No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Understanding the hepatitis C virus life cycle paves the way for highly effective therapies
Abstract
More than two decades of intense research has provided a detailed understanding of hepatitis C virus (HCV), which chronically infects 2% of the world's population. This effort has paved the way for the development of antiviral compounds to spare patients from life-threatening liver disease. An exciting new era in HCV therapy dawned with the recent approval of two viral protease inhibitors, used in combination with pegylated interferon-α and ribavirin; however, this is just the beginning. Multiple classes of antivirals with distinct targets promise highly efficient combinations, and interferon-free regimens with short treatment duration and fewer side effects are the future of HCV therapy. Ongoing and future trials will determine the best antiviral combinations and whether the current seemingly rich pipeline is sufficient for successful treatment of all patients in the face of major challenges, such as HCV diversity, viral resistance, the influence of host genetics, advanced liver disease and other co-morbidities.
... The genomic architecture of HCV exemplifies a Flaviviridae member in being a positivesense, single-stranded RNA genome, constituting a single ORF [74]. This ORF is responsible for the synthesis of a large single polyprotein, approximately 3000 amino acids in length, as delineated in the work by Li et al. and Scheel and Rice [75,76]. The processing of HCV polyprotein into its functional single proteins involves a series of cleavage events mediated by both host cellular proteases and virus-specific enzymes, yielding an array of structural and non-structural (NS) proteins critical for the viral life cycle [76,77]. ...
... This ORF is responsible for the synthesis of a large single polyprotein, approximately 3000 amino acids in length, as delineated in the work by Li et al. and Scheel and Rice [75,76]. The processing of HCV polyprotein into its functional single proteins involves a series of cleavage events mediated by both host cellular proteases and virus-specific enzymes, yielding an array of structural and non-structural (NS) proteins critical for the viral life cycle [76,77]. ...
... The NS2 protein, a cysteine protease, facilitates the cleavage of NS3 from the NS2-NS3 junction [83]. NS3, together with NS4A, constitutes a serine protease complex essential for the cleavage of subsequent NS proteins, regulating the sequential processing of the viral proteome [76]. NS4B serves as a membrane anchor for the viral replication complex, while NS5A, alongside NS4B, contributes to the formation of the endoplasmic reticulum (ER) membranous web, a structure essential for viral replication dynamics [76,84]. ...
Hepatitis C virus (HCV) remains a significant global health challenge. Approximately 50 million people were living with chronic hepatitis C based on the World Health Organization as of 2024, contributing extensively to global morbidity and mortality. The advent and approval of several direct-acting antiviral (DAA) regimens significantly improved HCV treatment, offering potentially high rates of cure for chronic hepatitis C. However, the promising aim of eventual HCV eradication remains challenging. Key challenges include the variability in DAA access across different regions, slightly variable response rates to DAAs across diverse patient populations and HCV genotypes/subtypes, and the emergence of resistance-associated substitutions (RASs), potentially conferring resistance to DAAs. Therefore, periodic reassessment of current HCV knowledge is needed. An up-to-date review on HCV is also necessitated based on the observed shifts in HCV epidemiological trends, continuous development and approval of therapeutic strategies, and changes in public health policies. Thus, the current comprehensive review aimed to integrate the latest knowledge on the epidemiology, pathophysiology, diagnostic approaches, treatment options and preventive strategies for HCV, with a particular focus on the current challenges associated with RASs and ongoing efforts in vaccine development. This review sought to provide healthcare professionals, researchers, and policymakers with the necessary insights to address the HCV burden more effectively. We aimed to highlight the progress made in managing and preventing HCV infection and to highlight the persistent barriers challenging the prevention of HCV infection. The overarching goal was to align with global health objectives towards reducing the burden of chronic hepatitis, aiming for its eventual elimination as a public health threat by 2030.
... The virus belongs to the genus Hepacivirus of the Flaviviridae family. It is an approximately 9,600 bp positive sense single stranded ribonucleic acid (RNA) virus with a long open reading frame (bordered by 5' and 3' untranslated regions) that codes for structural (core, E1 and E2) and nonstructural (p7, NS2, NS3, NS4A, NS4B NS5A and NS5B) proteins [2] (Figure 1A). At least 6 genotypes and more than 80 subtypes with varying global and regional distributions have been identified [3,4]. ...
... Upon transmission, HCV makes its way to the liver cells (hepatocytes) [5] and undergoes a replication cycle typical of a positive sense RNA virus, and ultimately produces ample viruses that infect other hepatocytes. Because the life cycle and pathogenesis of HCV has been well studied and depicted [2], an illustration to highlight the regions of the life cycle targeted by anti-HCV agents is presented ( Figure 1B). If left untreated, HCV infection can result in complications such as scarring of the liver (cirrhosis), hepatocellular carcinoma (liver cancer), liver failure, and even death. ...
... First report of DAA therapy in children and adolescents 2016 FDA approval of the first pan-genotypic fixed-dose combination tablet (consisting of sofosbuvir and velpatasvir) for the treatment of chronic hepatitis C infection by all six major HCV genotypes 2013-2020 Evaluation of all-oral DAA and other combination therapies, as well as advocacy for pan-genotypic regimens This table was compiled from the following references and resources: [1,2,34,50,[56][57][58][59][60][61][62][63][64][65][66][67]. that relapse rate was significantly reduced following 72 weeks of therapy [19]. ...
Remarkable scientific breakthroughs have been made in the stride towards the development of potent and tolerable hepatitis C regimens within the last three decades. Earlier approaches involved the use of pegylated interferon alfa and ribavirin as standard-of-care treatment. Treating genotype 1a infection with this regimen which was at that time considered the gold standard for hepatitis C virus therapy was rife with challenges; safety and toxicity issues necessitated a rigorous quest for alternative regimens. Deeper understanding of the pathogenesis of hepatitis C virus ushered in the era of direct acting antiviral agents. These agents have been the subject of intensive research in the last two decades, leading to the development of drug classes such as protease inhibitors (e.g., grazoprevir), NS5A inhibitors (e.g., daclatasvir) and NS5B inhibitors (e.g., sofosbuvir). While many are still under development, several have been approved for hepatitis C therapy. A number of studies investigating the combination of direct acting antiviral agents with or without pegylated interferon and/or ribavirin for the treatment of chronic hepatitis have demonstrated sustained virologic response of > 90%. Given the array of direct acting antiviral agents currently available, the present landscape of hepatitis C therapy is now characterized by a gradual transition to all-oral interferon-free regimens. Despite these milestones, the WHO global target of eliminating hepatitis C as a public health problem by 2030 seems uncertain. In this review, we provide a concise account of the evolution and advancements in the development of anti-HCV regimens.
... Over 30 years have passed since the discovery of hepatitis C virus (HCV) infection in 1989, which is one of the most common causes of liver disease worldwide (1). HCV infection causes acute and chronic hepatitis and is the main cause of post-transfusion hepatitis. ...
... HCV infection causes acute and chronic hepatitis and is the main cause of post-transfusion hepatitis. It is transmitted mainly by exposure to contaminated blood, contaminated needles or razors, hemodialysis, organ transplantation, and, to a lesser extent, by sexual intercourse (1,2). HCV is highly heterogeneous and is susceptible to ongoing mutation. ...
... HCV genotypes 1, 2, and 3 are found in the USA, Europe, and Japan, while genotypes 4 and 5 are found in Africa. Genotypes 3 and 6 are endemic to Asia, whereas genotype 7 is endemic to central Africa and has not been fully evaluated (1,2). This article discusses recent published literature on antiviral therapy for HCV infection, focusing on infection of the peripheral blood mononuclear cells (PBMCs), and the effects of HCV infection on the immune system. ...
Hepatitis C is a worldwide liver disease caused by hepatitis C virus (HCV) infection. The virus causes acute and chronic liver inflammation, and it is transmitted mainly by exposure to contaminated blood. HCV is capable of infecting hepatocytes and peripheral blood mononuclear cells, causing complications and disease progression. This mini review provides an overview of HCV infection, including details on the virological aspects, infection of the immune cells, and its impact on the immune system.
... 14 The end-product non-structural proteins include NS3/4A serine protease, NS5A, which is the regulator of replication and viral assembly, and NS5B RNA-dependent RNA polymerase. 15 Cellular proteases and viral NS3/4A protease cleave HCV polypeptide and release ten HCV proteins. 15 Due to the lack of proof-reading function in HCV replication machinery, viral mutations result in a high level of variation. ...
... 15 Cellular proteases and viral NS3/4A protease cleave HCV polypeptide and release ten HCV proteins. 15 Due to the lack of proof-reading function in HCV replication machinery, viral mutations result in a high level of variation. 16 These variations lead to eight major HCV genotypes that are different primarily based on geographic origin. ...
... 30 While disease association is largely similar across all HCV genotypes, treatment response varies. 15 Patients with cirrhosis and/or HCV genotype 3 are relatively difficult to treat compared to patients without cirrhosis or those with other HCV genotypes using currently available DAAs. 31 There are also fewer treatment options available for HCV genotype 3 compared to genotype 1. Other challenging clinical situations include patients who have undergone liver transplantation, patients at increased risk of rapid progression of liver fibrosis, people who inject drugs, and patients on hemodialysis. ...
Hepatitis C virus (HCV) infection has a significant global burden with complications that include hepatocellular carcinoma, decompensated cirrhosis, and the need for liver transplantation to avoid death. Although many treatment options are available for treatment of HCV infections, treatment often requires at least 12 weeks of therapy, which may be complicated by HCV genotype, the need for addition of ribavirin, presence of advanced liver disease, end-stage kidney disease, prior treatment failure, and presence of resistance-associated substitutions. Glecaprevir-pibrentasvir is a pangenotypic, ribavirin free treatment option approved for ages 3 years or older. This regimen can be used in patients with end-stage kidney disease, has a high-barrier to resistance, and has a shorter treatment duration of 8 weeks for most patients. The purpose of this review is to evaluate the safety and efficacy of this regimen in adults and children who are infected with HCV, which may be the only treatment option for certain patients.
... Approximately 130-170 million people worldwide have chronic HCV infection, and these patients are at high risk of progression to HCC and require early and effective antiviral therapy [99,100]. Treatment that induces a sustained viral response (SVR) is a reliable marker of HCV eradication and is associated with the inhibition of liver disease progression and the associated complications, including HCC development [101]. ...
... Further studies are needed to understand the molecular mechanisms underlying the risk of HCC progression after the HCV cure, but different HCV genotypes, subtypes, and gene mutations seem to play a role [100,103]. The recurrence of HCV-induced HCC is not only manifested in the progression to HCC after the cure of HCV infection, but also in the recurrence of HCC patients after surgical resection. ...
Simple Summary
Hepatocellular carcinoma (HCC) is a global health challenge. Hepatitis virus infection (including HBV, HCV and HDV) is one of the major risk factors for HCC development. These viruses induce hepatocyte cancer by causing chronic hepatitis and by a variety of complex mechanisms. Here we discuss the mechanisms by which several hepatitis viruses induce HCC, as well as new diagnostic and therapeutic approaches to HCC based on the findings of these mechanisms. Finally, we also discuss the potential relationship between HEV and HCC.
Abstract
Hepatocellular carcinoma (HCC) remains a global health challenge, causing 600,000 deaths each year. Infectious factors, including hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis D virus (HDV), have long been considered the major risk factors for the development and progression of HCC. These pathogens induce hepatocyte transformation through a variety of mechanisms, including insertional mutations caused by viral gene integration, epigenetic changes, and the induction of long-term immune dysfunction. The discovery of these mechanisms, while advancing our understanding of the disease, also provides targets for new diagnostic and therapeutic approaches. In addition, the discovery and research of chronic HEV infection over the past decade indicate that this common hepatitis virus also seems to have the potential to induce HCC. In this review, we provide an overview of recent studies on the link between hepatitis virus and HCC, as well as new diagnostic and therapeutic approaches to HCC based on these findings. Finally, we also discuss the potential relationship between HEV and HCC. In conclusion, these associations will further optimize the diagnosis and treatment of infection-associated HCC and call for better management policies.
... The use of direct acting antivirals with high barrier to resistance and a combination of several DAA classes can be beneficial in ensuring a successful achievement of sustained virological response (SVR). A combination of highly efficient pangenotypic DAA has reduced the need for baseline resistance testing [23][24][25][26]. However, current guidelines suggest testing for NS5A resistance associated substitutions (RAS) in the treatment of naïve patients with HCV GT3 and liver cirrhosis before treatment with sofosbuvir/velpatasvir since Y93H mutation was shown to reduce SVR to 84-88% in these patients [26,27]. ...
... Since every DAA class induces specific mutation profile characteristic for various HCV genotypes and subtypes, the use of combination therapy with agents targeting various viral proteins has been successful in obtaining high rates of SVR [23][24][25][26]. Only a few studies analysed the presence of RAS to all three DAA classes for every patient, mainly due to the lack of sequences spanning across all relevant genome regions. ...
Molecular epidemiology of hepatitis C virus (HCV) is exceptionally complex due to the highly diverse HCV genome. Genetic diversity, transmission dynamics, and epidemic history of the most common HCV genotypes were inferred by population sequencing of the HCV NS3, NS5A, and NS5B region followed by phylogenetic and phylodynamic analysis. The results of this research suggest high overall prevalence of baseline NS3 resistance associate substitutions (RAS) (33.0%), moderate prevalence of NS5A RAS (13.7%), and low prevalence of nucleoside inhibitor NS5B RAS (8.3%). Prevalence of RAS significantly differed according to HCV genotype, with the highest prevalence of baseline resistance to NS3 inhibitors and NS5A inhibitors observed in HCV subtype 1a (68.8%) and subtype 1b (21.3%), respectively. Phylogenetic tree reconstructions showed two distinct clades within the subtype 1a, clade I (62.4%) and clade II (37.6%). NS3 RAS were preferentially associated with clade I. Phylogenetic analysis demonstrated that 27 (9.0%) HCV sequences had a presumed epidemiological link with another sequence and classified into 13 transmission pairs or clusters which were predominantly comprised of subtype 3a viruses and commonly detected among intravenous drug users (IDU). Phylodynamic analyses highlighted an exponential increase in subtype 1a and 3a effective population size in the late 20th century, which is a period associated with an explosive increase in the number of IDU in Croatia.
... Received: January 14, 2022 Accepted: February 17, 2022 Published: March 15, 2022 Введение Ф ормирование системной патологии сопровождается адекватными и значимыми для растущего организма изменениями метаболизма. Не являются исключением и вирусные гепатиты (ВГ) у детей, при которых, кроме лабораторного детектирования виремии и титра специфических антител, характерны многочисленные разнонаправленные сдвиги метаболома крови [1][2][3][4]. Однако системный анализ ВГ проводится недостаточно, несмотря на значимость этих данных для разработки новых диагностических алгоритмов оценки степени тяжести поражений печени, наличия фибротических изменений в ней и т.п. [5]. ...
... В основу непосредственной оценки результатов дискриминантного анализа легли стандартизированные коэффициенты канонических дискриминантных функций, указывающие на вклад каждого значимого параметра в зависимый показатель, а также данные структурной ма-трицы, характеризующие модуль и направленность корреляции каждого лабораторного критерия с зависимой переменной [5,[15][16][17][18]. Основными зависимыми параметрами считали диагноз (наличие ВГ типа В или С) [2,3,[15][16][17][18][19][20][21] и наличие фиброза печени [6,11,12,18]. ...
The aim of the work is to form a mathematical model of viral hepatitis based on structural modelling, discriminant and factorial analysis of laboratory parameters of patients. Materials and methods. The data array included the results of a comprehensive laboratory testing of 109 children with viral hepatitis B and C (33 parameters). Results. Seven main factors in the pathogenesis of viral hepatitis in children reflect the severity of endogenous intoxication and disorders of protein metabolism, modification of the mode of functioning of enzymes, and supramolecular multi-enzyme complexes in conditions of progression of the first two components. Conclusion. The identified factors of the pathogenesis of viral hepatitis may reflect the endogenous intoxication during disease progression, the state of detoxification enzyme systems, and the further fibrotic changes in the liver.
... Replication of pathogens in the host relies on their own suite of enzymes, either encapsulated within the pathogen (e.g., bacteria [1], HIV [2]) or encoded in their genome and subsequently synthesized in host cells (e.g., SARS-CoV-2 [3], HCV [4]). The pathogen-derived enzymes have evolved to facilitate critical replication steps and thereby possess specialized activities, such as reverse transcription, DNA or RNA polymerization, and DNA topology adjustment (Table 1). ...
Pathogens encapsulate or encode their own suite of enzymes to facilitate replication in the host. The pathogen-derived enzymes possess specialized activities that are essential for pathogen replication and have naturally been candidates for drug targets. Phenotypic assays detecting the activities of pathogen-derived enzymes and characterizing their inhibition under drugs offer an opportunity for pathogen detection, drug resistance testing for individual patients, and as a research tool for new drug development. Here, we used HIV as an example to develop assays targeting the reverse transcriptase (RT) enzyme encapsulated in HIV for sensitive detection and phenotypic characterization, with the potential for point-of-care (POC) applications. Specifically, we targeted the complementary (cDNA) generation activity of the HIV RT enzyme by adding engineered RNA as substrates for HIV RT enzyme to generate cDNA products, followed by cDNA amplification and detection facilitated by loop-mediated isothermal amplification (LAMP) or CRISPR-Cas systems. To guide the assay design, we first used qPCR to characterize the cDNA generation activity of HIV RT enzyme. In the LAMP-mediated Product-Amplified RT activity assay (LamPART), the cDNA generation and LAMP amplification were combined into one pot with novel assay designs. When coupled with direct immunocapture of HIV RT enzyme for sample preparation and endpoint lateral flow assays for detection, LamPART detected as few as 20 copies of HIV RT enzyme spiked into 25μL plasma (fingerstick volume), equivalent to a single virion. In the Cas-mediated Product-Amplified RT activity assay (CasPART), we tailored the substrate design to achieve a LoD of 2e4 copies (1.67fM) of HIV RT enzyme. Furthermore, with its phenotypic characterization capability, CasPART was used to characterize the inhibition of HIV RT enzyme under antiretroviral drugs and differentiate between wild-type and mutant HIV RT enzyme for potential phenotypic drug resistance testing. Moreover, the CasPART assay can be readily adapted to target the activity of other pathogen-derived enzymes. As a proof-of-concept, we successfully adapted CasPART to detect HIV integrase with a sensitivity of 83nM. We anticipate the developed approach of detecting enzyme activity with product amplification has the potential for a wide range of pathogen detection and phenotypic characterization.
... When a virus attaches to a host cell, it penetrates the cell and uncoating (disassembly) takes place, allowing the virus to replicate and produce new virions, which are released from the infected cell and infect other cells [34]. In this study, time-of-addition method investigated at which stage the viral life cycle is disrupted by antiviral compound [35]. ...
This study aims to investigate the antiviral activities of methanolic extract of Syzygium polyanthum towards Herpes simplex virus type 1 (HSV-1). Plaque reduction assays were carried out to evaluate the antiviral activity of S. polyanthum extract against HSV-1. Time dependent studies were conducted to evaluate the effect of delayed treatment and also the effect of different time exposure to S. polyanthum methanol extract againstHSV-1 antiviral activity. The effect on replication phases of HSV-1 was determined by time-of-addition and time-of-removal assays. Time-of-addition assay showed the extract inhibits 70% of the virus at 2 hours post infection (hpi). In time-of-removal assay, treatment with extract caused 20% reduction of plaque formation as early as 2 hpi and reached more than 80% after 16 hpi. Gene expression analysis by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) using selected primers was done to investigate the level of gene transcript at different replication phases of HSV-1 which included immediate early genes (UL54), early gene (UL30), and late gene (UL27). The result showed that transcript levels of these genes in infected cells treated with S. polyanthum extract were reduced compared to infected cells without treatment. This study showed that S. polyanthum extract has potential as anti-HSV-1 by the following modes: interruption of virus attachment and penetration into cells, direct damage to viral particle, reduction of viral progeny infectivity, and reduction of expression of HSV-1 genes at different phases of viral replication.
... However, many individuals did not show sustained virological response (SVR) either because of infection with nonresponder genotypes or extreme side effects and compliance issues. 5 This necessitated interferon-free Direct Acting Antiviral Therapy (DAAT). Now, therapeutic management of HCV has switched from interferon-based therapies to interferon-free oral directacting antiviral (DAA) combination regimens. ...
Objective: To assess the impact and effectiveness of a pan-genotypic Directly Acting Antiviral (DAA) based regimen for patients with chronic Hepatitis-C (HCV) infection in our setup. Study Design: Cross-sectional study. Place and Duration of Study: Departments of Pathology and Medicine, Combined Military Hospital, Malir, Karachi, from Dec 2019 to Jan 2021. Methodology: Sofosbuvir and Daclatasvir were administered orally at 400mg and 60mg daily, respectively, with or without Ribavirin to treatment-naïve and treatment-experienced adult patients with chronic HCV infection. End-of-treatment response and Sustained virological response were determined among these patients by monitoring viral load using quantitative HCV RNA PCR and various genotypes. Results: Of the 59 patients, 39(66%) patients received sofosbuvir (SOF)+Daclatasvir (DCV), 12(20.3%) patients were placed on SOF+Ribavirin (Rib) and 8(13.5%) were given triple regime of SOF+DCV+Rib for 12 and 24 weeks respectively. Most of our patients were females, 32(54.23%), with a mean age of 31±17 years. Forty-eight patients (81.35%) were infected with genotype 3, followed by genotype 1 and 2, respectively. The rapid viral response was noted after four weeks of therapy among 27(45.76%) patients. The sustained viral response was noted in 38(64.40%) and 21(35.59%) patients after 12 and 24 weeks, respectively. The end-of-treatment response was observed in 40(67.8%) patients. Conclusion: Direct-acting antiviral-based regimens have shown favourable results with fewer adverse effects in our patients.
... The Flaviviridae family comprises enveloped, single-stranded positive-sense RNA viruses, including the Hepatitis C virus (HCV), which is the causative agent of chronic hepatitis. It encodes a large polyprotein precursor of approximately 3,000 amino acid residues, which is processed by viral proteases and cellular signalizes to generate three structural proteins (core, E1, and E2) and seven non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B; Scheel and Rice, 2013). The non-structural proteins NS3, NS4A, NS4B, NS5A, and NS5B form the viral replication machinery, while p7 and NS2 are crucial for the production of infectious viruses (Jones et al., 2007;Jirasko et al., 2010). ...
The endosomal sorting complex required for transport (ESCRT) is an essential molecular machinery in eukaryotic cells that facilitates the invagination of endosomal membranes, leading to the formation of multivesicular bodies (MVBs). It participates in various cellular processes, including lipid bilayer remodeling, cytoplasmic separation, autophagy, membrane fission and re-modeling, plasma membrane repair, as well as the invasion, budding, and release of certain enveloped viruses. The ESCRT complex consists of five complexes, ESCRT-0 to ESCRT-III and VPS4, along with several accessory proteins. ESCRT-0 to ESCRT-II form soluble complexes that shuttle between the cytoplasm and membranes, mainly responsible for recruiting and transporting membrane proteins and viral particles, as well as recruiting ESCRT-III for membrane neck scission. ESCRT-III, a soluble monomer, directly participates in vesicle scission and release, while VPS4 hydrolyzes ATP to provide energy for ESCRT-III complex disassembly, enabling recycling. Studies have confirmed the hijacking of ESCRT complexes by enveloped viruses to facilitate their entry, replication, and budding. Recent research has focused on the interaction between various components of the ESCRT complex and different viruses. In this review, we discuss how different viruses hijack specific ESCRT regulatory proteins to impact the viral life cycle, aiming to explore commonalities in the interaction between viruses and the ESCRT system.
... HCV is a positive-sense single-stranded RNA virus that belongs to the Hepacivirus genus of the Flaviviridae family. The HCV genome consists of 9.6-kb RNA encoding a single polyprotein of about 3,010 amino acids, which is processed by viral proteases and cellular signalases to produce three structural proteins (Core, E1, and E2) and seven nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (5). ...
We previously reported that hepatitis C virus (HCV) infection activates the reactive oxygen species (ROS)/c-Jun N-terminal kinase (JNK) signaling pathway. Activation of JNK contributes to the development of liver diseases, including metabolic disorders, steatosis, liver cirrhosis and hepatocellular carcinoma. JNK is known to have numerous target genes, including JunB, a member of activator protein-1 transcription factor family. However, the roles of JunB in the HCV life cycle and HCV-associated pathogenesis remain unclear. To clarify a physiological role of JunB in HCV infection, we investigated the phosphorylation of JunB in HCV J6/JFH1-infected Huh-7.5 cells. Immunoblot analysis revealed that HCV-induced ROS/JNK activation promoted phosphorylation of JunB. The small interfering RNA (siRNA) knockdown of JunB significantly increased the amount of intracellular HCV RNA as well as the intracellular and extracellular HCV infectivity titers. Conversely, overexpression of JunB significantly reduced the amount of intracellular HCV RNA and the intracellular and extracellular HCV infectivity titers. These results suggest that JunB plays a role in inhibiting HCV propagation. Additionally, HCV-mediated JunB activation promoted hepcidin promoter activity and hepcidin mRNA levels, a key factor in modulating iron homeostasis, suggesting that JunB is involved in HCV-induced transcriptional upregulation of hepcidin. Taken together, we propose that the HCV-induced ROS/JNK/JunB signaling pathway plays roles in inhibiting HCV replication and contributing to HCV-mediated iron metabolism disorder.
... The utilization of medication combinations with orally administered direct-acting antivirals (DAAs) has resulted in recent advancements in HCV treatment [32]. Two or more DAAs are used in highly successful HCV combination treatment to target various HCV drug target proteins that perform diverse roles in the viral life cycle [33,34]. DAAs that block the NS5B polymerase in HCV are some of the most therapeutically effective HCV treatments (Fig. 1). ...
About 130–150 million people worldwide are chronically infected with the hepatitis C virus (HCV), and this
infection can lead to serious liver problems such as cirrhosis, hepatocellular carcinoma, or liver cancer. One of
the most well researched therapeutic targets for HCV is the NS5B protein, which is used to find novel treatment
candidates to supplement currently approved combinations or multiple combinations of medications. As a result,
NS5B has been the focus of numerous intriguing medicinal chemistry projects over the past several years, which
has led to the appearance of promising preclinical therapeutic compounds. For all those medicinal chemist researchers dealing with HCV research programs, specifically pointing on NS5B and setting broad spectrum
identification of creative anti-HCV compounds, the discussion described in this particular review will undoubtedly be beneficial and essential which describes the recent synthesized NS5B inhibitors including patents,
structure-activity relationship and docking studies
... The Flaviviridae family comprises enveloped, single-stranded positive-sense RNA viruses, including the Hepatitis C virus (HCV), which is the causative agent of chronic hepatitis. It encodes a large polyprotein precursor of approximately 3,000 amino acid residues, which is processed by viral proteases and cellular signalises to generate three structural proteins (core, E1, and E2) and seven non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) 127 . The non-structural proteins NS3, NS4A, NS4B, NS5A, and NS5B form the viral replication machinery, while p7 and NS2 are crucial for the production of infectious viruses 128,129 . ...
The endosomal sorting complex required for transport (ESCRT) is an essential molecular machinery in eukaryotic cells that facilitates the invagination of endosomal membranes, leading to the formation of multivesicular bodies (MVBs). It participates in various cellular processes, including lipid bilayer remodeling, cytoplasmic separation, autophagy, membrane fission and remodeling, plasma membrane repair, as well as the invasion, budding, and release of certain enveloped viruses. The ESCRT complex consists of five complexes, ESCRT-0 to ESCRT-III and VPS4, along with several accessory proteins. ESCRT-0 to ESCRT-II form soluble complexes that shuttle between the cytoplasm and membranes, mainly responsible for recruiting and transporting membrane proteins and viral particles, as well as recruiting ESCRT-III for membrane neck scission. ESCRT-III, a soluble monomer, directly participates in vesicle scission and release, while VPS4 hydrolyzes ATP to provide energy for ESCRT-III complex disassembly, enabling recycling. Studies have confirmed the hijacking of ESCRT complexes by enveloped viruses to facilitate their entry, replication, and budding. Recent research has focused on the interaction between various components of the ESCRT complex and different viruses. In this review, we discuss how different viruses hijack specific ESCRT regulatory proteins to impact the viral life cycle, aiming to explore commonalities in the interaction between viruses and the ESCRT system.
... For assembly and release, HCV also makes advantage of fatty acid pathways and the creation of extremely low-density lipoprotein (VLDL). The HCV life cycle is shown in Figure 1 with an emphasis on the critical phases of Virus replication, such as HCV adherence and entry into the host organism, HCV RNA reproduction, viral assembling and release, and HCV RNA translation to generate a giant polyprotein that is translated into 10 HCV proteins [28]. Replication of hepatitis C virus inside the host cell [29]. ...
The spread of hepatitis C virus (HCV) infection is a worldwide crisis. Emerging evidence reveals the host-viral interactions govern the natural course and treatment response, individualy. For pegylated interferon plus ribavirin treatment, the response is most strongly predicted by the patient's HCV genotype. HCV genotype 1 with subtypes is the most important viral component in determining the success of direct-acting antiviral treatmenst. In addition to baseline viral load and HCV genomic heterogeneity, these two factors are linked with the treatment response. In chronic HCV infection, hosts’ genetic variations in immune responsive genes, have been identified as predictors of spontaneous disease progression and therapeutic outcome. In previous large genome-wide association studies, interferon3 gene polymorphisms have been shown to be linked with spontaneous clearance and treatment responsiveness. An inosine triphosphatase gene polymorphism has been shown to reduce the risk of anaemia and other side effects caused by the antiviral drug ribavirin. Hepatic steatosis and fibrosis are linked to a second genetic mutation in the patatin-like phospholipase domain containing 3 genes in HCV patients. This study examined the role of viral and host genetics in the natural history and treatment outcomes of HCV infection, and it is focused on the known viral and host variables linked with patient outcomes in HCV infection. This will result in fresh concepts of individualising, both the preventation to therapeutics.
... Therefore, factors of the host can also become potent targets for the therapy of anti-HCV. According to a recent study, at least two targeting agents of the host (HT) have clinically reached the stage of development, including microRNA122 antagonists and cyclophilin A inhibitors. 131,133,134 The mechanism involved is appropriate low-density lipoprotein synthesis/secretion in the production of infectious fragments of HCV. The Hepatitis C virus uses this lipoprotein biosynthetic pathway to generate mature particles of the virus and exports them. ...
Human viral oncogenesis is a complex phenomenon and a major contributor to the global cancer burden. Recent studies revealed several cellular events and molecular pathways taken over by viruses that promote the development and initiation of malignancy. The use of antiviral treatment to eliminate viral infections and prevent the activation of oncogenesis in the first place becomes a proactive approach. Understanding the molecular pathogenesis of various oncogenic viruses like Hepatitis virus, HIV, HPV, HSV, EBV, etc., against which efforts are being made to expand many potent antivirals that may escalate the apoptosis of infected malignant cells while sparing normal and healthy ones. Contemporary therapies like engineered antibodies, antiviral agents, signaling pathways, and cell biomarkers can inhibit viral oncogenesis. This review elaborates on the recent advances in both natural and synthetic antivirals to control viral oncogenesis. We also highlight the challenges and future perspectives of using antivirals in viral oncogenesis.
... [115][116][117][118] The role of each virus component in the virus life cycle, such as viral entry, uncoating, replication, virion assembly, and virion release, has also been unraveled for HBV and HCV over the years. [118][119][120] For instance, the HBV pre-S1 domain of L-HBs is primarily responsible for the virus binding and entry into the hepatocytes via the interaction with the bile transporter sodium taurocholate cotransporting peptide (NTCP). 121 Furthermore, the HCV virus entry is mediated by the interaction between hypervariable region 1 of the E2 protein and multiple hepatocyte membrane proteins, including claudin-1(CLDN1), occludin (OCLN), scavenger receptor class B type I (SRB1), and CD81. ...
Liver organoids are 3D cellular models of tissue in which cells interact to form unique structures in culture. Since their inception, liver organoids with various cellular compositions, structural features, and functional properties have been described over the past ten years. Methods to create these advanced human cell models range from simple tissue culture techniques to complex bioengineering approaches. These liver organoid culture platforms have been utilized in various liver research fields, including the modeling of liver diseases to regenerative therapy. This review will discuss how liver organoids are used to model diseases, including hereditary liver diseases, primary liver cancer, viral hepatitis, and nonalcoholic fatty liver disease. Specifically, we will focus on studies that utilized two widely adopted approaches: differentiation from pluripotent stem cells and epithelial organoids cultured from patient tissues. These approaches have enabled the generation of advanced human liver models and, more importantly, the establishment of patient-tailored models for evaluating individual-specific disease phenotypes and therapeutic responses.
... For assembly and release, HCV also makes advantage of fatty acid pathways and the creation of extremely low-density lipoprotein (VLDL). The HCV life cycle is shown in Figure 1 with an emphasis on the critical phases of Virus replication, such as HCV adherence and entry into the host organism, HCV RNA reproduction, viral assembling and release, and HCV RNA translation to generate a giant polyprotein that is translated into 10 HCV proteins [28]. ...
The spread of hepatitis C virus (HCV) infection is a worldwide crisis. Intricate host-viral interactions control the HCV infection's natural course and treatment response according to new research. The patient's HCV genotype is the best predictor of response to pegylated interferon plus ribavirin therapy. The most crucial viral factor in determining the efficacy of direct-acting antiviral therapy is the HCV genotype 1 subtype. In addition to baseline viral load and HCV genomic heterogeneity, these two factors are linked with the treatment response. In previous large genome-wide association studies, interferon3 gene polymorphisms have been shown to be linked with spontaneous clearance and treatment responsiveness. An inosine triphosphatase gene polymorphism has been shown to reduce the risk of anaemia and other side effects caused by the antiviral drug ribavirin. In HCV patients, a second genetic mutation in the three-gene patatin-like phospholipase domain is associated with hepatic steatosis and fibrosis. This study examined the effects of viral and host genetics on the course and results of HCV therapy while concentrating on the known viral and host variables linked to HCV patient outcomes. This will result in fresh concepts for individualising both preventative care and therapeutic treatment.
... The life cycle of HCV begins with its entry into cells. It is a highly elaborated multistep process that requires the interaction of the virus with host cellular molecules [26,27]. The initial step of HCV entry from blood stream to target cell involves the interaction of apoE found on the surface of HCV-LVP with glycosaminoglycans and LDL receptors (LDLr) [28,29]. ...
Hepatitis C virus (HCV) infection represents the major cause of chronic liver disease, leading to a wide range of hepatic diseases, including cirrhosis and hepatocellular carcinoma. It is the leading indication for liver transplantation worldwide. In addition, there is a growing body of evidence concerning the role of HCV in extrahepatic manifestations, including immune-related disorders and metabolic abnormalities, such as insulin resistance and steatosis. HCV depends on its host cells to propagate successfully, and every aspect of the HCV life cycle is closely related to human lipid metabolism. The virus circulates as a lipid-rich particle, entering the hepatocyte via lipoprotein cell receptors. It has also been shown to upregulate lipid biosynthesis and impair lipid degradation, resulting in significant intracellular lipid accumulation (steatosis) and circulating hypocholesterolemia. Patients with chronic HCV are at increased risk for hepatic steatosis, dyslipidemia, and cardiovascular disease, including accelerated atherosclerosis. This review aims to describe different aspects of the HCV viral life cycle as it impacts host lipoproteins and lipid metabolism. It then discusses the mechanisms of HCV-related hepatic steatosis, hypocholesterolemia, and accelerated atherosclerosis.
... HCV is highly dependent on the host's lipid metabolism to create a favorable environment for its replication in the liver [4][5][6][7]. Internalization of HCV into hepatocytes occurs via clathrin-mediated endocytosis, and the low pH of the endosomal compartment induces the fusion and replication of HCV [8]. HCV induces cytokine production and antibodies through the activation of the immune system. ...
We aimed to determine the biomarker performance of the proteolytic enzymes cathepsin B(Cat B) and plasma kallikrein (PKa) and transforming growth factor (TGF)-βto detect hepatic fibrosis(HF) in chronic hepatitis C (CHC) patients. We studied 53 CHC patients and 71 healthy controls(HCs). Hepatic-disease stage was determined by liver biopsies, aminotransferase:platelet ratio index(APRI) and Fibrosis (FIB)4. Hepatic inflammation and HF in CHC patients were stratified usingthe METAVIR scoring system. Cat-B and PKa activities were monitored fluorometrically. Serumlevels of TGF-β(total and its active form) were determined using ELISA-like fluorometric methods.Increased serum levels of Cat B and PKa were found (p< 0.0001) in CHC patients with clinicallysignificant HF and hepatic inflammation compared with HCs. Levels of total TGF-β(p< 0.0001) andactive TGF-β(p< 0.001) were increased in CHC patients compared with HCs. Cat-B levels correlatedstrongly with PKa levels (r = 0.903,p< 0.0001) in CHC patients but did not correlate in HCs. Levelsof Cat B, PKa and active TGF-βincreased with the METAVIR stage of HF. Based on analyses ofreceiver operating characteristic (ROC) curves, Cat B and PKa showed high diagnostic accuracy (areaunder ROC = 0.99±0.02 and 0.991±0.007, respectively) for distinguishing HF in CHC patientsfrom HCs. Taken together, Cat B and PKa could be used as circulating biomarkers to detect HF inHCV-infected patients.
... Chronic injuries are associated with increased risk for malignancy, which is highest in chronic viral infections. 9,10 Endogenous pathways regenerating damaged liver remain poorly characterized, and failures of understanding contribute to a lack of pro-regenerative clinical strategies. As the health and economic burden of liver diseases rapidly increase, 11,12 the absence of such repair strategies is critical. ...
Ideal therapies for regenerative medicine or healthy aging require healthy organ growth and rejuvenation, but no organ-level approach is currently available. Using Mycobacterium leprae (ML) with natural partial cellular reprogramming capacity and its animal host nine-banded armadillos, we present an evolutionarily refined model of adult liver growth and regeneration. In infected armadillos, ML reprogram the entire liver and significantly increase total liver/body weight ratio by increasing healthy liver lobules, including hepatocyte proliferation and proportionate expansion of vasculature, and biliary systems. ML-infected livers are microarchitecturally and functionally normal without damage, fibrosis, or tumorigenesis. Bacteria-induced reprogramming reactivates liver progenitor/developmental/fetal genes and upregulates growth-, metabolism-, and anti-aging-associated markers with minimal change in senescence and tumorigenic genes, suggesting bacterial hijacking of homeostatic, regeneration pathways to promote de novo organogenesis. This may facilitate the unraveling of endogenous pathways that effectively and safely re-engage liver organ growth, with broad therapeutic implications including organ regeneration and rejuvenation.
... The HCV nonstructural protein NS5A is one of the components of the viral RNA replication complex [83]. It has not yet been found to have enzymatic activity, but it is essential for HCV RNA replication and is also related to the INF response [84]. NS5A inhibitors may exert anti-HCV effects by inhibiting the hyperphosphorylation of NS5A or altering the subcellular localization of NS5A [85]. ...
Viral DNA and RNA polymerases are two kinds of very important enzymes that synthesize the genetic materials of the virus itself, and they have become extremely favorable targets for the development of antiviral drugs because of their relatively conserved characteristics. There are many similarities in the structure and function of different viral polymerases, so inhibitors designed for a certain viral polymerase have acted as effective universal inhibitors on other types of viruses. The present review describes the development of classical antiviral drugs targeting polymerases, summarizes a variety of viral polymerase inhibitors from the perspective of chemically synthesized drugs and natural product drugs, describes novel approaches, and proposes promising development strategies for antiviral drugs.
... A total of 80-90% HCV-related HCC cases occur in the setting of cirrhosis (7). Several stud-Intern Med 61: 2721-2729, 2022 DOI: 10.2169/internalmedicine.8456-21 ies have indicated the importance of HCV management in HCC therapeutic care and prevention (8,9). ...
Objective Owing to advances in direct-acting antiviral (DAA) therapy, a considerable number of patients with hepatitis C virus (HCV)-positive hepatocellular carcinoma (HCC) are now able to achieve a sustained viral response (SVR) after curative treatment of HCC. However, the beneficial effect of a DAA-SVR on the survival remains unclear.
Methods A total of 205 patients with HCC who were HCV-positive with Child-Pugh A at the onset from 2008 to 2018 were categorized into 2 groups: 140 patients untreated for HCV throughout the entire course after HCC development (untreated group) and 65 patients treated for HCV with DAAs following HCC treatment who achieved an SVR (SVR group). After propensity score matching, 63 patients from each group were selected. Using these patients, the survival and maintenance of Child-Pugh A after HCC treatment were compared between the untreated group and SVR group.
Results There was a significant difference in the overall survival (p<0.001) and the rate of maintaining Child-Pugh A (p<0.001) between the groups. The 5-year survival rates were 96% (SVR group) and 60% (untreated group), and the proportions of patients with Child-Pugh A at 5 years after HCC treatment were 96% (SVR group) and 38% (untreated group).
Conclusion In patients with HCV-positive HCC, achieving a DAA-SVR after HCC treatment significantly improved the overall survival rate compared with HCV-untreated patients. The contribution of DAA-SVR during the course of HCC treatment to a longer survival is mainly due to the prevention of the progression of Child-Pugh A to B/C. Further research is needed to determine whether aggressive antiviral therapy is also effective for HCC patients with Child-Pugh B/C.
... Infection with Hepatitis C Virus (HCV ** ) causes several liver related health problems including hepatocellular carcinoma (1). It (6,7). NS3 is a member of the DExH helicase family, which is under superfamily 2 (8). ...
Hepatitis C virus (HCV) is a major cause of liver-related diseases and hepatocellular carcinoma. The helicase domain of one of the non-structural proteins of HCV, NS3 (non-structural protein 3), is essential for viral replication; however, its specific biological role is still under investigation. Here, we set out to determine the interaction between a purified recombinant full length NS3 and synthetic guanine-rich substrates that represent the conserved G-quadruplex (G4)- forming sequences in the HCV positive and negative strands. We performed fluorescence anisotropy binding, G4 reporter duplex unwinding, and G4RNA trapping assays to determine the binding and G4 unfolding activity of NS3. Our data suggest that NS3 can unfold the conserved G4 structures present within the genome and the negative strand of HCV. Additionally, we found the activity of NS3 on a G4RNA was reduced significantly in the presence of a G4 ligand. The ability of NS3 to unfold HCV G4RNA could imply a novel biological role of the viral helicase in replication.
... HCV is transmitted between adults and is responsible for a high percentage of chronic infections with a major risk of liver cirrhosis and hepatocellular carcinoma [58]. The co-culture of monocyte-derived macrophages with HCV-infected hepatocytes induces M2 surface markers. ...
The role of macrophages in viral infections is well documented. Their activation status also called macrophage polarization categorized by the dichotomy of M1 and M2 phenotype remained poorly investigated. Recent studies have shown the complexity of macrophage polarization in response to viral infection and the limits of its use in infected individuals. The aim of this chapter is to reappraise the concept of macrophage polarization in viral infectious diseases, which are more complicated than the models of macrophage-virus interaction. If this concept has been largely used to describe activation status of myeloid cells in experimental conditions, it has to be assessed in light of high-throughput technologies at molecular and phenotypic levels. We update knowledge on macrophage polarization in viral infectious diseases with a special attention for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection leading to coronavirus disease (COVID-19). Hence, we propose an overview of the concept of macrophages as targets for therapeutic intervention in viral infectious disease. Finally, we tempted to focus our approach on patient investigation restricting the use of in vitro experiments and animal models to mechanistic questions.
... has led to the discovery of a number of targets for direct-acting antiviral (DAA) agents [4,5]. Several DAAs targeting different families of the virus including NS5B nucleotide inhibitors (NI), NS5A complex inhibitors and NS3/4A protease inhibitors (PI)] have been developed [6][7][8][9]. ...
Direct-acting antivirals (DAAs) have achieved a sustained virological response (SVR) rate of 95-99% in treating HCV. Several studies suggested that treatment with sofosbuvir (SOF), one type of DAAs, may be associated with increased risk of developing HCC. The aim of this study is to investigate the potential mechanisms of SOF on the development of HCC. OR-6 (harboring full-length genotype 1b HCV) and Huh 7.5.1 cells were used to examine the effects of SOF on cell proliferation and migration of HCC cells. SOF-upregulated genes in OR-6 cells were inspected using next generation sequencing (NGS)and the clinical significance of these candidate genes was analyzed using The Cancer Genome Atlas (TCGA) database. We found that SOF increased cell proliferation and cell migration in OR-6 and Huh 7.5.1 cells. Several SOF-upregulated genes screened from NGS were confirmed by real-time PCR in OR-6 cells. Among these genes, PHOSPHO2, KLHL23, TRIM39, TSNAX-DISC1 and RPP21 expression were significantly elevated in the tumor tissues compared with the non-tumor tissues of HCC according to TCGA database. High expression of PHOSPHO2 and RPP21 was associated with poor overall survival of HCC patients. Moreover, knockdown of PHOSPHO2-KLHL23, TSNAX-DISC1, TRIM39 and RPP21 diminished cell proliferation and migration increased by SOF in OR-6 and Huh 7.5.1 cells. In conclusion, SOF-upregulated genes promoted HCC cell proliferation and migration, which might be associated with the development of HCC.
... The 9.6-kb genome of HCV encodes a large polyprotein consisting of 3,010 amino acids. This polyprotein is cleaved by cellular and viral proteases into ten different proteins including the structural proteins (core, E1, and E2), the hydrophobic peptide p7, and the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) [5,6]. Various HCV proteins, including the core [7][8][9][10], envelope [11], and nonstructural proteins [12,13], have been reported to play an essential role in HCC development. ...
Introduction:
Accumulated studies have suggested that hepatitis C virus (HCV) infection is one of the leading causes for hepatocellular carcinoma (HCC). However, the mechanisms underlying the effect of HCV on the occurrence of HCC is still poorly understood.
Methods:
HCV infection datasets (GSE82177 and GSE17856) and HCC datasets (TCGA-LIHC (The Cancer Genome Atlas Liver Hepatocellular Carcinoma) and GSE89377) were downloaded from Gene Expression Omnibus (GEO) or TCGA for analysis. The common differentially expressed genes (DEGs) in the above four datasets were identified by R software. The expression of Ubiquitin D (UBD) in HCV infected HepG2 cells was detected by RT-qPCR and western blot, respectively. The interaction between NS3 and p53 was detected by Co-Immunoprecipitation. The influence of UBD on the proliferation and migration ability of HepG2 cells was evaluated by CCK-8 and wound healing assay, respectively.
Results:
UBD was upregulated in both HCV infected samples and HCC samples. HCV NS3 interacted with p53 and inhibited its expression. HCV NS3 induced UBD promoted the proliferation and migration of HepG2 cells.
Conclusion:
Our results suggest that HCV NS3 induced UBD is positively correlated with the development of HCV-related HCC during HCV infection. Targeting UBD could be a potential strategy for preventing and treating HCV-induced HCC.
... The hepatitis C virus (HCV) is a hepacivirus that belongs to the Flaviviridae family of (+)ssRNA viruses. The HCV genome encodes information for the synthesis of ten proteins: core (C), envelope glycoproteins (E1 and E2), an ion channel (p7), NS2 protease, protease/helicase NS3 (and its cofactor NS4A), membrane-associated protein NS4B, regulator of viral replication NS5A, and RNA-dependent RNA-polymerase NS5B [1]. ...
In the course of experiments aimed at deciphering the inhibition mechanism of mycophenolic acid and ribavirin in hepatitis C virus (HCV) infection, we observed an inhibitory effect of the nucleoside guanosine (Gua). Here, we report that Gua, and not the other standard nucleosides, inhibits HCV replication in human hepatoma cells. Gua did not directly inhibit the in vitro polymerase activity of NS5B, but it modified the intracellular levels of nucleoside di- and tri-phosphates (NDPs and NTPs), leading to deficient HCV RNA replication and reduction of infectious progeny virus production. Changes in the concentrations of NTPs or NDPs modified NS5B RNA polymerase activity in vitro , in particular de novo RNA synthesis and template switching. Furthermore, the Gua-mediated changes were associated with a significant increase in the number of indels in viral RNA, which may account for the reduction of the specific infectivity of the viral progeny, suggesting the presence of defective genomes. Thus, a proper NTP:NDP balance appears to be critical to ensure HCV polymerase fidelity and minimal production of defective genomes.
... HCV is a positive-sense single-stranded RNA virus that belongs to the genus Hepacivirus of the family Flaviviridae. The HCV genome consists of 9.6-kb RNA encoding a single polyprotein of about 3,010 amino acids (aa), which is processed by viral proteases and cellular signalases to produce three structural proteins (Core, E1, and E2) and seven nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (4). ...
We previously reported that hepatitis C virus (HCV) infection activates the reactive oxygen species (ROS)/c-Jun N-terminal kinase (JNK) signaling pathway. However, the roles of ROS/JNK activation in the HCV life cycle still remain unclear. We sought to identify a novel role of ROS/JNK signaling pathway in the HCV life cycle. Immunoblot analysis revealed that HCV-induced ROS/JNK activation promoted phosphorylation of Itch, a HECT-type E3 ubiquitin ligase, leading to activation of Itch. The siRNA-knockdown of Itch significantly reduced the extracellular HCV infectivity titers, HCV RNA, and HCV core protein without affecting intracellular HCV infectivity titers, HCV RNA, and HCV proteins, suggesting that Itch is involved in release of HCV particles. HCV-mediated JNK/Itch activation specifically promoted polyubiquitylation of an AAA-type ATPase VPS4A, but not VPS4B, required to form multivesicular bodies. Site-directed mutagenesis revealed that two lysine residues (K23 and K121) on VPS4A were important for VPS4A polyubiquitylation. The siRNA-knockdown of VPS4A, but not VPS4B, significantly reduced extracellular HCV infectivity titers. Co-immunoprecipitation analysis revealed that HCV infection specifically enhanced the interaction between CHMP1B, a subunit of endosomal sorting complexes required for transport (ESCRT)-III complex, and VPS4A, but not VPS4B, whereas VPS4A K23R/K121R greatly reduced the interaction with CHMP1B. HCV infection significantly increased ATPase activity of VPS4A, but not VPS4A K23R/K121R or VPS4B, suggesting that HCV-mediated polyubiquitylation of VPS4A contributes to activation of VPS4A. Taken together, we propose that HCV-induced ROS/JNK/Itch signaling pathway promotes VPS4A polyubiquitylation, leading to enhanced VPS4A-CHMP1B interaction and promotion of VPS4A ATPase activity, thereby promoting the release of HCV particles.
IMPORTANCE
ROS/JNK signaling pathway contributes to liver diseases, including steatosis, metabolic disorders, and hepatocellular carcinoma. We previously reported that HCV activates the ROS/JNK signaling pathway, leading to the enhancement of hepatic gluconeogenesis and apoptosis induction. This study further demonstrates that HCV-induced ROS/JNK signaling pathway activates the E3 ubiquitin ligase Itch to promote release of HCV particles via polyubiquitylation of VPS4A. We provide evidence suggesting that HCV infection promotes the ROS/JNK/Itch signaling pathway and ESCRT/VPS4A machinery to release infectious HCV particles. Our results may lead to a better understanding of the mechanistic details of HCV particle release.
... Indeed, nascent virions associate with pre-VLDL particles to form lipoviroparticles (LVPs) containing apolipoproteins (ApoB, apoE, apoC, and apoA-I), (Catanese et al., 2013;Nielsen et al., 2006). LVPs then pass through the Golgi and are released from the cells by the secretory pathway (Scheel and Rice, 2013). And given the role of septin 9 in regulating the morphology of Golgi apparatus and Golgi-dependent secretion of proteins, we could also suggest the role of septin 9 in HCV particles maturation and release ( Figure 38). ...
Septins belong to the family of protein GTPases that can form hetero-oligomeric structures and are capable of binding to actin and microtubules. Septins also associate with cell membranes and thus recruit cytosolic proteins and other cytoskeletal elements to control many cellular functions. In our team, we have shown that septin 9 is hijacked by the hepatitis C virus (HCV) to induce the perinuclear accumulation of lipid droplets (LDs) in a PtdIns5P and microtubule (MT) dependent mechanism. While the increase of LDs within cells is controlled by two major pathways of metabolism: the anabolism and catabolism pathway. The catabolism of LD closely related to lysosomes. Indeed, LDs accumulation is often associated with lysosome dysfunction, so we hypothesized that septin 9 could be involved in LDs accumulation by affecting lysosome function. In this work, we show that the cellular content and distribution of LDs are correlated with those of the lysosome and regulated by the addition of oleate to cells and by septin 9. High expression of septin 9 promotes perinuclear clustering of lysosomes that co-localize with the Golgi and not with the surrounding LDs. Conversely, inhibition of septin 9 expression by siRNAs results in the dispersion of both organelles which co-localize at the cell periphery. These results were also validated by Rab7 analysis. Similarly the addition of exogenous PtdIns5P to cells that bind to septin 9 or transfection of MTMR3 that produces PtdIns5P from PtdIns(3,5)P2 produce similar effects to septin 9 on the lysosome and LDs. In contrast, PtdIns(3,5)P2 promotes GL co-localization with lysosomes. Thus, our data reveal a mechanism of intracellular LDs accumulation dependent on the role of the PtdIns5P /septin 9 complex on lysosomes.
Significant scientific advances have been made through the use of In Vitro techniques (such as cell culture). However, the entire organism is needed to study physiological processes and relationships between different organs. Animal models of infectious diseases have a significant advantage over In Vitro studies because they help researchers comprehend the complex interplay between pathogens and the immune system.
Viral infections are among the most common causes of morbidity and mortality worldwide. Moreover, drug resistance is a key challenge that necessitates vigilant management of treatment regimens and the ongoing discovery of novel antiretroviral medications. It is crucial to understand the fundamentals of using model systems because evaluating new antiviral drugs and vaccines in animal models is necessary before conducting clinical trials in human patients. An animal model of human viral infection should ideally mirror the host-pathogen interactions and disease progression found in the natural disease course. A suitable animal model of viral infection should enable the analysis of numerous infection parameters, such as clinical symptoms, viral growth, clinicopathological parameters, cellular and humoral immune responses, and virus-host interactions.
In this chapter, we summarize the findings on the use of animal models for the development and evaluation of therapeutic approaches and vaccines to combat some of the most dangerous viral infections that still pose a burden to public health.
This reference presents information about models utilized in experimental medicine and pharmaceutical research and development for several human diseases. Written by experts in immunology, cancer biology and pharmacology, the book provides readers with handy notes and updated data on animal models that are critical to research planning and lab execution.
The main feature of the book is a set of 12 structured chapters that focus on a specific disease such as cancer, infectious diseases, autism, autoimmune diseases, Alzheimer's disease and anemia. The contributors have gathered information on a wide range of genetic and physiological animal models that are employed in research with comparative charts that highlight their main differences. The book also includes chapters for special topics like food allergies and dentistry. Additional features of the book are an explanation of disease mechanisms that give an easy understanding, notes for idiopathic models and specific clinical conditions, and a list of references for advanced readers.
Animal Models In Experimental Medicine is essential reading for scholars, graduate students and senior researchers in life sciences and clinical medicine. It also serves as a resource for professionals involved in bench-to-bedside pharmaceutical projects.
Hepatitis C virus (HCV) is a positive-sense, single-stranded RNA virus that causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The release of infectious HCV particles from infected hepatocytes is a crucial step in viral dissemination and disease progression. While the exact mechanisms of HCV particle release remain poorly understood, emerging evidence suggests that HCV utilizes intracellular membrane trafficking and secretory pathways. These pathways include the Golgi secretory pathway and the endosomal trafficking pathways, such as the recycling endosome pathway and the endosomal sorting complex required for transport (ESCRT)-dependent multivesicular bodies (MVBs) pathway. This review provides an overview of recent advances in understanding the release of infectious HCV particles, with a particular focus on the involvement of the host cell factors that participate in HCV particle release. By summarizing the current knowledge in this area, this review aims to contribute to a better understanding of endosomal pathways involved in the extracellular release of HCV particles and the development of novel antiviral strategies.
Hepatitis C virus (HCV) infection can regulate the number and dynamics of mitochondria, and is associated with a prominent hepatic mitochondrial injury. Mitochondrial distress conveys oxidative damage which is implicated in liver disease progression. The present study was conducted to assess the change of mitochondrial DNA (mtDNA) copy number in patients with HCV-related chronic liver disease and the impact of direct-acting antiviral (DAA) therapy. Whole blood mtDNA copy number was measured using real-time quantitative polymerase chain reaction at baseline and 12 weeks after the end of therapy in 50 treatment-naïve HCV-infected patients who achieved sustained viral response (SVR) after DAA therapy and 20 healthy controls. Whole blood mtDNA copy number appeared significantly lower in HCV-infected patients before therapy compared to healthy subjects ( P < 0.001). Post-treatment, there was significant increase of mtDNA copy number in HCV-infected patients at SVR12 compared to the pre-treatment values ( P < 0.001), meanwhile it didn’t differ significantly between HCV-infected patients after therapy and healthy subjects ( P = 0.059). Whole blood mtDNA copy number correlated inversely to the serum bilirubin in HCV-infected patients ( P = 0.013), however it didn’t correlate significantly to the serum aminotransferases, viral load or fibrosis-4 score ( P > 0.05). In conclusion, chronic HCV infection has been associated with a prominent mitochondrial injury which could mediate a progressive liver disease. The improved mtDNA content after DAA therapy highlights a possible potential of these drugs to alleviate mitochondrial damage in HCV-related liver disease.
Hepatitis C virus (HCV) is a globally prevalent and hazardous disorder that is responsible for inducing several persistent and potentially fatal liver diseases. Current treatment strategies offer limited efficacy, often accompanied by severe and debilitating adverse effects. Consequently, there is an urgent and compelling need to develop novel therapeutic interventions that can provide maximum efficacy in combating HCV while minimizing the burden of adverse effects on patients. One promising target against HCV is the NS3-4A serine protease, a complex composed of two HCV-encoded proteins. This non-covalent heterodimer is crucial in the viral life cycle and has become a primary focus for therapeutic interventions. Although peginterferon, combined with ribavirin, is commonly employed for HCV treatment, its efficacy is hampered by significant adverse effects that can profoundly impact patients' quality of life. In recent years, the development of direct-acting antiviral agents (DAAs) has emerged as a breakthrough in HCV therapy. These agents exhibit remarkable potency against the virus and have demonstrated fewer adverse effects when combined with other DAAs. However, it is important to note that there is a potential for developing resistance to DAAs due to alterations in the amino acid position of the NS3-4A protease. This emphasizes the need for ongoing research to identify strategies that can minimize the emergence of resistance and ensure long-term effectiveness. While the combination of DAAs holds promise for HCV treatment, it is crucial to consider the possibility of drug-drug interactions. These interactions may occur when different DAAs are used concurrently, potentially compromising their therapeutic efficacy. Therefore, carefully evaluating and monitoring potential drug interactions are vital to optimize treatment outcomes. In the pursuit of novel therapeutic interventions for HCV, the field of computational biology and bioinformatics has emerged as a valuable tool. These advanced technologies and methodologies enable the development and design of new drugs and therapeutic agents that exhibit maximum efficacy, reduced risk of resistance, and minimal adverse effects. By leveraging computational approaches, researchers can efficiently screen and optimize potential candidates, accelerating the discovery and development of highly effective treatments for HCV, treatments.
Globally, more than 58 million people are chronically infected with Hepatitis C virus (HCV) with 1.5 million new infections occurring each year. An effective vaccine for HCV is therefore a major unmet medical and public health need. Since HCV rapidly accumulates mutations, vaccines must elicit the production of broadly neutralising antibodies (bnAbs) in a reproducible fashion. Decades of research have generated a number of HCV vaccine candidates. Based on the available data and research through clinical development, a vaccine antigen based on the E1E2 glycoprotein complex appears to be the best choice, but robust induction of humoral and cellular responses leading to virus neutralisation has not yet been achieved. One issue that has arisen in developing an HCV vaccine (and many other vaccines as well) is the platform used for antigen delivery. The majority of viral vaccine trials have employed subunit vaccines. However, subunit vaccines often have limited immunogenicity, as seen for HCV, and thus multiple formats must be examined in order to elicit a robust anti-HCV immune response. Nanoparticle vaccines are gaining prominence in the field due to their ability to facilitate a controlled multivalent presentation and trafficking to lymph nodes, where they can interact with both arms of the immune system. This review discusses the potential for development of a nanoparticle-based HCV E1E2 vaccine, with an emphasis on the potential benefits of such an approach along with the major challenges facing the incorporation of E1E2 into nanoparticulate delivery systems and how those challenges can be addressed.
As noncellular organisms, viruses do not have their own metabolism and rely on the metabolism of host cells to provide energy and metabolic substances for their life cycles. Increasing evidence suggests that host cells infected with oncogenic viruses have dramatically altered metabolic requirements and that oncogenic viruses produce substances used for viral replication and virion production by altering host cell metabolism. We focused on the processes by which oncogenic viruses manipulate host lipid metabolism and the lipid metabolism disorders that occur in oncogenic virus-associated diseases. A deeper understanding of viral infections that cause changes in host lipid metabolism could help with the development of new antiviral agents as well as potential new therapeutic targets.
Entry of the hepatitis C virus (HCV) into host cells is a multistep process mediated by several host factors, including a tight junction protein claudin-1 (CLDN1). We repeatedly passaged HCV-JFH1-tau, an HCV substrain with higher infectivity, on Huh7.5.1-8 cells. A multi-passaged HCV-JFH1-tau lot was infectious to CLDN1-defective S7-A cells, non-permissive to original HCV-JFH1-tau infection. We identified a single mutation, M706L, in the E2 glycoprotein of the HCV-JFH1-tau lot as an essential mutation for infectivity to S7-A cells. The pseudovirus JFH1/M706L mutant could not infect human embryonic kidney 293 T (HEK293T) cells lacking CLDN family but infected HEK293T cells expressing CLDN1, CLDN6, or CLDN9. Thus, this mutant virus could utilize CLDN1, and other CLDN6 and CLDN9, making HCV possible to infect cells other than hepatocytes. iPS cells, one of the stem cells, do not express CLDN1 but express CLDN6 and other host factors required for HCV infection. We confirmed that the HCV-JFH1-tau-derived mutant with an M706L mutation infected iPS cells in a CLDN6-dependent manner. These results demonstrated that a missense mutation in E2 could broaden the CLDN member specificity for HCV infection. HCV may change its receptor requirement through a single amino acid mutation and infect non-hepatic cells.
Background Reliable real-world data on direct acting anti-retroviral (DAA) uptake and treatment outcomes are lacking for patients with hepatitis C virus (HCV) in Sub-Saharan Africa (SSA). This study provides data on HCV DAA-based treatment outcomes, mortality, loss-to-follow up, and associated factors among patients in Eritrea. Methodology: A multicenter retrospective observational cohort study was conducted in two tertiary hospitals in Asmara, Eritrea. A structured checklist was used to collect data from patients’ cards. Descriptive and inferential statistics used included means, medians, chi-squire, Kaplan–Meier estimates, and multivariate Cox proportional hazard models. Results Out of 238 patients enrolled in the study, 227 were initiated on treatment. 125 patients had viral load at 12 weeks EOT whereas 102 patients had no viral load at 12 weeks EOT. Out of these patients with HCV RNA data post EOT, 116 (92.8%) had SVR12. The prevalence of death and lost-to-follow up (LTFU) were (7.5%, 95% CI: 1.7–4.1) and 67 (28.1%, 95% CI: 22.3–33.9) and 1.1 (95%CI: 0.8–1.5) per 10,000 person days, respectively. Independent predictors of LTFU included the enrollment year (2020: aHR = 2.2, 95% CI: 1-4.7; p-value = 0.04); Hospital (Hospital B: aHR = 2.2, 95% CI: 1-4.7; p-value = 0.03) and the FIB-4 score (FIB-Score < 1.45: aHR = 3.7, 95% CI: 1.2–11.5; p-value = 0.02). Conclusion The SVR rates achieved in this cohort were high. However, high mortality, late presentation and suboptimal population screening/case finding were uncovered. These challenges can be addressed by test-and-treat programs that simultaneously prioritize programmatic screening, decentralization of care, and better patient tracking in the HCV care cascade.
Hepatitis C virus (HCV) is a common cause of liver disease and is associated with various extrahepatic manifestations (EHMs). This mini-review outlines the currently available treatments for HCV infection and their prognostic effect on hepatic manifestations and EHMs. Direct-acting antiviral (DAA) regimens are considered pan-genotypic as they achieve a sustained virological response (SVR) > 85% after 12 wk through all the major HCV genotypes, with high percentages of SVR even in advanced fibrosis and cirrhosis. The risk factors for DAA failure include old males, cirrhosis, and the presence of resistance-associated substitutions (RAS) in the region targeted by the received DAAs. The effectiveness of DAA regimens is reduced in HCV genotype 3 with baseline RAS like A30K, Y93H, and P53del. Moreover, the European Association for the Study of the Liver recommended the identification of baseline RAS for HCV genotype 1a. The higher rate of hepatocellular carcinoma (HCC) after DAA therapy may be related to the fact that DAA regimens are offered to patients with advanced liver fibrosis and cirrhosis, where interferon was contraindicated to those patients. The change in the growth of pre-existing subclinical, undetectable HCC upon DAA treatment might be also a cause. Furthermore, after DAA therapy, the T cell-dependent immune response is much weaker upon HCV clearance, and the down-regulation of TNF-α or the elevated neutrophil to lymphocyte ratio might increase the risk of HCC. DAAs can result in reactivation of hepatitis B virus (HBV) in HCV co-infected patients. DAAs are effective in treating HCV-associated mixed cryoglobulinemia, with clinical and immunological responses, and have rapid and high effectiveness in thrombocytopenia. DAAs improve insulin resistance in 90% of patients, increase glomerular filtration rate, and decrease proteinuria, hematuria and articular manifestations. HCV clearance by DAAs allows a significant improvement in atherosclerosis and metabolic and immunological conditions, with a reduction of major cardiovascular events. They also improve physical function, fatigue, cognitive impairment, and quality of life. Early therapeutic approach with DAAs is recommended as it cure many of the EHMs that are still in a reversible stage and can prevent others that can develop due to delayed treatment.
Paritaprevir and Ombitasvir are part of a triple combination first-generation direct acting anti-viral regimen as a fixed dose tablet. Commercially manufactured Form II of paritaprevir and Form I of ombitasvir are high stoichiometry crystalline hydrates with 2.5 and 4.5 molar equivalents of water respectively. The dominant (001) surface for paritaprevir interacts with adjacent non interlocking layers via weak dispersion forces due to large interlayer d-spacing. On the other hand, the dominant (001) surface of ombitasvir is characterized by interdigitated layers and 3-D H-bonding network albeit in a relatively low-density crystalline structure. A comparative analysis of hardness and Young’s modulus with a larger data-set of molecular crystals corroborates with the structural features, and helps quantify the extra-ordinarily soft viscoelastic nature of the dominant surface of paritaprevir Form II (H and E of 0.014 and 0.029 GPa) and the slightly softer and more elastic dominant surface of ombitasvir Form I (H and E of 0.29 and 6.6 GPa). The surface of paritaprevir can therefore undergo irreversible compression induced deformation, however its viscoelastic nature makes the extent of such deformation inconsequential for product quality during process relevant perturbations. The elastic features of the dominant surface of ombitasvir in combination with well-designed process controls allow crystals to heal from or avoid the loss of mechanical integrity when undergoing two solid-solid phase conversions that are an essential feature of commercial isolation process.
Infections caused by the Hepatitis C virus (HCV) affect around 70 million people worldwide, leading to serious liver problems such as fibrosis, steatosis, cirrhosis, in addition to progressing to hepatocellular carcinoma, and becoming globally the main cause of liver disease. Despite great therapeutic advances in obtaining pan-genotypic direct-acting antivirals (DAAs), around 5-10% of affected individuals are unable to eliminate the virus by their own immune system’s activity. Still, there are no licensed vaccines so far. In this context, the orchestrated process of virus entry into host cells is a crucial step in the life cycle and the infectivity capability of most viruses. In recent years, the entry of viruses has become one of the main druggable targets used for designing effective antiviral molecules. This goal has come to be widely studied to develop pharmacotherapeutic strategies against HCV, combined or not with DAAs in multitarget approaches. Among the inhibitors found in the literature, ITX 5061 corresponds to the most effective one, with EC50 and CC50 values of 0.25 nM and >10 µM (SI: 10,000), respectively. This SR-BI antagonist completed the phase I trial, constituting a promising compound against HCV. Interestingly, chlorcyclizine (an antihistamine drug) showed action both in E1 apolipoproteins (EC50 and CC50 values of 0.0331 and 25.1 µM, respectively), as well as in NPC1L1 (IC50 and CC50 values of 2.3 nM and > 15 µM, respectively). Thus, this review will be addressed to promising inhibitors targeting HCV entry, discussing their SAR analyzes, recent contributions and advances in this field.
Background & aims:
Lack of tractable immunocompetent animal models amenable to robust experimental challenge impedes vaccine efforts for hepatitis C virus (HCV). Infection with rodent hepacivirus from Rattus norvegicus (RHV-rn1) in rats shares HCV-defining characteristics, including liver tropism, chronicity, and pathology. RHV in vitro cultivation would facilitate genetic studies on particle production, host factor interactions, and evaluation of antibody neutralization guiding HCV vaccine approaches.
Approach & results:
We report an infectious reverse genetic cell culture system for RHV-rn1 utilizing highly permissive rat hepatoma cells and adaptive mutations in the E2, NS4B, and NS5A viral proteins. Cell culture derived RHV-rn1 particles (RHVcc) share hallmark biophysical characteristics of HCV and are infectious in mice and rats. Culture adaptive mutations attenuated RHVcc in immunocompetent rats and the mutations reverted following prolonged infection, but not in immunodeficient SCID mice, suggesting that adaptive immune pressure is a primary driver of reversion. Accordingly, sera from RHVcc-infected SCID mice or the early acute phase of immunocompetent mice and rats were infectious in culture. We further established an in vitro RHVcc neutralization assay, and observed neutralizing activity of rat sera specifically from the chronic phase of infection. Finally, we found that scavenger receptor class B type I promoted RHV-rn1 entry in vitro and in vivo.
Conclusions:
The RHV-rn1 infectious cell culture system enables studies of humoral immune responses against hepacivirus infection. Moreover, recapitulation of the entire RHV-rn1 infectious cycle in cell culture will facilitate reverse genetic studies and the exploration of tropism and virus-host interactions.
For more than 20 years, NS3 was known to participate in NS5A sequential phosphorylation. In the present study, we show for the first time that the ATP-binding domain of NS3 is involved in NS5A phosphorylation. In vitro assays showed that casein kinase Iα is a very potent kinase responsible for NS5A phosphorylation at serines 225, 232, and 235.
Recently, lipid membrane‐targeting strategies hold great promise for development of broad‐spectrum antivirals. However, it remains a big challenge to identify novel membrane‐based targets of viruses and virus‐infected cells for development of precision targeted approaches. Here, we discover that viroporins, viral‐encoded ion channels which have been reported to mediate release of hydrogen ions, trigger membrane acidification of virus‐infected cells. Through development of a fine‐scale library of gradient pH‐sensitive (GPS) polymeric nanoprobes, we measure the cellular membrane pH transition from pH 6.8‐7.1 (un‐infection) to pH 6.5‐6.8 (virus‐infection). In response to the subtle pH alterations, the GPS polymer with sharp response at pH 6.8 (GPS6.8) selectively binds to virus‐infected cell membranes or viral envelope, and even completely disrupts viral envelope. Accordingly, GPS6.8 treatment exerts suppressive effects on a wide variety of viruses including SARS‐CoV‐2 through triggering viral envelope lysis rather than affecting immune pathway or viability of host cells. Murine viral infection models exhibit that supplementation of GPS6.8 decreases viral titers and ameliorates inflammatory damage. Thus, the gradient pH‐sensitive nanotechnology offers a promising strategy for accurate detection of biological pH environments and robust interference with viruses. This article is protected by copyright. All rights reserved
Les virus de l’immunodéficience humaine (VIH) et de l’hépatite C (VHC) partagent des modes de transmission, la co-infection par le VIH et le VHC est donc fréquente. Ces deux virus interagissent et affectent négativement leurs histoires naturelles respectives. La co-infection est également associée à de nombreuses complications avec un risque de morbi-mortalité plus élevé par rapport aux patients mono-infectés historiquement.Chez les patients porteurs du VIH, l’infection par le VHC est associée à une restauration plus lente des capacités immunitaires après l’initiation d’un traitement antirétroviral (ARV). De plus, avec le développement des ARV, les causes de décès dans cette population ont été grandement modifiées, les causes hépatiques devenant l’une des principales causes de décès. L’infection par le VIH augmente le risque d’infection chronique par le VHC, de transmission, d’atteinte et d’événement hépatique et d’échec thérapeutique chez les patients mono-infectés VHC. Les patients porteurs du VIH et du VHC sont également plus à risque de complications telles que les événements cardiovasculaires ou les cancers que les patients porteurs du VIH ou du VHC seul.L’infection par le VHC a connu une modification majeure de son contexte avec l’arrivée des traitements par antiviraux à action directe (AAD). Ces traitements permettent d’obtenir une réponse virologique soutenue (RVS), chez plus de 90 % des patients traités. Contrairement aux générations antérieures de traitement, l’infection par le VIH ne semble pas affecter leur efficacité. L’effet bénéfique de la RVS sur le risque de complications liées au VHC a largement été montré. Cependant, l’impact de 1) l’infection par le VIH (co-infectés VIH/VHC) sur l’évolution de la maladie par rapport aux patients mono-infectés VHC et de 2) la co-infection par le VIH et le VHC puis de la RVS par rapport aux patients mono-infectés VIH n’a pas encore étudié à l’ère des AAD.L’objectif de cette thèse est donc d’étudier l’évolution des participants co-infectés par le VIH et le VHC après traitement par AAD ou RVS en termes de mortalités et de morbidités. Nous avons ainsi (1) estimé les incidences des complications liées au VHC et au VIH et identifié les facteurs les influençant chez les patients co-infectés par le VIH et le VHC après la RVS, évalué l’association (2) entre la co-infection par le VIH et les complications liées au VHC chez les patients porteurs du VHC traités par AAD et (3) entre la co-infection par le VHC et la RVS et les complications liées au VHC et au VIH chez les patients porteurs du VIH. Les participants de la cohorte ANRS CO13 HEPAVIH ont été inclus (1) et comparer aux participants de la cohorte ANRS CO22 HEPATHER (2) ainsi que les participants de la collaboration inter-cohortes de patients porteurs du VIH traités par ARV (ART-CC) (3).Les principaux résultats sont qu’après l’obtention de la RVS, l’atteinte hépatique était le principal facteur associé aux risques de complications hépatiques et de mortalité et les cancers sont plus fréquents que les événements hépatiques chez les patients co-infectés par le VIH et le VHC. D’ailleurs, alors que la co-infection par le VIH n’affectait pas le risque de complications hépatiques chez les patients porteurs du VHC, elle augmentait le risque de complications non-hépatiques et de cancers. Ce sur-risque pourrait être expliqué par la dérégulation immunitaire et l’inflammation chronique causée par l’infection par le VIH. De plus, chez les patients porteurs du VIH, l’infection par le VHC était associée à une augmentation du risque de cancers qui persiste après l’obtention de la RVS. Les patients co-infectés par le VHC avaient un risque de mortalité plus élevé, mais la RVS permettait de faire disparaître ce sur-risque.Nos résultats permettent de mieux comprendre l’évolution des patients porteurs du VIH et du VHC après l’obtention de la RVS en termes de morbi-mortalité ce qui est important pour leur prise en charge.
Hepatitis C virus (HCV) is a global public health problem involving chronic infection of the liver, which can cause liver disease and is linked with liver cancer. Viral innate immune evasion strategies and human genetic determinants underlie the transition of acute HCV infection to viral persistence and the support of chronic infection. Host genetic factors, such as sequence polymorphisms in IFNL3, a gene in the host interferon system, can influence both the outcome of the infection and the response to antiviral therapy. Recent insights into how HCV regulates innate immune signaling within the liver reveal a complex interaction of patient genetic background with viral and host factors of innate immune triggering and control that imparts the outcome of HCV infection and immunity.
Although there are over 1,150 bat species worldwide, the diversity of viruses harbored by bats has only recently come into focus as a result of expanded wildlife surveillance. Such surveys are of importance in determining the potential for novel viruses to emerge in humans, and for optimal management of bats and their habitats. To enhance our knowledge of the viral diversity present in bats, we initially surveyed 415 sera from African and Central American bats. Unbiased high-throughput sequencing revealed the presence of a highly diverse group of bat-derived viruses related to hepaciviruses and pegiviruses within the family Flaviridae. Subsequent PCR screening of 1,258 bat specimens collected worldwide indicated the presence of these viruses also in North America and Asia. A total of 83 bat-derived viruses were identified, representing an infection rate of nearly 5%. Evolutionary analyses revealed that all known hepaciviruses and pegiviruses, including those previously documented in humans and other primates, fall within the phylogenetic diversity of the bat-derived viruses described here. The prevalence, unprecedented viral biodiversity, phylogenetic divergence, and worldwide distribution of the bat-derived viruses suggest that bats are a major and ancient natural reservoir for both hepaciviruses and pegiviruses and provide insights into the evolutionary history of hepatitis C virus and the human GB viruses.
Background:
In phase 2 trials, the nucleotide polymerase inhibitor sofosbuvir was effective in previously untreated patients with chronic hepatitis C virus (HCV) genotype 1, 2, or 3 infection.
Methods:
We conducted two phase 3 studies in previously untreated patients with HCV infection. In a single-group, open-label study, we administered a 12-week regimen of sofosbuvir plus peginterferon alfa-2a and ribavirin in 327 patients with HCV genotype 1, 4, 5, or 6 (of whom 98% had genotype 1 or 4). In a noninferiority trial, 499 patients with HCV genotype 2 or 3 infection were randomly assigned to receive sofosbuvir plus ribavirin for 12 weeks or peginterferon alfa-2a plus ribavirin for 24 weeks. In the two studies, the primary end point was a sustained virologic response at 12 weeks after the end of therapy.
Results:
In the single-group study, a sustained virologic response was reported in 90% of patients (95% confidence interval, 87 to 93). In the noninferiority trial, a sustained response was reported in 67% of patients in both the sofosbuvir-ribavirin group and the peginterferon-ribavirin group. Response rates in the sofosbuvir-ribavirin group were lower among patients with genotype 3 infection than among those with genotype 2 infection (56% vs. 97%). Adverse events (including fatigue, headache, nausea, and neutropenia) were less common with sofosbuvir than with peginterferon.
Conclusions:
In a single-group study of sofosbuvir combined with peginterferon-ribavirin, patients with predominantly genotype 1 or 4 HCV infection had a rate of sustained virologic response of 90% at 12 weeks. In a noninferiority trial, patients with genotype 2 or 3 infection who received either sofosbuvir or peginterferon with ribavirin had nearly identical rates of response (67%). Adverse events were less frequent with sofosbuvir than with peginterferon. (Funded by Gilead Sciences; FISSION and NEUTRINO ClinicalTrials.gov numbers, NCT01497366 and NCT01641640, respectively.).
Unlabelled:
Hepatitis C virus (HCV) and human pegivirus (HPgV or GB virus C) are globally distributed and infect 2 to 5% of the human population. The lack of tractable-animal models for these viruses, in particular for HCV, has hampered the study of infection, transmission, virulence, immunity, and pathogenesis. To address this challenge, we searched for homologous viruses in small mammals, including wild rodents. Here we report the discovery of several new hepaciviruses (HCV-like viruses) and pegiviruses (GB virus-like viruses) that infect wild rodents. Complete genome sequences were acquired for a rodent hepacivirus (RHV) found in Peromyscus maniculatus and a rodent pegivirus (RPgV) found in Neotoma albigula. Unique genomic features and phylogenetic analyses confirmed that these RHV and RPgV variants represent several novel virus species in the Hepacivirus and Pegivirus genera within the family Flaviviridae. The genetic diversity of the rodent hepaciviruses exceeded that observed for hepaciviruses infecting either humans or non-primates, leading to new insights into the origin, evolution, and host range of hepaciviruses. The presence of genes, encoded proteins, and translation elements homologous to those found in human hepaciviruses and pegiviruses suggests the potential for the development of new animal systems with which to model HCV pathogenesis, vaccine design, and treatment.
Importance:
The genetic and biological characterization of animal homologs of human viruses provides insights into the origins of human infections and enhances our ability to study their pathogenesis and explore preventive and therapeutic interventions. Horses are the only reported host of nonprimate homologs of hepatitis C virus (HCV). Here, we report the discovery of HCV-like viruses in wild rodents. The majority of HCV-like viruses were found in deer mice (Peromyscus maniculatus), a small rodent used in laboratories to study viruses, including hantaviruses. We also identified pegiviruses in rodents that are distinct from the pegiviruses found in primates, bats, and horses. These novel viruses may enable the development of small-animal models for HCV, the most common infectious cause of liver failure and hepatocellular carcinoma after hepatitis B virus, and help to explore the health relevance of the highly prevalent human pegiviruses.
Pestiviruses, including bovine viral diarrhea virus, are important animal pathogens and are closely related to hepatitis C virus, which remains a major global health threat. They have an outer lipid envelope bearing two glycoproteins, E1 and E2, required for cell entry. They deliver their genome into the host cell cytoplasm by fusion of their envelope with a cellular membrane. The crystal structure of bovine viral diarrhea virus E2 reveals a unique protein architecture consisting of two Ig-like domains followed by an elongated β-stranded domain with a new fold. E2 forms end-to-end homodimers with a conserved C-terminal motif rich in aromatic residues at the contact. A disulfide bond across the interface explains the acid resistance of pestiviruses and their requirement for a redox activation step to initiate fusion. From the structure of E2, we propose alternative possible membrane fusion mechanisms. We expect the pestivirus fusion apparatus to be conserved in hepatitis C virus.
The nonstructural 5A (NS5A) protein is a target for drug development against hepatitis C virus (HCV). Interestingly, the NS5A inhibitor daclatasvir (BMS-790052) caused a decrease in serum HCV RNA levels by about two orders of magnitude within 6 h of administration. However, NS5A has no known enzymatic functions, making it difficult to understand daclatasvir's mode of action (MOA) and to estimate its antiviral effectiveness. Modeling viral kinetics during therapy has provided important insights into the MOA and effectiveness of a variety of anti-HCV agents. Here, we show that understanding the effects of daclatasvir in vivo requires a multiscale model that incorporates drug effects on the HCV intracellular lifecycle, and we validated this approach with in vitro HCV infection experiments. The model predicts that daclatasvir efficiently blocks two distinct stages of the viral lifecycle, namely viral RNA synthesis and virion assembly/secretion with mean effectiveness of 99% and 99.8%, respectively, and yields a more precise estimate of the serum HCV half-life, 45 min, i.e., around four times shorter than previous estimates. Intracellular HCV RNA in HCV-infected cells treated with daclatasvir and the HCV polymerase inhibitor NM107 showed a similar pattern of decline. However, daclatasvir treatment led to an immediate and rapid decline of extracellular HCV titers compared to a delayed (6-9 h) and slower decline with NM107, confirming an effect of daclatasvir on both viral replication and assembly/secretion. The multiscale modeling approach, validated with in vitro kinetic experiments, brings a unique conceptual framework for understanding the mechanism of action of a variety of agents in development for the treatment of HCV.
Background:
The standard treatment for hepatitis C virus (HCV) infection is interferon, which is administered subcutaneously and can have troublesome side effects. We evaluated sofosbuvir, an oral nucleotide inhibitor of HCV polymerase, in interferon-sparing and interferon-free regimens for the treatment of HCV infection.
Methods:
We provided open-label treatment to eight groups of patients. A total of 40 previously untreated patients with HCV genotype 2 or 3 infection were randomly assigned to four groups; all four groups received sofosbuvir (at a dose of 400 mg once daily) plus ribavirin for 12 weeks. Three of these groups also received peginterferon alfa-2a for 4, 8, or 12 weeks. Two additional groups of previously untreated patients with HCV genotype 2 or 3 infection received sofosbuvir monotherapy for 12 weeks or sofosbuvir plus peginterferon alfa-2a and ribavirin for 8 weeks. Two groups of patients with HCV genotype 1 infection received sofosbuvir and ribavirin for 12 weeks: 10 patients with no response to prior treatment and 25 with no previous treatment. We report the rate of sustained virologic response 24 weeks after therapy.
Results:
Of the 40 patients who underwent randomization, all 10 (100%) who received sofosbuvir plus ribavirin without interferon and all 30 (100%) who received sofosbuvir plus ribavirin for 12 weeks and interferon for 4, 8, or 12 weeks had a sustained virologic response at 24 weeks. For the other patients with HCV genotype 2 or 3 infection, all 10 (100%) who received sofosbuvir plus peginterferon alfa-2a and ribavirin for 8 weeks had a sustained virologic response at 24 weeks, as did 6 of 10 (60%) who received sofosbuvir monotherapy. Among patients with HCV genotype 1 infection, 21 of 25 previously untreated patients (84%) and 1 of 10 with no response to previous therapy (10%) had a sustained virologic response at 24 weeks. The most common adverse events were headache, fatigue, insomnia, nausea, rash, and anemia.
Conclusions:
Sofosbuvir plus ribavirin for 12 weeks may be effective in previously untreated patients with HCV genotype 1, 2, or 3 infection. (Funded by Pharmasset and Gilead Sciences; ClinicalTrials.gov number, NCT01260350.).
Enveloped viruses have developed various adroit mechanisms to invade their host cells. This process requires one or more viral envelope glycoprotein to achieve cell attachment and membrane fusion. Members of the Flaviviridae such as flaviviruses possess only one envelope glycoprotein, E, whereas pestiviruses and hepacivirus encode two glycoproteins, E1 and E2. Although E2 is involved in cell attachment, it has been unclear which protein is responsible for membrane fusion. We report the crystal structures of the homodimeric glycoprotein E2 from the pestivirus bovine viral diarrhea virus 1 (BVDV1) at both neutral and low pH. Unexpectedly, BVDV1 E2 does not have a class II fusion protein fold, and at low pH the N-terminal domain is disordered, similarly to the intermediate postfusion state of E2 from sindbis virus, an alphavirus. Our results suggest that the pestivirus and possibly the hepacivirus fusion machinery are unlike any previously observed.
Hepatitis C virus (HCV) replication is dependent on microRNA 122 (miR-122), a liver-specific microRNA that recruits Argonaute 2 to the 5' end of the viral genome, stabilizing it and slowing its decay both in cell-free reactions and in infected cells. Here we describe the RNA degradation pathways against which miR-122 provides protection. Transfected HCV RNA is degraded by both the 5' exonuclease Xrn1 and 3' exonuclease exosome complex, whereas replicating RNA within infected cells is degraded primarily by Xrn1 with no contribution from the exosome. Consistent with this, sequencing of the 5' and 3' ends of RNA degradation intermediates in infected cells confirmed that 5' decay is the primary pathway for HCV RNA degradation. Xrn1 knockdown enhances HCV replication, indicating that Xrn1 decay and the viral replicase compete to set RNA abundance within infected cells. Xrn1 knockdown and miR-122 supplementation have equal, redundant, and nonadditive effects on the rate of viral RNA decay, indicating that miR-122 protects HCV RNA from 5' decay. Nevertheless, Xrn1 knockdown does not rescue replication of a viral mutant defective in miR-122 binding, indicating that miR-122 has additional yet uncharacterized function(s) in the viral life cycle.
All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.
Eukaryotes possess seven different phosphoinositides (PIPs) that help form the unique signatures of various intracellular membranes. PIPs serve as docking sites for the recruitment of specific proteins to mediate membrane alterations and integrate various signaling cascades. The spatio-temporal regulation of PI kinases and phosphatases generates distinct intracellular hubs of PIP signaling. Hepatitis C virus (HCV), like other plus-strand RNA viruses, promotes the rearrangement of intracellular membranes to assemble viral replication complexes. HCV stimulates enrichment of phosphatidylinositol 4-phosphate (PI4P) pools near endoplasmic reticulum (ER) sites by activating PI4KIIIα, the kinase responsible for generation of ER-specific PI4P pools. Inhibition of PI4KIIIα abrogates HCV replication. PI4P, the most abundant phosphoinositide, predominantly localizes to the Golgi and plays central roles in Golgi secretory functions by recruiting effector proteins involved in transport vesicle generation. The PI4P effector proteins also include the lipid-transfer and structural proteins such as ceramide transfer protein (CERT), oxysterol binding protein (OSBP) and Golgi phosphoprotein 3 (GOLPH3) that help maintain Golgi-membrane composition and structure. Depletion of Golgi-specific PI4P pools by silencing PI4KIIIβ, expression of dominant negative CERT and OSBP mutants, or silencing GOLPH3 perturb HCV secretion. In this review we highlight the role of PIPs and specifically PI4P in the HCV life cycle.
Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by off target effects. Development of antiviral ribonucleosides for treatment of hepatitis C virus (HCV) infection has been hampered by appearance of toxicity during clinical trials that evaded detection during preclinical studies. It is well established that the human mitochondrial DNA polymerase is an off target for deoxyribonucleoside reverse transcriptase inhibitors. Here we test the hypothesis that triphosphorylated metabolites of therapeutic ribonucleoside analogues are substrates for cellular RNA polymerases. We have used ribonucleoside analogues with activity against HCV as model compounds for therapeutic ribonucleosides. We have included ribonucleoside analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents that are non-obligate chain terminators of the HCV RNA polymerase. We show that all of the anti-HCV ribonucleoside analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Unexpectedly, analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents were inhibitors of POLRMT and Pol II. Importantly, the proofreading activity of TFIIS was capable of excising these analogues from Pol II transcripts. Evaluation of transcription in cells confirmed sensitivity of POLRMT to antiviral ribonucleosides, while Pol II remained predominantly refractory. We introduce a parameter termed the mitovir (mitochondrial dysfunction caused by antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity.
Nonstructural protein 5B (NS5B) is essential for hepatitis C virus (HCV) replication as it carries the viral RNA-dependent
RNA polymerase enzymatic activity. HCV replication occurs in a membrane-associated multiprotein complex in which HCV NS5A
and host cyclophilin A (CypA) have been shown to be present together with the viral polymerase. We used NMR spectroscopy to
perform a per residue level characterization of the molecular interactions between the unfolded domains 2 and 3 of NS5A (NS5A-D2
and NS5A-D3), CypA, and NS5BΔ21. We show that three regions of NS5A-D2 (residues 250–262 (region A), 274–287 (region B), and 306–333 (region C)) interact
with NS5BΔ21, whereas NS5A-D3 does not. We show that both NS5BΔ21 and CypA share a common binding site on NS5A that contains residues Pro-306 to Glu-323. No direct molecular interaction has
been detected by NMR spectroscopy between HCV NS5BΔ21 and host CypA. We show that cyclosporine A added to a sample containing NS5BΔ21, NS5A-D2, and CypA specifically inhibits the interaction between CypA and NS5A-D2 without altering the one between NS5A-D2
and NS5BΔ21. A high quality heteronuclear NMR spectrum of HCV NS5BΔ21 has been obtained and was used to characterize the binding site on the polymerase of NS5A-D2. Moreover these data highlight
the potential of using NMR of NS5BΔ21 as a powerful tool to characterize in solution the interactions of the HCV polymerase with all kinds of molecules (proteins,
inhibitors, RNA). This work brings new insights into the comprehension of the molecular interplay between NS5B, NS5A, and
CypA, three essentials proteins for HCV replication.
Chronic infection with hepatitis C virus (HCV) is an important cause of end stage liver disease worldwide. In the United States, most HCV-related disease is associated with genotype 1 infection, which remains difficult to treat. Drug and vaccine development was hampered by inability to culture patient isolates representing HCV genotypes 1-7 and subtypes; only a recombinant 2a genome (strain JFH1) spontaneously replicated in vitro. Recently, we identified three mutations F1464L/A1672S/D2979G (LSG) in the nonstructural (NS) proteins, essential for development of full-length HCV 2a (J6) and 2b (J8) culture systems in Huh7.5 cells. Here, we developed a highly efficient genotype 1a (strain TN) full-length culture system. We initially found that the LSG substitutions conferred viability to an intergenotypic recombinant composed of TN 5' untranslated region (5'UTR)-NS5A and JFH1 NS5B-3'UTR; recovered viruses acquired two adaptive mutations located in NS3 and NS4B. Introduction of these changes into a replication-deficient TN full-length genome, harboring LSG, permitted efficient HCV production. Additional identified NS4B and NS5B mutations fully adapted the TN full-length virus. Thus, a TN genome with 8 changes (designated TN cell-culture derived, TNcc) replicated efficiently and released infectious particles of ∼5 log(10) focus-forming units per mL; passaged TNcc did not require additional changes. IFN-α and directly acting antivirals targeting the HCV protease, NS5A, and NS5B, each inhibited full-length TN infection dose-dependently. Given the unique importance of genotype 1 for pathogenesis, this infectious 1a culture system represents an important advance in HCV research. The approach used and the mutations identified might permit culture development for other HCV isolates, thus facilitating vaccine development and personalized treatment.
A hallmark of hepatitis C virus (HCV) particles is their association with host cell lipids, most notably lipoprotein components.
It is thought that this property accounts for the low density of virus particles and their large heterogeneity. However, the
composition of infectious virions and their biochemical and morphological properties are largely unknown. We developed a system
in which the envelope glycoprotein E2 was N-terminally tagged with a FLAG epitope. This virus, designated Jc1E2FLAG, produced infectivity titers to wild type levels and allowed affinity purification of virus particles that were analyzed
for their protein and lipid composition. By using mass spectrometry, we found the lipid composition of Jc1E2FLAG particles to resemble the one very low- and low density-lipoprotein with cholesteryl esters accounting for almost half of
the total HCV lipids. Thus, HCV particles possess a unique lipid composition that is very distinct from all other viruses
analyzed so far and from the human liver cells in which HCV was produced. By electron microscopy (EM), we found purified Jc1E2FLAG particles to be heterogeneous, mostly spherical structures, with an average diameter of about 73 nm. Importantly, the majority
of E2-containing particles also contained apoE on their surface as assessed by immuno-EM. Taken together, we describe a rapid
and efficient system for the production of large quantities of affinity-purified HCV allowing a comprehensive analysis of
the infectious virion, including the determination of its lipid composition.
Continuing Medical Education: Sofosbuvir for Hepatitis C Genotype 2 or 3 in Patients without Treatment Options CME, N Engl J Med 2013;368:1951. Question 1 should have begun, Which one of the following statements summarizes the results in the population of patients with hepatitis C virus (HCV) infection for whom interferon treatment was not an option and who were randomly assigned ... , rather than Which one of the following statements summarizes the results of the trial involving patients with hepatitis C virus (HCV) infection who were randomly assigned ... . In Choices C and D, after initiation of treatment should have been ... after the end of treatment. The examination is correct at NEJM.org.
Hepatitis C virus (HCV) infection is a global health problem affecting an estimated 170 million individuals worldwide. We report the identification of multiple independent adaptive mutations that cluster in the HCV nonstructural protein NS5A and confer increased replicative ability in vitro. Among these adaptive mutations were a single amino acid substitution that allowed HCV RNA replication in 10% of transfected hepatoma cells and a deletion of 47 amino acids encompassing the interferon (IFN) sensitivity determining region (ISDR). Independent of the ISDR, IFN-α rapidly inhibited HCV RNA replication in vitro. This work establishes a robust, cell-based system for genetic and functional analyses of HCV replication.
Background: Daclatasvir (DCV) plus sofosbuvir (SOF) RBV achieved high rates of sustained virologic response in HCV GT1-3-infected previously untreated patients (arms A-H). Additional arms (I, J) evaluated the efficacy of DCV+SOF RBV in patients who failed TVR or BOC.
Methods: A total of 41 GT1 non-cirrhotic patients with previous breakthrough (n=15), relapse (n=13), or nonresponse (n=14) to pegIFN/RBV+TVR (n=33) or BOC (n=9) (1 patient received both) were randomized 1:1 to DCV+SOF with or without RBV for 24 weeks. Patients who discontinued TVR or BOC due to adverse events were excluded. DCV and SOF were dosed orally at 60mg QD and 400mg QD, respectively. RBV was dosed BID at 1000-1200 mg/d. The primary end point was HCV RNA<25 IU/mL at 12 weeks post-treatment (SVR12).
Results: Most patients had HCV GT1a (83%), were IL28B non-CC (98%), and had estimated METAVIR stage ≥F2 (83%). Mean HCV RNA was ≥6 log IU/mL. HCV RNA <25 IU/mL was achieved in 40/41 patients by week 4 and in all patients by end of treatment. None had breakthrough or relapse and all patients with available data (40/41) achieved SVR12 (Table). The most common adverse events (>30% total) were fatigue and headache. There were no grade 3-4 hematologic or hepatic laboratory abnormalities.
Conclusion : The all-oral, once-daily combination of DCV+SOF with or without RBV for 24 weeks achieved SVR12 in 98% of non-cirrhotic GT1 prior TVR/BOC treatment failures. These data provide proof-of-concept that the combination of two potent direct-acting antivirals with different viral targets is effective in patients who failed pegIFN/RBV + a protease inhibitor.
HCV RNA < 25 IU/mL, mITT
I
DCV + SOF
x 24 weeks
(n = 21)
J
DCV + SOF + RBV
x 24 weeks
(n = 20)
Week 4
21 (100)
19 (95)a
End Of Treatment (Week 24)
21 (100)
20 (100)
SVR4
21 (100)
20 (100)
SVR12
21 (100)
19 (95)b
a 1 missing; b 1 missingpatient had HCV RNA undetectable at post-treatment week4 and post-treatment week 24 (preliminary)
Background: The IFN- and RBV-free regimen of DCV (NS5A inhibitor), ASV (protease inhibitor) and BMS-791325 (non-nucleoside NS5B inhibitor, 75mg BID) achieved sustained virologic response (SVR4, SVR12) >90% in treatment-naïve, hepatitis C virus (HCV) genotype (GT) 1 patients. We evaluated this regimen using two BMS-791325 doses (75 vs 150 mg BID).
Methods: HCV GT1, treatment-naïve, non-cirrhotic patients (N=32) were randomized 1:1 to DCV 60mg QD, ASV 200mg BID, and BMS-791325 75mg BID for 24 (Group 1) or 12 (Group 2) weeks. Subsequently, 34 additional patients were randomized to DCV, ASV, and BMS-791325 150mg BID for 24 (Group 3) or 12 (Group 4) weeks. The primary end point was HCV RNA <25 IU/mL at 12 weeks post-treatment (SVR12). Interim results are presented.
Results: Patients were mainly GT1a (74%), white (79%), and IL28Bnon-CC (70%). 64/66 patients had HCV RNA <25 IU/mL by Week 4 (Table). There was no difference in virologic response between 12 and 24 weeks of treatment. Overall, patients achieved SVR4 92% (46/50), SVR12 94% (30/32), and SVR24 94% (15/16). No patient discontinued for adverse events (AEs) related to DCV+ASV+BMS-791325. Most common AEs (≥10% total) were headache, asthenia, and gastrointestinal. Two serious AEs were reported, both unrelated to DCV+ASV+BMS-791325. No hepatotoxicity or Grade 3/4 elevations of ALT/AST or bilirubin were reported.
Conclusion: DCV+ASV+BMS-791325 achieved high rates of SVR4, SVR12, and SVR24 in treatment-naive GT1 patients, characterized by GT1a and IL28Bnon-CC. This regimen was well tolerated with no apparent safety signals. Expansion of the current study is underway to better define the efficacy and safety of this regimen.
Virologic Response During and After Treatment
BMS-791325 Dose
75 mg
150 mg
Duration
24 weeks
12 weeks
24 weeks
12 weeks
Group
1 (N=16)
2 (N=16)
3 (N=16)
4 (N=18)
HCV RNA <25IU/mL, n(%)
Week 4
16(100)
16(100)
16(100)
16(89)a
EOT/Last on-treatment
15(94)b
16(100)
-
17(94)c
SVR4
15(94)b
15(94)d
-
16(89)c,e
SVR12
15(94)b
15(94)d
-
-
SVR24
-
15(94)f
-
-
Virologic breakthrough, n(%)
0
0
1(7)
1(6)
Relapse, n(%)
0
0
-
1(6)
aOne patient with isolated HCV RNA of 43 IU/mL, one patient missing; b Patient withdrewconsent; cOne viral breakthrough; dOne missing; eOne relapse; fA second patient missing.
Telaprevir and boceprevir are the first direct-acting antiviral agents approved for use in HCV treatment and represent a significant advance in HCV therapy. However, these first-generation drugs also have significant limitations related to thrice-daily dosing, clinically challenging side-effect profiles, low barriers to resistance and a lack of pan-genotype activity. A second wave of protease inhibitors are in phase II and III trials and promise to provide a drug regimen with a better dosing schedule and improved tolerance. These second-wave protease inhibitors will probably be approved in combination with PEG-IFN and Ribavirin (RBV), as well as future all-oral regimens. The true second-generation protease inhibitors are in earlier stages of development and efficacy data are anxiously awaited as they may provide pan-genotypic antiviral activity and a high genetic barrier to resistance.
Despite major advances in the understanding and treatment of hepatitis C, a preventive vaccine remains elusive. The marked genetic diversity and multiple mechanisms of persistence of hepatitis C virus, combined with the relatively poor immune response of the infected host against the virus, are major barriers. The lack of robust and convenient model systems further hampers the effort to develop an effective vaccine. Advances in our understanding of virus-host interactions and protective immunity in hepatitis C virus infection provide an important roadmap to develop potent and broadly directed vaccine candidates targeting both humoral and cellular immune responses. Multiple approaches to generating and testing viral immunogens have met with variable success. Several candidates have advanced to clinical trials based on promising results in chimpanzees. The ultimate path to a successful preventive vaccine requires comprehensive evaluations of all aspects of protective immunity, innovative application of state-of-the-art vaccine technology and properly designed vaccine trials that can affirm definitive endpoints of efficacy.
We are entering an important new chapter in the story of hepatitis C virus (HCV) infection. There are clear challenges and opportunities. On the one hand, new HCV infections are still occurring, and an estimated 185 million people are or have previously been infected worldwide. Most HCV-infected persons are unaware of their status yet are at risk for life-threatening diseases such as cirrhosis and hepatocellular carcinoma (HCC), whose incidences are predicted to rise in the coming decade. On the other hand, new HCV infections can be prevented, and those that have already occurred can be detected and treated-viral eradication is even possible. How the story ends will largely be determined by the extent to which these rapidly advancing opportunities overcome the growing challenges and by the vigor of the public health response.
Background:
Patients chronically infected with hepatitis C virus (HCV) genotype 2 or 3 for whom treatment with peginterferon is not an option, or who have not had a response to prior interferon treatment, currently have no approved treatment options. In phase 2 trials, regimens including the oral nucleotide polymerase inhibitor sofosbuvir have shown efficacy in patients with HCV genotype 2 or 3 infection.
Methods:
We conducted two randomized, phase 3 studies involving patients with chronic HCV genotype 2 or 3 infection. In one trial, patients for whom treatment with peginterferon was not an option received oral sofosbuvir and ribavirin (207 patients) or matching placebo (71) for 12 weeks. In a second trial, patients who had not had a response to prior interferon therapy received sofosbuvir and ribavirin for 12 weeks (103 patients) or 16 weeks (98). The primary end point was a sustained virologic response at 12 weeks after therapy.
Results:
Among patients for whom treatment with peginterferon was not an option, the rate of a sustained virologic response was 78% (95% confidence interval [CI], 72 to 83) with sofosbuvir and ribavirin, as compared with 0% with placebo (P<0.001). Among previously treated patients, the rate of response was 50% with 12 weeks of treatment, as compared with 73% with 16 weeks of treatment (difference, -23 percentage points; 95% CI, -35 to -11; P<0.001). In both studies, response rates were lower among patients with genotype 3 infection than among those with genotype 2 infection and, among patients with genotype 3 infection, lower among those with cirrhosis than among those without cirrhosis. The most common adverse events were headache, fatigue, nausea, and insomnia; the overall rate of discontinuation of sofosbuvir was low (1 to 2%).
Conclusions:
In patients with HCV genotype 2 or 3 infection for whom treatment with peginterferon and ribavirin was not an option, 12 or 16 weeks of treatment with sofosbuvir and ribavirin was effective. Efficacy was increased among patients with HCV genotype 2 infection and those without cirrhosis. In previously treated patients with genotype 3 infection, 16 weeks of therapy was significantly more effective than 12 weeks. (Funded by Gilead Sciences; POSITRON and FUSION ClinicalTrials.gov numbers, NCT01542788 and NCT01604850, respectively.).
Background:
The stability and propagation of hepatitis C virus (HCV) is dependent on a functional interaction between the HCV genome and liver-expressed microRNA-122 (miR-122). Miravirsen is a locked nucleic acid-modified DNA phosphorothioate antisense oligonucleotide that sequesters mature miR-122 in a highly stable heteroduplex, thereby inhibiting its function.
Methods:
In this phase 2a study at seven international sites, we evaluated the safety and efficacy of miravirsen in 36 patients with chronic HCV genotype 1 infection. The patients were randomly assigned to receive five weekly subcutaneous injections of miravirsen at doses of 3 mg, 5 mg, or 7 mg per kilogram of body weight or placebo over a 29-day period. They were followed until 18 weeks after randomization.
Results:
Miravirsen resulted in a dose-dependent reduction in HCV RNA levels that endured beyond the end of active therapy. In the miravirsen groups, the mean maximum reduction in HCV RNA level (log10 IU per milliliter) from baseline was 1.2 (P=0.01) for patients receiving 3 mg per kilogram, 2.9 (P=0.003) for those receiving 5 mg per kilogram, and 3.0 (P=0.002) for those receiving 7 mg per kilogram, as compared with a reduction of 0.4 in the placebo group. During 14 weeks of follow-up after treatment, HCV RNA was not detected in one patient in the 5-mg group and in four patients in the 7-mg group. We observed no dose-limiting adverse events and no escape mutations in the miR-122 binding sites of the HCV genome.
Conclusions:
The use of miravirsen in patients with chronic HCV genotype 1 infection showed prolonged dose-dependent reductions in HCV RNA levels without evidence of viral resistance. (Funded by Santaris Pharma; ClinicalTrials.gov number, NCT01200420.).
Unlabelled:
In a sentinel cohort, hepatitis C virus (HCV) patients (primarily genotype [GT] 1a) were treated with daclatasvir (NS5A inhibitor) and asunaprevir (NS3 protease inhibitor). Preexistence, emergence, and persistence of resistance variants in patients who failed this treatment are described. HCV-infected null responders received daclatasvir (60 mg once daily) and asunaprevir (600 mg twice daily) alone (Group A, 11 patients) or with peginterferon alfa-2a and ribavirin (Group B, 10 patients) for 24 weeks. Resistance testing was performed on baseline samples and samples with HCV RNA ≥1,000 IU/mL at Week 1 through posttreatment Week 48. Resistance substitution susceptibility to inhibition by asunaprevir and daclatasvir was assessed using HCV replicon assays. In Group A, six GT1a patients experiencing viral breakthrough and one GT1a patient who relapsed had detectable NS5A (Q30E/R, L31V/M, Y93C/N) and NS3 (R155K, D168A/E/V/Y) resistance-associated variants at failure. Two of six viral breakthrough patients achieved SVR48 after treatment intensification with peginterferon alfa-2a and ribavirin. For 2/4 viral breakthrough patients not responding to treatment intensification, NS3 resistance variants changed (D168Y to D168T; R155K to V36M-R155K). At posttreatment Week 48, daclatasvir-resistant variants persisted while asunaprevir-resistant variants were generally replaced by wild-type sequences. The NS3 sequence remained unchanged in the one patient with NS3-R155K at baseline, relapse, and posttreatment Week 48. In Group B, no viral breakthrough was observed.
Conclusion:
The treatment failure of daclatasvir and asunaprevir in HCV GT1a patients was associated with both NS5A and NS3 resistance variants in prior null responders. NS5A resistance variants persisted while NS3 resistance variants generally decayed, suggesting a higher relative fitness of NS5A variants.
Unlabelled:
Intravenous silibinin (SIL) is an approved therapeutic that has recently been applied to patients with chronic hepatitis C, successfully clearing hepatitis C virus (HCV) infection in some patients even in monotherapy. Previous studies suggested multiple antiviral mechanisms of SIL; however, the dominant mode of action has not been determined. We first analyzed the impact of SIL on replication of subgenomic replicons from different HCV genotypes in vitro and found a strong inhibition of RNA replication for genotype 1a and genotype 1b. In contrast, RNA replication and infection of genotype 2a were minimally affected by SIL. To identify the viral target of SIL we analyzed resistance to SIL in vitro and in vivo. Selection for drug resistance in cell culture identified a mutation in HCV nonstructural protein (NS) 4B conferring partial resistance to SIL. This was corroborated by sequence analyses of HCV from a liver transplant recipient experiencing viral breakthrough under SIL monotherapy. Again, we identified distinct mutations affecting highly conserved amino acid residues within NS4B, which mediated phenotypic SIL resistance also in vitro. Analyses of chimeric viral genomes suggest that SIL might target an interaction between NS4B and NS3/4A. Ultrastructural studies revealed changes in the morphology of viral membrane alterations upon SIL treatment of a susceptible genotype 1b isolate, but not of a resistant NS4B mutant or genotype 2a, indicating that SIL might interfere with the formation of HCV replication sites.
Conclusion:
Mutations conferring partial resistance to SIL treatment in vivo and in cell culture argue for a mechanism involving NS4B. This novel mode of action renders SIL an attractive candidate for combination therapies with other directly acting antiviral drugs, particularly in difficult-to-treat patient cohorts.
Background
There is a need for interferon-free treatment regimens for hepatitis C virus (HCV) infection. The goal of this study was to evaluate ABT-450, a potent HCV NS3 protease inhibitor, combined with low-dose ritonavir (ABT-450/r), in addition to ABT-333, a nonnucleoside NS5B polymerase inhibitor, and ribavirin, for the treatment of HCV infection.
Methods
We conducted a 12-week, phase 2a, open-label study involving patients who had HCV genotype 1 infection without cirrhosis. All patients received ABT-333 (400 mg twice daily) and ribavirin (1000 to 1200 mg per day) and one of two daily doses of ABT-450/r. Groups 1 and 2 included previously untreated patients; group 1 received 250 mg of ABT-450 and 100 mg of ritonavir, and group 2 received 150 mg and 100 mg, respectively. Group 3, which included patients who had had a null or partial response to previous therapy with peginterferon and ribavirin, received daily doses of 150 mg of ABT-450 and 100 mg of ritonavir. The primary end point was an undetectable level of HCV RNA from week 4 through week 12 (extended rapid virologic response).
Results
A total of 17 of the 19 patients in group 1 (89%) and 11 of the 14 in group 2 (79%) had an extended rapid virologic response; a sustained virologic response 12 weeks after the end of treatment was achieved in 95% and 93% of the patients, respectively. In group 3, 10 of 17 patients (59%) had an extended rapid virologic response, and 8 (47%) had a sustained virologic response 12 weeks after therapy; 6 patients had virologic breakthrough, and 3 had a relapse. Adverse events included abnormalities in liver-function tests, fatigue, nausea, headache, dizziness, insomnia, pruritus, rash, and vomiting.
Conclusions
This preliminary study suggests that 12 weeks of therapy with a combination of a protease inhibitor, a nonnucleoside polymerase inhibitor, and ribavirin may be effective for treatment of HCV genotype 1 infection. (Funded by Abbott; ClinicalTrials.gov number, NCT01306617.)
Chronic hepatitis C virus (HCV) infection outcomes include liver failure, hepatocellular carcinoma (HCC), and liver-related death.
To assess the association between sustained virological response (SVR) and all-cause mortality in patients with chronic HCV infection and advanced hepatic fibrosis.
An international, multicenter, long-term follow-up study from 5 large tertiary care hospitals in Europe and Canada of 530 patients with chronic HCV infection who started an interferon-based treatment regimen between 1990 and 2003, following histological proof of advanced hepatic fibrosis or cirrhosis (Ishak score 4-6). Complete follow-up ranged between January 2010 and October 2011.
All-cause mortality. Secondary outcomes were liver failure, HCC, and liver-related mortality or liver transplantation.
The 530 study patients were followed up for a median (interquartile range [IQR]) of 8.4 (6.4-11.4) years. The baseline median (IQR) age was 48 (42-56) years and 369 patients (70%) were men. The Ishak fibrosis score was 4 in 143 patients (27%), 5 in 101 patients (19%), and 6 in 286 patients (54%). There were 192 patients (36%) who achieved SVR; 13 patients with SVR and 100 without SVR died (10-year cumulative all-cause mortality rate, 8.9% [95% CI, 3.3%-14.5%] with SVR and 26.0% [95% CI, 20.2%-28.4%] without SVR; P < .001). In time-dependent multivariate Cox regression analysis, SVR was associated with reduced risk of all-cause mortality (hazard ratio [HR], 0.26; 95% CI, 0.14-0.49; P < .001) and reduced risk of liver-related mortality or transplantation (HR, 0.06; 95% CI, 0.02-0.19; P < .001), the latter occurring in 3 patients with SVR and 103 without SVR. The 10-year cumulative incidence rate of liver-related mortality or transplantation was 1.9% (95% CI, 0.0%-4.1%) with SVR and 27.4% (95% CI, 22.0%-32.8%) without SVR (P < .001). There were 7 patients with SVR and 76 without SVR who developed HCC (10-year cumulative incidence rate, 5.1%; 95% CI, 1.3%-8.9%; vs 21.8%; 95% CI, 16.6%-27.0%; P < .001), and 4 patients with SVR and 111 without SVR experienced liver failure (10-year cumulative incidence rate, 2.1%; 95% CI, 0.0%-4.5%; vs 29.9%; 95% CI, 24.3%-35.5%; P < .001).
Among patients with chronic HCV infection and advanced hepatic fibrosis, sustained virological response to interferon-based treatment was associated with lower all-cause mortality.
A number of promising new hepatitis C virus (HCV) antiviral regimens have emerged during the last few years, with a trend toward increased efficacy, safety, and tolerability, when compared with currently available therapies. The focus of recent HCV antiviral drug development has been on inhibition of HCV replication, largely by targeting specific components of the HCV replication complex itself. A significant effort has been put into generating drugs that inhibit the NS5B polymerase. A number of such drugs have been developed, and NS5B polymerase inhibitors can be divided into nucleoside polymerase inhibitors and nonnucleoside polymerase inhibitors, with each group carrying specific pharmacologic and clinical characteristics. Additional research has explored the efficacy of drugs that inhibit the HCV replication complex via other mechanisms. Second-generation NS3-4A protease inhibitors have been developed, which have generally improved on the efficacy of the currently available FDA-approved first-generation agents. NS5A inhibitors have also been studied. These medications impede HCV replication and viral particle assembly and enhance host immune activation via novel mechanisms. Alternatively, medications that target a host protein, cyclophillin B, are under evaluation. These medications block HCV replication via modification of the effects of NS5B and via other poorly understood mechanisms. Detailed below are the most important HCV antiviral agents under development, many of which show promise for use within the next few years.
The recent development of small molecule compounds that directly inhibit the viral life cycle represents a major milestone for the treatment of chronic hepatitis C virus (HCV) infection. These new drugs that are collectively termed direct-acting antivirals (DAA) include a range of inhibitors of the non-structural (NS) 3/4A protease, NS5B polymerase and NS5A protein. Two NS3/4A protease inhibitors (boceprevir and telaprevir) in combination with pegylated interferon and ribavirin have now been approved for the treatment of chronic HCV genotype 1 infection and cure rates could be increased by 20-30%. However, the majority of DAAs is still in early clinical development. The rapid replication rate of HCV, along with the error-prone polymerase activity leads to a high genetic diversity among HCV virions that includes mutants with reduced susceptibility to DAA-therapy. These resistance-associated variants often occur at very low frequencies. However, during DAA-based treatment, rapid selection of resistance mutations may occur, eventually leading to viral break-through. A number of variants with different levels of resistance have been described in vitro and in vivo for virtually all DAAs. We review the parameters that determine DAA resistance as well as the clinical implications of resistance testing. In addition, the most recent literature and conference data on resistance profiles of DAAs in clinical development and future strategies to avoid the emergence of viral resistance are also discussed.
Telaprevir is a recently approved direct-acting antiviral against hepatitis C virus (HCV) that works through inhibition of the NS3/4A serine protease inhibitor. Phase 2b and 3 studies have shown marked increase in sustained virologic response rates in both treatment-naïve and treatment-experienced patients with HCV genotype 1 treated with a telaprevir-containing regimen compared with pegylated interferon (Peg-IFN) and ribavirin alone. The most commonly observed side effects of telaprevir therapy are anemia to a greater degree than that observed with Peg-IFN/ribavirin alone; eczematous rash, which can be severe in a minority of patients; and anorectal discomfort.
As a relatively simple virus, hepatitis C virus (HCV) depends extensively on its host to infect, replicate and disseminate. HCV has evolved host interactions that result in a restricted tropism, both in terms of cell type and species. Efforts into identifying and validating HCV-host interactions have been hampered by a limited number of infectious virus clones and cell lines that support HCV infection. Despite these limitations, consensus HCV-host interactions have emerged that help define the entry, replication, assembly, and tropism of HCV. This has had important implications in expanding our in vitro and in vivo systems to study HCV replication and pathogenesis. Additionally, a number of these host factors are being targeted for therapeutic development. In this review, we focus on medically relevant pro-viral host factors, their role in HCV biology, and their importance in expanding our model systems.