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Fateev IV, Sasmakov SA, Abdurakhmanov JM, Ziyaev AA, Khasanov SS, Eshboev FB, Ashirov ON, Frolova VD, Eletskaya BZ, Smirnova OS, Berzina MY, Arnautova AO, Abramchik YA, Kostromina MA, Kayushin AL, Antonov KV, Paramonov AS, Andronova VL, Galegov GA, Esipov RS, Azimova SS, Miroshnikov AI, Konstantinova ID. Synthesis of Substituted 1,2,4-Triazole-3-Thione Nucleosides Using E. coli Purine Nucleoside Phosphorylase. Biomolecules 2024; 14:745. [PMID: 39062460 PMCID: PMC11274511 DOI: 10.3390/biom14070745] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2024] [Revised: 06/18/2024] [Accepted: 06/19/2024] [Indexed: 07/28/2024] Open
Abstract
1,2,4-Triazole derivatives have a wide range of biological activities. The most well-known drug that contains 1,2,4-triazole as part of its structure is the nucleoside analogue ribavirin, an antiviral drug. Finding new nucleosides based on 1,2,4-triazole is a topical task. The aim of this study was to synthesize ribosides and deoxyribosides of 1,2,4-triazole-3-thione derivatives and test their antiviral activity against herpes simplex viruses. Three compounds from a series of synthesized mono- and disubstituted 1,2,4-triazole-3-thione derivatives were found to be substrates for E. coli purine nucleoside phosphorylase. Of six prepared nucleosides, the riboside and deoxyriboside of 3-phenacylthio-1,2,4-triazole were obtained at good yields. The yields of the disubstituted 1,2,4-triazol-3-thiones were low due to the effect of bulky substituents at the C3 and C5 positions on the selectivity of enzymatic glycosylation for one particular nitrogen atom in the triazole ring. The results of cytotoxic and antiviral studies on acyclovir-sensitive wild-type strain HSV-1/L2(TK+) and acyclovir-resistant strain (HSV-1/L2/RACV) in Vero E6 cell culture showed that the incorporation of a thiobutyl substituent into the C5 position of 3-phenyl-1,2,4-triazole results in a significant increase in the cytotoxicity of the base and antiviral activity. The highest antiviral activity was observed in the 3-phenacylthio-1-(β-D-ribofuranosyl)-1,2,4-triazole and 5-butylthio-1-(2-deoxy-β-D-ribofuranosyl)-3-phenyl-1,2,4-triazole nucleosides, with their selectivity indexes being significantly higher than that of ribavirin. It was also found that with the increasing lipophilicity of the nucleosides, the activity and toxicity of the tested compounds increased.
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Affiliation(s)
- Ilya V. Fateev
- Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, Russia; (V.D.F.); (B.Z.E.); (O.S.S.); (M.Y.B.); (A.O.A.); (Y.A.A.); (M.A.K.); (A.L.K.); (K.V.A.); (A.S.P.); (R.S.E.); (A.I.M.); (I.D.K.)
| | - Sobirdjan A. Sasmakov
- Acad. S.Yu. Yunusov Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Mirzo Ulugbek Str. 77, 100170 Tashkent, Uzbekistan; (J.M.A.); (A.A.Z.); (Sh.Sh.K.); (F.B.E.); (O.N.A.); (S.S.A.)
| | - Jaloliddin M. Abdurakhmanov
- Acad. S.Yu. Yunusov Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Mirzo Ulugbek Str. 77, 100170 Tashkent, Uzbekistan; (J.M.A.); (A.A.Z.); (Sh.Sh.K.); (F.B.E.); (O.N.A.); (S.S.A.)
| | - Abdukhakim A. Ziyaev
- Acad. S.Yu. Yunusov Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Mirzo Ulugbek Str. 77, 100170 Tashkent, Uzbekistan; (J.M.A.); (A.A.Z.); (Sh.Sh.K.); (F.B.E.); (O.N.A.); (S.S.A.)
| | - Shukhrat Sh. Khasanov
- Acad. S.Yu. Yunusov Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Mirzo Ulugbek Str. 77, 100170 Tashkent, Uzbekistan; (J.M.A.); (A.A.Z.); (Sh.Sh.K.); (F.B.E.); (O.N.A.); (S.S.A.)
| | - Farkhod B. Eshboev
- Acad. S.Yu. Yunusov Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Mirzo Ulugbek Str. 77, 100170 Tashkent, Uzbekistan; (J.M.A.); (A.A.Z.); (Sh.Sh.K.); (F.B.E.); (O.N.A.); (S.S.A.)
| | - Oybek N. Ashirov
- Acad. S.Yu. Yunusov Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Mirzo Ulugbek Str. 77, 100170 Tashkent, Uzbekistan; (J.M.A.); (A.A.Z.); (Sh.Sh.K.); (F.B.E.); (O.N.A.); (S.S.A.)
| | - Valeriya D. Frolova
- Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, Russia; (V.D.F.); (B.Z.E.); (O.S.S.); (M.Y.B.); (A.O.A.); (Y.A.A.); (M.A.K.); (A.L.K.); (K.V.A.); (A.S.P.); (R.S.E.); (A.I.M.); (I.D.K.)
| | - Barbara Z. Eletskaya
- Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, Russia; (V.D.F.); (B.Z.E.); (O.S.S.); (M.Y.B.); (A.O.A.); (Y.A.A.); (M.A.K.); (A.L.K.); (K.V.A.); (A.S.P.); (R.S.E.); (A.I.M.); (I.D.K.)
| | - Olga S. Smirnova
- Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, Russia; (V.D.F.); (B.Z.E.); (O.S.S.); (M.Y.B.); (A.O.A.); (Y.A.A.); (M.A.K.); (A.L.K.); (K.V.A.); (A.S.P.); (R.S.E.); (A.I.M.); (I.D.K.)
| | - Maria Ya. Berzina
- Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, Russia; (V.D.F.); (B.Z.E.); (O.S.S.); (M.Y.B.); (A.O.A.); (Y.A.A.); (M.A.K.); (A.L.K.); (K.V.A.); (A.S.P.); (R.S.E.); (A.I.M.); (I.D.K.)
| | - Alexandra O. Arnautova
- Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, Russia; (V.D.F.); (B.Z.E.); (O.S.S.); (M.Y.B.); (A.O.A.); (Y.A.A.); (M.A.K.); (A.L.K.); (K.V.A.); (A.S.P.); (R.S.E.); (A.I.M.); (I.D.K.)
| | - Yulia A. Abramchik
- Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, Russia; (V.D.F.); (B.Z.E.); (O.S.S.); (M.Y.B.); (A.O.A.); (Y.A.A.); (M.A.K.); (A.L.K.); (K.V.A.); (A.S.P.); (R.S.E.); (A.I.M.); (I.D.K.)
| | - Maria A. Kostromina
- Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, Russia; (V.D.F.); (B.Z.E.); (O.S.S.); (M.Y.B.); (A.O.A.); (Y.A.A.); (M.A.K.); (A.L.K.); (K.V.A.); (A.S.P.); (R.S.E.); (A.I.M.); (I.D.K.)
| | - Alexey L. Kayushin
- Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, Russia; (V.D.F.); (B.Z.E.); (O.S.S.); (M.Y.B.); (A.O.A.); (Y.A.A.); (M.A.K.); (A.L.K.); (K.V.A.); (A.S.P.); (R.S.E.); (A.I.M.); (I.D.K.)
| | - Konstantin V. Antonov
- Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, Russia; (V.D.F.); (B.Z.E.); (O.S.S.); (M.Y.B.); (A.O.A.); (Y.A.A.); (M.A.K.); (A.L.K.); (K.V.A.); (A.S.P.); (R.S.E.); (A.I.M.); (I.D.K.)
| | - Alexander S. Paramonov
- Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, Russia; (V.D.F.); (B.Z.E.); (O.S.S.); (M.Y.B.); (A.O.A.); (Y.A.A.); (M.A.K.); (A.L.K.); (K.V.A.); (A.S.P.); (R.S.E.); (A.I.M.); (I.D.K.)
| | - Valeria L. Andronova
- D. I. Ivanovsky Institute of Virology (N. F. Gamaleya Research Center of Epidemiology and Microbiology, Ministry of Healthcare of the Russian Federation), Gamaleya St. 18, 123098 Moscow, Russia; (V.L.A.); (G.A.G.)
| | - Georgiy A. Galegov
- D. I. Ivanovsky Institute of Virology (N. F. Gamaleya Research Center of Epidemiology and Microbiology, Ministry of Healthcare of the Russian Federation), Gamaleya St. 18, 123098 Moscow, Russia; (V.L.A.); (G.A.G.)
| | - Roman S. Esipov
- Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, Russia; (V.D.F.); (B.Z.E.); (O.S.S.); (M.Y.B.); (A.O.A.); (Y.A.A.); (M.A.K.); (A.L.K.); (K.V.A.); (A.S.P.); (R.S.E.); (A.I.M.); (I.D.K.)
| | - Shakhnoz S. Azimova
- Acad. S.Yu. Yunusov Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Mirzo Ulugbek Str. 77, 100170 Tashkent, Uzbekistan; (J.M.A.); (A.A.Z.); (Sh.Sh.K.); (F.B.E.); (O.N.A.); (S.S.A.)
| | - Anatoly I. Miroshnikov
- Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, Russia; (V.D.F.); (B.Z.E.); (O.S.S.); (M.Y.B.); (A.O.A.); (Y.A.A.); (M.A.K.); (A.L.K.); (K.V.A.); (A.S.P.); (R.S.E.); (A.I.M.); (I.D.K.)
| | - Irina D. Konstantinova
- Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, Russia; (V.D.F.); (B.Z.E.); (O.S.S.); (M.Y.B.); (A.O.A.); (Y.A.A.); (M.A.K.); (A.L.K.); (K.V.A.); (A.S.P.); (R.S.E.); (A.I.M.); (I.D.K.)
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Reingardt DE, Ostankova YV, Lyalina LV, Anufrieva EV, Semenov AV, Totolian AA. Distribution of hepatitis С virus drug resistance mutations among patients with recurrence of the disease during therapy with direct antiviral drugs. HIV INFECTION AND IMMUNOSUPPRESSIVE DISORDERS 2024; 15:86-93. [DOI: 10.22328/2077-9828-2023-15-4-86-93] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2025]
Abstract
The aim of the study was to identify the prevalence of drug resistance mutations in the hepatitis C virus among patients with relapse of the disease on therapy with direct antiviral drugs.Materials and methods. The study material included 31 blood plasma samples from patients with chronic hepatitis C with relapse of the disease on therapy with direct antiviral drugs. Samples were screened for the presence of HCV RNA. In case of detection of HCV RNA, amplification was carried out using a set of primers jointly flanking the NS3, NS5A, NS5B genes. After sequencing the nucleotide sequences of these genes, the subtype of the virus was determined and drug resistance mutations were identified.Results and discussion. The age of the patients ranged from 33 to 62 and averaged 45.8±8.38 years. The number of men in the group prevailed compared to women — 21 (67%) and 10 (33%), respectively. Viral load determination results ranged from 3.1×103 to 4.2×107 IU/ml. The distribution of genotypes was as follows: 1a — 26% (n=8), 1b — 29% (n=9), 3a — 45% (n=14). The nucleotide sequence of the NS3, NS5A, NS5B regions was determined in all samples. Mutations associated with drug resistance were detected in 87% (n=27). In all identified cases, the mutations resulted in viral resistance to at least one drug included in the patient’s current treatment regimen. In one patient, amino acid substitutions were found in three regions at once, which led to the emergence of resistance to two drugs in the regimen.Conclusion. Conducting a preliminary examination of patients to identify mutations of drug resistance to direct antiviral drugs can affect the effectiveness of the planned treatment and the choice of the optimal regimen.
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Affiliation(s)
| | | | | | | | - A. V. Semenov
- State Scientific Center of Virology and Biotechnology «Vector»
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Cancela F, Rendon-Marin S, Quintero-Gil C, Houston DR, Gumbis G, Panzera Y, Pérez R, Arbiza J, Mirazo S. Modelling of Hepatitis E virus RNA-dependent RNA polymerase genotype 3 from a chronic patient and in silico interaction analysis by molecular docking with Ribavirin. J Biomol Struct Dyn 2023; 41:705-721. [PMID: 34861797 DOI: 10.1080/07391102.2021.2011416] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Hepatitis E Virus (HEV) infection is an emergent zoonotic disease, where chronic hepatitis E associated to solid organ transplant (SOT) recipients, related to genotype 3, is the clinical manifestation of major concern. In this setting, ribavirin (RBV) treatment is the only available therapy, though drug-resistant variants could emerge leading to a therapeutic failure. Crystallographic structures have not been reported for most of the HEV proteins, including the RNA-polymerase (RdRp). Therefore, the mechanism of action of RBV against HEV and the molecular interactions between this drug and RdRp are largely unknown. In this work, we aimed to model in silico the 3 D structure of a novel HEV3 RdRp (HEV_C1_Uy) from a chronically HEV infected-SOT recipient treated with RBV and to perform a molecular docking simulation between RBV triphosphate (RBVT), 7-methyl-guanosine-5'-triphosphate and the modelled protein. The models were generated using I-TASSER server and validated with multiple bioinformatics tools. The docking analysis were carried out with AutoDock Vina and LeDock software. We obtained a suitable model for HEV_C1_Uy (C-Score=-1.33, RMSD = 10.4 ± 4.6 Å). RBVT displayed a binding affinity of -7.6 ± 0.2 Kcal/mol by molecular docking, mediated by 6 hydrogen-bonds (Q195-O14, S198-O11, E257-O13, S260-O2, O3, S311-O11) between the finger's-palm-domains and a free binding energy of 31.26 ± 16.81 kcal/mol by molecular dynamics simulations. We identified the possible HEV RdRp interacting region for incoming nucleotides or analogs and provide novel insights that will contribute to better understand the molecular interactions of RBV and the enzyme and the mechanism of action of this antiviral drug.Communicated by Ramaswamy H. Sarma.
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Affiliation(s)
- Florencia Cancela
- Sección Virología, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
| | - Santiago Rendon-Marin
- Grupo de Investigación en Ciencias Animales - GRICA, Facultad de Medicina Veterinaria y Zootecnia, Universidad Cooperativa de Colombia, sede Bucaramanga, Bucaramanga, Colombia
| | - Carolina Quintero-Gil
- Grupo de Investigación en Ciencias Animales - GRICA, Facultad de Medicina Veterinaria y Zootecnia, Universidad Cooperativa de Colombia, sede Bucaramanga, Bucaramanga, Colombia
| | - Douglas R Houston
- Institute of Quantitative Biology, Biochemistry and Biotechnology, The University of Edinburgh, Edinburgh, UK
| | - Gediminas Gumbis
- Institute of Quantitative Biology, Biochemistry and Biotechnology, The University of Edinburgh, Edinburgh, UK
| | - Yanina Panzera
- Sección Genética Evolutiva, Instituto de Biología, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
| | - Ruben Pérez
- Sección Genética Evolutiva, Instituto de Biología, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
| | - Juan Arbiza
- Sección Virología, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
| | - Santiago Mirazo
- Sección Virología, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay.,Departamento de Bacteriología y Virología, Instituto de Higiene, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
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Atypical Mutational Spectrum of SARS-CoV-2 Replicating in the Presence of Ribavirin. Antimicrob Agents Chemother 2023; 67:e0131522. [PMID: 36602354 PMCID: PMC9872624 DOI: 10.1128/aac.01315-22] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
We report that ribavirin exerts an inhibitory and mutagenic activity on SARS-CoV-2-infecting Vero cells, with a therapeutic index higher than 10. Deep sequencing analysis of the mutant spectrum of SARS-CoV-2 replicating in the absence or presence of ribavirin indicated an increase in the number of mutations, but not in deletions, and modification of diversity indices, expected from a mutagenic activity. Notably, the major mutation types enhanced by replication in the presence of ribavirin were A→G and U→C transitions, a pattern which is opposite to the dominance of G→A and C→U transitions previously described for most RNA viruses. Implications of the inhibitory activity of ribavirin, and the atypical mutational bias produced on SARS-CoV-2, for the search for synergistic anti-COVID-19 lethal mutagen combinations are discussed.
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Mechanisms and Consequences of Genetic Variation in Hepatitis C Virus (HCV). Curr Top Microbiol Immunol 2023; 439:237-264. [PMID: 36592248 DOI: 10.1007/978-3-031-15640-3_7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Chronic infection with hepatitis C virus (HCV) is an important contributor to the global incidence of liver diseases, including liver cirrhosis and hepatocellular carcinoma. Although common for single-stranded RNA viruses, HCV displays a remarkable high level of genetic diversity, produced primarily by the error-prone viral polymerase and host immune pressure. The high genetic heterogeneity of HCV has led to the evolution of several distinct genotypes and subtypes, with important consequences for pathogenesis, and clinical outcomes. Genetic variability constitutes an evasion mechanism against immune suppression, allowing the virus to evolve epitope escape mutants that avoid immune recognition. Thus, heterogeneity and variability of the HCV genome represent a great hindrance for the development of vaccines against HCV. In addition, the high genetic plasticity of HCV allows the virus to rapidly develop antiviral resistance mutations, leading to treatment failure and potentially representing a major hindrance for the cure of chronic HCV patients. In this chapter, we will present the central role that genetic diversity has in the viral life cycle and epidemiology of HCV. Incorporation errors and recombination, both the result of HCV polymerase activity, represent the main mechanisms of HCV evolution. The molecular details of both mechanisms have been only partially clarified and will be presented in the following sections. Finally, we will discuss the major consequences of HCV genetic diversity, namely its capacity to rapidly evolve antiviral and immunological escape variants that represent an important limitation for clearance of acute HCV, for treatment of chronic hepatitis C and for broadly protective vaccines.
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Younas S, Sumrin A, Hussain N, Bilal M. Identification of NS5B Resistance against SOFOSBUVIR in Hepatitis C Virus Genotype 3a, naive and treated Patients. J Appl Microbiol 2022; 133:2826-2834. [PMID: 35916643 DOI: 10.1111/jam.15754] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2022] [Revised: 07/28/2022] [Accepted: 07/29/2022] [Indexed: 11/29/2022]
Abstract
AIMS Pakistan has the second highest prevalence of HCV with genotype 3a (GT-3a) being the most frequently circulating genotype. Currently resistance associated substitutions (RASs) are a major challenge in HCV treatment with direct acting antivirals (DAAs). Sofosbuvir (SOF) is an FDA-approved NS5B nucleotide inhibitor. The aim of this study was to identify these RASs in the NS5B gene in naive and treated Pakistani HCV 3a isolates against SOF. METHODS AND RESULTS Blood samples were collected from anti-HCV positive patients, followed by HCV RNA isolation and real time PCR quantification. HCV positive patients were processed for HCV RNA genotyping, Patients with genotype 3a were processed for NS5B gene amplification and sequencing. GT-3a was the most prevalent genotype (62.2%). S282T was identified in 2 (8.7%) patients, C316Y/G/R in 3 (13%), V321A, and L320P in 1 (4.3%) each in SOF/RBV resistant patients. Variants of S282 were detected in 3 (13%) of SOF/RBV treated patients. While INF/RBV associated mutations were also analyzed, D244N, A333R, and A334E were identified in 2 (9.5%), 3 (14.2%), and 7 (33.3%) in treatment-naive and 15 (65.2%), 7 (30.4%), and 5 (21.7%) treated patients respectively. Q309R was observed only in one treatment experienced patients. Some substitutions were present at higher frequency in both groups like N307G, K304R, A272D and R345H, considered that they do not have any role in Sofosbuvir resistance. CONCLUSION It was concluded that Sofosbuvir RASs are present in Pakistani HCV GT-3a isolates, and they should be monitored carefully, especially in treatment-experienced patients, for further selection of treatment regimens. SIGNIFICANCE AND IMPACT OF STUDY HCV RASs have been studied very well across the world but there is scarcity of data regarding this topic in Pakistani population, this study provides data regarding prevalence of these RASs in Pakistani HCV isolates emphasizing the fact that these RASs must be carefully monitored before starting HCV treatment especially in treatment failure patients.
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Affiliation(s)
- Saima Younas
- Centre for Applied Molecular Biology (CAMB), University of the Punjab Lahore, Pakistan
| | - Aleena Sumrin
- Centre for Applied Molecular Biology (CAMB), University of the Punjab Lahore, Pakistan
| | - Nazim Hussain
- Centre for Applied Molecular Biology (CAMB), University of the Punjab Lahore, Pakistan
| | - Muhammad Bilal
- School of Life Science and Food Engineering, Huaiyin Institute of Technology, Huaian, China
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Valutite D, Ostankova Y, Semenov A, Lyalina L, Totolian A. Distribution of Primary Resistance Mutations in Saint Petersburg in Patients with Chronic Hepatitis C. Diagnostics (Basel) 2022; 12:diagnostics12051054. [PMID: 35626210 PMCID: PMC9139387 DOI: 10.3390/diagnostics12051054] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2022] [Revised: 04/18/2022] [Accepted: 04/21/2022] [Indexed: 11/17/2022] Open
Abstract
The advent of direct-acting antiviral drugs (DAAs) was a breakthrough in the treatment of patients with chronic hepatitis C, yet high viral replication errors can lead to the development of resistance associated variants (RAVs). Thus, assessment of RAV in infected patients is necessary to monitor treatment effectiveness. The aim of our study was to investigate the presence of primary resistance mutations in the NS3 and NS5 regions of HCV in treatment-naive patients. Samples were taken from 42 patients with HCV who had not previously received DAA treatment. In the present study, we used the method for determining drug resistance mutations based on direct sequencing of the NS3, NS5A, and NS5B genes developed by the Saint Petersburg Pasteur Institute. Primary mutations associated with resistance were detected in 5 patients (12%). According to the Geno2pheno [hcv] 0.92 database, nucleotide substitutions were identified in various viral genes conferring resistance or decreased sensitivity to the respective inhibitors. This study has shown different mutations in the analyzed genes in patients with HCV who had not previously received DAA treatment. These mutations may increase the likelihood of treatment failure in the future.
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Affiliation(s)
- Diana Valutite
- Saint Petersburg Pasteur Institute, 197101 St. Petersburg, Russia; (Y.O.); (L.L.); (A.T.)
- Correspondence: ; Tel.: +7-95-2395-8965
| | - Yulia Ostankova
- Saint Petersburg Pasteur Institute, 197101 St. Petersburg, Russia; (Y.O.); (L.L.); (A.T.)
| | - Alexandr Semenov
- FSBI State Scientific Center of Virology and Biotechnology «Vector» of the Federal Service for Surveillance of Consumer Rights Protection and Human Welfare (Rospotrebnadzor), 620030 Ekaterinburg, Russia;
| | - Liudmila Lyalina
- Saint Petersburg Pasteur Institute, 197101 St. Petersburg, Russia; (Y.O.); (L.L.); (A.T.)
| | - Areg Totolian
- Saint Petersburg Pasteur Institute, 197101 St. Petersburg, Russia; (Y.O.); (L.L.); (A.T.)
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Sabariegos R, Ortega-Prieto AM, Díaz-Martínez L, Grande-Pérez A, García Crespo C, Gallego I, de Ávila AI, Albentosa-González L, Soria ME, Gastaminza P, Domingo E, Perales C, Mas A. Guanosine inhibits hepatitis C virus replication and increases indel frequencies, associated with altered intracellular nucleotide pools. PLoS Pathog 2022; 18:e1010210. [PMID: 35085375 PMCID: PMC8794218 DOI: 10.1371/journal.ppat.1010210] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2021] [Accepted: 12/16/2021] [Indexed: 12/30/2022] Open
Abstract
In the course of experiments aimed at deciphering the inhibition mechanism of mycophenolic acid and ribavirin in hepatitis C virus (HCV) infection, we observed an inhibitory effect of the nucleoside guanosine (Gua). Here, we report that Gua, and not the other standard nucleosides, inhibits HCV replication in human hepatoma cells. Gua did not directly inhibit the in vitro polymerase activity of NS5B, but it modified the intracellular levels of nucleoside di- and tri-phosphates (NDPs and NTPs), leading to deficient HCV RNA replication and reduction of infectious progeny virus production. Changes in the concentrations of NTPs or NDPs modified NS5B RNA polymerase activity in vitro, in particular de novo RNA synthesis and template switching. Furthermore, the Gua-mediated changes were associated with a significant increase in the number of indels in viral RNA, which may account for the reduction of the specific infectivity of the viral progeny, suggesting the presence of defective genomes. Thus, a proper NTP:NDP balance appears to be critical to ensure HCV polymerase fidelity and minimal production of defective genomes.
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Affiliation(s)
- Rosario Sabariegos
- Laboratorio de Virología Molecular, Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Albacete, Spain
- Facultad de Medicina, Universidad de Castilla-La Mancha, Albacete, Spain
- Unidad de Biomedicina UCLM-CSIC, Albacete, Spain
| | - Ana María Ortega-Prieto
- Centro de Biología Molecular “Severo Ochoa”, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), Campus de Cantoblanco, Madrid, Spain
| | - Luis Díaz-Martínez
- Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora," Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHMS-UMA-CSIC), Málaga, Spain
- Área de Genética, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain
| | - Ana Grande-Pérez
- Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora," Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHMS-UMA-CSIC), Málaga, Spain
- Área de Genética, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain
| | - Carlos García Crespo
- Centro de Biología Molecular “Severo Ochoa”, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), Campus de Cantoblanco, Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
| | - Isabel Gallego
- Centro de Biología Molecular “Severo Ochoa”, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), Campus de Cantoblanco, Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
| | - Ana I. de Ávila
- Centro de Biología Molecular “Severo Ochoa”, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), Campus de Cantoblanco, Madrid, Spain
| | - Laura Albentosa-González
- Laboratorio de Virología Molecular, Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Albacete, Spain
| | - María Eugenia Soria
- Centro de Biología Molecular “Severo Ochoa”, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), Campus de Cantoblanco, Madrid, Spain
- Department of Clinical Microbiology, IIS-Fundación Jiménez Díaz, UAM, Madrid, Spain
| | - Pablo Gastaminza
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
- Department of Cellular and Molecular Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, Madrid, Spain
| | - Esteban Domingo
- Unidad de Biomedicina UCLM-CSIC, Albacete, Spain
- Centro de Biología Molecular “Severo Ochoa”, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), Campus de Cantoblanco, Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
- * E-mail: (AM); (CP); (ED)
| | - Celia Perales
- Centro de Biología Molecular “Severo Ochoa”, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), Campus de Cantoblanco, Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
- Department of Clinical Microbiology, IIS-Fundación Jiménez Díaz, UAM, Madrid, Spain
- * E-mail: (AM); (CP); (ED)
| | - Antonio Mas
- Laboratorio de Virología Molecular, Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Albacete, Spain
- Unidad de Biomedicina UCLM-CSIC, Albacete, Spain
- Facultad de Farmacia, Universidad de Castilla-La Mancha, Albacete, Spain
- * E-mail: (AM); (CP); (ED)
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9
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Adeboyejo K, Grosche VR, José DP, Ferreira GM, Shimizu JF, King BJ, Tarr AW, Soares MMCN, Ball JK, McClure CP, Jardim ACG. Simultaneous determination of HCV genotype and NS5B resistance associated substitutions using dried serum spots from São Paulo state, Brazil. Access Microbiol 2022; 4:000326. [PMID: 35693474 PMCID: PMC9175972 DOI: 10.1099/acmi.0.000326] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2021] [Accepted: 12/30/2021] [Indexed: 11/26/2022] Open
Abstract
Hepatitis C virus (HCV) is responsible for more than 180 million infections worldwide, and about 80 % of infections are reported in Low and Middle-income countries (LMICs). Therapy is based on the administration of interferon (INF), ribavirin (RBV) or more recently Direct-Acting Antivirals (DAAs). However, amino acid substitutions associated with resistance (RAS) have been extensively described and can contribute to treatment failure, and diagnosis of RAS requires considerable infrastructure, not always locally available. Dried serum spots (DSS) sampling is an alternative specimen collection method, which embeds drops of serum onto filter paper to be transported by posting to a centralized laboratory. Here, we assessed feasibility of genotypic analysis of HCV from DSS in a cohort of 80 patients from São Paulo state Brazil. HCV RNA was detected on DSS specimens in 83 % of samples of HCV infected patients. HCV genotypes 1a, 1b, 2a, 2c and 3a were determined using the sequence of the palm domain of NS5B region, and RAS C316N/Y, Q309R and V321I were identified in HCV 1b samples. Concerning therapy outcome, 75 % of the patients who used INF +RBV as a previous protocol of treatment did not respond to DAAs, and 25 % were end-of-treatment responders. It suggests that therapy with INF plus RBV may contribute for non-response to a second therapeutic protocol with DAAs. One patient that presented RAS (V321I) was classified as non-responder, and combination of RAS C316N and Q309R does not necessarily imply in resistance to treatment in this cohort of patients. Data presented herein highlights the relevance of studying circulating variants for a better understanding of HCV variability and resistance to the therapy. Furthermore, the feasibility of carrying out genotyping and RAS phenotyping analysis by using DSS card for the potential of informing future treatment interventions could be relevant to overcome the limitations of processing samples in several location worldwide, especially in LMICs.
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Affiliation(s)
- Kazeem Adeboyejo
- NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust and the University of Nottingham, Nottingham, UK.,School of Life Sciences, University of Nottingham, Nottingham, UK
| | - Victória Riquena Grosche
- Institute of Biomedical Sciences, Federal University of Uberlândia, Uberlândia, Minas Gerais, Brazil.,Institute of Bioscience, Language and Exact Sciences, São Paulo State University, São José do Rio Preto, São Paulo, Brazil
| | | | - Giulia Magalhães Ferreira
- Institute of Biomedical Sciences, Federal University of Uberlândia, Uberlândia, Minas Gerais, Brazil
| | - Jacqueline Farinha Shimizu
- Institute of Biomedical Sciences, Federal University of Uberlândia, Uberlândia, Minas Gerais, Brazil.,Institute of Bioscience, Language and Exact Sciences, São Paulo State University, São José do Rio Preto, São Paulo, Brazil
| | - Barnabas J King
- NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust and the University of Nottingham, Nottingham, UK.,School of Life Sciences, University of Nottingham, Nottingham, UK.,MRC/EPSRC Nottingham Molecular Pathology Node, University of Nottingham, Nottingham, UK
| | - Alexander W Tarr
- NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust and the University of Nottingham, Nottingham, UK.,School of Life Sciences, University of Nottingham, Nottingham, UK.,MRC/EPSRC Nottingham Molecular Pathology Node, University of Nottingham, Nottingham, UK
| | | | - Jonathan K Ball
- NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust and the University of Nottingham, Nottingham, UK.,School of Life Sciences, University of Nottingham, Nottingham, UK.,MRC/EPSRC Nottingham Molecular Pathology Node, University of Nottingham, Nottingham, UK
| | - C Patrick McClure
- NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust and the University of Nottingham, Nottingham, UK.,School of Life Sciences, University of Nottingham, Nottingham, UK.,MRC/EPSRC Nottingham Molecular Pathology Node, University of Nottingham, Nottingham, UK
| | - Ana Carolina Gomes Jardim
- Institute of Biomedical Sciences, Federal University of Uberlândia, Uberlândia, Minas Gerais, Brazil.,Institute of Bioscience, Language and Exact Sciences, São Paulo State University, São José do Rio Preto, São Paulo, Brazil
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10
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Population Disequilibrium as Promoter of Adaptive Explorations in Hepatitis C Virus. Viruses 2021; 13:v13040616. [PMID: 33916702 PMCID: PMC8067247 DOI: 10.3390/v13040616] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2021] [Revised: 03/24/2021] [Accepted: 03/30/2021] [Indexed: 02/07/2023] Open
Abstract
Replication of RNA viruses is characterized by exploration of sequence space which facilitates their adaptation to changing environments. It is generally accepted that such exploration takes place mainly in response to positive selection, and that further diversification is boosted by modifications of virus population size, particularly bottleneck events. Our recent results with hepatitis C virus (HCV) have shown that the expansion in sequence space of a viral clone continues despite prolonged replication in a stable cell culture environment. Diagnosis of the expansion was based on the quantification of diversity indices, the occurrence of intra-population mutational waves (variations in mutant frequencies), and greater individual residue variations in mutant spectra than those anticipated from sequence alignments in data banks. In the present report, we review our previous results, and show additionally that mutational waves in amplicons from the NS5A-NS5B-coding region are equally prominent during HCV passage in the absence or presence of the mutagenic nucleotide analogues favipiravir or ribavirin. In addition, by extending our previous analysis to amplicons of the NS3- and NS5A-coding region, we provide further evidence of the incongruence between amino acid conservation scores in mutant spectra from infected patients and in the Los Alamos National Laboratory HCV data banks. We hypothesize that these observations have as a common origin a permanent state of HCV population disequilibrium even upon extensive viral replication in the absence of external selective constraints or changes in population size. Such a persistent disequilibrium—revealed by the changing composition of the mutant spectrum—may facilitate finding alternative mutational pathways for HCV antiviral resistance. The possible significance of our model for other genetically variable viruses is discussed.
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11
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El-Sokkary MMA, Gotina L, Al-Sanea MM, Pae AN, Elbargisy RM. Molecular Characterization of Hepatitis C Virus for Developed Antiviral Agents Resistance Mutations and New Insights into in-silico Prediction Studies. Infect Drug Resist 2020; 13:4235-4248. [PMID: 33262618 PMCID: PMC7696641 DOI: 10.2147/idr.s267809] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2020] [Accepted: 09/24/2020] [Indexed: 02/05/2023] Open
Abstract
Background Identification and characterization of developed antiviral drug resistance mutations are key to the success of antiviral therapies against hepatitis C virus (HCV), which remains a worldwide highly prevalent pathogenic disease. Although most studies focus on HCV genotypes 1, 2 or 3, the investigation of drug resistance in HCV genotype 4, predominant in North Africa, is especially significant in Egypt. Methods We performed mutational and genotypic analysis of the untranslated region (UTR) and nonstructural protein 5B (NS5B) drug resistance-associated regions of HCV for patients in the surrounding villages of Mansoura city, who were not responding to different antiviral treatments (sofosbuvir (SOF), ribavirin, and interferon). Furthermore, molecular modelling approaches (homology modelling and docking studies) were used to investigate the significance of the identified NS5B mutations for SOF and ribavirin binding in the HCV genotype 4a NS5B active site. Results Genotypic analysis confirmed all samples to have genotype 4 with sub-genotype 4a predominant. Partial sequencing of the UTR and NS5B resistance-associated regions identified D258E, T282S and A307G mutations in all isolates of NS5B. The UTR mutation site at position 243 was associated with interferon resistance, whereas the NS5B T282S mutation was considered as significant for SOF and ribavirin resistance. Docking studies in the HCV genotype 4a homology model predict SOF and ribavirin to accommodate a nucleotide-like binding mode, in which the T282 residue does interfere with the binding as it would in HCV genotypes 1 and 2. Mutation energy calculations predict T282S to moderately destabilize the binding of SOF and ribavirin by 0.57 and 0.47 kcal/mol, respectively. Conclusion The performed study identified and characterized several antiviral drug resistance mutations of HCV genotype 4a and proposed a mechanism by which the T282S mutation may contribute to SOF and ribavirin resistance.
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Affiliation(s)
- Mohamed M Adel El-Sokkary
- Department of Microbiology and Immunology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt
| | - Lizaveta Gotina
- Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology, Seongbuk-gu, Seoul 02792, Republic of Korea.,Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology (UST), Daejeon, Korea
| | - Mohammad M Al-Sanea
- Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Sakaka, Al-Jouf, Saudi Arabia
| | - Ae Nim Pae
- Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology, Seongbuk-gu, Seoul 02792, Republic of Korea.,Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology (UST), Daejeon, Korea
| | - Rehab Mohammed Elbargisy
- Department of Microbiology and Immunology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt.,Department of Pharmaceutics, College of Pharmacy, Jouf University, Sakaka, Al-Jouf, Saudi Arabia
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12
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Soria ME, García-Crespo C, Martínez-González B, Vázquez-Sirvent L, Lobo-Vega R, de Ávila AI, Gallego I, Chen Q, García-Cehic D, Llorens-Revull M, Briones C, Gómez J, Ferrer-Orta C, Verdaguer N, Gregori J, Rodríguez-Frías F, Buti M, Esteban JI, Domingo E, Quer J, Perales C. Amino Acid Substitutions Associated with Treatment Failure for Hepatitis C Virus Infection. J Clin Microbiol 2020; 58:e01985-20. [PMID: 32999010 PMCID: PMC7685896 DOI: 10.1128/jcm.01985-20] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2020] [Accepted: 09/21/2020] [Indexed: 02/07/2023] Open
Abstract
Despite the high virological response rates achieved with current directly acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral response. The identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultradeep sequencing (UDS) methods, for HCV characterization and patient management. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV, which are not bona fide resistance-associated substitutions (RAS), from 220 patients who failed therapy. They were present frequently in basal and posttreatment virus of patients who failed different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. They also have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value for treatment response, are discussed.
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Affiliation(s)
- María Eugenia Soria
- Department of Clinical Microbiology, IIS-Fundación Jiménez Díaz, Madrid, Spain
- Liver Unit, Internal Medicine Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain
- Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain
| | - Carlos García-Crespo
- Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain
| | - Brenda Martínez-González
- Department of Clinical Microbiology, IIS-Fundación Jiménez Díaz, Madrid, Spain
- Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain
| | - Lucía Vázquez-Sirvent
- Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain
| | - Rebeca Lobo-Vega
- Department of Clinical Microbiology, IIS-Fundación Jiménez Díaz, Madrid, Spain
- Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain
| | - Ana Isabel de Ávila
- Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain
| | - Isabel Gallego
- Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
| | - Qian Chen
- Liver Unit, Internal Medicine Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
| | - Damir García-Cehic
- Liver Unit, Internal Medicine Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
| | - Meritxell Llorens-Revull
- Liver Unit, Internal Medicine Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
| | - Carlos Briones
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
- Centro de Astrobiología (CAB, CSIC-INTA), Torrejón de Ardoz, Madrid, Spain
| | - Jordi Gómez
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
- Instituto de Parasitología y Biomedicina 'López-Neyra' (CSIC), Parque Tecnológico Ciencias de la Salud, Armilla, Granada, Spain
| | - Cristina Ferrer-Orta
- Structural Biology Department, Institut de Biología Molecular de Barcelona CSIC, Barcelona, Spain
| | - Nuria Verdaguer
- Structural Biology Department, Institut de Biología Molecular de Barcelona CSIC, Barcelona, Spain
| | - Josep Gregori
- Liver Unit, Internal Medicine Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
- Roche Diagnostics, S.L., Barcelona, Spain
| | - Francisco Rodríguez-Frías
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
- Biochemistry and Microbiology Departments, VHIR-HUVH, Barcelona, Spain
| | - María Buti
- Liver Unit, Internal Medicine Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
| | - Juan Ignacio Esteban
- Liver Unit, Internal Medicine Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
| | - Esteban Domingo
- Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
| | - Josep Quer
- Liver Unit, Internal Medicine Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
| | - Celia Perales
- Department of Clinical Microbiology, IIS-Fundación Jiménez Díaz, Madrid, Spain
- Liver Unit, Internal Medicine Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain
- Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
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Mutations Identified in the Hepatitis C Virus (HCV) Polymerase of Patients with Chronic HCV Treated with Ribavirin Cause Resistance and Affect Viral Replication Fidelity. Antimicrob Agents Chemother 2020; 64:AAC.01417-20. [PMID: 32928732 DOI: 10.1128/aac.01417-20] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2020] [Accepted: 09/07/2020] [Indexed: 12/11/2022] Open
Abstract
Ribavirin has been used for 25 years to treat patients with chronic hepatitis C virus (HCV) infection; however, its antiviral mechanism of action remains unclear. Here, we studied virus evolution in a subset of samples from a randomized 24-week trial of ribavirin monotherapy versus placebo in chronic HCV patients, as well as the viral resistance mechanisms of the observed ribavirin-associated mutations in cell culture. Thus, we performed next-generation sequencing of the full-length coding sequences of HCV recovered from patients at weeks 0, 12, 20, 32 and 40 and analyzed novel single nucleotide polymorphisms (SNPs), diversity, and mutation-linkage. At week 20, increased genetic diversity was observed in 5 ribavirin-treated compared to 4 placebo-treated HCV patients due to new synonymous SNPs, particularly G-to-A and C-to-U ribavirin-associated transitions. Moreover, emergence of 14 nonsynonymous SNPs in HCV nonstructural 5B (NS5B) occurred in treated patients, but not in placebo controls. Most substitutions located close to the NS5B polymerase nucleotide entry site. Linkage analysis showed that putative resistance mutations were found in the majority of genomes in ribavirin-treated patients. Identified NS5B mutations from genotype 3a patients were further introduced into the genotype 3a cell-culture-adapted DBN strain for studies in Huh7.5 cells. Specific NS5B substitutions, including DBN-D148N+I363V, DBN-A150V+I363V, and DBN-T227S+S183P, conferred resistance to ribavirin in long-term cell culture treatment, possibly by reducing the HCV polymerase error rate. In conclusion, prolonged exposure of HCV to ribavirin in chronic hepatitis C patients induces NS5B resistance mutations leading to increased polymerase fidelity, which could be one mechanism for ribavirin resistance.
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14
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Primary HCV Drug Resistance Mutations in Patients with Newly Diagnosed HIV Infection. PROBLEMS OF PARTICULARLY DANGEROUS INFECTIONS 2020. [DOI: 10.21055/0370-1069-2020-3-97-105] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Objective of our work was to assess prevalence of the primary HCV drug resistance mutations in the NS5b gene in patients with newly diagnosed HIV infection.Materials and methods. The study material was 196 blood plasma samples from patients living in the North-Western Federal District with newly diagnosed HIV. Samples were examined for the anti-HCV antibodies and HCV RNA presence. If HCV RNA was detected, amplification was performed using three primers pairs that co-flanked the NS5b gene. After sequencing the indicated gene nucleotide sequence, the virus subtype was determined and drug resistance mutations were detected.Results and discussion. Antibodies to HCV were detected in 18.87 % of HIV-infected individuals. HCV RNA was detected in 18.36 % of the patients, including 89.18 % anti-HCV-positive and 1.88 % anti-HCV-negative. It was shown that co-infection is more common in men (77.8 %) compared to women (22.2 %) – χ2 = 3.996 at p = 0.0456, df = 2. The difference in the HIV viral load between the groups with HIV monoinfection and with HIV + HCV coinfection was demonstrated (χ2 = 6.284 at p = 0.0432, df = 2). A significant difference between the groups by the CD4 + lyphocytes number was shown. In the phylogenetic analysis, the HCV subtypes are distributed as follows: HCV 1b – 47.2 %, HCV 3a – 30.6 %, HCV 1a – 13.9 %, HCV 2a – 5.5 % and only one sample was defined as HCV 2k – 2.8 %, respectively. Nine samples (25 %) presented NS5b mutations in the positions related to the development of drug resistance of HCV, including two samples among HCV genotypes 1a and 3a (i.e., 5.6 % of the total HIV + HCV group), as well as five samples among HCV 1b (13.9 % of the total group). Mutations among HCV 1a were C316Y and N444D substitutions. Among HCV 1b, C316N, C451S, S556N/G substitutions were identified. Among patients with HCV 3a, 2 samples (5.6 %) with a D310N mutation associated with an unfavorable disease prognosis were found. The introduction of direct sequencing of HCV nucleotide sequences into the routine laboratory diagnostics will allow us to estimate the primary drug resistance mutations prevalence in risk groups to predict the HCV life-threatening complications development – fibrosis, cirrhosis, hepatocellular carcinoma, as well as the outcome of antiviral therapy prognosis. The data obtained can be rationally used to assess the dynamics of the HCV primary pharmacoresistance prevalence among HIV-infected individuals.
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15
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El-Tahan RR, Ghoneim AM, Zaghloul H. Dissection of two drug-targeted regions of Hepatitis C virus subtype 4a infecting Egyptian patients. Virus Genes 2020; 56:564-581. [PMID: 32572756 PMCID: PMC7307947 DOI: 10.1007/s11262-020-01776-y] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2019] [Accepted: 06/16/2020] [Indexed: 12/11/2022]
Abstract
Recently, treatment of HCV infection has been improved after the development of direct acting antivirals (DAAs) which target different viral proteins (NS3-4A, NS5A and NS5B). The activity and effectiveness of these DAAs are affected by the presence of resistance associated substitutions (RASs). This study aimed to characterize HCV genotypes circulating among Egyptian HCV patients, to dissect the full sequences of HCV NS3-4A and NS5B regions, and to characterize RASs associated with NS3-4A and NS5B inhibitors in HCV treatment-naïve patients. Genotyping of 80 HCV samples from treatment-naïve patients was done using restriction fragment length polymorphism and phylogenetic analysis based on some full NS5B sequences. Results showed the prevalence of HCV subtype 4a. Twenty four new full sequences of NS3-4A and NS5B regions of subtype 4a were deposited in the GenBank database. In general, the substitutions associated with NS3-4A-targeting drugs were absent predicting possible responsiveness of Egyptian HCV patients to these drugs. In addition, the absence of amino acid substitutions associated with resistance to Sofosbuvir may predict good response to treatment with Sofosbuvir. Some amino acid substitutions associated with resistance to different classes of non-nucleoside inhibitors were detected. Further investigations on treated Egyptian HCV patients may evaluate the effectiveness of the massively used drugs. Many predicted T-cell-binding epitopes in NS3-4A and NS5B regions were found to be highly conserved in the currently studied isolates; a finding that might be important for HCV vaccine development. We demonstrated potential NS3 epitopes that could be used in engineering T cells against HCV epitopes.
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Affiliation(s)
- Radwa R. El-Tahan
- Zoology Department, Faculty of Science, Damietta University, New Damietta, P.O. 34517, Damietta, Egypt
| | - Ahmed M. Ghoneim
- Zoology Department, Faculty of Science, Damietta University, New Damietta, P.O. 34517, Damietta, Egypt
| | - Hosam Zaghloul
- Clinical Pathology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt
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16
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Diversity of the hepatitis C virus NS5B gene during HIV co-infection. PLoS One 2020; 15:e0237162. [PMID: 32750098 PMCID: PMC7402467 DOI: 10.1371/journal.pone.0237162] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2020] [Accepted: 07/20/2020] [Indexed: 01/06/2023] Open
Abstract
Viral diversity is an important feature of hepatitis C virus (HCV) infection and an important predictor of disease progression and treatment response. HIV/HCV co-infection is associated with enhanced HCV replication, increased fibrosis, and the development of liver disease. HIV also increases quasispecies diversity of HCV structural genes, although limited data are available regarding the impact of HIV on non-structural genes of HCV, particularly in the absence of direct-acting therapies. The genetic diversity and presence of drug resistance mutations within the RNA-dependent RNA polymerase (NS5B) gene were examined in 3 groups of women with HCV genotype 1a infection, including those with HCV mono-infection, antiretroviral (ART)-naïve women with HIV/HCV co-infection and CD4 cell count <350 cells/mm3, and ART-naïve women with HIV/HCV co-infection and CD4 cell count ≥350 cells/mm3. None had ever been treated for HCV infection. There was evidence of significant diversity across the entire NS5B gene in all women. There were several nucleotides and amino acids with distinct distributions across the three study groups, although no obvious clustering of NS5B sequences was observed based on HIV co-infection or CD4 cell count. Polymorphisms at amino acid positions associated with resistance to dasabuvir and sofosbuvir were limited, although the Q309R variant associated with ribavirin resistance was present in 12 individuals with HCV mono-infection, 8 HIV/HCV co-infected individuals with CD4 <350 cells/mm3, and 12 HIV/HCV co-infected individuals with CD4 ≥350 cells/mm3. Previously reported fitness altering mutations were rare. CD8+ T cell responses against the human leukocyte antigen (HLA) B57-restricted epitopes NS5B2629-2637 and NS5B2936-2944 are critical for HCV control and were completely conserved in 44 (51.8%) and 70 (82.4%) study participants. These data demonstrate extensive variation across the NS5B gene. Genotypic variation may have a profound impact on HCV replication and pathogenesis and deserves careful evaluation.
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17
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Feasibility of Known RNA Polymerase Inhibitors as Anti-SARS-CoV-2 Drugs. Pathogens 2020; 9:pathogens9050320. [PMID: 32357471 PMCID: PMC7281371 DOI: 10.3390/pathogens9050320] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2020] [Revised: 04/23/2020] [Accepted: 04/24/2020] [Indexed: 12/22/2022] Open
Abstract
Coronaviruses (CoVs) are positive-stranded RNA viruses that infect humans and animals. Infection by CoVs such as HCoV-229E, -NL63, -OC43 and -HKU1 leads to the common cold, short lasting rhinitis, cough, sore throat and fever. However, CoVs such as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and the newest SARS-CoV-2 (the causative agent of COVID-19) lead to severe and deadly diseases with mortality rates ranging between ~1 to 35% depending on factors such as age and pre-existing conditions. Despite continuous global health threats to humans, there are no approved vaccines or drugs targeting human CoVs, and the recent outbreak of COVID-19 emphasizes an urgent need for therapeutic interventions. Using computational and bioinformatics tools, here we present the feasibility of reported broad-spectrum RNA polymerase inhibitors as anti- SARS-CoV-2 drugs targeting its main RNA polymerase, suggesting that investigational and approved nucleoside RNA polymerase inhibitors have potential as anti-SARS-CoV-2 drugs. However, we note that it is also possible for SARS-CoV-2 to evolve and acquire drug resistance mutations against these nucleoside inhibitors.
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18
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Nitta S, Takahashi K, Kawai-Kitahata F, Tsuchiya J, Sato A, Miyoshi M, Murakawa M, Itsui Y, Nakagawa M, Azuma S, Kakinuma S, Watanabe M, Asahina Y. Time course alterations of virus sequences and immunoglobulin titers in a chronic hepatitis E patient. Hepatol Res 2020; 50:524-531. [PMID: 31883166 DOI: 10.1111/hepr.13480] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/06/2019] [Revised: 12/04/2019] [Accepted: 12/23/2019] [Indexed: 02/08/2023]
Abstract
AIM Hepatitis E virus (HEV) can cause chronic infection in immunocompromised hosts. However, the dynamics of HEV during persistent infection is not well understood. To elucidate time course alterations in virus sequences and anti-HEV antibodies during persistent infection, we analyzed the HEV sequences and titers of anti-HEV antibodies from a chronic hepatitis E patient. METHODS Serum samples were obtained from a chronic hepatitis E patient under corticosteroid therapy for neurological disease. The titers of anti-HEV antibodies (immunoglobulin A, immunoglobulin M, and immunoglobulin G) in serum samples were detected by enzyme immunoassay. The full or near-full nucleotide sequences of HEV isolated from consecutive serum samples were identified and compared. Phylogenetic analysis was also performed. RESULTS Alterations of anti-HEV antibodies from a chronic hepatitis E patient were different from those previously reported in acute hepatitis E patients. The virus sequence was unchanged in the period without treatment, but nucleotide mutations were observed after ribavirin treatment was started. In addition, the sequence of this strain had extremely high identity to that isolated from swine liver in Japan. CONCLUSIONS Virus mutations in HEV emerged after ribavirin treatment was started. Sequence analysis may useful for deciding the treatment strategy for chronic hepatitis E patients who did not eliminate the virus with 3 months of RBV treatment and inferring the origin of the infection. This report provides insights into the chronicity of hepatitis E, and the impact of persistent infection and ribavirin treatment on the emergence of virus mutations.
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Affiliation(s)
- Sayuri Nitta
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan
| | - Kazuaki Takahashi
- Department of Medical Sciences, Tokyo-Shinagawa Hospital, Tokyo, Japan.,Division of Infectious Diseases, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan
| | - Fukiko Kawai-Kitahata
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan
| | - Jun Tsuchiya
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan
| | - Ayako Sato
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan
| | - Masato Miyoshi
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan
| | - Miyako Murakawa
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan
| | - Yasuhiro Itsui
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan
| | - Mina Nakagawa
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan
| | - Seishin Azuma
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan
| | - Sei Kakinuma
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan.,Department of Liver Disease Control, Tokyo Medical and Dental University, Tokyo, Japan
| | - Mamoru Watanabe
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan.,TMDU Advanced Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
| | - Yasuhiro Asahina
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan.,Department of Liver Disease Control, Tokyo Medical and Dental University, Tokyo, Japan
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19
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20
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Saito Y, Imamura M, Uchida T, Osawa M, Teraoka Y, Fujino H, Nakahara T, Ono A, Murakami E, Kawaoka T, Miki D, Tsuge M, Serikawa M, Aikata H, Abe-Chayama H, Hayes CN, Chayama K. Ribavirin induces hepatitis C virus genome mutations in chronic hepatitis patients who failed to respond to prior daclatasvir plus asunaprevir therapy. J Med Virol 2019; 92:210-218. [PMID: 31584207 DOI: 10.1002/jmv.25602] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2019] [Accepted: 09/30/2019] [Indexed: 12/17/2022]
Abstract
Ribavirin (RBV) induces nucleotide (nt) substitutions in hepatitis C virus (HCV) genome nonstructural (NS) regions. Although emergence of drug resistance-associated variants is associated with direct-acting antiviral treatment failure, the effect of RBV on genome substitutions in such patients is unknown. Genotype 1b HCV subgenomic replicon cells were treated with RBV for 120 hours. Six patients with chronic genotype 1b with HCV-infected patients who failed to respond to prior daclatasvir plus asunaprevir (DCV/ASV) therapy were treated with 12 weeks of sofosbuvir and ledipasvir plus RBV after 4 weeks of RBV monotherapy. RBV-induced genome mutations in the HCV NS region (nt3493-9301) in replicon cells and in patients during 4 weeks of RBV monotherapy were analyzed by deep sequencing. RBV-associated G-to-A and C-to-U transitions increased in a dose-dependent manner in HCV replicon cells after the RBV treatment. In patients with prior DCV/ASV treatment failures, the median serum HCV RNA level was 6.25 ± 0.31 log IU/mL at the start of RBV therapy and decreased significantly to 5.95 ± 0.4 log IU/mL (P = .03) after 4 weeks of RBV monotherapy. Although predominant HCV genome substitutions rates were similar between nontreatment and RBV-treatment periods (0.042 and 0.031 per base pair, respectively; P = .248), the frequencies of G-to-A and C-to-U transitions significantly increased after RBV monotherapy. These transitions were enriched, particularly within the HCV NS3 region in all patients. RBV treatment induces G-to-A and C-to-U transitions in the HCV genome even in chronic patients with hepatitis C with prior DCV/ASV treatment failures.
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Affiliation(s)
- Yuhei Saito
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Michio Imamura
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Takuro Uchida
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Mitsutaka Osawa
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Yuji Teraoka
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Hatsue Fujino
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Takashi Nakahara
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Atsushi Ono
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Eisuke Murakami
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Tomokazu Kawaoka
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Daiki Miki
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Masataka Tsuge
- Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan.,Natural Science Center for Basic Research and Development, Hiroshima University, Hiroshima, Japan
| | - Masahiro Serikawa
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Hiroshi Aikata
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Hiromi Abe-Chayama
- Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan.,Center for Medical Specialist Graduate Education and Research, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - C Nelson Hayes
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Kazuaki Chayama
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
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21
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Synergistic lethal mutagenesis of hepatitis C virus. Antimicrob Agents Chemother 2019:AAC.01653-19. [PMID: 31570400 DOI: 10.1128/aac.01653-19] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Lethal mutagenesis is an antiviral approach that consists in extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagenic agent, often a nucleotide analogue. One of its advantages is its broad spectrum nature that renders the strategy potentially effective against emergent RNA viral infections. Here we describe synergistic lethal mutagenesis of hepatitis C virus (HCV) by a combination of favipiravir (T-705) and ribavirin. Synergy has been documented over a broad range of analogue concentrations using the Chou-Talalay method as implemented in the CompuSyn graphics, with average dose reduction index (DRI) above 1 (68.02±101.6 for favipiravir, and 5.83±6.07 for ribavirin), and average combination indices (CI) below 1 (0.52±0.28). Furthermore, analogue concentrations that individually did not extinguish high fitness HCV in ten serial infections, when used in combination they extinguished high fitness HCV in one to two passages. Although both analogues display a preference for G→A and C→U transitions, deep sequencing analysis of mutant spectra indicated a different preference of the two analogues for the mutation sites, thus unveiling a new possible synergy mechanism in lethal mutagenesis. Prospects of synergy among mutagenic nucleotides as a strategy to confront emerging viral infections are discussed.
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22
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Espy N, Nagle E, Pfeffer B, Garcia K, Chitty AJ, Wiley M, Sanchez-Lockhart M, Bavari S, Warren T, Palacios G. T-705 induces lethal mutagenesis in Ebola and Marburg populations in macaques. Antiviral Res 2019; 170:104529. [PMID: 31195019 DOI: 10.1016/j.antiviral.2019.06.001] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2019] [Accepted: 06/02/2019] [Indexed: 10/26/2022]
Abstract
Nucleoside analogues (NA) disrupt RNA viral RNA-dependent RNA polymerase (RdRP) function and fidelity for multiple viral families. The mechanism of action (MOA) of T-705 has been attributed alternatively or concurrently to chain termination and lethal mutagenesis depending on the viral species during in vitro studies. In this study, we evaluated the effect of T-705 on the viral population in non-human primates (NHPs) after challenge with Ebola virus (EBOV) or Marburg virus (MARV) to identify the predominant in vivo MOA. We used common virological assays in conjunction with deep sequencing to characterize T-705 effects. T-705 exhibited antiviral activity that was associated with a reduction in specific infectivity and an accumulation of low frequency nucleotide variants in plasma samples collected day 7 post infection. Stranded analysis of deep sequencing data to identify chain termination demonstrated no change in the transcriptional gradient in negative stranded viral reads and minimal changes in positive stranded viral reads in T-705 treated animals, questioning as a MOA in vivo. These findings indicate that lethal mutagenesis is a MOA of T-705 that may serve as an indication of therapeutic activity of NAs for evaluation in clinical settings. This study expands our understanding of MOAs of these compounds for the Filovirus family and provides further evidence that lethal mutagenesis could be a preponderant MOA for this class of therapeutic compounds.
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Affiliation(s)
- Nicole Espy
- United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA
| | - Elyse Nagle
- United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA
| | - Brad Pfeffer
- United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA
| | - Karla Garcia
- United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA
| | - Alex J Chitty
- United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA
| | - Michael Wiley
- United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA
| | - Mariano Sanchez-Lockhart
- United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA
| | - Sina Bavari
- United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA
| | - Travis Warren
- United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA
| | - Gustavo Palacios
- United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA.
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23
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Resistance of high fitness hepatitis C virus to lethal mutagenesis. Virology 2018; 523:100-109. [PMID: 30107298 DOI: 10.1016/j.virol.2018.07.030] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2018] [Revised: 07/30/2018] [Accepted: 07/30/2018] [Indexed: 01/07/2023]
Abstract
Viral fitness quantifies the degree of virus adaptation to a given environment. How viral fitness can influence the mutant spectrum complexity of a viral quasispecies subjected to lethal mutagenesis has not been investigated. Here we document that two high fitness hepatitis C virus populations display higher resistance to the mutagenic nucleoside analogues favipiravir and ribavirin than their parental, low fitness HCV. All populations, however, exhibited a mutation transition bias indicative of active mutagenesis. Resistance to the analogues was associated with a limited expansion of mutant spectrum complexity, as evidenced by several diversity indices used to characterize mutant spectra. The results are consistent with a replicative site-drug competition mechanism that was previously proposed for HCV fitness-associated resistance to non-mutagenic inhibitors. Other alternative, non-mutually exclusive mechanisms are considered. The results introduce viral fitness as a relevant parameter to evaluate the response of viruses to lethal mutagenesis, with implications for antiviral designs.
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24
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Altraif IH, Sanai F, Babatin MA, Alalwan AA, Abdo AA, Alhamoudi W, Peedikayil M, Alghamdi H, Alsohaibani F, Alswat KA, Murtaza S, Alghamdi A, Altraif S, Aljumah A, Handoo FA, AlBekairy AM, Al-Ashgar HI, Alquaiz M, Alblawi MA, AlTamimi W, Loustaud-Ratti V, Marquett P. Pharmacokinetics-Based Adjusted Versus Standard Dose of Ribavirin Does Not Improve Virologic Response Rates in Chronic Hepatitis C Genotype 4 Patients: A Randomized Controlled Trial. J Interferon Cytokine Res 2018; 37:488-493. [PMID: 29135370 DOI: 10.1089/jir.2017.0054] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Optimal doses of Ribavirin (RBV) for hepatitis C virus (HCV) treatment are not known. To assess the safety and efficacy of PegIFNalfa-2a in combination with an adjusted (ADJ) RBV dose based on early pharmacokinetics versus a fixed standard (STD) dose of RBV in chronic HCV genotype (GT) 4-naive patients in a randomized trial. One hundred eighty-one patients were randomized. The baseline variables were similar in both arms and females were 50.3% of the patients, 76.5% had minimal-moderate fibrosis (F0-2). Sustained virologic response (SVR) was achieved in 99 (54.7%) subjects. SVR was seen in 50/90 (55.6%) of ADJ dose of RBV and 49/91 (53.9%) of STD dose subjects. Prematurely withdrawal or discontinuation of treatment prematurely in the ADJ RBV arm occurred in 11/90 patients (12.2%) compared with 6/91 subjects (6.6%) in the STD arm (P = 0.214). Similarly, virologic relapse was seen in 14/90 (15.6%) patients of the ADJ arm and 12/91 (13.2%) of the STD arm. Anemia grade 3-4 was seen in 36.7% in ADJ versus 17.6% in STD arm (P = 0.003). Occurrence of rapid virologic response and absences of F4 fibrosis predicted SVR in a univariate analysis. However, age, gender, weight, presence of diabetes, baseline alanine aminotransferase, and vitamin D levels were not significantly different in patients achieving SVR. ADJ higher doses of RBV based on its early pharmacokinetics-based RBV do not improve SVR rates in HCV GT4 treated in combination with peg-IFN alpha-2-a versus STD therapy. Patients on ADJ higher doses of RBV experienced higher rates of anemia and require more erythropoietin without increasing SVR.
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Affiliation(s)
- Ibrahim H Altraif
- 1 Division of Hepatology, Department of Organ Transplant and Hepatobiliary Sciences, Ministry of National Guard Health Affairs, King Abdullah International Medical Research Center/King Saud Bin Abdulaziz University for Health Sciences , Riyadh, Saudi Arabia
| | - Faisal Sanai
- 2 Gastroenterology Unit, Department of Medicine, King Abdulaziz Medical City , Jeddah, Saudi Arabia
| | - Mohammed A Babatin
- 3 Gastroenterology Unit, Department of Medicine, King Fahad General Hospital , Jeddah, Saudi Arabia
| | - Abduljaleel A Alalwan
- 1 Division of Hepatology, Department of Organ Transplant and Hepatobiliary Sciences, Ministry of National Guard Health Affairs, King Abdullah International Medical Research Center/King Saud Bin Abdulaziz University for Health Sciences , Riyadh, Saudi Arabia
| | - Ayman A Abdo
- 4 Liver Disease Research Center, College of Medicine, King Saud University , Riyadh, Saudi Arabia
| | - Waleed Alhamoudi
- 4 Liver Disease Research Center, College of Medicine, King Saud University , Riyadh, Saudi Arabia
| | - Musthafa Peedikayil
- 5 Gastroenterology Section, Department of Medicine, King Faisal Specialist Hospital and Research Center , Riyadh, Saudi Arabia
| | - Hamdan Alghamdi
- 1 Division of Hepatology, Department of Organ Transplant and Hepatobiliary Sciences, Ministry of National Guard Health Affairs, King Abdullah International Medical Research Center/King Saud Bin Abdulaziz University for Health Sciences , Riyadh, Saudi Arabia
| | - Fahad Alsohaibani
- 5 Gastroenterology Section, Department of Medicine, King Faisal Specialist Hospital and Research Center , Riyadh, Saudi Arabia
| | - Khalid A Alswat
- 4 Liver Disease Research Center, College of Medicine, King Saud University , Riyadh, Saudi Arabia
| | - Shazia Murtaza
- 1 Division of Hepatology, Department of Organ Transplant and Hepatobiliary Sciences, Ministry of National Guard Health Affairs, King Abdullah International Medical Research Center/King Saud Bin Abdulaziz University for Health Sciences , Riyadh, Saudi Arabia
| | - Abdullah Alghamdi
- 3 Gastroenterology Unit, Department of Medicine, King Fahad General Hospital , Jeddah, Saudi Arabia
| | - Sara Altraif
- 1 Division of Hepatology, Department of Organ Transplant and Hepatobiliary Sciences, Ministry of National Guard Health Affairs, King Abdullah International Medical Research Center/King Saud Bin Abdulaziz University for Health Sciences , Riyadh, Saudi Arabia
| | - Abdulrahman Aljumah
- 1 Division of Hepatology, Department of Organ Transplant and Hepatobiliary Sciences, Ministry of National Guard Health Affairs, King Abdullah International Medical Research Center/King Saud Bin Abdulaziz University for Health Sciences , Riyadh, Saudi Arabia
| | - Fayaz A Handoo
- 1 Division of Hepatology, Department of Organ Transplant and Hepatobiliary Sciences, Ministry of National Guard Health Affairs, King Abdullah International Medical Research Center/King Saud Bin Abdulaziz University for Health Sciences , Riyadh, Saudi Arabia
| | - Abdulkareem M AlBekairy
- 6 Department of Pharmaceutical Care, Ministry of National Guard Health Affairs, King Abdullah International Medical Research Center/King Saud Bin Abdulaziz University for Health Sciences , Riyadh, Saudi Arabia
| | - Hamad I Al-Ashgar
- 7 Gastroenterology Section, Department of Medicine, King Faisal Specialist Hospital and Research Center, Alfaisal University , Riyadh, Saudi Arabia
| | - Mohammed Alquaiz
- 5 Gastroenterology Section, Department of Medicine, King Faisal Specialist Hospital and Research Center , Riyadh, Saudi Arabia
| | - Mohammed A Alblawi
- 8 Department of Pathology and Laboratory Medicine, Ministry of National Guard Health Affairs, King Abdullah International Medical Research Center/King Saud Bin Abdulaziz University for Health Sciences , Riyadh, Saudi Arabia
| | - Waleed AlTamimi
- 8 Department of Pathology and Laboratory Medicine, Ministry of National Guard Health Affairs, King Abdullah International Medical Research Center/King Saud Bin Abdulaziz University for Health Sciences , Riyadh, Saudi Arabia
| | | | - Pierre Marquett
- 10 Department of Pharmacology and Toxicology, CHU Limoges , Limoges, France
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25
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Mejer N, Fahnøe U, Galli A, Ramirez S, Benfield T, Bukh J. Ribavirin-induced mutagenesis across the complete open reading frame of hepatitis C virus genotypes 1a and 3a. J Gen Virol 2018; 99:1066-1077. [PMID: 29927371 DOI: 10.1099/jgv.0.001095] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Ribavirin (RBV) has been used for the last 20 years to treat patients with chronic hepatitis C virus (HCV) infection. This pluripotent drug is believed to induce mutagenesis in HCV RNA. However, for cell-cultured HCV (HCVcc) this phenomenon has only been investigated in genotype 2a recombinants. Here we studied the mutations that developed in HCVcc of genotypes 1a and 3a treated with RBV or ribavirin triphosphate (RBV-TP) compared to non-treated controls. Analysis was performed on the amplified full-length open reading frame (ORF) of recovered viruses following next-generation sequencing and clonal analyses. Compared to non-treated controls, the spread of TNcc(1a) and DBN3acc(3a) HCVcc was delayed by RBV and RBV-TP at concentrations of 40 µM or higher. The delay in HCVcc spread was associated with increased new single-nucleotide polymorphisms (SNP). Significantly higher numbers of new SNP were observed in TNcc(1a) viruses treated with RBV or RBV-TP compared to matched non-treated controls. RBV or RBV-TP treatment led to significantly increased proportions of new G-to-A and C-to-U SNP compared to non-treated TNcc(1a). Clonal analyses confirmed a significantly increased mutation rate in RBV-treated TNcc(1a). Synonymous pairwise distances increased in both viruses across the complete ORF under RBV and RBV-TP treatment compared to controls. Consensus-shifts in single samples of RBV- or RBV-TP-treated TNcc(1a) viruses occurred in proteins E1, p7, NS3 and NS4B. No non-synonymous consensus changes were observed in DBN3acc(3a). This study supports a biased G-to-A and C-to-U mutagenic effect of RBV and RBV-TP throughout the entire ORF of HCV genotypes 1a and 3a.
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Affiliation(s)
- Niels Mejer
- 1Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.,2Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
| | - Ulrik Fahnøe
- 1Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.,2Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
| | - Andrea Galli
- 1Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.,2Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
| | - Santseharay Ramirez
- 1Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.,2Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
| | - Thomas Benfield
- 2Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark.,3Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Jens Bukh
- 1Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.,2Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
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Díaz-Martínez L, Brichette-Mieg I, Pineño-Ramos A, Domínguez-Huerta G, Grande-Pérez A. Lethal mutagenesis of an RNA plant virus via lethal defection. Sci Rep 2018; 8:1444. [PMID: 29362502 PMCID: PMC5780445 DOI: 10.1038/s41598-018-19829-6] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2017] [Accepted: 01/09/2018] [Indexed: 01/28/2023] Open
Abstract
Lethal mutagenesis is an antiviral therapy that relies on increasing the viral mutation rate with mutagenic nucleoside or base analogues. Currently, the molecular mechanisms that lead to virus extinction through enhanced mutagenesis are not fully understood. Increasing experimental evidence supports the lethal defection model of lethal mutagenesis of RNA viruses, where replication-competent-defectors drive infective virus towards extinction. Here, we address lethal mutagenesis in vivo using 5-fluorouracil (5-FU) during the establishment of tobacco mosaic virus (TMV) systemic infections in N. tabacum. The results show that 5-FU decreased the infectivity of TMV without affecting its viral load. Analysis of molecular clones spanning two genomic regions showed an increase of the FU-related base transitions A → G and U → C. Although the mutation frequency or the number of mutations per molecule did not increase, the complexity of the mutant spectra and the distribution of the mutations were altered. Overall, our results suggest that 5-FU antiviral effect on TMV is associated with the perturbation of the mutation-selection balance in the genomic region of the RNA-dependent RNA polymerase (RdRp). Our work supports the lethal defection model for lethal mutagenesis in vivo in a plant RNA virus and opens the way to study lethal mutagens in plant-virus systems.
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Affiliation(s)
- Luis Díaz-Martínez
- Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Consejo Superior de Investigaciones Científicas-Universidad de Málaga, Área de Genética, Facultad de Ciencias, Campus de Teatinos, 29071, Málaga, Spain
| | - Isabel Brichette-Mieg
- Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Consejo Superior de Investigaciones Científicas-Universidad de Málaga, Área de Genética, Facultad de Ciencias, Campus de Teatinos, 29071, Málaga, Spain
| | - Axier Pineño-Ramos
- Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Consejo Superior de Investigaciones Científicas-Universidad de Málaga, Área de Genética, Facultad de Ciencias, Campus de Teatinos, 29071, Málaga, Spain
| | - Guillermo Domínguez-Huerta
- Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Consejo Superior de Investigaciones Científicas-Universidad de Málaga, Área de Genética, Facultad de Ciencias, Campus de Teatinos, 29071, Málaga, Spain
- Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Consejo Superior de Investigaciones Científicas-Universidad de Málaga, Estación Experimental "La Mayora", 29750, Algarrobo-Costa, Málaga, Spain
| | - Ana Grande-Pérez
- Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Consejo Superior de Investigaciones Científicas-Universidad de Málaga, Área de Genética, Facultad de Ciencias, Campus de Teatinos, 29071, Málaga, Spain.
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Quer J, Rodríguez-Frias F, Gregori J, Tabernero D, Soria ME, García-Cehic D, Homs M, Bosch A, Pintó RM, Esteban JI, Domingo E, Perales C. Deep sequencing in the management of hepatitis virus infections. Virus Res 2017; 239:115-125. [PMID: 28040474 DOI: 10.1016/j.virusres.2016.12.020] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2016] [Revised: 11/10/2016] [Accepted: 12/22/2016] [Indexed: 02/07/2023]
Abstract
The hepatitis viruses represent a major public health problem worldwide. Procedures for characterization of the genomic composition of their populations, accurate diagnosis, identification of multiple infections, and information on inhibitor-escape mutants for treatment decisions are needed. Deep sequencing methodologies are extremely useful for these viruses since they replicate as complex and dynamic quasispecies swarms whose complexity and mutant composition are biologically relevant traits. Population complexity is a major challenge for disease prevention and control, but also an opportunity to distinguish among related but phenotypically distinct variants that might anticipate disease progression and treatment outcome. Detailed characterization of mutant spectra should permit choosing better treatment options, given the increasing number of new antiviral inhibitors available. In the present review we briefly summarize our experience on the use of deep sequencing for the management of hepatitis virus infections, particularly for hepatitis B and C viruses, and outline some possible new applications of deep sequencing for these important human pathogens.
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Affiliation(s)
- Josep Quer
- Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Internal Medicine Department, Vall d'Hebron Institut Research-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Universitat Autonoma de Barcelona, 08035, Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain; Universitat Autònoma de Barcelona, Barcelona, Spain.
| | - Francisco Rodríguez-Frias
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain; Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
| | - Josep Gregori
- Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Internal Medicine Department, Vall d'Hebron Institut Research-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Universitat Autonoma de Barcelona, 08035, Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain; Roche Diagnostics, S.L., Sant Cugat del Vallés, Spain
| | - David Tabernero
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain; Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
| | - Maria Eugenia Soria
- Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Internal Medicine Department, Vall d'Hebron Institut Research-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Universitat Autonoma de Barcelona, 08035, Barcelona, Spain
| | - Damir García-Cehic
- Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Internal Medicine Department, Vall d'Hebron Institut Research-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Universitat Autonoma de Barcelona, 08035, Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain
| | - Maria Homs
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain; Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
| | - Albert Bosch
- Department of Microbiology, Enteric Virus Laboratory, University of Barcelona, Diagonal 645, 08028 Barcelona, Spain
| | - Rosa María Pintó
- Department of Microbiology, Enteric Virus Laboratory, University of Barcelona, Diagonal 645, 08028 Barcelona, Spain
| | - Juan Ignacio Esteban
- Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Internal Medicine Department, Vall d'Hebron Institut Research-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Universitat Autonoma de Barcelona, 08035, Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain; Universitat Autònoma de Barcelona, Barcelona, Spain
| | - Esteban Domingo
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain; Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain
| | - Celia Perales
- Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Internal Medicine Department, Vall d'Hebron Institut Research-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Universitat Autonoma de Barcelona, 08035, Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain; Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain
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Raj VS, Hundie GB, Schürch AC, Smits SL, Pas SD, Le Pogam S, Janssen HLA, de Knegt RJ, Osterhaus ADME, Najera I, Boucher CA, Haagmans BL. Identification of HCV Resistant Variants against Direct Acting Antivirals in Plasma and Liver of Treatment Naïve Patients. Sci Rep 2017; 7:4688. [PMID: 28680115 PMCID: PMC5498547 DOI: 10.1038/s41598-017-04931-y] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2017] [Accepted: 05/22/2017] [Indexed: 02/06/2023] Open
Abstract
Current standard-of-care treatment of chronically infected hepatitis C virus (HCV) patients involves direct-acting antivirals (DAA). However, concerns exist regarding the emergence of drug -resistant variants and subsequent treatment failure. In this study, we investigate potential natural drug-resistance mutations in the NS5B gene of HCV genotype 1b from treatment-naïve patients. Population-based sequencing and 454 deep sequencing of NS5B gene were performed on plasma and liver samples obtained from 18 treatment- naïve patients. The quasispecies distribution in plasma and liver samples showed a remarkable overlap in each patient. Although unique sequences in plasma or liver were observed, in the majority of cases the most dominant sequences were shown to be identical in both compartments. Neither in plasma nor in the liver codon changes were detected at position 282 that cause resistance to nucleos(t)ide analogues. However, in 10 patients the V321I change conferring resistance to nucleos(t)ide NS5B polymerase inhibitors and in 16 patients the C316N/Y/H non-nucleoside inhibitors were found mainly in liver samples. In conclusion, 454-deep sequencing of liver and plasma compartments in treatment naïve patients provides insight into viral quasispecies and the pre-existence of some drug-resistant variants in the liver, which are not necessarily present in plasma.
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Affiliation(s)
- V Stalin Raj
- Department of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands
| | | | - Anita C Schürch
- Department of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands
| | - Saskia L Smits
- Department of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands.,Viroclinics Biosciences BV, Rotterdam, The Netherlands
| | - Suzan D Pas
- Department of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands
| | - Sophie Le Pogam
- Virology Discovery, Pharma Research Early Development Hoffmann La Roche, Nutley, NJ, USA
| | - Harry L A Janssen
- Department of Gastroenterology and Hepatology, Erasmus Medical Center, Rotterdam, The Netherlands.,Division of Gastroenterology, University Health Network, Toronto, Canada
| | - Rob J de Knegt
- Department of Gastroenterology and Hepatology, Erasmus Medical Center, Rotterdam, The Netherlands
| | - Albert D M E Osterhaus
- Artemis One health, Utrecht, The Netherlands.,Center for Infection Medicine and Zoonoses Research, University of Veterinary Medicine, Hannover, Germany
| | - Isabel Najera
- Virology Discovery, Pharma Research Early Development Hoffmann La Roche, Nutley, NJ, USA
| | - Charles A Boucher
- Department of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands
| | - Bart L Haagmans
- Department of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands.
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29
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Prevalence of NS5B Resistance Mutations in Hepatitis C Virus (HCV) Treatment Naive South Africans. HEPATITIS MONTHLY 2017. [DOI: 10.5812/hepatmon.14248] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/13/2023]
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30
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Debing Y, Ramière C, Dallmeier K, Piorkowski G, Trabaud MA, Lebossé F, Scholtès C, Roche M, Legras-Lachuer C, de Lamballerie X, André P, Neyts J. Hepatitis E virus mutations associated with ribavirin treatment failure result in altered viral fitness and ribavirin sensitivity. J Hepatol 2016; 65:499-508. [PMID: 27174035 DOI: 10.1016/j.jhep.2016.05.002] [Citation(s) in RCA: 101] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/13/2015] [Revised: 05/02/2016] [Accepted: 05/02/2016] [Indexed: 12/13/2022]
Abstract
BACKGROUND & AIMS Ribavirin monotherapy is the preferred treatment for chronic hepatitis E, although occasional treatment failure occurs. We present a patient with chronic hepatitis E experiencing ribavirin treatment failure with a completely resistant phenotype. We aimed to identify viral mutations associated with treatment failure and explore the underlying mechanisms. METHODS Viral genomes were deep-sequenced at different time points and the role of identified mutations was assessed in vitro using mutant replicons, antiviral assays, cell culture of patient-derived virus and deep-sequencing. RESULTS Ribavirin resistance was associated with Y1320H, K1383N and G1634R mutations in the viral polymerase, but also an insertion in the hypervariable region comprising a duplication and a polymerase-derived fragment. Analysis of these genome alterations in vitro revealed replication-increasing roles for Y1320H and G1634R mutations and the hypervariable region insertion. In contrast, the K1383N mutation in the polymerase F1-motif suppressed viral replication and increased the in vitro sensitivity to ribavirin, contrary to the clinical phenotype. Analysis of the replication of mutant full-length virus and in vitro culturing of patient-derived virus confirmed that sensitivity to ribavirin was retained. Finally, deep-sequencing of hepatitis E virus genomes revealed that ribavirin is mutagenic to viral replication in vitro and in vivo. CONCLUSIONS Mutations Y1320H, G1634R and the hypervariable region insertion compensated for K1383N-associated replication defects. The specific role of the K1383N mutation remains enigmatic, but it appears to be of importance for the ribavirin resistant phenotype in this patient. LAY SUMMARY Ribavirin is the most common treatment for chronic hepatitis E and is mostly effective, although some cases of ribavirin treatment failure have been described. Here, we report on a particular case of ribavirin resistance and investigate the underlying causes of treatment failure. Mutations in the viral polymerase, an essential enzyme for viral replication, appear to be responsible.
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Affiliation(s)
- Yannick Debing
- Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, Leuven, Belgium
| | - Christophe Ramière
- Laboratoire de Virologie, Hôpital de la Croix-Rousse, Hospices Civils de Lyon, Lyon, France; Centre International de Recherche en Infectiologie (CIRI) (Inserm U1111, CNRS UMR 5308), Lyon, France; Université Claude Bernard Lyon 1, F-69100 Villeurbanne, France; Université de Lyon, F-69000 Lyon, France
| | - Kai Dallmeier
- Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, Leuven, Belgium
| | - Géraldine Piorkowski
- Aix Marseille Université, IRD French Institute of Research for Development, EHESP French School of Public Health, EPV UMR_D 190 "Emergence des Pathologies Virales", 13385 Marseille, France
| | - Mary-Anne Trabaud
- Laboratoire de Virologie, Hôpital de la Croix-Rousse, Hospices Civils de Lyon, Lyon, France
| | - Fanny Lebossé
- Université Claude Bernard Lyon 1, F-69100 Villeurbanne, France; Université de Lyon, F-69000 Lyon, France; Service d'Hépato-Gastroentérologie, Hôpital de la Croix-Rousse, Hospices Civils de Lyon, Lyon, France; INSERM U1052, CRCL (Centre de Recherche en Cancérologie de Lyon), Lyon, France
| | - Caroline Scholtès
- Laboratoire de Virologie, Hôpital de la Croix-Rousse, Hospices Civils de Lyon, Lyon, France; Centre International de Recherche en Infectiologie (CIRI) (Inserm U1111, CNRS UMR 5308), Lyon, France; Université Claude Bernard Lyon 1, F-69100 Villeurbanne, France; Université de Lyon, F-69000 Lyon, France
| | | | - Catherine Legras-Lachuer
- ViroScan3D, Trevoux, France; UMR CNRS 5557 UCBL USC INRA 1193 ENVL, Dynamique Microbienne et Transmission Virale, Lyon, France
| | - Xavier de Lamballerie
- Aix Marseille Université, IRD French Institute of Research for Development, EHESP French School of Public Health, EPV UMR_D 190 "Emergence des Pathologies Virales", 13385 Marseille, France; Institut Hospitalo-Universitaire Méditerranée Infection, Marseille, France
| | - Patrice André
- Laboratoire de Virologie, Hôpital de la Croix-Rousse, Hospices Civils de Lyon, Lyon, France; Centre International de Recherche en Infectiologie (CIRI) (Inserm U1111, CNRS UMR 5308), Lyon, France; Université Claude Bernard Lyon 1, F-69100 Villeurbanne, France; Université de Lyon, F-69000 Lyon, France
| | - Johan Neyts
- Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, Leuven, Belgium.
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Trends in Antiviral Strategies. VIRUS AS POPULATIONS 2016. [PMCID: PMC7149557 DOI: 10.1016/b978-0-12-800837-9.00009-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/03/2022]
Abstract
Viral populations are true moving targets regarding the genomic sequences to be targeted in antiviral designs. Experts from different fields have expressed the need of new paradigms for antiviral interventions and viral disease control. This chapter reviews several strategies that aim at counteracting the adaptive capacity of viral quasispecies. The proposed designs are based on combinations of different antiviral drugs and immune modulators, or in the administration of virus-specific mutagenic agents, in an approach termed lethal mutagenesis of viruses. It consists of decreasing viral fitness by an excess of mutations that render viral proteins sub-optimal or non-functional. Viral extinction by lethal mutagenesis involves several sequential, overlapping steps that recapitulate the major concepts of intra-population interactions and genetic information stability discussed in preceding chapters. Despite the magnitude of the challenge, the chapter closes with some optimistic prospects for an effective control of viruses displaying error-prone replication, based on the combined targeting of replication fidelity and the induction of the innate immune response.
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Perales C, Quer J, Gregori J, Esteban JI, Domingo E. Resistance of Hepatitis C Virus to Inhibitors: Complexity and Clinical Implications. Viruses 2015; 7:5746-66. [PMID: 26561827 PMCID: PMC4664975 DOI: 10.3390/v7112902] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2015] [Revised: 10/23/2015] [Accepted: 10/26/2015] [Indexed: 12/20/2022] Open
Abstract
Selection of inhibitor-resistant viral mutants is universal for viruses that display quasi-species dynamics, and hepatitis C virus (HCV) is no exception. Here we review recent results on drug resistance in HCV, with emphasis on resistance to the newly-developed, directly-acting antiviral agents, as they are increasingly employed in the clinic. We put the experimental observations in the context of quasi-species dynamics, in particular what the genetic and phenotypic barriers to resistance mean in terms of exploration of sequence space while HCV replicates in the liver of infected patients or in cell culture. Strategies to diminish the probability of viral breakthrough during treatment are briefly outlined.
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Affiliation(s)
- Celia Perales
- Liver Unit, Internal Medicine, Laboratory of Malalties Hepàtiques, Vall d'Hebron Institut de Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Universitat Autònoma de Barcelona, 08035 Barcelona, Spain.
- Centro de Biologia Molecular "Severo Ochoa" (CSIC-UAM), Cantoblanco, 28049 Madrid, Spain.
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), 08035 Barcelona, Spain.
| | - Josep Quer
- Liver Unit, Internal Medicine, Laboratory of Malalties Hepàtiques, Vall d'Hebron Institut de Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Universitat Autònoma de Barcelona, 08035 Barcelona, Spain.
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), 08035 Barcelona, Spain.
- Universitat Autònoma de Barcelona, Bellaterra 08193, Spain.
| | - Josep Gregori
- Liver Unit, Internal Medicine, Laboratory of Malalties Hepàtiques, Vall d'Hebron Institut de Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Universitat Autònoma de Barcelona, 08035 Barcelona, Spain.
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), 08035 Barcelona, Spain.
- Roche Diagnostics SL, 08174 Sant Cugat del Vallès, Spain.
| | - Juan Ignacio Esteban
- Liver Unit, Internal Medicine, Laboratory of Malalties Hepàtiques, Vall d'Hebron Institut de Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Universitat Autònoma de Barcelona, 08035 Barcelona, Spain.
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), 08035 Barcelona, Spain.
- Universitat Autònoma de Barcelona, Bellaterra 08193, Spain.
| | - Esteban Domingo
- Centro de Biologia Molecular "Severo Ochoa" (CSIC-UAM), Cantoblanco, 28049 Madrid, Spain.
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), 08035 Barcelona, Spain.
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Lack of mutational hot spots during decitabine-mediated HIV-1 mutagenesis. Antimicrob Agents Chemother 2015; 59:6834-43. [PMID: 26282416 DOI: 10.1128/aac.01644-15] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2015] [Accepted: 08/10/2015] [Indexed: 01/01/2023] Open
Abstract
Decitabine has previously been shown to induce lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1). However, the factors that determine the susceptibilities of individual sequence positions in HIV-1 to decitabine have not yet been defined. To investigate this, we performed Illumina high-throughput sequencing of multiple amplicons prepared from proviral DNA that was recovered from decitabine-treated cells infected with HIV-1. We found that decitabine induced an ≈4.1-fold increase in the total mutation frequency of HIV-1, primarily due to a striking ≈155-fold increase in the G-to-C transversion frequency. Intriguingly, decitabine also led to an ≈29-fold increase in the C-to-G transversion frequency. G-to-C frequencies varied substantially (up to ≈80-fold) depending upon sequence position, but surprisingly, mutational hot spots (defined as upper outliers within the mutation frequency distribution) were not observed. We further found that every single guanine position examined was significantly susceptible to the mutagenic effects of decitabine. Taken together, these observations demonstrate for the first time that decitabine-mediated HIV-1 mutagenesis is promiscuous and occurs in the absence of a clear bias for mutational hot spots. These data imply that decitabine-mediated G-to-C mutagenesis is a highly effective antiviral mechanism for extinguishing HIV-1 infectivity.
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Dengue Virus Evolution under a Host-Targeted Antiviral. J Virol 2015; 89:5592-601. [PMID: 25762732 DOI: 10.1128/jvi.00028-15] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2015] [Accepted: 03/02/2015] [Indexed: 12/19/2022] Open
Abstract
UNLABELLED The host-targeted antiviral drug UV-4B reduces viral replication and promotes survival in a mouse model of experimental dengue virus (DENV) infection. UV-4B is an iminosugar that inhibits the α-glucosidase family of enzymes and subsequently the folding of glycosylated proteins, both viral and host. Here, we utilized next-generation sequencing to investigate evolution of a flavivirus under selective pressure by a host-targeted antiviral in vivo. In viral populations recovered from UV-4B-treated mice, there was a significant increase in the number of single-nucleotide polymorphisms (SNPs) and the ratio of nonsynonymous to synonymous SNPs compared to findings in viral populations from vehicle-treated mice. The strongest evidence of positive selection was in the glycosylated membrane protein, thereby providing in vivo validation of the mechanism of action of an iminosugar. In addition, mutations in glycosylated proteins were present only in drug-treated mice after a single passage. However, the bulk of the other mutations were present in both populations, indicating nonspecific selective pressure. Together with the continued control of viremia by UV-4B, these findings are consistent with the previously predicted high genetic barrier to escape mutations in host-targeted antivirals. IMPORTANCE Although hundreds of millions of people are infected with DENV every year, there is currently no approved vaccine or antiviral therapy. UV-4B has demonstrated antiviral activity against DENV and is expected to enter clinical trials soon. Therefore, it is important to understand the mechanisms of DENV resistance to UV-4B. Host-targeted antivirals are thought to have a higher genetic barrier to escape mutants than directly acting antivirals, yet there are very few published studies of viral evolution under host-targeted antivirals. No study to date has described flavivirus evolution in vivo under selective pressure by a host-based antiviral drug. We present the first in vivo study of the sequential progression of viral evolution under selective pressure by a host-targeted antiviral compound. This study bolsters support for the clinical development of UV-4B as an antiviral drug against DENV, and it provides a framework to compare how treatment with other host-targeted antiflaviviral drugs in humans and different animal models influence viral genetic diversity.
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Development of antiviral agents toward enterovirus 71 infection. JOURNAL OF MICROBIOLOGY, IMMUNOLOGY, AND INFECTION = WEI MIAN YU GAN RAN ZA ZHI 2015; 48:1-8. [DOI: 10.1016/j.jmii.2013.11.011] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/31/2013] [Accepted: 11/16/2013] [Indexed: 01/20/2023]
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Ji H, Kozak RA, Biondi MJ, Pilon R, Vallee D, Liang BB, La D, Kim J, Van Domselaar G, Leonard L, Sandstrom P, Brooks J. Next generation sequencing of the hepatitis C virus NS5B gene reveals potential novel S282 drug resistance mutations. Virology 2015; 477:1-9. [PMID: 25600207 DOI: 10.1016/j.virol.2014.12.037] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2014] [Revised: 11/11/2014] [Accepted: 12/23/2014] [Indexed: 01/06/2023]
Abstract
Identifying HCV drug resistance mutations (DRMs) is increasingly important as new direct acting antiviral therapies (DAA) become available. Tagged pooled pyrosequencing (TPP) was originally developed as cost-effective approach for detecting low abundance HIV DRMs. Using 127 HCV-positive samples from a Canadian injection drug user cohort, we demonstrated the suitability and efficiency of TPP for evaluating DRMs in HCV NS5B gene. At a mutation identification threshold of 1%, no nucleoside inhibitor DRMs were detected among these DAA naïve subjects. Clinical NS5B resistance to non-nucleoside inhibitors and interferon/ribavirin was predicted to be low within this cohort. S282T mutation, the primary mutation selected by sofosbuvir in vitro, was not identified while S282G/C/R variants were detected in 9 subjects. Further characterization on these new S282 variants using in silico molecular modeling implied their potential association with resistance. Combining TPP with in silico analysis detects NS5B polymorphisms that may explain differences in treatment outcomes.
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Affiliation(s)
- Hezhao Ji
- National HIV & Retrovirology Laboratories, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada
| | - Robert A Kozak
- Department of Pathobiology, University of Guelph, Guelph, Canada
| | - Mia J Biondi
- Arthur Labatt Family School of Nursing, Western University, London, Canada
| | - Richard Pilon
- National HIV & Retrovirology Laboratories, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada
| | - Dominic Vallee
- National HIV & Retrovirology Laboratories, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada
| | - Ben Binhua Liang
- Bioinformatics Core, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada
| | - David La
- Bioinformatics Core, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada
| | - John Kim
- National HIV & Retrovirology Laboratories, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada
| | - Gary Van Domselaar
- Bioinformatics Core, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada
| | - Lynne Leonard
- Department of Epidemiology and Community Medicine, University of Ottawa, Ottawa, Canada
| | - Paul Sandstrom
- National HIV & Retrovirology Laboratories, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada
| | - James Brooks
- National HIV & Retrovirology Laboratories, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada.
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Arias A, Thorne L, Goodfellow I. Favipiravir elicits antiviral mutagenesis during virus replication in vivo. eLife 2014; 3:e03679. [PMID: 25333492 PMCID: PMC4204012 DOI: 10.7554/elife.03679] [Citation(s) in RCA: 117] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2014] [Accepted: 09/24/2014] [Indexed: 12/17/2022] Open
Abstract
Lethal mutagenesis has emerged as a novel potential therapeutic approach to treat viral infections. Several studies have demonstrated that increases in the high mutation rates inherent to RNA viruses lead to viral extinction in cell culture, but evidence during infections in vivo is limited. In this study, we show that the broad-range antiviral nucleoside favipiravir reduces viral load in vivo by exerting antiviral mutagenesis in a mouse model for norovirus infection. Increased mutation frequencies were observed in samples from treated mice and were accompanied with lower or in some cases undetectable levels of infectious virus in faeces and tissues. Viral RNA isolated from treated animals showed reduced infectivity, a feature of populations approaching extinction during antiviral mutagenesis. These results suggest that favipiravir can induce norovirus mutagenesis in vivo, which in some cases leads to virus extinction, providing a proof-of-principle for the use of favipiravir derivatives or mutagenic nucleosides in the clinical treatment of noroviruses. DOI:http://dx.doi.org/10.7554/eLife.03679.001 Viruses can infect, take control of and replicate themselves inside the living cells of other organisms. Some viral diseases can be treated with antiviral drugs, which stop viral infections either by making it more difficult for viruses to enter cells or by preventing the virus replicating once inside. As antiviral drugs are currently only available to treat a handful of viral infections, efforts are underway to develop and test experimental antiviral drugs. One such experimental drug is called favipiravir, which is proving to be effective against several viruses that store their genetic information in the form of RNA molecules. These viruses include those that cause diseases such as influenza, gastroenteritis, and Ebola. Along with ongoing work determining how safe and effective favipiravir is for treating viral infections, researchers are also attempting to better understand how favipiravir works. Whenever a strand of RNA is copied to allow a new virus to form, there is a risk that mistakes—or mutations—that could harm the virus are introduced into the genetic code. Previous experiments performed on cells grown in the laboratory suggested that favipiravir works against RNA viruses by increasing how often these mutations occur. RNA viruses naturally experience a large number of mutations and the ability to make mutations is in fact a benefit for viruses as it allows them to evolve rapidly and to escape immune responses. However, there is a limit to how many mutations can be tolerated in the viral genome before it can no longer replicate. Therefore, a slight increase in how often mutations occur—as thought to be caused by favipiravir—is able to stop the RNA virus replicating and halt the infection. However, favipiravir's mode of action had yet to be confirmed in living animals. Using mice, Arias et al. tested favipiravir's ability to treat a persistent infection by norovirus—the most common cause of viral gastroenteritis in humans and also responsible for life-threatening chronic diarrhoea in immunodeficient patients. Treatment increased the number of mutations that occurred when the viral RNA replicated and could reduce the amount of virus in the mice to undetectable levels. In addition, favipiravir did not show toxicity in mice after 8 weeks of treatment. This suggests that favipiravir has the potential to be used safely and effectively to treat norovirus and other RNA viruses, although further studies are required before it can be developed into a clinical treatment. DOI:http://dx.doi.org/10.7554/eLife.03679.002
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Affiliation(s)
- Armando Arias
- Division of Virology, Addenbrooke's Hospital, University of Cambridge, Cambridge, United Kingdom
| | - Lucy Thorne
- Division of Virology, Addenbrooke's Hospital, University of Cambridge, Cambridge, United Kingdom
| | - Ian Goodfellow
- Division of Virology, Addenbrooke's Hospital, University of Cambridge, Cambridge, United Kingdom
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Ribavirin at the Era of Novel Direct Antiviral Agents for the Treatment of Hepatitis C Virus Infection: Relevance of Pharmacological Monitoring. ACTA ACUST UNITED AC 2014. [DOI: 10.1155/2014/493087] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Ribavirin is often used for the treatment of hepatitis C virus (HCV) infection. Although its mechanisms of action remain to be clearly elucidated, ribavirin plays a beneficial role for achieving virological response and decreasing the rate of virological relapse after treatment cessation. However, ribavirin may induce side effects leading to early treatment discontinuation. Among them, hemolytic anemia is the most frequent and results from intraerythrocyte accumulation. Pharmacological studies have shown that early ribavirin exposure assessed by the area under the curve (AUC) at day 0 and ribavirin trough concentration during the first three months of therapy were correlated with sustained virological response (SVR). These studies highlighted the relevance of ribavirin pharmacologic monitoring and early dose adaptation during therapy. Although the role of ribavirin within new direct acting antiviral (DAA) combinations will probably decrease in the future, its potential benefit in difficult-to-treat patients such as patients with severe hepatopathy or patients who failed triple therapy including patients with multiresistance will need to be further investigated.
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Quiles-Pérez R, Muñoz-de-Rueda P, Maldonado AML, Martín-Álvarez A, Quer J, Salmerón J. Effects of ribavirin monotherapy on the viral population in patients with chronic hepatitis C genotype 1: direct sequencing and pyrosequencing of the HCV regions. J Med Virol 2014; 86:1886-97. [PMID: 25091333 DOI: 10.1002/jmv.24035] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/04/2014] [Indexed: 11/11/2022]
Abstract
Ribavirin remains essential to chronic hepatitis C treatment. This paper investigates the influence of ribavirin priming to steady state before combined pegylated-interferon/ribavirin treatment on viral kinetics, ribavirin trough concentrations, genetic variability within HCV-core, -NS5B and -NS5A, and response to antiviral therapy. A prospective cohort study was made of 27 chronic hepatitis C genotype 1 naïve patients who received four weeks of ribavirin followed by pegIFN-α-2a/ribavirin for 48 weeks (Group A). The results obtained were compared with those for a control/historical group (Group B). In addition, direct sequencing and pyrosequencing were applied to determine ribavirin monotherapy-induced sequence changes. The rapid, early, and sustained virological response values obtained were 48%, 89%, and 52%, respectively, in Group A, and 52%, 90%, and 52% in Group B (P > 0.05). In the four-week combined treatment, the Group A patients showed a greater decrease in HCV-RNA (2.3 log10 IU/ml vs. 1.2 log10 IU/ml; P = 0.04), lower alanine aminotransferase levels (23.5 ± 1.33 U/L vs. 60.11 ± 18 U/L; P < 0.001) and higher mean ribavirin trough concentrations (3.28 ± 1.26 mg/L vs. 1.74 ± 0.7 mg/L; P = 0.001). No general increase in rates of nucleotide substitutions in the ribavirin monotherapy-treated patients was observed in NS5B, ISDR, or PKRbd, but there was a decrease in silent mutations in the HCV core (P = 0.04). This result was confirmed by pyrosequencing in the NS5A region. It is concluded that the ribavirin priming combined treatment with pegIFN-α-2a does not improve sustained virological response rates in HCV genotype 1 naïve infected patients. However, the greater reductions in viral load and alanine aminotransferase levels, together with the higher ribavirin trough concentration values obtained, could reflect the greater effectiveness of the treatment. Ribavirin does not have a mutagenic effect on the virus in patients with chronic hepatitis C.
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Affiliation(s)
- R Quiles-Pérez
- Research Support Unit, UNAI, San Cecilio University Hospital, Granada, Spain; CIBEREHD, Spain
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Ali M, Rehman IU, Idrees M. Emergence of genetically variant Hepatitis C virus population in response to increased antiviral drug pressure, Pakistan. Virus Genes 2014; 48:543-9. [PMID: 24532035 DOI: 10.1007/s11262-014-1047-y] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2013] [Accepted: 01/30/2014] [Indexed: 01/06/2023]
Abstract
Mutations in NS5B gene of Hepatitis C virus (HCV) have been reported in patients undergoing antiviral therapy. In the present study, we report emerging clade of HCV-3a in patients administered with IFN plus ribavirin therapy for 24 weeks and having low viral loads (<250 IU/mL). Mutations D/N244E, K304R, N/K307G, Q/T329V, and A338V were found associated with these emerging strains. This distinct HCV could be associated with the increased antiviral drug pressure.
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Affiliation(s)
- Muhammad Ali
- Division of Molecular Virology, National Centre of Excellence in Molecular Biology, University of the Punjab, 87-West Canal bank Road, Thokar Niaz baig, Lahore, 53700, Pakistan,
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Mihm U, Welker MW, Teuber G, Wedemeyer H, Berg T, Sarrazin C, Böhm S, Alshuth U, Herrmann E, Zeuzem S. Impact of ribavirin priming on viral kinetics and treatment response in chronic hepatitis C genotype 1 infection. J Viral Hepat 2014; 21:42-52. [PMID: 24329856 DOI: 10.1111/jvh.12124] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/23/2012] [Accepted: 03/24/2013] [Indexed: 12/23/2022]
Abstract
Ribavirin amplifies the interferon-alpha (IFN) signalling cascade. As ribavirin needs 4 weeks to reach steady state, ribavirin priming may optimize hepatic IFN sensitivity before starting a pegylated (PEG)-IFN/ribavirin combination therapy. This study investigated potential benefits of ribavirin priming prior to PEG-IFN2a/ribavirin combination therapy on viral kinetics, on-treatment and sustained virological response (SVR) in chronic hepatitis C virus (HCV) genotype 1 infection. Sixty-eight treatment naive patients were randomized 2:2:1 to ribavirin (ribavirin arm) or placebo (placebo arm) or PEG-IFN2a (PEG-IFN2a arm) for 6 weeks prior to 12 weeks of PEG-IFN2a/ribavirin combination therapy within a double-blind, placebo-controlled trial. Then, standard PEG-IFN2a/ribavirin combination therapy according to the German guidelines was continued under the responsibility of the investigators. Ribavirin was given according to body weight and PEG-IFN2a at a dose of 180 μg subcutaneously once/week. During ribavirin priming, HCV RNA showed a decline of -0.58 log10 IU/mL (P < 0.001) that was unrelated to the IL28B rs12979860 genotype (CC vs CT/TT, P = 0.244). Ribavirin priming did neither increase the PEG-IFN2a-induced first- or second-phase viral decline (P values >0.100) nor on-treatment response or SVR (HCV RNA undetectable at week 12 of combination therapy: ribavirin arm 56%, placebo arm 38%, PEG-IFN2a arm 50%; SVR: ribavirin arm 41%, placebo arm 54%, PEG-IFN2a arm 50%; P values >0.300). In conclusion, ribavirin monotherapy showed a significant antiviral activity that was not influenced by the IL28B genotype. Ribavirin priming prior to PEG-IFN2a/ribavirin combination therapy did neither increase the first- or second-phase viral decline nor on-treatment response or SVR.
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Affiliation(s)
- U Mihm
- Medizinische Klinik I, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt/Main, Germany
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Ortega-Prieto AM, Sheldon J, Grande-Pérez A, Tejero H, Gregori J, Quer J, Esteban JI, Domingo E, Perales C. Extinction of hepatitis C virus by ribavirin in hepatoma cells involves lethal mutagenesis. PLoS One 2013; 8:e71039. [PMID: 23976977 PMCID: PMC3745404 DOI: 10.1371/journal.pone.0071039] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2013] [Accepted: 06/26/2013] [Indexed: 12/14/2022] Open
Abstract
Lethal mutagenesis, or virus extinction produced by enhanced mutation rates, is under investigation as an antiviral strategy that aims at counteracting the adaptive capacity of viral quasispecies, and avoiding selection of antiviral-escape mutants. To explore lethal mutagenesis of hepatitis C virus (HCV), it is important to establish whether ribavirin, the purine nucleoside analogue used in anti-HCV therapy, acts as a mutagenic agent during virus replication in cell culture. Here we report the effect of ribavirin during serial passages of HCV in human hepatoma Huh-7.5 cells, regarding viral progeny production and complexity of mutant spectra. Ribavirin produced an increase of mutant spectrum complexity and of the transition types associated with ribavirin mutagenesis, resulting in HCV extinction. Ribavirin-mediated depletion of intracellular GTP was not the major contributory factor to mutagenesis since mycophenolic acid evoked a similar decrease in GTP without an increase in mutant spectrum complexity. The intracellular concentration of the other nucleoside-triphosphates was elevated as a result of ribavirin treatment. Mycophenolic acid extinguished HCV without an intervening mutagenic activity. Ribavirin-mediated, but not mycophenolic acid-mediated, extinction of HCV occurred via a decrease of specific infectivity, a feature typical of lethal mutagenesis. We discuss some possibilities to explain disparate results on ribavirin mutagenesis of HCV.
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Affiliation(s)
- Ana M Ortega-Prieto
- Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, Madrid, Spain
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Anjum S, Afzal MS, Ahmad T, Aslam B, Waheed Y, Shafi T, Qadri I. Mutations in the STAT1‑interacting domain of the hepatitis C virus core protein modulate the response to antiviral therapy. Mol Med Rep 2013; 8:487-492. [PMID: 23799612 DOI: 10.3892/mmr.2013.1541] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2012] [Accepted: 01/31/2013] [Indexed: 02/07/2023] Open
Abstract
RNA viruses, such as hepatitis C virus (HCV), have markedly error-prone replication, resulting in high rates of mutagenesis. In addition, the standard treatment includes ribavirin, a base analog that is likely to cause mutations in different regions of the HCV genome, resulting in deleterious effects on HCV itself. The N-terminal region of the core protein is reported to block interferon (IFN) signaling by interaction with the STAT1‑SH2 domain, resulting in HCV resistance to IFN therapy. In this study, mutations in the HCV core protein from IFN/ribavirin‑treated patients were analyzed, with particular focus on the N‑terminal domain of the HCV core which is reported to interact with STAT1. HCV PCR positive patients enrolled in this study were either undergoing pegylated IFN/ribavirin bitherapy and had completed 12 weeks of initial treatment or were treatment‑naïve patients. The HCV core protein was cloned and sequenced from these patients and mutations observed in the STAT1‑interacting domain of the core protein from treated patients were characterized using in silico interaction to depict the role of these mutations in disease outcomes. Our results suggest that the amino acids at positions 2, 3, 8, 16 and 23 of the HCV core protein are critical for core-STAT1 interaction and ribavirin-induced mutations at these positions interfere with the interaction, resulting in a better response of the treated patients. In conclusion, this study anticipates that HCV core residues 2, 3, 8, 16 and 23 directly interact with STAT1. We propose that IFN/ribavirin bitherapy‑induced mutations in the STAT1‑interacting domain of the HCV core protein may be responsible for the improved therapeutic response and viral clearance, thus amino acids 1-23 of the N-terminus of the core protein are an ideal antiviral target. However, this treatment may give rise to resistant variants that are able to escape the current therapy. We propose similar studies in responsive and non-responsive genotypes in order to gain a broader picture of this proposed mechanism of viral clearance.
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Affiliation(s)
- Sadia Anjum
- Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Islamabad 44000, Pakistan.
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Arias A, Isabel de Ávila A, Sanz-Ramos M, Agudo R, Escarmís C, Domingo E. Molecular dissection of a viral quasispecies under mutagenic treatment: positive correlation between fitness loss and mutational load. J Gen Virol 2013; 94:817-830. [DOI: 10.1099/vir.0.049171-0] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Low fidelity replication and the absence of error-repair activities in RNA viruses result in complex and adaptable ensembles of related genomes in the viral population, termed quasispecies, with important implications for natural infections. Theoretical predictions suggested that elevated replication error rates in RNA viruses might be near to a maximum compatible with viral viability. This fact encouraged the use of mutagenic nucleosides as a new antiviral strategy to induce viral extinction through increased replication error rates. Despite extensive evidence of lethal mutagenesis of RNA viruses by different mutagenic compounds, a detailed picture of the infectivity of individual genomes and its relationship with the mutations accumulated is lacking. Here, we report a molecular analysis of a foot-and-mouth disease virus population previously subjected to heavy mutagenesis to determine whether a correlation between increased mutagenesis and decreased fitness existed. Plaque-purified viruses isolated from a ribavirin-treated quasispecies presented decreases of up to 200-fold in infectivity relative to clones in the reference population, associated with an overall eightfold increase in the mutation frequency. This observation suggests that individual infectious genomes of a quasispecies subjected to increased mutagenesis lose infectivity by their continuous mutagenic ‘poisoning’. These results support the lethal defection model of virus extinction and the practical use of chemical mutagens as antiviral treatment. Even when extinction is not achieved, mutagenesis can decrease the infectivity of surviving virus, and facilitate their clearance by host immune responses or complementing antiviral approaches.
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Affiliation(s)
- Armando Arias
- Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 1TN, UK
- Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, Madrid, Spain
| | - Ana Isabel de Ávila
- Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, Madrid, Spain
| | - Marta Sanz-Ramos
- Division of Virology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
- Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, Madrid, Spain
| | - Rubén Agudo
- Fachbereich Chemie, Philipps Universität Marburg, Hans-Meerwein-Strasse, 35032 Marburg, Germany
- Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, Madrid, Spain
| | - Cristina Escarmís
- Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, Madrid, Spain
| | - Esteban Domingo
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain
- Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, Madrid, Spain
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Deep sequencing reveals mutagenic effects of ribavirin during monotherapy of hepatitis C virus genotype 1-infected patients. J Virol 2013; 87:6172-81. [PMID: 23536652 DOI: 10.1128/jvi.02778-12] [Citation(s) in RCA: 82] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
The preeminent mode of action of the broad-spectrum antiviral nucleoside ribavirin in the therapy of chronic hepatitis C is currently unresolved. Particularly under contest are possible mutagenic effects of ribavirin that may lead to viral extinction by lethal mutagenesis of the hepatitis C virus (HCV) genome. We applied ultradeep sequencing to determine ribavirin-induced sequence changes in the HCV coding region (nucleotides [nt] 330 to 9351) of patients treated with 6-week ribavirin monotherapy (n = 6) in comparison to placebo (n = 6). Baseline HCV RNA levels maximally declined on average by -0.8 or -0.1 log10 IU/ml in ribavirin- versus placebo-treated patients. No general increase in rates of nucleotide substitutions in ribavirin-treated patients was observed. However, more HCV genome positions with high G-to-A and C-to-U transition rates were detected between baseline and treatment week 6 in ribavirin-treated patients in comparison to placebo-treated patients (rate of 0.0041 transitions per base pair versus rate of 0.0022 transitions per base pair; P = 0.049). Similarly, the sensitive detection of low-frequency minority variants by statistical filtering indicated significantly more positions with G-to-A and C-to-U transitions in ribavirin-treated patients than in placebo-treated patients (rate of 0.0331 transitions versus rate of 0.0186 transitions per G/C-containing position at baseline; P = 0.018). In contrast, non-ribavirin-associated A-to-G and U-to-C transitions were not enriched in the ribavirin group (P = 0.152). We conclude that ribavirin exerts a mutagenic effect on the virus in patients with chronic hepatitis C by facilitating G-to-A and C-to-U nucleotide transitions.
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Kozuka R, Enomoto M, Hai H, Ogawa T, Nakaya M, Hagihara A, Fujii H, Kobayashi S, Iwai S, Morikawa H, Tamori A, Kawada N. Changes in sequences of core region, interferon sensitivity-determining region and interferon and ribavirin resistance-determining region of hepatitis C virus genotype 1 during interferon-alpha and ribavirin therapy, and efficacy of retreatment. Hepatol Res 2012; 42:1157-1167. [PMID: 22672644 DOI: 10.1111/j.1872-034x.2012.01046.x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
AIM Some regions associated with sensitivity to interferon-α and ribavirin have been identified in the hepatitis C virus (HCV) genome, including amino acid 70 in the core region (core a.a. 70), a.a. 2209-2248 (interferon sensitivity-determining region, ISDR) and a.a. 2334-2379 (interferon and ribavirin resistance-determining region, IRRDR). METHODS We examined changes in the sequences of these regions in 25 patients with chronic HCV genotype 1 infection who had not had sustained virological response (SVR) to interferon-α and ribavirin for 24-48 weeks and subsequently received retreatment for 48-72 weeks. RESULTS At baseline, the core a.a. 70 was mutant (resistant) type in seven patients. At the start of retreatment, the core a.a. 70 had changed from sensitive to resistant type in 2 patients, and SVR was not achieved by retreatment. The ISDR variations were resistant type (0-1 mutations) in 17 patients at baseline. After 2 weeks of treatment, amino acid change was found in two patients; in one, the substitutions returned to baseline status after treatment, and in the other, the substitution persisted. At the start of retreatment, ISDR sequences had changed from resistant to sensitive type in two patients and SVR was achieved and from sensitive to resistant type in three patients and SVR was not achieved. The IRRDR variations were resistant type (<6 mutations) in 19 patients at baseline and at the start of retreatment. CONCLUSION Sequences of the core region and ISDR sometimes change during anti-HCV therapy, potentially affecting the outcomes of retreatment.
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Affiliation(s)
- Ritsuzo Kozuka
- Department of Hepatology, Osaka City University Graduate School of Medicine, Osaka Center for the Advancement of Higher Education, Faculty of Engineering, Kinki University, Hiroshima, Japan
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47
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Castilho MCB, Martins AN, Horbach IS, Perez RDM, Figueiredo FAF, Pinto PDTA, Nabuco LC, Lima DBD, Tanuri A, Porto LC, Ferreira Júnior ODC. Association of hepatitis C virus NS5B variants with resistance to new antiviral drugs among untreated patients. Mem Inst Oswaldo Cruz 2012; 106:968-75. [PMID: 22241118 DOI: 10.1590/s0074-02762011000800011] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2011] [Accepted: 09/08/2011] [Indexed: 01/06/2023] Open
Abstract
Mutations located in the 109-amino acid fragment of NS5B are typically associated with resistance to interferon (IFN) and ribavirin (RIB) and to new antiviral drugs. The prevalence of these mutations was examined in 69 drug-naïve individuals with hepatitis C virus (HCV) infections in Rio de Janeiro, Brazil. Mutations related to non-response to IFN/RIB were observed in all subtypes studied (1a, 1b, 2b, 3a and 4). The most common mutation was Q309R, present in all subtypes, except subtype 2b with frequency above 20%. D244N was detected only in subtype 3a and A333E was detected only in subtype 2b. We did not detect the S282T, S326G or T329I mutations in any of the samples analysed. Of note, the C316N mutation, previously related to a new non-nucleoside compound (HCV796 and AG-021541), was observed in only eight of 33 (24%) samples from subtype 1b. Site 316 was under positive selection in this HCV variant. Our data highlight the presence of previously described resistance mutations in HCV genotypes from drug-naïve patients.
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Affiliation(s)
- Magda Cristina Bernardino Castilho
- Laboratório de Histocompatibilidade e Criopreservação, Policlínica Piquet Carneiro, Departamento de Histologia e Embriologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.
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48
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Jaspe RC, Sulbarán YF, Sulbarán MZ, Loureiro CL, Rangel HR, Pujol FH. Prevalence of amino acid mutations in hepatitis C virus core and NS5B regions among Venezuelan viral isolates and comparison with worldwide isolates. Virol J 2012; 9:214. [PMID: 22995142 PMCID: PMC3511240 DOI: 10.1186/1743-422x-9-214] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2012] [Accepted: 09/18/2012] [Indexed: 12/11/2022] Open
Abstract
BACKGROUND Recent reports show that R70Q and L/C91M amino acid substitutions in the core from different hepatitis C virus (HCV) genotypes have been associated with variable responses to interferon (IFN) and ribavirin (RBV) therapy, as well to an increase of hepatocellular carcinoma (HCC) risk, liver steatosis and insulin resistance (IR). Mutations in NS5B have also been associated to IFN, RBV, nucleoside and non-nucleoside inhibitors drug resistance. The prevalence of these mutations was studied in HCV RNA samples from chronically HCV-infected drug-naïve patients. METHODS After amplification of core and NS5B region by nested-PCR, 12 substitutions were analyzed in 266 Venezuelan HCV isolates subtype 1a, 1b, 2a, 2c, 2b, 2j (a subtype frequently found in Venezuela) and 3a (n = 127 and n = 228 for core and NS5B respectively), and compared to isolates from other countries (n = 355 and n = 646 for core and NS5B respectively). RESULTS R70Q and L/C91M core substitutions were present exclusively in HCV G1b. Both substitutions were more frequent in American isolates compared to Asian ones (69% versus 26%, p < 0.001 and 75% versus 45%, p < 0.001 respectively). In Venezuelan isolates NS5B D310N substitution was detected mainly in G3a (100%) and G1a (13%), this later with a significantly higher prevalence than in Brazilian isolates (p = 0.03). The NS5B mutations related to IFN/RBV treatment D244N was mainly found in G3a, and Q309R was present in all genotypes, except G2. Resistance to new NS5B inhibitors (C316N) was only detected in 18% of G1b, with a significantly lower prevalence than in Asian isolates, where this polymorphism was surprisingly frequent (p < 0.001). CONCLUSIONS Genotypical, geographical and regional differences were found in the prevalence of substitutions in HCV core and NS5B proteins. The substitutions found in the Venezuelan G2j type were similar to that found in G2a and G2c isolates. Our results suggest a high prevalence of the R70Q and L/C91M mutations of core protein for G1b and D310N substitution of NS5B protein for the G3a. C316N polymorphism related with resistance to new NS5B inhibitors was only found in G1b. Some of these mutations could be associated with a worse prognosis of the disease in HCV infected patients.
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Affiliation(s)
- Rossana C Jaspe
- Laboratorio de Virología Molecular, Centro de Microbiología y Biología Celular, Instituto Venezolano de Investigaciones Científicas, Apdo 20632, Caracas 1020-A, Venezuela
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49
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Palmer M, Rubin R, Rustgi V. Randomised clinical trial: pre-dosing with taribavirin before starting pegylated interferon vs. standard combination regimen in treatment-naïve patients with chronic hepatitis C genotype 1. Aliment Pharmacol Ther 2012; 36:370-8. [PMID: 22708649 PMCID: PMC3492905 DOI: 10.1111/j.1365-2036.2012.05188.x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/22/2012] [Revised: 04/05/2012] [Accepted: 05/30/2012] [Indexed: 12/16/2022]
Abstract
BACKGROUND Combination therapy with the ribavirin (RBV) prodrug taribavirin (TBV) and pegylated interferon (PIFN) has produced lower rates of anaemia than with RBV and PIFN. Studies have demonstrated that the sharpest decline in viral load during TBV therapy occurs at Weeks 4 through 6, when TBV reaches steady-state blood levels. AIM The current proof-of-concept study was conducted to examine whether first-order viral kinetics could be influenced by pre-dosing TBV to steady state before introducing PIFN. METHODS Therapy-naïve patients with chronic hepatitis C virus (HCV) genotype 1 (G1) were randomised to receive (i) TBV 600 mg BID monotherapy for 4 weeks followed by combination therapy with PIFN [pre-dosing arm (n = 23)] or (ii) TBV administered concurrently with PIFN [standard dosing arm (n = 19)]. RESULTS More patients achieved undetectable virus or a ≥2-log(10) reduction of HCV RNA at Week 4 in the pre-dosing vs. the standard dosing arm [33% vs. 22% (P = 0.497)]. There was also a trend towards greater reduction in mean log(10) change in HCV RNA in the pre-dosing vs. the standard dosing arm, which was statistically significant at Day 1 [-0.34 ± 0.46 vs. 0.09 ± 0.32 (P < 0.003)] but not at other time points up to Week 24. No significant difference was observed in the rates of anaemia (haemoglobin <10 g/dL) between study arms (4.5% vs. 5.3%). CONCLUSIONS Pre-dosing TBV prior to starting PIFN produces a trend towards improved efficacy although statistical significance was not reached in this small patient population. These results warrant larger clinical trials of TBV pre-dosing.
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Affiliation(s)
- M Palmer
- Liver Center of Long IslandPlainview, NY, USA
| | - R Rubin
- Liver Center of Long IslandPlainview, NY, USA
| | - V Rustgi
- Liver Center of Long IslandPlainview, NY, USA
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50
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Tokumoto Y, Hiasa Y, Uesugi K, Watanabe T, Mashiba T, Abe M, Kumagi T, Ikeda Y, Matsuura B, Onji M. Ribavirin regulates hepatitis C virus replication through enhancing interferon-stimulated genes and interleukin 8. J Infect Dis 2012; 205:1121-30. [PMID: 22357660 DOI: 10.1093/infdis/jis025] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
BACKGROUND The manner in which ribavirin (RBV) enhances the antiviral effects of interferon (IFN) against hepatitis C virus (HCV) remains unknown. We investigated whether RBV modifies IFN-stimulated genes (ISGs) in vivo and in vitro. METHODS We measured the messenger RNA (mRNA) levels of ISGs in T lymphocytes from patients with HCV infection who were receiving IFN-α therapy with or without RBV. We added RBV and/or IFN-α to a plasmid-based HCV replication system containing a full-length HCV genotype 1a sequence in HepG2 and Huh7 cell lines and the JFH-1 HCV genotype 2a sequence in Huh7 cell lines and measured levels of ISGs and autocrine IFN-β. RESULTS The expression of protein kinase R and myxovirus resistance A mRNA was enhanced more with IFN-α and RBV than by IFN-α alone in assays in vivo and in vitro. Such enhancement depended on autocrine IFN-β being enhanced by RBV. RBV upregulated interleukin 8 (IL-8) in the absence of IFN-α. The IL-8 upregulation induced by RBV was responsible for the activation of activator protein 1 (AP-1). CONCLUSIONS Ribavirin augments the anti-HCV effects of IFN-α induced by ISGs through enhancing autocrine IFN-β. Moreover, RBV can enhance IL-8 through activating AP-1. Improved understanding of ISG modulation by RBV would help to establish a means of eliminating HCV.
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Affiliation(s)
- Yoshio Tokumoto
- Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan
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