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Shafqat A, Li M, Zakirullah, Liu F, Tong Y, Fan J, Fan H. A comprehensive review of research advances in the study of lactoferrin to treat viral infections. Life Sci 2025; 361:123340. [PMID: 39730037 DOI: 10.1016/j.lfs.2024.123340] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2024] [Revised: 12/17/2024] [Accepted: 12/23/2024] [Indexed: 12/29/2024]
Abstract
Lactoferrin (Lf) is a naturally occurring glycoprotein known for its antiviral and antibacterial properties and is present in various physiological fluids. Numerous studies have demonstrated its antiviral effectiveness against multiple viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus (IFV), herpes simplex virus (HSV), hepatitis B virus (HBV), and human immunodeficiency virus (HIV). Lf, a vital component of the mucosal defense system, plays a crucial role in inhibiting viral infection by binding to both host cells and viral particles, such as the Hepatitis C virus (HCV). This interaction enables Lf to keep viral particles away from their target cells, emphasizing its significance as a fundamental element of mucosal defense against viral infections. Additionally, Lf has the ability to modulate cytokine expression and enhance cellular immune responses. In the innate immune system, Lf serves as a unique iron transporter and helps suppress various pathogens like bacteria, fungi, and viruses. This article summarises the potential antiviral properties of Lf against various viruses, along with its other mentioned functions. The advancement of Lf-based therapies supports the homology of food and medicine, providing a promising avenue to address viral infections and other public health challenges.
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Affiliation(s)
- Amna Shafqat
- College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China
| | - Maochen Li
- College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China
| | - Zakirullah
- College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China
| | - Feitong Liu
- H&H Group, H&H Research, China Research and Innovation, Guangzhou, China.
| | - Yigang Tong
- College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China.
| | - Junfen Fan
- Institute of Cerebrovascular Diseases Research and Department of Neurology, Xuanwu Hospital of Capital Medical University, Beijing, China.
| | - Huahao Fan
- School of Life Sciences, Tianjin University, Tianjin, China.
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Sistayanarain A, Kunthalert D. Molecular characterization of the nonstructural 5A (NS5A) region of hepatitis C virus in Thai blood donors. Arch Microbiol 2024; 206:215. [PMID: 38619622 DOI: 10.1007/s00203-024-03950-4] [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: 02/16/2024] [Revised: 03/27/2024] [Accepted: 04/01/2024] [Indexed: 04/16/2024]
Abstract
Direct acting antivirals (DAAs) have been developed for hepatitis C virus (HCV) therapy, and they are usually effective, however resistance to DAA regimens has also been reported to have a significant impact. Resistance associated substitutions (RASs) in the NS5A region are known to be correlated with failure of DAA therapy. HCV genotypes 3a and 1 are the most prevalent genotypes in Thailand. This study analyzed the type and frequency of RASs associated with DAA failure, focusing on the NS5A region. Serum samples of HCV genotype 3a, 1a, and 1b infection from Thai blood donors were selected. The NS5A region was amplified using reverse transcription-polymerase chain reaction (RT-PCR). A phylogenetic tree was constructed to identify the genotypes of HCV. Nucleotide sequencing and amino acid sequencing were conducted to determine the prevalence of RASs. Construction of the phylogenetic tree indicated that 29 samples were genotype 3a, 11 samples were genotype 1a, and 9 were genotype 1b. Both HCV genotypes 1a and 3a can be categorized into two subclades. Results showed that the NS5A substitutions A30V/K, A62T/V/I/M/P/S/L, and S98G were present in HCV genotype 3a. In HCV genotype 1a, only NS5A RASs H54Y was detected. NS5A amino acid substitutions Q54H and P58L were found in HCV genotype 1b. In conclusion, NS5A RASs at amino acid positions 30, 62, 54, 58, and 98 are present within HCV genotypes 3a and 1. While keeping in mind that additional information was not available on the anonymous blood donors tested in this study, these findings can contribute to understand the NS5A mutation. Further study with known patients under drug treatment is recommended.
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Affiliation(s)
- Anchalee Sistayanarain
- Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand.
| | - Duangkamol Kunthalert
- Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand
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Sharma S, Thomas E, Caputi M, Asghar W. RT-LAMP-Based Molecular Diagnostic Set-Up for Rapid Hepatitis C Virus Testing. BIOSENSORS 2022; 12:298. [PMID: 35624599 PMCID: PMC9138684 DOI: 10.3390/bios12050298] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 03/31/2022] [Revised: 04/23/2022] [Accepted: 04/29/2022] [Indexed: 06/15/2023]
Abstract
Hepatitis C virus (HCV) infections occur in approximately 3% of the world population. The development of an enhanced and extensive-scale screening is required to accomplish the World Health Organization's (WHO) goal of eliminating HCV as a public health problem by 2030. However, standard testing methods are time-consuming, expensive, and challenging to deploy in remote and underdeveloped areas. Therefore, a cost-effective, rapid, and accurate point-of-care (POC) diagnostic test is needed to properly manage the disease and reduce the economic burden caused by high case numbers. Herein, we present a fully automated reverse-transcription loop-mediated isothermal amplification (RT-LAMP)-based molecular diagnostic set-up for rapid HCV detection. The set-up consists of an automated disposable microfluidic chip, a small surface heater, and a reusable magnetic actuation platform. The microfluidic chip contains multiple chambers in which the plasma sample is processed. The system utilizes SYBR green dye to detect the amplification product with the naked eye. The efficiency of the microfluidic chip was tested with human plasma samples spiked with HCV virions, and the limit of detection observed was 500 virions/mL within 45 min. The entire virus detection process was executed inside a uniquely designed, inexpensive, disposable, and self-driven microfluidic chip with high sensitivity and specificity.
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Affiliation(s)
- Sandhya Sharma
- Department of Electrical Engineering and Computer Science, Florida Atlantic University, Boca Raton, FL 33431, USA;
- Asghar-Lab: Micro and Nanotechnology in Medicine, College of Engineering and Computer Science, Boca Raton, FL 33431, USA
| | - Emmanuel Thomas
- Department of Microbiology and Immunology and Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, FL 33136, USA;
| | - Massimo Caputi
- Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, FL 33431, USA;
| | - Waseem Asghar
- Department of Electrical Engineering and Computer Science, Florida Atlantic University, Boca Raton, FL 33431, USA;
- Asghar-Lab: Micro and Nanotechnology in Medicine, College of Engineering and Computer Science, Boca Raton, FL 33431, USA
- Department of Biological Sciences (Courtesy Appointment), Florida Atlantic University, Boca Raton, FL 33431, USA
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Abstract
In the 1970s, an unknown virus was suspected for documented cases of transfusion-associated hepatitis, a phenomenon called non-A, non-B hepatitis. In 1989, the infectious transmissible agent was identified and named hepatitis C virus (HCV) and, soon enough, the first diagnostic HCV antibody test was developed, which led to a dramatic decrease in new infections. Today, HCV infection remains a global health burden and a major cause of liver cirrhosis, hepatocellular carcinoma and liver transplantation. However, tremendous advances have been made over the decades, and HCV became the first curable, chronic viral infection. The introduction of direct antiviral agents revolutionized antiviral treatment, leading to viral eradication in more than 98% of all patients infected with HCV. This Perspective discusses the history of HCV research, which reads like a role model for successful translational research: starting from a clinical observation, specific therapeutic agents were developed, which finally were implemented in national and global elimination programmes.
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Affiliation(s)
- Michael P. Manns
- grid.10423.340000 0000 9529 9877Hannover Medical School, Hannover, Germany
| | - Benjamin Maasoumy
- grid.10423.340000 0000 9529 9877Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
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5
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Khuroo MS, Sofi AA. The Discovery of Hepatitis Viruses: Agents and Disease. J Clin Exp Hepatol 2020; 10:391-401. [PMID: 32655240 PMCID: PMC7335725 DOI: 10.1016/j.jceh.2020.04.006] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/03/2020] [Accepted: 04/08/2020] [Indexed: 02/06/2023] Open
Abstract
Discovery of five hepatitis viruses A to E has followed distinctive definable phases. Human experiments at Willowbrook identified two forms of hepatitis namely infectious hepatitis and serum hepatitis. The discovery of Australia antigen in 1965 led to rapid scientific developments in viral hepatitis. SH antigen was detected in sera of patients with serum hepatitis and soon SH antigen and Australia antigen were found to be identical and selectively associated with serum hepatitis. In 1970, 42-nm Dane particles were detected in Australia antigen positive sera and linked to the virus of serum hepatitis. Subsequently, a new antigen-antibody system (e-antigen/antibody) was detected in such patients and associated with infectivity. Then, DNA polymerase was found in concentrated pellets containing Australia antigen. Hepatitis B virus (HBV) DNA cloning and sequencing of HBV followed these developments. In 1973, 27 nm hepatitis A virus (HAV)-like particles were visualized in stool samples obtained during acute phase of illness after inoculation of MS-1 strain in volunteers. Cloning and sequencing of HAV followed. In 1977, a new antigen-antibody system (δ antigen-antibody system) was identified by chance associated with HBV. Based on animal transmission studies, δ agent was found to be another virus called hepatitis D virus that is defective, requires the helper functions of HBV and interferes with HBV replication. The search for hepatitis C virus started when non-A, non-B hepatitis was recognised in multiply transfused patients with subsequent successful animal transmission. HCV was identified by a novel immunoscreening approach involving screening of cDNA libraries from infectious sera. The story of hepatitis E is historically linked to discovery of waterborne epidemic non-A, non-B hepatitis from Kashmir, India. Virus-like-particles of the agent were identified in stool samples of a human volunteer after a self-experimentation. HEV cDNA was detected in bile-enriched infectious samples and full-length HEV RNA genome was subsequently cloned and sequenced.
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Affiliation(s)
- Mohammad S. Khuroo
- Digestive Diseases Centre, Dr Khuroo Medical Clinic, Srinagar, Kashmir, J&K (UT), India
- Sher-I-Kashmir Institute of Medical Sciences, Soura, Srinagar, Kashmir, J&K (UT), India
| | - Ahmad A. Sofi
- Digestive Diseases Centre, Dr Khuroo Medical Clinic, Srinagar, Kashmir, J&K (UT), India
- Burn Hall School, Gupkar Road, Sonwar, Srinagar, Kashmir, J&K (UT), India
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Oancea CN, Butaru AE, Streba CT, Pirici D, Rogoveanu I, Diculescu MM, Gheonea DI. Global hepatitis C elimination: history, evolution, revolutionary changes and barriers to overcome. ROMANIAN JOURNAL OF MORPHOLOGY AND EMBRYOLOGY = REVUE ROUMAINE DE MORPHOLOGIE ET EMBRYOLOGIE 2020; 61:643-653. [PMID: 33817705 PMCID: PMC8112794 DOI: 10.47162/rjme.61.3.02] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/10/2020] [Accepted: 03/25/2021] [Indexed: 02/05/2023]
Abstract
The fundamental discovery of the hepatitis C virus (HCV) in 1989 has led to winning this year's Nobel Prize in Medicine. This achievement guided all the steps in identifying the elements of the virus, in order to develop the treatment and to increase the screening solutions, which have slowed the exposure to the virus. The management of infection started with interferon-alpha (IFN-α), which has later enhanced by adding Ribavirin. Nowadays, HCV treatment is based on direct-acting antiviral agents (DAAs). Currently, HCV infection benefits of curative treatment, with which most patients can be cured. When speaking about hepatitis C future, we can say it is looking bright, considering all the progress that has been made in recent years and all the options that we have for curing all genotypes of HCV infection. The aim of this review is to sum up the historical characteristics of HCV discovery, the evolution of treatment and screening actions, gaps, and stages for achieving the international elimination target of the World Health Organization.
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Affiliation(s)
- Carmen Nicoleta Oancea
- Department of Scientific Research Methodology and Department of Pulmonology, University of Medicine and Pharmacy of Craiova, Romania;
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Romero-López C, Berzal-Herranz A. The Role of the RNA-RNA Interactome in the Hepatitis C Virus Life Cycle. Int J Mol Sci 2020; 21:1479. [PMID: 32098260 PMCID: PMC7073135 DOI: 10.3390/ijms21041479] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2020] [Revised: 02/18/2020] [Accepted: 02/19/2020] [Indexed: 02/05/2023] Open
Abstract
RNA virus genomes are multifunctional entities endowed with conserved structural elements that control translation, replication and encapsidation, among other processes. The preservation of these structural RNA elements constraints the genomic sequence variability. The hepatitis C virus (HCV) genome is a positive, single-stranded RNA molecule with numerous conserved structural elements that manage different steps during the infection cycle. Their function is ensured by the association of protein factors, but also by the establishment of complex, active, long-range RNA-RNA interaction networks-the so-called HCV RNA interactome. This review describes the RNA genome functions mediated via RNA-RNA contacts, and revisits some canonical ideas regarding the role of functional high-order structures during the HCV infective cycle. By outlining the roles of long-range RNA-RNA interactions from translation to virion budding, and the functional domains involved, this work provides an overview of the HCV genome as a dynamic device that manages the course of viral infection.
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Affiliation(s)
- Cristina Romero-López
- Instituto de Parasitología y Biomedicina López-Neyra (IPBLN-CSIC), Av. Conocimiento 17, Armilla, 18016 Granada, Spain
| | - Alfredo Berzal-Herranz
- Instituto de Parasitología y Biomedicina López-Neyra (IPBLN-CSIC), Av. Conocimiento 17, Armilla, 18016 Granada, Spain
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Study of multiple genetic variations caused by persistent hepatitis C virus replication in long-term cell culture. Arch Virol 2019; 165:331-343. [PMID: 31832864 DOI: 10.1007/s00705-019-04461-0] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2019] [Accepted: 10/04/2019] [Indexed: 12/23/2022]
Abstract
The most characteristic feature of the hepatitis C virus (HCV) genome in patients with chronic hepatitis C is its remarkable variability and diversity. To better understand this feature, we performed genetic analysis of HCV replicons recovered from two human hepatoma HuH-7-derived cell lines after 1, 3, 5, 7, and 9 years in culture: The cell lines 50-1 and sO harbored HCV 1B-1 and O strain-derived HCV replicons established in 2002 and 2003, respectively. The results revealed that genetic variations in both replicons accumulated in a time-dependent manner at a constant rate despite the maintenance of moderate diversity (less than 1.8% difference) between the clones and that the mutation rate in the 50-1 and sO replicons was 2.5 and 2.9 × 10-3 base substitutions/site/year, respectively. We found that the genetic distance of both replicons increased from 7.9% to 10.5% after 9 years in culture. In addition, we observed that the guanine + cytosine (GC) content of both replicon RNAs increased in a time-dependent manner, as observed in our previous studies. Finally, we demonstrated that the high sensitivity of both replicons to direct-acting antivirals was maintained even after 9 years in culture. Our results suggest that long-term cultured HCV replicon-harboring cells are a useful model for understanding the variability and diversity of the HCV genome and the drug sensitivity of HCV in patients with chronic hepatitis C.
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Wrensch F, Ligat G, Heydmann L, Schuster C, Zeisel MB, Pessaux P, Habersetzer F, King BJ, Tarr AW, Ball JK, Winkler M, Pöhlmann S, Keck ZY, Foung SK, Baumert TF. Interferon-Induced Transmembrane Proteins Mediate Viral Evasion in Acute and Chronic Hepatitis C Virus Infection. Hepatology 2019; 70:1506-1520. [PMID: 31062385 PMCID: PMC6819197 DOI: 10.1002/hep.30699] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/07/2018] [Accepted: 04/30/2019] [Indexed: 02/07/2023]
Abstract
Although adaptive immune responses against hepatitis C virus (HCV) infection have been studied in great detail, the role of innate immunity in protection against HCV infection and immune evasion is only partially understood. Interferon-induced transmembrane proteins (IFITMs) are innate effector proteins restricting host cell entry of many enveloped viruses, including HCV. However, the clinical impact of IFITMs on HCV immune escape remains to be determined. Here, we show that IFITMs promote viral escape from the neutralizing antibody (nAb) response in clinical cohorts of HCV-infected patients. Using pseudoparticles bearing HCV envelope proteins from acutely infected patients, we show that HCV variants isolated preseroconversion are more sensitive to the antiviral activity of IFITMs than variants from patients isolated during chronic infection postseroconversion. Furthermore, HCV variants escaping nAb responses during liver transplantation exhibited a significantly higher resistance to IFITMs than variants that were eliminated posttransplantation. Gain-of-function and mechanistic studies revealed that IFITMs markedly enhance the antiviral activity of nAbs and suggest a cooperative effect of human monoclonal antibodies and IFITMs for antibody-mediated neutralization driving the selection pressure in viral evasion. Perturbation studies with the IFITM antagonist amphotericin B revealed that modulation of membrane properties by IFITM proteins is responsible for the IFITM-mediated blockade of viral entry and enhancement of antibody-mediated neutralization. Conclusion: Our results indicate IFITM proteins as drivers of viral immune escape and antibody-mediated HCV neutralization in acute and chronic HCV infection. These findings are of clinical relevance for the design of urgently needed HCV B-cell vaccines and might help to increase the efficacy of future vaccine candidates.
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Affiliation(s)
- Florian Wrensch
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000 Strasbourg, France,Université de Strasbourg, 67000 Strasbourg, France
| | - Gaëtan Ligat
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000 Strasbourg, France,Université de Strasbourg, 67000 Strasbourg, France
| | - Laura Heydmann
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000 Strasbourg, France,Université de Strasbourg, 67000 Strasbourg, France
| | - Catherine Schuster
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000 Strasbourg, France,Université de Strasbourg, 67000 Strasbourg, France
| | - Mirjam B. Zeisel
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000 Strasbourg, France,Université de Strasbourg, 67000 Strasbourg, France,Inserm U1052, CNRS UMR 5286, Cancer Research Center of Lyon (CRCL), Université de Lyon (UCBL), 69373 Lyon, France
| | - Patrick Pessaux
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000 Strasbourg, France,Université de Strasbourg, 67000 Strasbourg, France,Institut Hospitalo-Universitaire, Pôle Hépato-digestif, Hôpitaux Universitaires de Strasbourg, 67000 Strasbourg, France
| | - François Habersetzer
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000 Strasbourg, France,Université de Strasbourg, 67000 Strasbourg, France,Institut Hospitalo-Universitaire, Pôle Hépato-digestif, Hôpitaux Universitaires de Strasbourg, 67000 Strasbourg, France
| | - Barnabas J. King
- School of Life Sciences, The University of Nottingham, Nottingham NG7 2UH, UK,NIHR Nottingham BRC, Nottingham University Hospitals NHS Trust and the University of Nottingham, Nottingham NG7 2UH, UK
| | - Alexander W. Tarr
- School of Life Sciences, The University of Nottingham, Nottingham NG7 2UH, UK,NIHR Nottingham BRC, Nottingham University Hospitals NHS Trust and the University of Nottingham, Nottingham NG7 2UH, UK
| | - Jonathan K. Ball
- School of Life Sciences, The University of Nottingham, Nottingham NG7 2UH, UK,NIHR Nottingham BRC, Nottingham University Hospitals NHS Trust and the University of Nottingham, Nottingham NG7 2UH, UK
| | - Michael Winkler
- Infection Biology Unit, German Primate Center–Leibniz Institute for Primate Research, 37077 Göttingen, Germany
| | - Stefan Pöhlmann
- Infection Biology Unit, German Primate Center–Leibniz Institute for Primate Research, 37077 Göttingen, Germany,Faculty of Biology and Psychology, University of Göttingen, 37073 Göttingen, Germany
| | - Zhen-yong Keck
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305
| | - Steven K.H. Foung
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305
| | - Thomas F. Baumert
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000 Strasbourg, France,Université de Strasbourg, 67000 Strasbourg, France,Institut Hospitalo-Universitaire, Pôle Hépato-digestif, Hôpitaux Universitaires de Strasbourg, 67000 Strasbourg, France,Institut Universitaire de France, 75231 Paris, France
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Sezaki H, Suzuki F, Hosaka T, Fujiyama S, Kawamura Y, Akuta N, Kobayashi M, Suzuki Y, Saitoh S, Arase Y, Ikeda K, Kobayashi M, Kumada H. Initial- and re-treatment effectiveness of glecaprevir and pibrentasvir for Japanese patients with chronic hepatitis C virus-genotype 1/2/3 infections. J Gastroenterol 2019; 54:916-927. [PMID: 30903385 DOI: 10.1007/s00535-019-01575-9] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/29/2019] [Accepted: 03/17/2019] [Indexed: 02/04/2023]
Abstract
BACKGROUND Glecaprevir and pibrentasvir (GLE/PIB) are potent antiviral agents for hepatitis C virus (HCV) pan-genotypic infections; however, their clinical effectiveness and safety remain limited in the real-world. This study aimed to evaluate viral responses and the safety of GLE/PIB for patients with chronic HCV-1/2/3 infections during both initial- (Arm A) and re-treatment (Arm B) with all-oral direct-acting antiviral agents (DAAs). METHODS This prospective-observational cohort study included Japanese patients with chronic HCV-1/2/3 infections (n = 271: 183 in Arm A and 83 in Arm B), who had started receiving GLE/PIB. Primary end point was a sustained virological response (SVR) rate at week 12 (SVR12) after the end of GLE/PIB treatment (EOT). RESULTS SVR12 was achieved by 99.4% of patients (180/181: modified intention-to-treat (mITT) analysis excluding 2 patients lost to follow-up) in Arm A. One patient with an HCV-3b infection who discontinued at week 8 failed to achieve SVR12. SVR12 was achieved by 97.7% of patients (85/87: mITT excluding 1 patient lost to follow-up) in Arm B. Virological relapse occurred in 2 patients with HCV-1b, presenting common 5 loci of resistance-associated substitutions (RASs) including A92 RASs in the NS5A lesion at baseline. Any adverse events (AEs) (grade ≥ 3) occurred in 8 patients (3.0%). 8 patients (3.0%) discontinued due to AEs, however, all of them achieved SVR12. CONCLUSIONS Initial and re-treatment with GLE/PIB are effective and safe for Japanese patients with HCV-1/2/3 in real-life settings. Further studies are required to elucidate the mechanism underlying treatment failures of GLE/PIB to completely eradicate HCV worldwide.
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Affiliation(s)
- Hitomi Sezaki
- Department of Hepatology, Toranomon Hospital, 2-2-2 Toranomon, Minato-ku, Tokyo, 105-8470, Japan.
| | - Fumitaka Suzuki
- Department of Hepatology, Toranomon Hospital, 2-2-2 Toranomon, Minato-ku, Tokyo, 105-8470, Japan
| | - Tetsuya Hosaka
- Department of Hepatology, Toranomon Hospital, 2-2-2 Toranomon, Minato-ku, Tokyo, 105-8470, Japan
| | - Shunichirou Fujiyama
- Department of Hepatology, Toranomon Hospital, 2-2-2 Toranomon, Minato-ku, Tokyo, 105-8470, Japan
| | - Yusuke Kawamura
- Department of Hepatology, Toranomon Hospital, 2-2-2 Toranomon, Minato-ku, Tokyo, 105-8470, Japan
| | - Norio Akuta
- Department of Hepatology, Toranomon Hospital, 2-2-2 Toranomon, Minato-ku, Tokyo, 105-8470, Japan
| | - Masahiro Kobayashi
- Department of Hepatology, Toranomon Hospital, 2-2-2 Toranomon, Minato-ku, Tokyo, 105-8470, Japan
| | - Yoshiyuki Suzuki
- Department of Hepatology, Toranomon Hospital, 2-2-2 Toranomon, Minato-ku, Tokyo, 105-8470, Japan
| | - Satoshi Saitoh
- Department of Hepatology, Toranomon Hospital, 2-2-2 Toranomon, Minato-ku, Tokyo, 105-8470, Japan
| | - Yasuji Arase
- Department of Hepatology, Toranomon Hospital, 2-2-2 Toranomon, Minato-ku, Tokyo, 105-8470, Japan
| | - Kenji Ikeda
- Department of Hepatology, Toranomon Hospital, 2-2-2 Toranomon, Minato-ku, Tokyo, 105-8470, Japan
| | - Mariko Kobayashi
- Research Institute for Hepatology, Toranomon Hospital, Tokyo, Japan
| | - Hiromitsu Kumada
- Department of Hepatology, Toranomon Hospital, 2-2-2 Toranomon, Minato-ku, Tokyo, 105-8470, Japan
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11
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Sano T, Akuta N, Suzuki F, Kasuya K, Fujiyama S, Kawamura Y, Sezaki H, Hosaka T, Saitoh S, Kobayashi M, Suzuki Y, Kobayashi M, Arase Y, Ikeda K, Kumada H. Role of NS5A-L31/Y93 Double Wild-type in Failure of Glecaprevir/Pibrentasvir Double Therapy in Two Patients with a History of Direct-acting Antiviral Agent Failure: An Ultra-deep Sequencing Analysis. Intern Med 2019; 58:2657-2662. [PMID: 31178495 PMCID: PMC6794189 DOI: 10.2169/internalmedicine.2604-18] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/30/2018] [Accepted: 03/24/2019] [Indexed: 01/26/2023] Open
Abstract
We experienced two cases of hepatitis C virus (HCV) eradication failure in patients with a history of non-responsiveness to previous treatments with direct-acting antiviral agents (DAAs) who were subsequently treated with the combination of glecaprevir and pibrentasvir (GLE/PIB). Direct sequencing at commencement of GLE/PIB therapy showed non-structural protein (NS) 5A-P32 deletion in the first patient and NS5A-R30E/Q54H/A92K in the second patient (both genotype 1b). The common point was that L31/Y93 was double wild-type, and the IL28B polymorphism was non-TT type. Even when L31/Y93 is double wild-type, other NS5A mutations may affect the DAA re-treatment outcome. We analyzed the transition of amino acid mutations at NS5A by ultra-deep sequencing.
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Affiliation(s)
- Tomoya Sano
- Department of Hepatology, Toranomon Hospital, Japan
| | - Norio Akuta
- Department of Hepatology, Toranomon Hospital, Japan
| | | | | | | | | | | | | | | | | | | | | | - Yasuji Arase
- Department of Hepatology, Toranomon Hospital, Japan
| | - Kenji Ikeda
- Department of Hepatology, Toranomon Hospital, Japan
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12
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Takeuchi Y, Akuta N, Sezaki H, Suzuki F, Fujiyama S, Kawamura Y, Hosaka T, Kobayashi M, Kobayashi M, Saitoh S, Suzuki Y, Arase Y, Ikeda K, Kumada H. Efficacy and safety of elbasvir plus grazoprevir combination therapy in Japanese patients infected with hepatitis C virus genotype 1b. Hepatol Res 2019; 49:256-263. [PMID: 30125438 DOI: 10.1111/hepr.13242] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/29/2018] [Revised: 07/20/2018] [Accepted: 08/14/2018] [Indexed: 02/08/2023]
Abstract
AIM Treatment with all-oral direct-acting antiviral agents (DAAs) elbasvir/grazoprevir (EBR/GZR) is associated with high sustained virologic response (SVR). The aim of this study was to evaluate the safety and treatment efficacy of EBR/GZR in hepatitis C virus (HCV)-infected patients. METHODS This retrospective cohort study included 147 consecutive patients with chronic HCV genotype 1b infection who were treated with EBR (50 mg) plus GZR (100 mg) once daily for 12 weeks. The rates of SVR at 12 weeks after the end of treatment (SVR12) were evaluated based on patient baseline characteristics. Treatment efficacy was analyzed according to background chronic kidney disease (CKD), and retreatment efficacy in patients who failed to respond to previous DAAs. RESULTS The SVR12 was 94% (138 of 147 patients), based on intention-to-treat analysis. Rates of SVR12 were 97% (131 of 135) and 58% (7 of 12) in cases naïve to DAA treatment and failure to respond to prior DAAs, respectively. The SVR12 rates in patients with CKD stage 4-5 was 100% (8 of 8). All patients (4 of 4 patients) with stage 4-5 and advanced fibrosis (Fibrosis-4 index ≥3.25) also achieved SVR12. Multivariate analysis that included the above variables identified pretreatment with other DAAs as an independent factor that was significantly and independently associated with non-SVR12 (odds ratio, 97.5; P < 0.001). Relapsers of first DAAs, excluding the combination of ledipasvir and sofosbuvir, achieved SVR12. Characteristic novel non-structural protein 5A substitutions were not detected after failure of retreatment with EBR/GZR. CONCLUSION Treatment with EBR/GZR was highly efficacious with acceptable safety, even in patients with CKD stage 4-5. Retreatment of relapsers to prior DAAs, excluding ledipasvir and sofosbuvir, achieved SVR12.
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Affiliation(s)
- Yasue Takeuchi
- Department of Hepatology, Toranomon Hospital, Okinaka Memorial Institute for Medical Research, Tokyo, Japan
| | - Norio Akuta
- Department of Hepatology, Toranomon Hospital, Okinaka Memorial Institute for Medical Research, Tokyo, Japan
| | - Hitomi Sezaki
- Department of Hepatology, Toranomon Hospital, Okinaka Memorial Institute for Medical Research, Tokyo, Japan
| | - Fumitaka Suzuki
- Department of Hepatology, Toranomon Hospital, Okinaka Memorial Institute for Medical Research, Tokyo, Japan
| | - Shunichiro Fujiyama
- Department of Hepatology, Toranomon Hospital, Okinaka Memorial Institute for Medical Research, Tokyo, Japan
| | - Yusuke Kawamura
- Department of Hepatology, Toranomon Hospital, Okinaka Memorial Institute for Medical Research, Tokyo, Japan
| | - Tetsuya Hosaka
- Department of Hepatology, Toranomon Hospital, Okinaka Memorial Institute for Medical Research, Tokyo, Japan
| | - Masahiro Kobayashi
- Department of Hepatology, Toranomon Hospital, Okinaka Memorial Institute for Medical Research, Tokyo, Japan
| | | | - Satoshi Saitoh
- Department of Hepatology, Toranomon Hospital, Okinaka Memorial Institute for Medical Research, Tokyo, Japan
| | - Yoshiyuki Suzuki
- Department of Hepatology, Toranomon Hospital, Okinaka Memorial Institute for Medical Research, Tokyo, Japan
| | - Yasuji Arase
- Department of Hepatology, Toranomon Hospital, Okinaka Memorial Institute for Medical Research, Tokyo, Japan
| | - Kenji Ikeda
- Department of Hepatology, Toranomon Hospital, Okinaka Memorial Institute for Medical Research, Tokyo, Japan
| | - Hiromitsu Kumada
- Department of Hepatology, Toranomon Hospital, Okinaka Memorial Institute for Medical Research, Tokyo, Japan
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13
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Jansons J, Sominskaya I, Petrakova N, Starodubova ES, Smirnova OA, Alekseeva E, Bruvere R, Eliseeva O, Skrastina D, Kashuba E, Mihailova M, Kochetkov SN, Ivanov AV, Isaguliants MG. The Immunogenicity in Mice of HCV Core Delivered as DNA Is Modulated by Its Capacity to Induce Oxidative Stress and Oxidative Stress Response. Cells 2019; 8:208. [PMID: 30823485 PMCID: PMC6468923 DOI: 10.3390/cells8030208] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2018] [Revised: 02/06/2019] [Accepted: 02/20/2019] [Indexed: 12/16/2022] Open
Abstract
HCV core is an attractive HCV vaccine target, however, clinical or preclinical trials of core-based vaccines showed little success. We aimed to delineate what restricts its immunogenicity and improve immunogenic performance in mice. We designed plasmids encoding full-length HCV 1b core and its variants truncated after amino acids (aa) 60, 98, 152, 173, or up to aa 36 using virus-derived or synthetic polynucleotides (core191/60/98/152/173/36_191v or core152s DNA, respectively). We assessed their level of expression, route of degradation, ability to trigger the production of reactive oxygen species/ROS, and to activate the components of the Nrf2/ARE antioxidant defense pathway heme oxygenase 1/HO-1 and NAD(P)H: quinone oxidoreductase/Nqo-1. All core variants with the intact N-terminus induced production of ROS, and up-regulated expression of HO-1 and Nqo-1. The capacity of core variants to induce ROS and up-regulate HO-1 and Nqo-1 expression predetermined their immunogenicity in DNA-immunized BALB/c and C57BL/6 mice. The most immunogenic was core 152s, expressed at a modest level and inducing moderate oxidative stress and oxidative stress response. Thus, immunogenicity of HCV core is shaped by its ability to induce ROS and oxidative stress response. These considerations are important in understanding the mechanisms of viral suppression of cellular immune response and in HCV vaccine design.
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Affiliation(s)
- Juris Jansons
- Department of Pathology, Riga Stradins University, LV-1007 Riga, Latvia.
- Latvian Biomedical Research and Study Centre, LV-1067 Riga, Latvia.
- Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
| | - Irina Sominskaya
- Latvian Biomedical Research and Study Centre, LV-1067 Riga, Latvia.
| | - Natalia Petrakova
- N.F. Gamaleya Research Center of Epidemiology and Microbiology, Ministry of Health of the Russian Federation, 123098 Moscow, Russia.
| | - Elizaveta S Starodubova
- N.F. Gamaleya Research Center of Epidemiology and Microbiology, Ministry of Health of the Russian Federation, 123098 Moscow, Russia.
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia.
| | - Olga A Smirnova
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia.
| | - Ekaterina Alekseeva
- Department of Pathology, Riga Stradins University, LV-1007 Riga, Latvia.
- Latvian Biomedical Research and Study Centre, LV-1067 Riga, Latvia.
| | - Ruta Bruvere
- Latvian Biomedical Research and Study Centre, LV-1067 Riga, Latvia.
| | - Olesja Eliseeva
- N.F. Gamaleya Research Center of Epidemiology and Microbiology, Ministry of Health of the Russian Federation, 123098 Moscow, Russia.
| | - Dace Skrastina
- Department of Pathology, Riga Stradins University, LV-1007 Riga, Latvia.
- Latvian Biomedical Research and Study Centre, LV-1067 Riga, Latvia.
| | - Elena Kashuba
- Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
- RE Kavetsky Institite of Experimental Pathology, Oncology and Radiobiology, The National Academy of Sciences of Ukraine, 03022 Kyiv, Ukraine.
| | - Marija Mihailova
- Latvian Biomedical Research and Study Centre, LV-1067 Riga, Latvia.
| | - Sergey N Kochetkov
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia.
| | - Alexander V Ivanov
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia.
| | - Maria G Isaguliants
- Department of Pathology, Riga Stradins University, LV-1007 Riga, Latvia.
- Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
- N.F. Gamaleya Research Center of Epidemiology and Microbiology, Ministry of Health of the Russian Federation, 123098 Moscow, Russia.
- MP Chumakov Center for Research and Development of Immune and Biological Preparations of RAS, 108819 Moscow, Russia.
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14
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Mailly L, Wrensch F, Heydmann L, Fauvelle C, Brignon N, Zeisel MB, Pessaux P, Keck ZY, Schuster C, Fuerst TR, Foung SKH, Baumert TF. In vivo combination of human anti-envelope glycoprotein E2 and -Claudin-1 monoclonal antibodies for prevention of hepatitis C virus infection. Antiviral Res 2018; 162:136-141. [PMID: 30599173 DOI: 10.1016/j.antiviral.2018.12.018] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2018] [Revised: 11/14/2018] [Accepted: 12/28/2018] [Indexed: 12/20/2022]
Abstract
Despite the development of direct-acting antivirals (DAAs), hepatitis C virus (HCV) infection remains a major cause for liver disease and cancer worldwide. Entry inhibitors block virus host cell entry and, therefore, prevent establishment of chronic infection and liver disease. Due to their unique mechanism of action, entry inhibitors provide an attractive antiviral strategy in organ transplantation. In this study, we developed an innovative approach in preventing HCV infection using a synergistic combination of a broadly neutralizing human monoclonal antibody (HMAb) targeting the HCV E2 protein and a host-targeting anti-claudin 1 (CLDN1) humanized monoclonal antibody. An in vivo proof-of-concept study in human liver-chimeric FRG-NOD mice proved the efficacy of the combination therapy at preventing infection by an HCV genotype 1b infectious serum. While administration of individual antibodies at lower doses only showed a delay in HCV infection, the combination therapy was highly protective. Furthermore, the combination proved to be effective in preventing infection of primary human hepatocytes by neutralization-resistant HCV escape variants selected during liver transplantation, suggesting that a combination therapy is suited for the neutralization of difficult-to-treat variants. In conclusion, our findings suggest that the combination of two HMAbs targeting different steps of virus entry improves treatment efficacy while simultaneously reducing treatment duration and costs. Our approach not only provides a clinical perspective to employ HMAb combination therapies to prevent graft re-infection and its associated liver disease but may also help to alleviate the urgent demand for organ transplants by allowing the transplantation of organs from HCV-positive donors.
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Affiliation(s)
- Laurent Mailly
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000, Strasbourg, France; Université de Strasbourg, 67000, Strasbourg, France
| | - Florian Wrensch
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000, Strasbourg, France; Université de Strasbourg, 67000, Strasbourg, France
| | - Laura Heydmann
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000, Strasbourg, France; Université de Strasbourg, 67000, Strasbourg, France
| | - Catherine Fauvelle
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000, Strasbourg, France; Université de Strasbourg, 67000, Strasbourg, France
| | - Nicolas Brignon
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000, Strasbourg, France; Université de Strasbourg, 67000, Strasbourg, France
| | - Mirjam B Zeisel
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000, Strasbourg, France; Université de Strasbourg, 67000, Strasbourg, France; Inserm U1052, CNRS UMR 5286, Cancer Research Center of Lyon (CRCL), Université de Lyon (UCBL), Lyon, France
| | - Patrick Pessaux
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000, Strasbourg, France; Université de Strasbourg, 67000, Strasbourg, France; Institut Hospitalo-Universitaire, Pôle Hépato-digestif, Hôpitaux Universitaires de Strasbourg, Strasbourg, France
| | - Zhen-Yong Keck
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA
| | - Catherine Schuster
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000, Strasbourg, France; Université de Strasbourg, 67000, Strasbourg, France
| | - Thomas R Fuerst
- University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, MD, USA; Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA
| | - Steven K H Foung
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA
| | - Thomas F Baumert
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000, Strasbourg, France; Université de Strasbourg, 67000, Strasbourg, France; Institut Hospitalo-Universitaire, Pôle Hépato-digestif, Hôpitaux Universitaires de Strasbourg, Strasbourg, France; Institut Universitaire de France, Paris, France.
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15
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Provazzi PJS, Rossi LMG, Carneiro BM, Miura VC, Rosa PCR, de Carvalho LR, de Andrade STQ, Fachini RM, Grotto RMT, Silva GF, Valêncio CR, Neto PS, Cordeiro JA, Nogueira ML, Rahal P. Hierarchical assessment of host factors influencing the spontaneous resolution of hepatitis C infection. Braz J Microbiol 2018; 50:147-155. [PMID: 30637644 DOI: 10.1007/s42770-018-0008-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2017] [Accepted: 04/06/2018] [Indexed: 11/29/2022] Open
Abstract
BACKGROUND Hepatitis C virus (HCV) infection is associated with chronic liver disease, resulting in cirrhosis and hepatocellular carcinoma. Approximately 20% of HCV infections are spontaneously resolved. Here, we assessed the hierarchical relevance of host factors contributing to viral clearance. METHODS DNA samples from 40 resolved infections and 40 chronic HCV patients paired by age were analyzed. Bivariate analysis was performed to rank the importance of each contributing factor in spontaneous HCV clearance. RESULTS Interestingly, 63.6% of patients with resolved infections exhibited the protective genotype CC for SNP rs12979860. Additionally, 59.3% of patients with resolved infections displayed the protective genotype TT/TT for SNP ss469415590. Moreover, a ranking of clearance factors was estimated. In order of importance, the IL28B CC genotype (OR 0.197, 95% CI 0.072-0.541) followed by the INFL4 TT/TT genotype (OR 0.237, 95% CI 0.083-0.679), and female gender (OR 0.394, 95% CI 0.159-0.977) were the main predictors for clearance of HCV infection. CONCLUSIONS HCV clearance is multifactorial and the contributing factors display a hierarchical order. Identifying all elements playing role in HCV clearance is of the most importance for HCV-related disease management. Dissecting the relevance of each contributing factor will certainly improve our understanding of the pathogenesis of HCV infection.
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Affiliation(s)
| | | | - Bruno Moreira Carneiro
- Department of Biology, São Paulo State University - UNESP, São José do Rio Preto, SP, 15054-000, Brazil
| | - Valeria Chamas Miura
- Department of Biology, São Paulo State University - UNESP, São José do Rio Preto, SP, 15054-000, Brazil
| | | | | | | | - Roberta Maria Fachini
- Department of Hepatology, São José do Rio Preto Medical School, São José do Rio Preto, SP, 15090-000, Brazil
| | | | - Giovanni Faria Silva
- Department of Internal Medicine, São Paulo State University - UNESP, Botucatu, SP, 18618-970, Brazil
| | - Carlos Roberto Valêncio
- Department of Computer Science and Statistics, São Paulo State University - UNESP, São José do Rio Preto, SP, 15054-000, Brazil
| | - Paulo Scarpelini Neto
- Department of Computer Science and Statistics, São Paulo State University - UNESP, São José do Rio Preto, SP, 15054-000, Brazil
| | - José Antonio Cordeiro
- Department of Computer Science and Statistics, São Paulo State University - UNESP, São José do Rio Preto, SP, 15054-000, Brazil
| | - Mauricio Lacerda Nogueira
- Laboratory of Virology, São José do Rio Preto Medical School, São José do Rio Preto, SP, 15090-000, Brazil
| | - Paula Rahal
- Department of Biology, São Paulo State University - UNESP, São José do Rio Preto, SP, 15054-000, Brazil.
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16
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Yerukhimovich MM, Marohnic CC, Frick DN. Role of the Conserved DECH-Box Cysteine in Coupling Hepatitis C Virus Helicase-Catalyzed ATP Hydrolysis to RNA Unwinding. Biochemistry 2018; 57:6247-6255. [PMID: 30281972 DOI: 10.1021/acs.biochem.8b00796] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
DECH-box proteins are a subset of DExH/D-box superfamily 2 helicases possessing a conserved Asp-Glu-Cys-His motif in their ATP binding site. The conserved His helps position the Asp and Glu residues, which coordinate the divalent metal cation that connects the protein to ATP and activate the water molecule needed for ATP hydrolysis, but the role of the Cys is still unclear. This study uses site-directed mutants of the model DECH-box helicase encoded by the hepatitis C virus (HCV) to examine the role of the Cys in helicase action. Proteins lacking a Cys unwound DNA less efficiently than wild-type proteins did. For example, at low protein concentrations, a helicase harboring a Gly instead of the DECH-box Cys unwound DNA more slowly than the wild-type helicase did, but at higher protein concentrations, the two proteins unwound DNA at similar rates. All HCV proteins analyzed had similar affinities for ATP and nucleic acids and hydrolyzed ATP in the presence of RNA at similar rates. However, in the absence of RNA, all proteins lacking a DECH-box cysteine hydrolyzed ATP 10-15 times faster with higher Km values, and lower apparent affinities for metal ions, compared to those observed with wild-type proteins. These differences were observed with proteins isolated from HCV genotypes 2a and 1b, suggesting that this role is conserved. These data suggest the helicase needs Cys292 to bind ATP in a state where ATP is not hydrolyzed until RNA binds.
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Affiliation(s)
- Mark M Yerukhimovich
- Department of Chemistry & Biochemistry , University of Wisconsin-Milwaukee , Milwaukee , Wisconsin 53211 , United States
| | - Christopher C Marohnic
- Abbott Laboratories , 100 Abbott Park Road , Abbott Park , Illinois 60064 , United States
| | - David N Frick
- Department of Chemistry & Biochemistry , University of Wisconsin-Milwaukee , Milwaukee , Wisconsin 53211 , United States
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17
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Gauna A, Losada S, Lorenzo M, Toledo M, Bermúdez H, D'Angelo P, Sánchez D, Noya O. Use of Synthetic Peptides and Multiple Antigen Blot Assay in the Immunodiagnosis of Hepatitis C Virus Infection. Viral Immunol 2018; 31:568-574. [PMID: 30256730 DOI: 10.1089/vim.2018.0023] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Acute hepatitis C virus (HCV) infection is usually asymptomatic, therefore, early diagnosis is rare. It may remain undiagnosed in individuals who progress to chronic infection, often until serious liver damage has developed. To incorporate the diagnosis of this viral disease in a multiple-diagnostic assay, we first analyzed by immunoinformatics the HCV subtype 1a polyprotein (specifically Core, E2, NS3, NS5A proteins) to select antigenic peptides to be tested initially by the Pepscan technique. Next, we performed the immunodiagnosis of HCV infection, using the Multiple Antigen Blot Assay (MABA). In 22 patients' sera included in this study, a 20-mer linear peptide belonging to the N-terminus of the worldwide conserved Core protein showed 100% sensitivity and specificity; other sequences showed different levels of antibody recognition. The use of MABA in combination with synthetic peptides as a source of multiple, specific, and nonexpensive antigens for other infectious diseases could represent a rapid, integrated, and inexpensive diagnostic methodology.
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Affiliation(s)
- Adriana Gauna
- 1 Programa de Doctorado en Biotecnología, Pontificia Universidad Católica de Valparaíso/Universidad Técnica Federico Santa María , Valparaíso, Chile
| | - Sandra Losada
- 2 Sección de Biohelmintiasis, Instituto de Medicina Tropical , Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela
| | - María Lorenzo
- 2 Sección de Biohelmintiasis, Instituto de Medicina Tropical , Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela
| | - Marilyan Toledo
- 3 Cátedra de Parasitología, Escuela de Medicina "Luis Razetti," Universidad Central de Venezuela , Caracas, Venezuela
| | - Henry Bermúdez
- 2 Sección de Biohelmintiasis, Instituto de Medicina Tropical , Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela
| | - Pierina D'Angelo
- 4 Laboratorio de Programas Especiales-Hepatitis y SIDA, Dpto de Virología, Gerencia Sectorial de Diagnóstico y Vigilancia Epidemiológica, Instituto Nacional de Higiene "Rafael Rangel ," Caracas, Venezuela
| | - Doneyla Sánchez
- 4 Laboratorio de Programas Especiales-Hepatitis y SIDA, Dpto de Virología, Gerencia Sectorial de Diagnóstico y Vigilancia Epidemiológica, Instituto Nacional de Higiene "Rafael Rangel ," Caracas, Venezuela
| | - Oscar Noya
- 2 Sección de Biohelmintiasis, Instituto de Medicina Tropical , Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela .,5 Centro para Estudios Sobre Malaria, Instituto de Altos Estudios "Dr. Arnoldo Gabaldón" Instituto Nacional de Higiene-Ministerio del Poder Popular para la Salud , Caracas, Venezuela
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18
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Akuta N, Sezaki H, Suzuki F, Fujiyama S, Kawamura Y, Hosaka T, Kobayashi M, Kobayashi M, Saitoh S, Suzuki Y, Arase Y, Ikeda K, Kumada H. Favorable efficacy of glecaprevir plus pibrentasvir as salvage therapy for HCV failures to prior direct‐acting antivirals regimens. J Med Virol 2018; 91:102-106. [DOI: 10.1002/jmv.25278] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2018] [Revised: 07/04/2018] [Accepted: 07/19/2018] [Indexed: 12/24/2022]
Affiliation(s)
- Norio Akuta
- Department of HepatologyToranomon Hospital, and Okinaka Memorial Institute for Medical ResearchTokyo Japan
| | - Hitomi Sezaki
- Department of HepatologyToranomon Hospital, and Okinaka Memorial Institute for Medical ResearchTokyo Japan
| | - Fumitaka Suzuki
- Department of HepatologyToranomon Hospital, and Okinaka Memorial Institute for Medical ResearchTokyo Japan
| | - Shunichiro Fujiyama
- Department of HepatologyToranomon Hospital, and Okinaka Memorial Institute for Medical ResearchTokyo Japan
| | - Yusuke Kawamura
- Department of HepatologyToranomon Hospital, and Okinaka Memorial Institute for Medical ResearchTokyo Japan
| | - Tetsuya Hosaka
- Department of HepatologyToranomon Hospital, and Okinaka Memorial Institute for Medical ResearchTokyo Japan
| | - Masahiro Kobayashi
- Department of HepatologyToranomon Hospital, and Okinaka Memorial Institute for Medical ResearchTokyo Japan
| | | | - Satoshi Saitoh
- Department of HepatologyToranomon Hospital, and Okinaka Memorial Institute for Medical ResearchTokyo Japan
| | - Yoshiyuki Suzuki
- Department of HepatologyToranomon Hospital, and Okinaka Memorial Institute for Medical ResearchTokyo Japan
| | - Yasuji Arase
- Department of HepatologyToranomon Hospital, and Okinaka Memorial Institute for Medical ResearchTokyo Japan
| | - Kenji Ikeda
- Department of HepatologyToranomon Hospital, and Okinaka Memorial Institute for Medical ResearchTokyo Japan
| | - Hiromitsu Kumada
- Department of HepatologyToranomon Hospital, and Okinaka Memorial Institute for Medical ResearchTokyo Japan
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19
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Mattar EH, Almehdar HA, Uversky VN, Redwan EM. Virucidal activity of human α- and β-defensins against hepatitis C virus genotype 4. MOLECULAR BIOSYSTEMS 2017; 12:2785-97. [PMID: 27327492 DOI: 10.1039/c6mb00283h] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Hepatitis C virus (HCV) is the major etiological agent of human non-A and non-B hepatitis affecting about 180 million people worldwide. The goal of the current study was to find effective anti-HCV proteins. As a result, defensins were selected as promising candidates due to their well-known anti-viral potential and small size. We conducted in vitro evaluation of two kinds of defensins (human α- and β-defensins and synthetic linear avian α-defensins) using tissue culture combined with reverse transcription nested PCR (RT-nested-PCR) and real-time PCR. Human α- and β-defensins showed strong anti-HCV activity in experiments on cellular protection, neutralization, and treatment at all concentrations used (10, 20 and 50 μg). The synthetic linear defensins could reach similar anti-HCV potential only at a noticeably higher concentration (250 μg) and do not show noticeable activity at 10 and 20 μg. This study suggests that defensins are potent anti-HCV agents.
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Affiliation(s)
- Ehab H Mattar
- Department of Biological Sciences, Faculty of Sciences, King Abdulaziz University, P.O. Box 80203, Jeddah, Saudi Arabia.
| | - Hussein A Almehdar
- Department of Biological Sciences, Faculty of Sciences, King Abdulaziz University, P.O. Box 80203, Jeddah, Saudi Arabia.
| | - Vladimir N Uversky
- Department of Biological Sciences, Faculty of Sciences, King Abdulaziz University, P.O. Box 80203, Jeddah, Saudi Arabia. and Department of Molecular Medicine and USF Health Byrd Alzheimer's Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL, USA and Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russian Federation
| | - Elrashdy M Redwan
- Department of Biological Sciences, Faculty of Sciences, King Abdulaziz University, P.O. Box 80203, Jeddah, Saudi Arabia. and Therapeutic and Protective Proteins Laboratory, Protein Research Department, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications, New Borg EL-Arab 21934, Alexandria, Egypt
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20
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Evaluation of preclinical antimalarial drugs, which can overcome direct-acting antivirals-resistant hepatitis C viruses, using the viral reporter assay systems. Virus Res 2017; 235:37-48. [PMID: 28322919 DOI: 10.1016/j.virusres.2017.03.015] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2017] [Revised: 03/15/2017] [Accepted: 03/16/2017] [Indexed: 11/23/2022]
Abstract
Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a major global health problem. Recently developed treatments with direct-acting antivirals (DAAs) have largely improved the sustained virologic response rate of patients with chronic hepatitis C. However, this approach is still hindered by its great expense and the problem of drug resistance. Using our cell-based HCV assay systems, we reported that the preclinical antimalarial drugs N-89 and N-251 exhibited potent anti-HCV activities. In this study we used our assay systems to evaluate the anti-HCV activities of six kinds of DAAs individually or in combination with N-89 or N-251. The results showed that the DAAs had potent anti-HCV activities and N-89 or N-251 contributed additive or synergistic effect. Using DAA-resistant HCV-RNA-replicating cells, which were prepared by continuous treatment with each DAA, we demonstrated that N-89 and N-251 could overcome all of the DAA-resistant HCVs. These preclinical drugs would have been potential as components of a therapeutic regimen that also included combinations of various DAAs. In addition, sequence analysis of the NS3-NS5B regions of the DAA-resistant HCV genomes newly found several amino acid (aa) substitutions that were suggested to contribute to DAA-resistance in addition to the aa substitutions already known to cause DAA-resistance. Among these new aa substitutions, we found that two substitutions in the NS3 region (D79G and S174Y) contributed to simeprevir- and/or asunaprevir-resistance.
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Sohda T, Yamauchi E, Anan A, Yokoyama K, Fukunaga A, Yamauchi R, Fukuda S, Takata K, Tanaka T, Hanano T, Kitamura Y, Morihara D, Takeyama Y, Irie M, Shakado S, Sakisaka S. Non-response to daclatasvir and asunaprevir therapy in patients co-infected with hepatitis C virus genotypes 1 and 2. Hepatol Res 2017; 47:364-367. [PMID: 27260815 DOI: 10.1111/hepr.12758] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/25/2015] [Revised: 06/01/2016] [Accepted: 06/02/2016] [Indexed: 02/08/2023]
Abstract
Direct-acting antiviral agents for hepatitis C virus (HCV) have been developed such as combined daclatasvir (DCV) and asunaprevir (ASV) treatment. This typically enables HCV serotype 1 patients to achieve a high sustained virological response rate, but a small number of such patients fail to respond to therapy. We investigated three HCV patients who showed no response to DCV and ASV therapy. Hepatitis C genotyping was undertaken in the three patients using nested polymerase chain reaction and polymerase chain reaction direct sequencing in the core region of the HCV genome. All three patients possessed HCV serotype 1, and no mutations were identified in either the non-structural protein 3 or 5A region. The three patients were shown to be co-infected with HCV genotypes 1 and 2 because genotypes 2a and 2b were also identified. This is the first report into failed response to DCV and ASV therapy in patients co-infected with HCV genotypes 1 and 2.
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Affiliation(s)
- Tetsuro Sohda
- Department of Gastroenterology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
| | - Eri Yamauchi
- Department of Gastroenterology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
| | - Akira Anan
- Department of Gastroenterology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
| | - Keiji Yokoyama
- Department of Gastroenterology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
| | - Atsushi Fukunaga
- Department of Gastroenterology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
| | - Ryo Yamauchi
- Department of Gastroenterology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
| | - Sho Fukuda
- Department of Gastroenterology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
| | - Kazuhide Takata
- Department of Gastroenterology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
| | - Takashi Tanaka
- Department of Gastroenterology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
| | - Takayuki Hanano
- Department of Gastroenterology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
| | - Yuji Kitamura
- Department of Gastroenterology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
| | - Daisuke Morihara
- Department of Gastroenterology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
| | - Yasuaki Takeyama
- Department of Gastroenterology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
| | - Makoto Irie
- Department of Gastroenterology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
| | - Satoshi Shakado
- Department of Gastroenterology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
| | - Shotaro Sakisaka
- Department of Gastroenterology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
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22
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Smith DB, Becher P, Bukh J, Gould EA, Meyers G, Monath T, Muerhoff AS, Pletnev A, Rico-Hesse R, Stapleton JT, Simmonds P. Proposed update to the taxonomy of the genera Hepacivirus and Pegivirus within the Flaviviridae family. J Gen Virol 2016; 97:2894-2907. [PMID: 27692039 PMCID: PMC5770844 DOI: 10.1099/jgv.0.000612] [Citation(s) in RCA: 123] [Impact Index Per Article: 13.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Proposals are described for the assignment of recently reported viruses, infecting rodents, bats and other mammalian species, to new species within the Hepacivirus and Pegivirus genera (family Flaviviridae). Assignments into 14 Hepacivirus species (Hepacivirus A–N) and 11 Pegivirus species (Pegivirus A–K) are based on phylogenetic relationships and sequence distances between conserved regions extracted from complete coding sequences for members of each proposed taxon. We propose that the species Hepatitis C virus is renamed Hepacivirus C in order to acknowledge its unique historical position and so as to minimize confusion. Despite the newly documented genetic diversity of hepaciviruses and pegiviruses, members of these genera remain phylogenetically distinct, and differ in hepatotropism and the possession of a basic core protein; pegiviruses in general lack these features. However, other characteristics that were originally used to support their division into separate genera are no longer definitive; there is overlap between the two genera in the type of internal ribosomal entry site and the presence of miR-122 sites in the 5′ UTR, the predicted number of N-linked glycosylation sites in the envelope E1 and E2 proteins, the presence of poly U tracts in the 3′ UTR and the propensity of viruses to establish a persistent infection. While all classified hepaciviruses and pegiviruses have mammalian hosts, the recent description of a hepaci-/pegi-like virus from a shark and the likely existence of further homologues in other non-mammalian species indicate that further species or genera remain to be defined in the future.
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Affiliation(s)
- Donald B Smith
- Centre for Immunity, Infection and Evolution, University of Edinburgh, Scotland, UK
| | - Paul Becher
- Institute of Virology, University of Veterinary Medicine, Hannover, Germany
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, Denmark.,Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
| | - Ernest A Gould
- EHESP French School of Public Health, French Institute of Research for Development (IRD), Aix Marseille Université, EPV UMR_D 190 Emergence des Pathologies Virales, Marseille, France
| | - Gregor Meyers
- Institut für Immunologie, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany
| | - Thomas Monath
- Hookipa Biotech AG, Vienna, Austria.,PaxVax Inc., Menlo Park and Redwood City, CA, USA
| | - A Scott Muerhoff
- Abbott Diagnostics Research and Development, Abbott Park, IL, USA
| | - Alexander Pletnev
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, USA
| | - Rebecca Rico-Hesse
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA.,Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA
| | - Jack T Stapleton
- Medical Service, Iowa City Veterans Affairs Medical Center, Iowa City, IA, USA.,Department of Internal Medicine, University of Iowa, Iowa City, IA, USA.,Department of Microbiology, University of Iowa, Iowa City, IA, USA
| | - Peter Simmonds
- Centre for Immunity, Infection and Evolution, University of Edinburgh, Scotland, UK.,Nuffield Department of Medicine, University of Oxford, Oxford, UK
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23
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Meanwell NA. 2015 Philip S. Portoghese Medicinal Chemistry Lectureship. Curing Hepatitis C Virus Infection with Direct-Acting Antiviral Agents: The Arc of a Medicinal Chemistry Triumph. J Med Chem 2016; 59:7311-51. [PMID: 27501244 DOI: 10.1021/acs.jmedchem.6b00915] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The development of direct-acting antiviral agents that can cure a chronic hepatitis C virus (HCV) infection after 8-12 weeks of daily, well-tolerated therapy has revolutionized the treatment of this insidious disease. In this article, three of Bristol-Myers Squibb's HCV programs are summarized, each of which produced a clinical candidate: the NS3 protease inhibitor asunaprevir (64), marketed as Sunvepra, the NS5A replication complex inhibitor daclatasvir (117), marketed as Daklinza, and the allosteric NS5B polymerase inhibitor beclabuvir (142), which is in late stage clinical studies. A clinical study with 64 and 117 established for the first time that a chronic HCV infection could be cured by treatment with direct-acting antiviral agents alone in the absence of interferon. The development of small molecule HCV therapeutics, designed by medicinal chemists, has been hailed as "the arc of a medical triumph" but may equally well be described as "the arc of a medicinal chemistry triumph".
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Affiliation(s)
- Nicholas A Meanwell
- Department of Discovery Chemistry, Bristol-Myers Squibb Research & Development , Wallingford, Connecticut 06492, United States
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24
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Schwartz RE, Bram Y, Frankel A. Pluripotent Stem Cell-Derived Hepatocyte-like Cells: A Tool to Study Infectious Disease. CURRENT PATHOBIOLOGY REPORTS 2016; 4:147-156. [PMID: 29910973 DOI: 10.1007/s40139-016-0113-7] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Purpose of Review Liver disease is an important clinical and global problem and is the 16th leading cause of death worldwide and responsible for 1 million deaths worldwide each year. Infectious disease is a major cause of liver disease specifically and overall is even a greater cause of patient morbidity and mortality. Tools to study human liver disease and infectious disease have been lacking which has significantly hampered the study of liver disease generally and hepatotropic pathogens more specifically. Historically, hepatoma cell lines have been used for in vitro cell culture models to study infectious disease. Significant differences between human hepatoma cell lines and the human hepatocyte has hampered our understanding of hepatocyte pathogen infection and hepatocyte--pathogen interactions. Recent Findings Despite these limitations, great progress was made in the understanding of specific aspects of the life cycle of the canonical hepatocyte viral pathogen, Hepatitis C Virus. Over time various specific drugs targeting various proteins of the HCV virion or aspects of the HCV viral life cycle have been created that enable almost complete elimination of the virus in vitro and clinically. These drugs, direct-acting antivirals have enabled achieving sustained virologic response in over 90-95 percent of patients. Summary Despite the development of direct-acting antivirals and the extreme success in achieving sustained virologic response, there has only been limited success elucidating host-pathogen interactions largely due to the poor nature of the hepatoma platform. Alternative approaches are needed. Pluripotent stem cells are renewable, can be derived from a single donor and can be efficiently and reproducibly differentiated towards many cell types including ectodermal-, endodermal-, and mesodermal-derived lineages. The development of pluripotent stem cell-derived hepatocyte-like cells (iHLCS) changes the paradigm as robust cells with the phenotype and function of hepatocytes can be readily created on demand with a variety of genetic background or alterations. iHLCs are readily used as models to study human drug metabolism, human liver disease, and human hepatotropic infectious disease. In this review, we discuss the biology of the HCV virus, the use of iHLCs as models to study human liver disease, and review the current work on using iHLCs to study HCV infection.
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Affiliation(s)
| | - Yaron Bram
- Weill Cornell School of Medicine, New York, NY, USA
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25
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T- and B-cell responses to multivalent prime-boost DNA and viral vectored vaccine combinations against hepatitis C virus in non-human primates. Gene Ther 2016; 23:753-759. [PMID: 27416077 PMCID: PMC7091906 DOI: 10.1038/gt.2016.55] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2016] [Revised: 05/09/2016] [Accepted: 06/20/2016] [Indexed: 12/17/2022]
Abstract
Immune responses against multiple epitopes are required for the prevention of hepatitis C virus (HCV) infection, and the progression to phase I trials of candidates may be guided by comparative immunogenicity studies in non-human primates. Four vectors, DNA, SFV, human serotype 5 adenovirus (HuAd5) and Modified Vaccinia Ankara (MVA) poxvirus, all expressing hepatitis C virus Core, E1, E2 and NS3, were combined in three prime-boost regimen, and their ability to elicit immune responses against HCV antigens in rhesus macaques was explored and compared. All combinations induced specific T-cell immune responses, including high IFN-γ production. The group immunized with the SFV+MVA regimen elicited higher E2-specific responses as compared with the two other modalities, while animals receiving HuAd5 injections elicited lower IL-4 responses as compared with those receiving MVA. The IFN-γ responses to NS3 were remarkably similar between groups. Only the adenovirus induced envelope-specific antibody responses, but these failed to show neutralizing activity. Therefore, the two novel regimens failed to induce superior responses as compared with already existing HCV vaccine candidates. Differences were found in response to envelope proteins, but the relevance of these remain uncertain given the surprisingly poor correlation with immunogenicity data in chimpanzees, underlining the difficulty to predict efficacy from immunology studies.
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26
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Chadha S, Sharma U, Chaudhary A, Prakash C, Gupta S, Venkatesh S. Molecular epidemiological analysis of three hepatitis C virus outbreaks in Jammu and Kashmir State, India. J Med Microbiol 2016; 65:804-813. [PMID: 27357565 DOI: 10.1099/jmm.0.000284] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Outbreaks of hepatitis C virus (HCV) infection are associated with unsafe injection practices, intravenous drug abuse and other exposure to blood and body fluids. We report here three outbreaks of HCV infection from Jammu and Kashmir (J&K) State, India, which occurred over a period of 3 years and in which molecular epidemiological investigations identified a presumptive common source of infection, most likely a single healthcare venue. Representative blood samples collected from cases of hepatitis C were sent to the National Centre for Disease Control (NCDC) for molecular characterization. These samples were positive by HCV ELISA. Subsequently, specimens were also tested for the presence of HCV RNA by RT-PCR. Sequencing was carried out for all positive samples. A total of 812 cases were laboratory confirmed by HCV ELISA; a total of 115 samples were sent to the NCDC for RT-PCR, and 77 were positive. Subtype 3a of HCV was found in all samples from Anantnag (February 2013); and for subtype 3b, in all samples from Srinagar (May 2015). Subtypes 3a and 3g were identified from two samples from the Kulgam outbreak (July 2014). A detailed epidemiological investigation should be conducted whenever a cluster of HCV cases is revealed, as this potentially allows for the identification of larger outbreaks. Epidemiological investigations of outbreaks should be further supported by inclusion of molecular tests. Efforts to limit therapeutic injections to only those cases having strong medical/surgical indications and to restrict the use of non-sterile needles are essential to prevent transmission of HCV.
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Affiliation(s)
- Sanjim Chadha
- Division of Biotechnology and Molecular Diagnostics, National Centre for Disease Control, Directorate General of Health Services, Ministry of Health & Family Welfare, Government of India, 22-Sham Nath Marg, Delhi 110054, India
| | - Uma Sharma
- Division of Biotechnology and Molecular Diagnostics, National Centre for Disease Control, Directorate General of Health Services, Ministry of Health & Family Welfare, Government of India, 22-Sham Nath Marg, Delhi 110054, India
| | - Artee Chaudhary
- Division of Biotechnology and Molecular Diagnostics, National Centre for Disease Control, Directorate General of Health Services, Ministry of Health & Family Welfare, Government of India, 22-Sham Nath Marg, Delhi 110054, India
| | - Charu Prakash
- Division of Biotechnology and Molecular Diagnostics, National Centre for Disease Control, Directorate General of Health Services, Ministry of Health & Family Welfare, Government of India, 22-Sham Nath Marg, Delhi 110054, India
| | - Sunil Gupta
- Division of Biotechnology and Molecular Diagnostics, National Centre for Disease Control, Directorate General of Health Services, Ministry of Health & Family Welfare, Government of India, 22-Sham Nath Marg, Delhi 110054, India
| | - S Venkatesh
- Division of Biotechnology and Molecular Diagnostics, National Centre for Disease Control, Directorate General of Health Services, Ministry of Health & Family Welfare, Government of India, 22-Sham Nath Marg, Delhi 110054, India
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27
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Abstract
Hepatitis C virus (HCV) is the major cause of transfusion-associated hepatitis and accounts for a significant proportion of hepatitis cases worldwide. Most, if not all, infections become persistent and about 60% of cases develop chronic liver disease with various outcomes ranging from an asymptomatic carrier state to chronic active hepatitis and liver cirrhosis, which is strongly associated with the development of hepatocellular carcinoma. Since the initial cloning of the viral genome in 1989, our knowledge of the molecular biology of HCV has increased rapidly and led to the identification of several potential targets for antiviral intervention. In contrast, the low replication of the virus in cell culture, the lack of convenient animal models and the high genome variability present major challenges for drug development. This review will describe candidate drug targets and summarize ‘classical’ and ‘novel’ approaches currently being pursued to develop efficient HCV-specific therapies.
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Affiliation(s)
- R Bartenschlager
- Institute for Virology, Johannes-Gutenberg University of Mainz, Obere Zahlbacher Strasse 67, 55131 Mainz, Germany
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28
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Elevated Concentration of Defensins in Hepatitis C Virus-Infected Patients. J Immunol Res 2016; 2016:8373819. [PMID: 27413763 PMCID: PMC4931052 DOI: 10.1155/2016/8373819] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2016] [Revised: 04/17/2016] [Accepted: 04/27/2016] [Indexed: 12/23/2022] Open
Abstract
Hepatitis C virus (HCV) is the major etiological agent of human non-A and non-B hepatitis, affecting around 180 million people worldwide. Defensins, small cysteine-rich cationic peptides, are shown to have potent antibacterial, antiviral, and antifungal properties. Defensins can be found in both normal and microbial infected patients, at variable concentrations. Notably, viral infections are often associated with elevated concentrations of defensins. The current study aimed to estimate the concentrations of total, α-, and β-defensins in serum taken from normal and HCV-infected patients. 12 healthy (noninfected) and 34 HCV-infected patients were enrolled. Standardized immunoassay kits were used to obtain serum concentrations of defensins. The obtained results were calibrated against kit standard reagents. Total defensin concentrations in HCV-infected patients were significantly higher (2- to 105-fold) compared to healthy individuals. The concentrations of α-defensins were also significantly elevated in the HCV-infected patients (31–1398 ng/50 μL). However, concentrations of β-defensins ranged from 44.5 ng/50 μL to 1056 ng/50 μL. The results did not reveal differences in serum defensin concentration between male and female HCV-infected patients. A-defensin concentration of ≥250 ng/50 μL was found to contain more β-defensins than total defensins and α-defensins. This study concludes, for the first time, that serum defensin levels are elevated in HCV-infected patients.
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29
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Provazzi PJS, Mukherjee S, Hanson AM, Nogueira ML, Carneiro BM, Frick DN, Rahal P. Analysis of the Enzymatic Activity of an NS3 Helicase Genotype 3a Variant Sequence Obtained from a Relapse Patient. PLoS One 2015; 10:e0144638. [PMID: 26658750 PMCID: PMC4684341 DOI: 10.1371/journal.pone.0144638] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2014] [Accepted: 11/20/2015] [Indexed: 12/15/2022] Open
Abstract
The hepatitis C virus (HCV) is a species of diverse genotypes that infect over 170 million people worldwide, causing chronic inflammation, cirrhosis and hepatocellular carcinoma. HCV genotype 3a is common in Brazil, and it is associated with a relatively poor response to current direct-acting antiviral therapies. The HCV NS3 protein cleaves part of the HCV polyprotein, and cellular antiviral proteins. It is therefore the target of several HCV drugs. In addition to its protease activity, NS3 is also an RNA helicase. Previously, HCV present in a relapse patient was found to harbor a mutation known to be lethal to HCV genotype 1b. The point mutation encodes the amino acid substitution W501R in the helicase RNA binding site. To examine how the W501R substitution affects NS3 helicase activity in a genotype 3a background, wild type and W501R genotype 3a NS3 alleles were sub-cloned, expressed in E. coli, and the recombinant proteins were purified and characterized. The impact of the W501R allele on genotype 2a and 3a subgenomic replicons was also analyzed. Assays monitoring helicase-catalyzed DNA and RNA unwinding revealed that the catalytic efficiency of wild type genotype 3a NS3 helicase was more than 600 times greater than the W501R protein. Other assays revealed that the W501R protein bound DNA less than 2 times weaker than wild type, and both proteins hydrolyzed ATP at similar rates. In Huh7.5 cells, both genotype 2a and 3a subgenomic HCV replicons harboring the W501R allele showed a severe defect in replication. Since the W501R allele is carried as a minor variant, its replication would therefore need to be attributed to the trans-complementation by other wild type quasispecies.
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Affiliation(s)
- Paola J. S. Provazzi
- São Paulo State University - UNESP, Department of Biology, São José do Rio Preto/SP, CEP: 15054–000, Brazil
- * E-mail:
| | - Sourav Mukherjee
- University of Wisconsin- Milwaukee, Department of Chemistry & Biochemistry, Milwaukee, WI, 53217, United States of America
| | - Alicia M. Hanson
- University of Wisconsin- Milwaukee, Department of Chemistry & Biochemistry, Milwaukee, WI, 53217, United States of America
| | - Mauricio L. Nogueira
- São José do Rio Preto Medical School, Laboratory of Virology, São José do Rio Preto/SP, CEP: 15090–000, Brazil
| | - Bruno M. Carneiro
- São Paulo State University - UNESP, Department of Biology, São José do Rio Preto/SP, CEP: 15054–000, Brazil
| | - David N. Frick
- University of Wisconsin- Milwaukee, Department of Chemistry & Biochemistry, Milwaukee, WI, 53217, United States of America
| | - Paula Rahal
- São Paulo State University - UNESP, Department of Biology, São José do Rio Preto/SP, CEP: 15054–000, Brazil
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30
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Akuta N, Suzuki F, Sezaki H, Hosaka T, Kobayashi M, Suzuki Y, Saitoh S, Kobayashi M, Ikeda K, Kumada H. Evolution of simeprevir-resistant variants in virological non-responders infected with HCV genotype 1b. J Med Virol 2015; 87:609-18. [PMID: 25655809 DOI: 10.1002/jmv.24113] [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: 11/04/2014] [Indexed: 01/06/2023]
Abstract
The present study was designed to assess the evolution of simeprevir-resistant variants (amino acid substitutions of aa80, aa155, aa156, and aa168 positions in HCV NS3 region) over time in virological non-responders (patients with positive HCV-RNA during and at end of treatment). The study enrolled 136 patients infected with HCV genotype 1b who received 12-week simeprevir-PEG-IFN-ribavirin therapy, and data of 87 patients were available for analysis. Twelve patients (14%) were considered virological non-responders, including 9 (75%) who showed absolute no-response (HCV RNA: ≥3.0 log IU/ml at 12 weeks after start of therapy). Multivariate analysis of these patients identified lack of response to prior treatment, use of low ribavirin dose, and old age as independent and significant determinants of virological non-response. Using ultra-deep sequencing, de novo variants of D168 were detected in all of 9 absolute non-responders. The majority of these variants emerged within 5 weeks of triple therapy. In comparison, de novo variants of Q80 were detected in only 3 of 9 absolute non-responders and emerged at 6-12 weeks. Variants of Q80 detected at baseline increased during the course of treatment in 5 of 9 absolute non-responders, while no such increase was noted in variants of R155 and/or A156 detected at baseline during the 12-week course. De novo variants of R155 and/or A156 were not detected in this study. The results demonstrated the emergence of simeprevir-resistant variants during the early stage of triple therapy.
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Affiliation(s)
- Norio Akuta
- Department of Hepatology, Toranomon Hospital, and Okinaka Memorial Institute for Medical Research, Tokyo, Japan
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31
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Nakamoto S, Imazeki F, Arai M, Yasui S, Nakamura M, Haga Y, Sasaki R, Kanda T, Shirasawa H, Yokosuka O. Effect of Hepatitis C Virus Genotype 1b Core and NS5A Mutations on Response to Peginterferon Plus Ribavirin Combination Therapy. Int J Mol Sci 2015; 16:21177-21190. [PMID: 26370958 PMCID: PMC4613248 DOI: 10.3390/ijms160921177] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2015] [Revised: 08/21/2015] [Accepted: 08/21/2015] [Indexed: 12/16/2022] Open
Abstract
We examined whether hepatitis C virus (HCV) genotype 1b core- and NS5A-region mutations are associated with response to peginterferon α-2b plus ribavirin combination therapy. A total of 103 patients with high HCV genotype 1b viral loads (≥ 100 KIU/mL) were treated with the combination therapy. Pretreatment mutations in the core region and interferon sensitivity determining region (ISDR) in the NS5A region were analyzed. In univariate analysis, arginine and leucine at positions 70 and 91 in the core region, defined as double wild (DW)-type, were associated with early virologic response (p = 0.002), sustained virologic response (SVR) (p = 0.004), and non-response (p = 0.005). Non-threonine at position 110 was associated with SVR (p = 0.004). Multivariate analysis showed the following pretreatment predictors of SVR: hemoglobin level ≥ 14 g/dL (odds ratio (OR) 6.2, p = 0.04); platelet count ≥ 14 × 10⁴/mm³ (OR 5.2, p = 0.04); aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio < 0.9 (OR 6.17, p = 0.009); DW-type (OR 6.8, p = 0.02); non-threonine at position 110 (OR 14.5, p = 0.03); and ≥ 2 mutations in the ISDR (OR 12.3, p = 0.02). Patients with non-DW-type, non-threonine at position 110, and < 2 ISDR mutations showed significantly lower SVR rates than others (11/45 (24.4%) vs. 27/37 (73.0%), respectively; p < 0.001). SVR can be predicted through core and NS5A region mutations and host factors like hemoglobin, platelet count, and AST/ALT ratio in HCV genotype 1b-infected patients treated with peginterferon and ribavirin combination therapy.
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Affiliation(s)
- Shingo Nakamoto
- Department of Gastroenterology and Nephrology, Graduate School of Medicine, Chiba University, Chiba 260-8677, Japan.
| | - Fumio Imazeki
- Department of Gastroenterology and Nephrology, Graduate School of Medicine, Chiba University, Chiba 260-8677, Japan.
| | - Makoto Arai
- Department of Gastroenterology and Nephrology, Graduate School of Medicine, Chiba University, Chiba 260-8677, Japan.
| | - Shin Yasui
- Department of Gastroenterology and Nephrology, Graduate School of Medicine, Chiba University, Chiba 260-8677, Japan.
| | - Masato Nakamura
- Department of Gastroenterology and Nephrology, Graduate School of Medicine, Chiba University, Chiba 260-8677, Japan.
| | - Yuki Haga
- Department of Gastroenterology and Nephrology, Graduate School of Medicine, Chiba University, Chiba 260-8677, Japan.
| | - Reina Sasaki
- Department of Gastroenterology and Nephrology, Graduate School of Medicine, Chiba University, Chiba 260-8677, Japan.
| | - Tatsuo Kanda
- Department of Gastroenterology and Nephrology, Graduate School of Medicine, Chiba University, Chiba 260-8677, Japan.
| | - Hiroshi Shirasawa
- Department of Gastroenterology and Nephrology, Graduate School of Medicine, Chiba University, Chiba 260-8677, Japan.
| | - Osamu Yokosuka
- Department of Gastroenterology and Nephrology, Graduate School of Medicine, Chiba University, Chiba 260-8677, Japan.
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Abstract
Hepatitis C virus (HCV) is a leading cause of chronic hepatitis and infects approximately three to four million people per year, about 170 million infected people in total, making it one of the major global health problems. In a minority of cases HCV is cleared spontaneously, but in most of the infected individuals infection progresses to a chronic state associated with high risk to develop liver cirrhosis, hepatocellular cancer, or liver failure. The treatment of HCV infection has evolved over the years. Interferon (IFN)-α in combination with ribavirin has been used for decades as standard therapy. More recently, a new standard-of-care treatment has been approved based on a triple combination with either HCV protease inhibitor telaprevir or boceprevir. In addition, various options for all-oral, IFN-free regimens are currently being evaluated. Despite substantial improvement of sustained virological response rates, some intrinsic limitations of these new direct-acting antivirals, including serious side effects, the risk of resistance development and high cost, urge the development of alternative or additional therapeutic strategies. Gene therapy represents a feasible alternative treatment. Small RNA technology, including RNA interference (RNAi) techniques and antisense approaches, is one of the potentially promising ways to investigate viral and host cell factors that are involved in HCV infection and replication. With this, newly developed gene therapy regimens will be provided to treat HCV. In this chapter, a comprehensive overview guides you through the current developments and applications of RNAi and microRNA-based gene therapy strategies in HCV treatment.
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A Structural Overview of RNA-Dependent RNA Polymerases from the Flaviviridae Family. Int J Mol Sci 2015; 16:12943-57. [PMID: 26062131 PMCID: PMC4490480 DOI: 10.3390/ijms160612943] [Citation(s) in RCA: 76] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2015] [Revised: 05/27/2015] [Accepted: 05/28/2015] [Indexed: 12/18/2022] Open
Abstract
RNA-dependent RNA polymerases (RdRPs) from the Flaviviridae family are representatives of viral polymerases that carry out RNA synthesis through a de novo initiation mechanism. They share a ≈ 600-residue polymerase core that displays a canonical viral RdRP architecture resembling an encircled right hand with palm, fingers, and thumb domains surrounding the active site. Polymerase catalytic motifs A-E in the palm and motifs F/G in the fingers are shared by all viral RdRPs with sequence and/or structural conservations regardless of the mechanism of initiation. Different from RdRPs carrying out primer-dependent initiation, Flaviviridae and other de novo RdRPs utilize a priming element often integrated in the thumb domain to facilitate primer-independent initiation. Upon the transition to the elongation phase, this priming element needs to undergo currently unresolved conformational rearrangements to accommodate the growth of the template-product RNA duplex. In the genera of Flavivirus and Pestivirus, the polymerase module in the C-terminal part of the RdRP protein may be regulated in cis by the N-terminal region of the same polypeptide. Either being a methyltransferase in Flavivirus or a functionally unclarified module in Pestivirus, this region could play auxiliary roles for the canonical folding and/or the catalysis of the polymerase, through defined intra-molecular interactions.
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Ali A, Nisar M, Idrees M, Rafique S, Iqbal M. Expression of Hepatitis C Virus Core and E2 antigenic recombinant proteins and their use for development of diagnostic assays. Int J Infect Dis 2015; 34:84-9. [DOI: 10.1016/j.ijid.2015.03.010] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2014] [Revised: 02/25/2015] [Accepted: 03/13/2015] [Indexed: 01/28/2023] Open
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Rossi LMG, Escobar-Gutierrez A, Rahal P. Advanced molecular surveillance of hepatitis C virus. Viruses 2015; 7:1153-88. [PMID: 25781918 PMCID: PMC4379565 DOI: 10.3390/v7031153] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2014] [Revised: 02/05/2015] [Accepted: 02/20/2015] [Indexed: 12/12/2022] Open
Abstract
Hepatitis C virus (HCV) infection is an important public health problem worldwide. HCV exploits complex molecular mechanisms, which result in a high degree of intrahost genetic heterogeneity. This high degree of variability represents a challenge for the accurate establishment of genetic relatedness between cases and complicates the identification of sources of infection. Tracking HCV infections is crucial for the elucidation of routes of transmission in a variety of settings. Therefore, implementation of HCV advanced molecular surveillance (AMS) is essential for disease control. Accounting for virulence is also important for HCV AMS and both viral and host factors contribute to the disease outcome. Therefore, HCV AMS requires the incorporation of host factors as an integral component of the algorithms used to monitor disease occurrence. Importantly, implementation of comprehensive global databases and data mining are also needed for the proper study of the mechanisms responsible for HCV transmission. Here, we review molecular aspects associated with HCV transmission, as well as the most recent technological advances used for virus and host characterization. Additionally, the cornerstone discoveries that have defined the pathway for viral characterization are presented and the importance of implementing advanced HCV molecular surveillance is highlighted.
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Affiliation(s)
- Livia Maria Gonçalves Rossi
- Department of Biology, Institute of Bioscience, Language and Exact Science, Sao Paulo State University, Sao Jose do Rio Preto, SP 15054-000, Brazil.
| | | | - Paula Rahal
- Department of Biology, Institute of Bioscience, Language and Exact Science, Sao Paulo State University, Sao Jose do Rio Preto, SP 15054-000, Brazil.
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Bhat P, Shwetha S, Sharma DK, Joseph AP, Srinivasan N, Das S. The beta hairpin structure within ribosomal protein S5 mediates interplay between domains II and IV and regulates HCV IRES function. Nucleic Acids Res 2015; 43:2888-901. [PMID: 25712089 PMCID: PMC4357715 DOI: 10.1093/nar/gkv110] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Translation initiation in Hepatitis C Virus (HCV) is mediated by Internal Ribosome Entry Site (IRES), which is independent of cap-structure and uses a limited number of canonical initiation factors. During translation initiation IRES–40S complex formation depends on high affinity interaction of IRES with ribosomal proteins. Earlier, it has been shown that ribosomal protein S5 (RPS5) interacts with HCV IRES. Here, we have extensively characterized the HCV IRES–RPS5 interaction and demonstrated its role in IRES function. Computational modelling and RNA–protein interaction studies demonstrated that the beta hairpin structure within RPS5 is critically required for the binding with domains II and IV. Mutations disrupting IRES–RPS5 interaction drastically reduced the 80S complex formation and the corresponding IRES activity. Computational analysis and UV cross-linking experiments using various IRES-mutants revealed interplay between domains II and IV mediated by RPS5. In addition, present study demonstrated that RPS5 interaction is unique to HCV IRES and is not involved in 40S–3′ UTR interaction. Further, partial silencing of RPS5 resulted in preferential inhibition of HCV RNA translation. However, global translation was marginally affected by partial silencing of RPS5. Taken together, results provide novel molecular insights into IRES–RPS5 interaction and unravel its functional significance in mediating internal initiation of translation.
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Affiliation(s)
- Prasanna Bhat
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India
| | - Shivaprasad Shwetha
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India
| | - Divya Khandige Sharma
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India
| | | | | | - Saumitra Das
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India
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Satoh S, Mori K, Ueda Y, Sejima H, Dansako H, Ikeda M, Kato N. Establishment of hepatitis C virus RNA-replicating cell lines possessing ribavirin-resistant phenotype. PLoS One 2015; 10:e0118313. [PMID: 25699517 PMCID: PMC4336140 DOI: 10.1371/journal.pone.0118313] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2014] [Accepted: 01/12/2015] [Indexed: 12/12/2022] Open
Abstract
BACKGROUND Ribavirin (RBV) is a potential partner of interferon-based therapy and recently approved therapy using direct acting antivirals for patients with chronic hepatitis C. However, the precise mechanisms underlying RBV action against hepatitis C virus (HCV) replication are not yet understood. To clarify this point, we attempted to develop RBV-resistant cells from RBV-sensitive HCV RNA-replicating cells. METHODOLOGY/PRINCIPAL FINDINGS By repetitive RBV (100 μM) treatment (10 weeks) of 3.5-year-cultured OL8 cells, in which genome-length HCV RNA (O strain of genotype 1b) efficiently replicates, dozens of colonies that survived RBV treatment were obtained. These colonies were mixed together and further treated with high doses of RBV (up to 200 μM). By such RBV treatment, we successfully established 12 RBV-survived genome-length HCV RNA-replicating cell lines. Among them, three representative cell lines were characterized. HCV RNA replication in these cells resisted RBV significantly more than that in the parental OL8 cells. Genetic analysis of HCV found several common and conserved amino acid substitutions in HCV proteins among the three RBV-resistant cell species. Furthermore, using cDNA microarray and quantitative RT-PCR analyses, we identified 5 host genes whose expression levels were commonly altered by more than four-fold among these RBV-resistant cells compared with the parental cells. Moreover, to determine whether viral or host factor contributes to RBV resistance, we developed newly HCV RNA-replicating cells by introducing total RNAs isolated from RBV-sensitive parental cells or RBV-resistant cells into the HCV RNA-cured-parental or -RBV-resistant cells using an electroporation method, and evaluated the degrees of RBV resistance of these developed cells. Consequently, we found that RBV-resistant phenotype was conferred mainly by host factor and partially by viral factor. CONCLUSIONS/SIGNIFICANCE These newly established HCV RNA-replicating cell lines should become useful tools for further understanding the anti-HCV mechanisms of RBV.
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Affiliation(s)
- Shinya Satoh
- Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
| | - Kyoko Mori
- Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
| | - Youki Ueda
- Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
| | - Hiroe Sejima
- Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
| | - Hiromichi Dansako
- Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
| | - Masanori Ikeda
- Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
| | - Nobuyuki Kato
- Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
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Ariumi Y. Multiple functions of DDX3 RNA helicase in gene regulation, tumorigenesis, and viral infection. Front Genet 2014; 5:423. [PMID: 25538732 PMCID: PMC4257086 DOI: 10.3389/fgene.2014.00423] [Citation(s) in RCA: 99] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2014] [Accepted: 11/19/2014] [Indexed: 12/11/2022] Open
Abstract
The DEAD-box RNA helicase DDX3 is a multifunctional protein involved in all aspects of RNA metabolism, including transcription, splicing, mRNA nuclear export, translation, RNA decay and ribosome biogenesis. In addition, DDX3 is also implicated in cell cycle regulation, apoptosis, Wnt-β-catenin signaling, tumorigenesis, and viral infection. Notably, recent studies suggest that DDX3 is a component of anti-viral innate immune signaling pathways. Indeed, DDX3 contributes to enhance the induction of anti-viral mediators, interferon (IFN) regulatory factor 3 and type I IFN. However, DDX3 seems to be an important target for several viruses, such as human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV), hepatitis B virus (HBV), and poxvirus. DDX3 interacts with HIV-1 Rev or HCV Core protein and modulates its function. At least, DDX3 is required for both HIV-1 and HCV replication. Therefore, DDX3 could be a novel therapeutic target for the development of drug against HIV-1 and HCV.
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Affiliation(s)
- Yasuo Ariumi
- Ariumi Project Laboratory, Center for AIDS Research - International Research Center for Medical Sciences, Kumamoto University Kumamoto, Japan
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Jiang X, Kanda T, Wu S, Nakamoto S, Wakita T, Shirasawa H, Yokosuka O. Hepatitis C virus nonstructural protein 5A inhibits thapsigargin-induced apoptosis. PLoS One 2014; 9:e113499. [PMID: 25409163 PMCID: PMC4237446 DOI: 10.1371/journal.pone.0113499] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2014] [Accepted: 10/24/2014] [Indexed: 12/17/2022] Open
Abstract
BACKGROUND We previously reported that the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) down-regulates TLR4 signaling and lipopolysaccharide-induced apoptosis of hepatocytes. There have been several reports regarding the association between HCV infection and endoplasmic reticulum (ER) stress. Here, we examined the regulation of HCV NS5A on the apoptosis of hepatocytes induced by thapsigargin, an inducer of ER stress. METHODS The apoptotic response to thapsigargin and the expression of molecules involved in human hepatocyte apoptotic pathways were examined in the presence or absence of HCV NS5A expression. RESULTS HCV JFH1 infection induced ER stress in the Huh7 cell line. HCV NS5A protected HepG2 cells against thapsigargin-induced apoptosis, the effect of which was linked to the enhanced expression of the 78-kDa glucose-regulated protein/immunoglobulin heavy-chain binding protein (GRP78). Consistent with a conferred pro-survival advantage, HCV NS5A reduced poly(adenosine diphosphate-ribose) polymerase cleavage and activation of caspases-3, -7 and -9, and Bax expression, while increasing the expressions of the anti-apoptotic molecules XIAP and c-FLIP. HCV NS5A weakly interacts with GRP78 and enhances GRP78 expression in hepatocytes. CONCLUSION HCV NS5A enhances GRP78 expression, resulting in the inhibition of apoptotic properties, and inhibits thapsigargin-induced apoptotic pathways in human hepatocytes, suggesting that disruption of ER stress-mediated apoptosis may have a role in the pathogenesis of HCV infection. Thus, HCV NS5A might engender the survival of HCV-infected hepatocytes contributing to the establishment of persistent infection.
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Affiliation(s)
- Xia Jiang
- Department of Gastroenterology and Nephrology, Chiba University, Graduate School of Medicine, Chiba, Japan
| | - Tatsuo Kanda
- Department of Gastroenterology and Nephrology, Chiba University, Graduate School of Medicine, Chiba, Japan
| | - Shuang Wu
- Department of Gastroenterology and Nephrology, Chiba University, Graduate School of Medicine, Chiba, Japan
| | - Shingo Nakamoto
- Department of Molecular Virology, Chiba University, Graduate School of Medicine, Chiba, Japan
| | - Takaji Wakita
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Hiroshi Shirasawa
- Department of Molecular Virology, Chiba University, Graduate School of Medicine, Chiba, Japan
| | - Osamu Yokosuka
- Department of Gastroenterology and Nephrology, Chiba University, Graduate School of Medicine, Chiba, Japan
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Application of a high-resolution melting technique for the rapid detection of partial replacement of HCV-1b by HCV-1a after PEG-IFNα/RBV therapy. J Appl Genet 2014; 56:271-5. [PMID: 25380691 PMCID: PMC4412693 DOI: 10.1007/s13353-014-0256-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2014] [Revised: 10/14/2014] [Accepted: 10/23/2014] [Indexed: 02/05/2023]
Abstract
A modified method which can be used for the rapid screening of mutations in the protein kinase R-binding domain (PKR-BD) region and the hypervariable region 1 (HVR1) of hepatitis C virus (HCV) is described. This method is based on a high-resolution melting (HRM) technique used for genotyping single nucleotide polymorphisms and allows the detection of single nucleotide substitutions in the DNA sequence by measuring its Tm. The modified method, in addition to precisely measuring the Tm, allows the recording of the melting curve of the investigated cDNA fragment, which can provide provisional information about the number of different quasi-species present in the sample. The HRM analysis of the amplified cDNAs encoding the PKR-BD and HVR1 allowed the detection of partial replacement of HCV-1b by HCV-1a subspecies in one of our patients, as well as evaluation of the effectiveness of pegylated interferon α/ribavirin (PEG-IFNα/RBV) therapy. The HRM technique has never been used for the rapid screening of sequence variations in these regions and may be used for a similar purpose in any viral genome.
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Fattahi M, Malekpour A, Mortazavi M, Safarpour A, Naseri N. The characteristics of rare codon clusters in the genome and proteins of hepatitis C virus; a bioinformatics look. Middle East J Dig Dis 2014; 6:214-27. [PMID: 25349685 PMCID: PMC4208930] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/03/2014] [Accepted: 09/12/2014] [Indexed: 11/16/2022] Open
Abstract
BACKGROUND Recent studies suggest that rare codon clusters are functionally important for protein activity. METHODS Here, for the first time we analyzed and reported rare codon clusters in Hepatitis C Virus (HCV) genome and then identified the location of these rare codon clusters in the structure of HCV protein. This analysis was performed using the Sherlocc program that detects statistically relevant conserved rare codon clusters. RESULTS By this program, we identified the rare codon cluster in three regions of HCV genome; NS2, NS3, and NS5A coding sequence of HCV genome. For further understanding of the role of these rare codon clusters, we studied the location of these rare codon clusters and critical residues in the structure of NS2, NS3 and NS5A proteins. We identified some critical residues near or within rare codon clusters. It should be mentioned that characteristics of these critical residues such as location and situation of side chains are important in assurance of the HCV life cycle. CONCLUSION The characteristics of these residues and their relative status showed that these rare codon clusters play an important role in proper folding of these proteins. Thus, it is likely that these rare codon clusters may have an important role in the function of HCV proteins. This information is helpful in development of new avenues for vaccine and treatment protocols.
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Affiliation(s)
- Mohammadreza Fattahi
- 1 Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
,Corresponding Author: Mohammadreza Fattahi, MD Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, P.O. Box: 71935-1311, Shiraz, Iran Telefax: + 98 71 36474263
| | - Abdorrasoul Malekpour
- 1 Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Mojtaba Mortazavi
- 2 Department of Biotechnology, Institute of Science and High Technology and Environmental Science, Graduate University of Advanced Technology, Kerman, Iran
| | - Alireza Safarpour
- 1 Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Nasrin Naseri
- 1 Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
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Core amino acid variation at position 110 is associated with sustained virological response in Caucasian patients with chronic hepatitis C virus 1b infection. Arch Virol 2014; 159:3345-51. [PMID: 25161034 DOI: 10.1007/s00705-014-2209-x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2014] [Accepted: 08/19/2014] [Indexed: 01/17/2023]
Abstract
The aim of this study was to analyze the impact of core variations on sustained virological response (SVR) to pegylated interferon plus ribavirin (PEG-IFN/RBV) and its association with predictive factors of response in Caucasian patients infected with genotype 1 hepatitis C virus (HCV-1). Full-length core sequences were analyzed in 100 Caucasian HCV-1-infected patients who received therapy with PEG-IFN/RBV. The associations between variations in the core protein and SVR, as well as with predictors of SVR, were analyzed. Variations at core 62, 70 and 110 were selected as candidates. There were almost no variations at these positions among patients harboring HCV-1a. However, they were identified in 10 (30.3 %), 21 (63.6 %) and 13 (39.4 %) subjects with HCV-1b, respectively. Among the HCV-1b patients, 39.1 % individuals carrying core R62 and 70 % subjects with core R62G showed SVR (p = 0.141), and 66.7 % of HCV-1b patients harboring core R70 and 38.1 % with core R70Q achieved SVR (p = 0.157), whereas the rate of SVR was 70 % for individuals with core T110 and 15.4 % for those with core T110N (p = 0.004). No statistical interaction between core variations and IL28B genotype was observed. Patients with R70 showed higher median (interquartile range) baseline plasma levels of low-density-lipoprotein cholesterol (LDL-C) than those with R70Q (96 [86-118] mg/dL vs. 76 [54-88] mg/dL, p = 0.014). We concluded that a substitution at core 110 is associated with a lower rate of SVR in Caucasian HCV-1b-infected patients receiving PEG-IFN/RBV. Furthermore, the variation at the core 70 position is related to plasma levels of LDL-C in these patients.
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Redwan EM, EL-Fakharany EM, Uversky VN, Linjawi MH. Screening the anti infectivity potentials of native N- and C-lobes derived from the camel lactoferrin against hepatitis C virus. BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE 2014; 14:219. [PMID: 24993815 PMCID: PMC4086701 DOI: 10.1186/1472-6882-14-219] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/02/2014] [Accepted: 06/30/2014] [Indexed: 02/06/2023]
Abstract
BACKGROUND Hepatitis C virus (HCV) infection represents a worldwide health threat that still needs efficient protective vaccine and/or effective drug. The traditional medicine, such as camel milk, is heavily used by the large sector of HCV patients to control the infection due to the high cost of the available standard therapy. Camel milk contains lactoferrin, which plays an important and multifunctional role in innate immunity and specific host defense against microbial infection. Continuing the analysis of the effectiveness of camel lactoferrin against HCV, the current study aimed to separate and purify the native N- and C-lobes from the proteolytically cleaved camel lactoferrin (cLF) and to compare their in vitro activities against the HCV infection in Huh7.5 cells in order to determine the most active domain. METHODS Lactoferrin and its digested N- and C-lobes were purified by Mono S 5/50 GL column and Superdex 200 5/150 column. The purified proteins were assessed through three venues: 1. To inhibit intracellular replication, HCV infected cells were treated with the proteins at different concentrations and time intervals; 2. The proteins were directly incubated with the viral particles (neutralization) and then such neutralized viruses were used to infect cells; 3. The cells were protected with proteins before exposure to the virus. The antiviral potentials of the cLf and its lobes were determined using three techniques: 1. RT-nested PCR, 2. Real-time PCR, and 3. Flow cytometry. RESULTS N- and C-lobes were purified in two consecutive steps; using Mono-S and Superdex 200 columns. The molecular mass of N- and C-lobes was about 40 kDa. cLF and its lobes could prevent HCV entry into Huh 7.5 cells with activity reached 100% through direct interaction with the virus. The inhibition of intracellular viral replication by N-lobe is 2-fold and 3-fold more effective than that of the cLF and C-lobe, respectively. CONCLUSION Generated native N- and C-lobes from camel lactoferrin demonstrated a range of noticeably different potentials against HCV cellular infectivity. The anti-HCV activities were sorted as N-lobe > cLf > C-lobe.
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Affiliation(s)
- Elrashdy M Redwan
- Biological Science Department, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Kingdom of Saudi Arabia
- Therapeutic and Protective Proteins Laboratory, Protein Research Department, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications, New Borg EL-Arab, Alexandria 21394, Egypt
| | - Esmail M EL-Fakharany
- Therapeutic and Protective Proteins Laboratory, Protein Research Department, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications, New Borg EL-Arab, Alexandria 21394, Egypt
| | - Vladimir N Uversky
- Biological Science Department, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Kingdom of Saudi Arabia
- Department of Molecular Medicine, USF Health Byrd Alzheimer’s Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA
| | - Mustafa H Linjawi
- College of Applied Medical Sciences, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Kingdom of Saudi Arabia
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Toyoda H, Kumada T, Shimada N, Takaguchi K, Ide T, Sata M, Ginba H, Matsuyama K, Izumi N. Characteristics and Outcomes of HCV Genotype-1-Infected Patients Treated with Peginterferon and Ribavirin Combination Therapy with Discordant HCV Responses 4 and 12 Weeks after Starting Therapy. Intervirology 2014; 57:289-96. [DOI: 10.1159/000362721] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2013] [Accepted: 04/08/2014] [Indexed: 11/19/2022] Open
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Romero-López C, Berzal-Herranz A. Structure-function relationship in viral RNA genomes: The case of hepatitis C virus. World J Med Genet 2014; 4:6-18. [DOI: 10.5496/wjmg.v4.i2.6] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/10/2013] [Revised: 01/23/2014] [Accepted: 04/03/2014] [Indexed: 02/06/2023] Open
Abstract
The acquisition of a storage information system beyond the nucleotide sequence has been a crucial issue for the propagation and dispersion of RNA viruses. This system is composed by highly conserved, complex structural units in the genomic RNA, termed functional RNA domains. These elements interact with other regions of the viral genome and/or proteins to direct viral translation, replication and encapsidation. The genomic RNA of the hepatitis C virus (HCV) is a good model for investigating about conserved structural units. It contains functional domains, defined by highly conserved structural RNA motifs, mostly located in the 5’-untranslatable regions (5’UTRs) and 3’UTR, but also occupying long stretches of the coding sequence. Viral translation initiation is mediated by an internal ribosome entry site located at the 5’ terminus of the viral genome and regulated by distal functional RNA domains placed at the 3’ end. Subsequent RNA replication strongly depends on the 3’UTR folding and is also influenced by the 5’ end of the HCV RNA. Further increase in the genome copy number unleashes the formation of homodimers by direct interaction of two genomic RNA molecules, which are finally packed and released to the extracellular medium. All these processes, as well as transitions between them, are controlled by structural RNA elements that establish a complex, direct and long-distance RNA-RNA interaction network. This review summarizes current knowledge about functional RNA domains within the HCV RNA genome and provides an overview of the control exerted by direct, long-range RNA-RNA contacts for the execution of the viral cycle.
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Arai M, Tsukiyama-Kohara K, Takagi A, Tobita Y, Inoue K, Kohara M. Resistance to cyclosporin A derives from mutations in hepatitis C virus nonstructural proteins. Biochem Biophys Res Commun 2014; 448:56-62. [PMID: 24751518 DOI: 10.1016/j.bbrc.2014.04.053] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2014] [Accepted: 04/10/2014] [Indexed: 01/27/2023]
Abstract
Cyclosporine A (CsA) is an immunosuppressive drug that targets cyclophilins, cellular cofactors that regulate the immune system. Replication of hepatitis C virus (HCV) is suppressed by CsA, but the molecular basis of this suppression is still not fully understood. To investigate this suppression, we cultured HCV replicon cells (Con1, HCV genotype 1b, FLR-N cell) in the presence of CsA and obtained nine CsA-resistant FLR-N cell lines. We determined full-length HCV sequences for all nine clones, and chose two (clones #6 and #7) of the nine clones that have high replication activity in the presence of CsA for further analysis. Both clones showed two consensus mutations, one in NS3 (T1280V) and the other in NS5A (D2292E). Characterization of various mutants indicated that the D2292E mutation conferred resistance to high concentrations of CsA (up to 2 μM). In addition, the missense mutation T1280V contributed to the recovery of colony formation activity. The effects of these mutations are also evident in two established HCV replicon cell lines-HCV-RMT ([1], genotype 1a) and JFH1 (genotype 2a). Moreover, three other missense mutations in NS5A-D2303H, S2362G, and E2414K-enhanced the resistance to CsA conferred by D2292E; these double or all quadruple mutants could resist approximately 8- to 25-fold higher concentrations of CsA than could wild-type Con1. These four mutations, either as single or combinations, also made Con1 strain resistant to two other cyclophilin inhibitors, N-methyl-4-isoleucine-cyclosporin (NIM811) or Debio-025. Interestingly, the changes in IC50 values that resulted from each of these mutations were the lowest in the Debio-025-treated cells, indicating its highest resistant activity against the adaptive mutation.
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Affiliation(s)
- Masaaki Arai
- Advanced Medical Research Laboratory, Mitsubishi Tanabe Pharma Corporation, 1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa 227-0033, Japan; Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Kyoko Tsukiyama-Kohara
- Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan
| | - Asako Takagi
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Yoshimi Tobita
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Kazuaki Inoue
- Division of Gastroenterology, Showa University Fujigaoka Hospital, 1-30, Aoba-ku, Fujigaoka, Yokohama 227-8501, Japan
| | - Michinori Kohara
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
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Akuta N, Suzuki F, Sezaki H, Suzuki Y, Hosaka T, Kobayashi M, Kobayashi M, Saitoh S, Ikeda K, Kumada H. Evolution of simeprevir-resistant variants over time by ultra-deep sequencing in HCV genotype 1b. J Med Virol 2014; 86:1314-22. [PMID: 24797623 DOI: 10.1002/jmv.23966] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/08/2014] [Indexed: 12/17/2022]
Abstract
Using ultra-deep sequencing technology, the present study was designed to investigate the evolution of simeprevir-resistant variants (amino acid substitutions of aa80, aa155, aa156, and aa168 positions in HCV NS3 region) over time. In Toranomon Hospital, 18 Japanese patients infected with HCV genotype 1b, received triple therapy of simeprevir/PEG-IFN/ribavirin (DRAGON or CONCERT study). Sustained virological response rate was 67%, and that was significantly higher in patients with IL28B rs8099917 TT than in those with non-TT. Six patients, who did not achieve sustained virological response, were tested for resistant variants by ultra-deep sequencing, at the baseline, at the time of re-elevation of viral loads, and at 96 weeks after the completion of treatment. Twelve of 18 resistant variants, detected at re-elevation of viral load, were de novo resistant variants. Ten of 12 de novo resistant variants become undetectable over time, and that five of seven resistant variants, detected at baseline, persisted over time. In one patient, variants of Q80R at baseline (0.3%) increased at 96-week after the cessation of treatment (10.2%), and de novo resistant variants of D168E (0.3%) also increased at 96-week after the cessation of treatment (9.7%). In conclusion, the present study indicates that the emergence of simeprevir-resistant variants after the start of treatment could not be predicted at baseline, and the majority of de novo resistant variants become undetectable over time. Further large-scale prospective studies should be performed to investigate the clinical utility in detecting simeprevir-resistant variants.
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Affiliation(s)
- Norio Akuta
- Department of Hepatology, Toranomon Hospital and Okinaka Memorial Institute for Medical Research, Tokyo, Japan
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Ueda Y, Mori K, Satoh S, Dansako H, Ikeda M, Kato N. Anti-HCV activity of the Chinese medicinal fungus Cordyceps militaris. Biochem Biophys Res Commun 2014; 447:341-5. [DOI: 10.1016/j.bbrc.2014.03.150] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2014] [Accepted: 03/31/2014] [Indexed: 01/27/2023]
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Kato N, Sejima H, Ueda Y, Mori K, Satoh S, Dansako H, Ikeda M. Genetic characterization of hepatitis C virus in long-term RNA replication using Li23 cell culture systems. PLoS One 2014; 9:e91156. [PMID: 24625789 PMCID: PMC3953375 DOI: 10.1371/journal.pone.0091156] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2013] [Accepted: 02/10/2014] [Indexed: 12/17/2022] Open
Abstract
BACKGROUND The most distinguishing genetic feature of hepatitis C virus (HCV) is its remarkable diversity and variation. To understand this feature, we previously performed genetic analysis of HCV in the long-term culture of human hepatoma HuH-7-derived HCV RNA-replicating cell lines. On the other hand, we newly established HCV RNA-replicating cell lines using human hepatoma Li23 cells, which were distinct from HuH-7 cells. METHODOLOGY/PRINCIPAL FINDINGS Li23-derived HCV RNA-replicating cells were cultured for 4 years. We performed genetic analysis of HCVs recovered from these cells at 0, 2, and 4 years in culture. Most analysis was performed in two separate parts: one part covered from the 5'-terminus to NS2, which is mostly nonessential for RNA replication, and the other part covered from NS3 to NS5B, which is essential for RNA replication. Genetic mutations in both regions accumulated in a time-dependent manner, and the mutation rates in the 5'-terminus-NS2 and NS3-NS5B regions were 4.0-9.0×10(-3) and 2.7-4.0×10(-3) base substitutions/site/year, respectively. These results suggest that the variation in the NS3-NS5B regions is affected by the pressure of RNA replication. Several in-frame deletions (3-105 nucleotides) were detected in the structural regions of HCV RNAs obtained from 2-year or 4-year cultured cells. Phylogenetic tree analyses clearly showed that the genetic diversity of HCV was expanded in a time-dependent manner. The GC content of HCV RNA was significantly increased in a time-dependent manner, as previously observed in HuH-7-derived cell systems. This phenomenon was partially due to the alterations in codon usages for codon optimization in human cells. Furthermore, we demonstrated that these long-term cultured cells were useful as a source for the selection of HCV clones showing resistance to anti-HCV agents. CONCLUSIONS/SIGNIFICANCE Long-term cultured HCV RNA-replicating cells are useful for the analysis of evolutionary dynamics and variations of HCV and for drug-resistance analysis.
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Affiliation(s)
- Nobuyuki Kato
- Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Shikata-cho, Okayama, Japan
- * E-mail:
| | - Hiroe Sejima
- Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Shikata-cho, Okayama, Japan
| | - Youki Ueda
- Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Shikata-cho, Okayama, Japan
| | - Kyoko Mori
- Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Shikata-cho, Okayama, Japan
| | - Shinya Satoh
- Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Shikata-cho, Okayama, Japan
| | - Hiromichi Dansako
- Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Shikata-cho, Okayama, Japan
| | - Masanori Ikeda
- Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Shikata-cho, Okayama, Japan
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50
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In vitro selection of the 3′-untranslated regions of the human liver mRNA that bind to the HCV nonstructural protein 5B. Virology 2014; 450-451:13-23. [DOI: 10.1016/j.virol.2013.11.036] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2013] [Revised: 11/12/2013] [Accepted: 11/21/2013] [Indexed: 12/21/2022]
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