3) in detecting mutations associated with hepatitis B antiviral resistance. J Hepatol 2009; 50:42-8. [PMID: 19019484 DOI: 10.1016/j.jhep.2008.08.020] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/18/2008] [Revised: 07/18/2008] [Accepted: 08/25/2008] [Indexed: 02/06/2023]
Abstract
BACKGROUND/AIMS Early detection of antiviral drug-resistant mutations enables prompt initiation of rescue therapy. The aim of this study was to determine the accuracy and sensitivity of a new line probe assay in the detection of antiviral drug-resistant HBV mutations. METHODS One-hundred samples from 54 patients with virologic breakthrough during entecavir, lamivudine or adefovir treatment and 21 samples from 21 nucleoside-naïve patients were tested by direct sequencing and an updated line probe assay (Innogenetics, HBV DR v.3) which incorporates probes that can detect mutations at 11 positions of the reverse transcriptase region of the HBV polymerase gene. RESULTS Complete concordance between line probe and sequencing results was observed for 90/121 samples (74.3%) and 1291/1331 amino acid positions (96.9%). Testing of follow-up samples and clonal analysis of discordant samples confirmed the presence of mutations where line probe assay but not direct sequencing detected mutations. HBV DR v.3 assay consistently detected mutations present in > or = 5% of the virus population when HBV DNA concentration was > or = 4 log10copies/mL. CONCLUSIONS The updated version of the line probe assay (HBV DR v.3) has high concordance with direct sequencing in detecting antiviral drug-resistant mutations but its sensitivity in detecting mutations at some positions needs to be improved.
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Affiliation(s)
- Bulent Degertekin
- Division of Gastroenterology, University of Michigan Medical Center, 3912 Taubman Center, SPC 5362, Ann Arbor, MI 48109, USA
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40
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Chevaliez S, Pawlotsky JM. Diagnosis and management of chronic viral hepatitis: antigens, antibodies and viral genomes. Best Pract Res Clin Gastroenterol 2008; 22:1031-48. [PMID: 19187865 DOI: 10.1016/j.bpg.2008.11.004] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Virological tools, including serological and molecular tools, are needed to diagnose chronic hepatitis B and C infections. They may also be useful to establish their prognosis, but they have found their principal application in guiding treatment decisions and assessing the virological responses to therapy. The goal of chronic hepatitis B therapy is to prevent progression of liver disease. This is achieved if HBV replication is durably abolished or significantly reduced. In HBeAg-positive patients, HBeAg clearance followed by the HBe seroconversion phase can be achieved. In HBeAg-negative patients, long-term antiviral suppression of viral replication is needed. The loss of HBsAg, eventually associated with an HBs seroconversion, is the most desirable endpoint of therapy but is rarely achieved. The efficacy endpoint of chronic hepatitis C treatment is the sustained virological response, defined by an undetectable HCV RNA in serum with a sensitive assay 24 weeks after the end of treatment. The HCV genotype and on-treatment viral kinetics can be used to tailor treatment dosages and duration.
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Affiliation(s)
- Stéphane Chevaliez
- Department of Virology & INSERM U955, French National Reference Centre for Viral Hepatitis B, C and delta, Hôpital Henri Mondor, Université Paris, Créteil, France
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41
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Abstract
Both serologic and molecular tools are useful for the diagnosis, monitoring and therapeutic management of viral hepatitis associated with hepatitis B virus (HBV). Compared with the standard tests to detect viral antigens and the antibodies directed against them, real-time PCR techniques today make it possible to quantify viral DNA more sensitively and more precisely. They thus replace the techniques previously used in most virology laboratories. New markers, such as the HBV genotype or the amino-acid substitution profile associated with HBV resistance to nucleoside and nucleotide analogs (NRTIs), can also be characterized by techniques based on sequencing or reverse hybridization. There is not currently any consensus about the first-line treatment for chronic HBV infection. Patients with a wild-type virus (HBe antigen-positive), a moderate viral load, and elevated serum alanine aminotransferase (from 2 to 5 times the upper limit of normal) are good candidates for treatment with pegylated interferon-alpha. Patients who are HBe antigen-positive and do not respond to this first-line treatment, as well as patients with chronic e-antigen-negative hepatitis B, are candidates for prolonged treatment, probably lifelong, by nucleoside or nucleotide analogs. Whatever the HBe serologic status and treatment, the efficacy of antiviral treatment is assessed by repeated measurements of viral load and serum alanine aminotransferase levels, in principle, every 12-24 months. In patients who are HBe antigen-positive, the efficacy of antiviral treatment is shown by the loss of HBe antigen, followed by the appearance of anti-HBe antibodies (HBe seroconversion), a reduction in viral load below 2x10(4)IU/mL and normalization of serum aminotransferase. In patients who are HBe antigen-negative or HBe antigen-positive but do not seroconvert after short-term treatment and who receive nucleoside or nucleotide analogs, the treatment objective is for HBV DNA to become undetectable by real-time PCR. Nonetheless, this objective is not always met, and it is recommended that viral replication be maintained at the lowest possible level for the longest possible time (ideally, lifelong). In all cases, when a virologic response to treatment is observed (significant reduction in viral load) and is followed by a relapse characterized by a viral load augmentation of at least 1 Log(10)IU/mL relative to the nadir, viral resistance to treatment must be suspected, after treatment adherence has been verified.
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Affiliation(s)
- J-M Pawlotsky
- Centre National de Référence des Hépatites B, C et delta, Laboratoire de Virologie, INSERM U841, Hôpital Henri Mondor, Université Paris 12, 51, avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France.
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42
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Ijaz S, Arnold C, Dervisevic S, Mechurova J, Tatman N, Tedder RS, Naoumov NV. Dynamics of lamivudine-resistant hepatitis B virus during adefovir monotherapy versus lamivudine plus adefovir combination therapy. J Med Virol 2008; 80:1160-70. [PMID: 18461609 DOI: 10.1002/jmv.21206] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Adefovir dipivoxil has been used alone or together with lamivudine to suppress lamivudine-resistant hepatitis B virus (HBV). However, the dynamics of HBV populations under different selection pressures and their impact on treatment outcome are poorly understood. Pyrosequencing was applied to quantify longitudinally the evolution of wild type and lamivudine/adefovir-resistant HBV. Eight patients, with lamivudine-resistant HBV, were randomized to receive adefovir monotherapy or adefovir/lamivudine combination therapy for a median of 79 and 71 weeks, respectively. Pyrosequencing proved highly sensitive with a lower limit of quantitation of minor HBV populations of 2% irrespective of viraemia levels. Adefovir/lamivudine treatment resulted in greater viraemia reduction than adefovir monotherapy. During combination therapy, lamivudine-resistant HBV populations (codons 180 and 204) remained dominant (>90%) and no adefovir-resistance developed. During adefovir monotherapy, reversion to wild-type HBV was detected in two patients with one patient accumulating rapidly adefovir-resistant HBV along with increased viraemia. In conclusion, the dynamics of drug-resistant HBV strains vary under different selection pressures which have a significant impact on the success of rescue therapy, as well as for the selection of new mutations. The use of techniques such as Pyrosequencing provides an evidence-based approach for successful management of drug-resistant HBV.
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Affiliation(s)
- Samreen Ijaz
- Virus Reference Department, Centre for Infections, Health Protection Agency, London, United Kingdom.
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43
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Villet S, Pichoud C, Billioud G, Barraud L, Durantel S, Trépo C, Zoulim F. Impact of hepatitis B virus rtA181V/T mutants on hepatitis B treatment failure. J Hepatol 2008; 48:747-55. [PMID: 18331765 DOI: 10.1016/j.jhep.2008.01.027] [Citation(s) in RCA: 160] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/31/2007] [Revised: 12/20/2007] [Accepted: 01/07/2008] [Indexed: 12/17/2022]
Abstract
BACKGROUND/AIMS Recent clinical observations reported the occurrence of amino acid substitutions at position 181 of the HBV polymerase, associated with a viral breakthrough under lamivudine or adefovir therapy. In this study, we characterized the main variants harboring the rtA181T/V mutation isolated from 10 consecutive patients who developed lamivudine and/or adefovir resistance. METHODS We performed a clonal analysis of the HBV polymerase gene amplified by PCR from serum samples during viral breakthrough. The main mutants were then tested after transfection of Huh7 cells for their resistance profile to nucleoside analogs. RESULTS Clonal analysis revealed the co-localization on the same HBV genome of rtA181T/V with rtN236T, but not with rtM204V/I mutations following lamivudine, adefovir or lamivudine+adefovir breakthrough. In cell culture, the rtA181T/V mutation induced a decreased susceptibility to lamivudine (<10-fold), adefovir (2- to 8-fold) and tenofovir (2- to 3-fold). Interestingly, the association of rtA181T with rtN236T on one clinical isolate genome increased the resistance to these three drugs. All the tested mutants remained sensitive to entecavir. CONCLUSIONS Our observations suggest that a single amino acid change at position rt181 may induce cross-resistance to lamivudine and adefovir. These data emphasize the clinical relevance of genotypic and phenotypic analysis in the management of antiviral drug resistance.
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Affiliation(s)
- Stéphanie Villet
- INSERM U871, Laboratoire des virus hépatiques et pathologies associées, 151 cours Albert Thomas, Lyon cedex 03, France
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44
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Larrat S, Hilleret MN, Germi R, Lupo J, Nicod S, Zarski JP, Seigneurin JM, Morand P. Sustained virological and biochemical responses to lamivudine and adefovir dipivoxil combination in a chronic hepatitis B infection despite mutations conferring resistance to both drugs. COMPARATIVE HEPATOLOGY 2008; 7:3. [PMID: 18336716 PMCID: PMC2322943 DOI: 10.1186/1476-5926-7-3] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/22/2007] [Accepted: 03/12/2008] [Indexed: 02/06/2023]
Abstract
Background Sequential monotherapies of nucleotide analogs used in chronic hepatitis B treatment can lead to the selection of a resistance mutation to each antiviral drug. Case presentation A patient with chronic hepatitis B was successively treated with lamivudine monotherapy, lamivudine-adefovir dual therapy, adefovir monotherapy and again with an adefovir-lamivudine dual therapy. Lamivudine-associated mutations (rtL180M and rtM204V/I) followed by adefovir-associated mutations (rtN236T and rtA181V) emerged during the two monotherapy regimens. Despite the presence of rtM204V/I, rtA181V, and rtN236T mutations at the beginning of the second dual therapy, sustained biochemical and virological responses have been observed thus far after 23 months. Conclusion This case illustrates that rtM204V/I, rtA181V, and rtN236T resistance mutations can coexist in a patient but do not preclude the recycling of lamivudine and adefovir in combination therapy, when no other therapeutic choices are available.
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Affiliation(s)
- Sylvie Larrat
- Laboratoire de Virologie moléculaire et structurale, CHU de Grenoble BP 217, 38043 Grenoble cedex 09, France.
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45
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Férir G, Kaptein S, Neyts J, De Clercq E. Antiviral treatment of chronic hepatitis B virus infections: the past, the present and the future. Rev Med Virol 2008; 18:19-34. [PMID: 17966115 DOI: 10.1002/rmv.554] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
A decade ago, standard therapy against chronic hepatitis B virus infections only consisted of lamivudine or IFN-alpha. Treatment with lamivudine and IFN has been compounded by, respectively, the emergence of drug-resistant virus strains and the appearance of serious side effects. In the last 10 years, hepatitis B treatment has made much progress. Several treatments are now licensed for the treatment of patients with chronic hepatitis B and others are under development. Here, we provide an overview of the potential and mode of action of anti-HBV agents that are currently available, and/or may become available in the near future. Foremost among these newer compounds are adefovir dipivoxil, entecavir and telbivudine.
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Affiliation(s)
- Geoffrey Férir
- Rega Institute for Medical Research, KULeuven, Leuven, Belgium
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46
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Pawlotsky JM, Dusheiko G, Hatzakis A, Lau D, Lau G, Liang TJ, Locarnini S, Martin P, Richman DD, Zoulim F. Virologic monitoring of hepatitis B virus therapy in clinical trials and practice: recommendations for a standardized approach. Gastroenterology 2008; 134:405-15. [PMID: 18242209 PMCID: PMC2676233 DOI: 10.1053/j.gastro.2007.11.036] [Citation(s) in RCA: 173] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/16/2007] [Accepted: 11/01/2007] [Indexed: 02/07/2023]
Abstract
Treatment of chronic hepatitis B virus (HBV) infection is aimed at suppressing viral replication to the lowest possible level, and thereby to halt the progression of liver disease and prevent the onset of complications. Two categories of drugs are used in HBV therapy: the interferons, including standard interferon alfa or pegylated interferon alfa, and specific nucleoside or nucleotide HBV inhibitors that target the reverse-transcriptase function of HBV-DNA polymerase. The reported results of clinical trials have used varying definitions of efficacy, failure, and resistance based on different measures of virologic responses. This article discusses HBV virologic markers and tests, and their optimal use both for planning and reporting clinical trials and in clinical practice.
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Affiliation(s)
- Jean-Michel Pawlotsky
- French National Reference Center for Viral Hepatitis B, C and delta, Department of Virology, Henri Mondor Hospital, University of Paris XII, Créteil, France.
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47
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Abstract
The global prevalence of chronic hepatitis B and its associated serious sequelae demand technologically advanced techniques of management. Nucleic acid testing (NAT) plays a key role in the diagnosis, surveillance, and treatment of chronic hepatitis B. NAT includes quantitative PCR-based HBV DNA assays, HBV genotyping, tests for mutations associated with resistance to antiviral medications, and assays to detect precore and core promoter mutations. This article reviews the uses of NAT in the diagnosis and management of chronic hepatitis B.
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Affiliation(s)
- Robert G Gish
- California Pacific Medical Center, 2340 Clay St., Room 223, San Francisco, CA 94115, USA.
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48
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Libbrecht E, Doutreloigne J, Van De Velde H, Yuen MF, Lai CL, Shapiro F, Sablon E. Evolution of primary and compensatory lamivudine resistance mutations in chronic hepatitis B virus-infected patients during long-term lamivudine treatment, assessed by a line probe assay. J Clin Microbiol 2007; 45:3935-41. [PMID: 17913933 PMCID: PMC2168556 DOI: 10.1128/jcm.00020-07] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
With the availability of more potent nucleotide/nucleoside analogues, the early detection of drug-resistant mutants of hepatitis B virus (HBV) is important for the strategic treatment of chronic hepatitis B. We studied 336 serum samples from 80 patients chronically infected with HBV who were receiving lamivudine treatment for the presence of lamivudine resistance mutations at codons 80, 173, 180, and 204 of the HBV polymerase. The sequencing data were compared with the results generated with the INNO-LiPA HBV DR (drug resistance) v2 strip, a line probe assay (LiPA) covering wild-type and mutant motifs, for resistance mutations to lamivudine and adefovir dipivoxil. This method provided at least the same information as sequencing for 99.1% of all codons analyzed. On the basis of the LiPA results, 20 of 80 patients developed a lamivudine resistance mutation after 1 year. In all 20 patients, the mutation occurred in the YMDD motif at reverse transcriptase position 204 (rt204; M204V/I) either with or without the compensatory mutation at position rt180 (L180M). A compensatory mutation at position rt80 (L80V/I) was detected in half of these patients. After 36 months, a compensatory mutation was seen at position rt173 (V173L) in 3/15 patients. Time-to-event survival analysis indicated a 2.8 times greater chance for LiPA to detect a given mutation than sequencing at any moment in time (hazard ratio, 2.8, 95% confidence interval, 1.79, 4.41; P < 0.0001). These results demonstrate that a highly sensitive and specific assay such as the INNO-LiPA HBV DR v2 can precociously detect and monitor the emergence of primary and compensatory lamivudine resistance mutations in patients chronically infected with HBV and is more sensitive than sequencing.
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Affiliation(s)
- Evelien Libbrecht
- Innogenetics NV, Industriepark Zwijnaarde 7/4, B-9052, Ghent, Belgium
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49
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Valsamakis A. Molecular testing in the diagnosis and management of chronic hepatitis B. Clin Microbiol Rev 2007; 20:426-39, table of contents. [PMID: 17630333 PMCID: PMC1932755 DOI: 10.1128/cmr.00009-07] [Citation(s) in RCA: 102] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Hepatitis B virus (HBV) is an enveloped virus with a small (3.2-kb) partially double-stranded DNA genome that causes acute and chronic infections. The impact of these infections on public health worldwide is enormous, with an estimated prevalence of 2 billion acute infections and 360 million chronic infections globally. This review focuses on chronic hepatitis B and the molecular assays used in its diagnosis and management. Background information, including that about features of the hepatitis B virion, viral replication, and epidemiology of infection, that is important for understanding chronic hepatitis B and molecular diagnostic tests for HBV is provided. To facilitate an understanding of the utility of molecular testing for chronic hepatitis B, the four stages of chronic hepatitis B infection that are currently recognized, as well as an additional entity, occult hepatitis B, that can be diagnosed only by sensitive nucleic acid amplification methods, are reviewed in detail, including available therapeutic agents. The molecular diagnostic content focuses on tests for HBV DNA quantification, genotyping, and mutation detection (including precore/core promoter and antiviral resistance mutations). The discussion of these tests encompasses their current utility and performance characteristics, drawing from current clinical guidelines and other studies from the literature. In recognition of the continual evolution of this field, the final section describes emerging molecular markers with future diagnostic potential.
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Affiliation(s)
- Alexandra Valsamakis
- Department of Pathology, The Johns Hopkins Hospital, 600 North Wolfe St., Meyer B1-193, Baltimore, MD 21287, USA.
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50
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Villet S, Ollivet A, Pichoud C, Barraud L, Villeneuve JP, Trépo C, Zoulim F. Stepwise process for the development of entecavir resistance in a chronic hepatitis B virus infected patient. J Hepatol 2007; 46:531-8. [PMID: 17239478 DOI: 10.1016/j.jhep.2006.11.016] [Citation(s) in RCA: 126] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/19/2006] [Revised: 11/22/2006] [Accepted: 11/24/2006] [Indexed: 01/04/2023]
Abstract
BACKGROUND/AIMS Complex mutants may be selected under sequential anti-VHB pressures. We analyzed the genotypic and phenotypic evolution of the viral quasi-species of a patient who developed resistance to entecavir following lamivudine breakthrough. METHODS The polymerase gene was amplified, cloned and sequenced at different time points. Hepatoma cell lines were transfected to compare the replication capacity of HBV mutants and their drug susceptibility. RESULTS A mixture of lamivudine-resistant HBV strains coexisted following viral breakthrough to lamivudine, all harboring the rtM204V mutation. The rtV173L+L180M+M204V dominant mutant displayed strong lamivudine-resistance and the highest replication capacity. Following the switch to entecavir, the viral load dropped but the lamivudine-resistant strains continued to be selected. Three years later, the viral load rose again, and a complex mixture of entecavir-resistant strains, all harboring the lamivudine-resistance signature rtL180M+M204V and the rtS202G mutation were observed. Although the rtL180M+S202G+M204V variant, that prevailed at the end of entecavir therapy, did not show the highest viral genome replication capacity, it conferred one of the strongest resistance levels to entecavir. CONCLUSIONS We report the selection of complex HBV mutants that escaped lamivudine and entecavir antiviral pressures. Genotypic and phenotypic analysis provided additional information to understand the process of HBV variant selection.
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Affiliation(s)
- Stéphanie Villet
- INSERM, U271, Laboratoire des virus hépatiques et pathologies associées, Lyon F-69003, France
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