Hepatitis C Virus Replication Research Articles

Molecular Insights into The Replication and Pathogenesis of Hepatitis C Virus

Hepatitis C virus (HCV) is a major infectious disease problem worldwide, resulting in serious liver disease, including cirrhosis and hepatocellular carcinoma. This review article acts as a general overview of molecular aspects of HCV replication and pathogenesis emphasizing the virus structure and genome, entry mechanisms, the replication cycle, and HCV interactions with the host immune system. It also examines the progression of disease causing HCV infections and the clinical symptoms. Antiviral treatments have improved patient outcomes, but they remain far from an effective vaccine, because viral diversity, lack of access to health care, remain. Research in the future should look to increase healthcare accessibility, to understand long term outcomes after treatment, and to develop the vaccine. Solution to these challenges will enable us to diminish the worldwide burden of HCV and better patient health results.

Roseoside Is a Bioactive Compound in Kirengeshoma koreana Nakai Extract with Potent In Vitro Antiviral Activity Against Hepatitis C Virus.

Hepatitis C virus (HCV) is a pathogen that causes cirrhosis and hepatocellular carcinoma through chronic hepatitis C. This study focused on the anti-HCV activity of a 70% ethanol extract of Kirengeshoma koreana Nakai (KKE) and its bioactive chemical constituent(s). The KKE and its n-butanol (n-BuOH) fraction induced a significant reduction in HCV RNA levels without inducing cytotoxicity. A high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis revealed the presence of roseoside in the n-butanol fraction of the KKE, which inhibited HCV RNA replication in a concentration- and time-dependent manner without exerting cytotoxicity. Consistent with in silico molecular docking analysis data, roseoside targets and inhibits HCV NS5A/B replicase. Collectively, our findings demonstrate that roseoside is a chemical constituent in KKE that interferes with HCV replication by targeting NS5A/B replicase.

Prospective Assessment of Serum Lipid Alterations in Chronic Hepatitis C Patients Treated with Direct Acting Antivirals: Insights Six Months Post Sustained Virological Response.

Background and Objectives: Chronic hepatitis C virus (HCV) infection is intricately linked with dysregulation of lipid metabolism. In particular, cholesterol plays a crucial role in HCV replication. Direct-acting antiviral agents (DAAs) therapy has revolutionized the hepatitis C treatment landscape, achieving high rates of sustained virological response (SVR). However, viral clearance comes with some alterations in lipid-related markers. This prospective study aimed to evaluate the impact of HCV clearance on lipid homeostasis and non-invasive liver fibrosis markers in hepatitis C patients treated with DAAs. Material and Methods: Fifty-two patients with varying degrees of fibrosis treated with DAAs therapy were evaluated at baseline and 24 weeks post-SVR. Lipid profiles and non-invasive liver fibrosis markers were assessed. Results: Our findings revealed an increase in total cholesterol, triglyceride, and LDLc (low-density lipoprotein cholesterol) levels at 24 weeks post-SVR, alongside an improvement in serum liver enzymes. Although improvements in liver stiffness were observed in non-invasive tests, there was an increase in lipid-related markers post-SVR. Conclusions: This suggests a potential increased cardiovascular risk despite improvements in liver function and fibrosis, highlighting the necessity for statin therapy in some cases and extended follow-ups for these patients. These findings underscore the importance of closely monitoring lipid profiles in chronic hepatitis C patients post-SVR, as well as the potential need for statin therapy to mitigate cardiovascular risk. Additionally, extended follow-up is essential to assess long-term outcomes and ensure the optimal management of these patients.

Poly(rC)-Binding Protein 2 Does Not Directly Participate in HCV Translation or Replication, but Rather Modulates Genome Packaging.

The hepatitis C virus (HCV) co-opts many cellular factors-including proteins and microRNAs-to complete its life cycle. A cellular RNA-binding protein, poly(rC)-binding protein 2 (PCBP2), was previously shown to bind to the hepatitis C virus (HCV) genome; however, its precise role in the viral life cycle remained unclear. Herein, using the HCV cell culture (HCVcc) system and assays that isolate each step of the viral life cycle, we found that PCBP2 does not have a direct role in viral entry, translation, genome stability, or HCV RNA replication. Rather, our data suggest that PCBP2 depletion only impacts viral RNAs that can undergo genome packaging. Taken together, our data suggest that endogenous PCBP2 modulates the early steps of genome packaging, and therefore only has an indirect effect on viral translation and RNA replication, likely by increasing the translating/replicating pool of viral RNAs to the detriment of virion assembly.

The Impact of Drugs and Substance Abuse on Viral Pathogenesis-A South African Perspective.

Illicit drug and alcohol abuse have significant negative consequences for individuals who inject drugs/use drugs (PWID/UDs), including decreased immune system function and increased viral pathogenesis. PWID/UDs are at high risk of contracting or transmitting viral illnesses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV). In South Africa, a dangerous drug-taking method known as "Bluetoothing" has emerged among nyaope users, whereby the users of this drug, after injecting, withdraw blood from their veins and then reinject it into another user. Hence, the transmission of blood-borne viruses (BBVs) is exacerbated by this "Bluetooth" practice among nyaope users. Moreover, several substances of abuse promote HIV, HBV, and HCV replication. With a specific focus on the nyaope drug, viral replication, and transmission, we address the important influence of abused addictive substances and polysubstance use in this review.

Hepatitis C Virus Dysregulates Polyamine and Proline Metabolism and Perturbs the Urea Cycle.

Hepatitis C virus (HCV) is an oncogenic virus that causes chronic liver disease in more than 80% of patients. During the last decade, efficient direct-acting antivirals were introduced into clinical practice. However, clearance of the virus does not reduce the risk of end-stage liver diseases to the level observed in patients who have never been infected. So, investigation of HCV pathogenesis is still warranted. Virus-induced changes in cell metabolism contribute to the development of HCV-associated liver pathologies. Here, we studied the impact of the virus on the metabolism of polyamines and proline as well as on the urea cycle, which plays a crucial role in liver function. It was found that HCV strongly suppresses the expression of arginase, a key enzyme of the urea cycle, leading to the accumulation of arginine, and up-regulates proline oxidase with a concomitant decrease in proline concentrations. The addition of exogenous proline moderately suppressed viral replication. HCV up-regulated transcription but suppressed protein levels of polyamine-metabolizing enzymes. This resulted in a decrease in polyamine content in infected cells. Finally, compounds targeting polyamine metabolism demonstrated pronounced antiviral activity, pointing to spermine and spermidine as compounds affecting HCV replication. These data expand our understanding of HCV's imprint on cell metabolism.

PSPC1 Binds to HCV IRES and Prevents Ribosomal Protein S5 Binding, Inhibiting Viral RNA Translation.

Hepatitis C virus (HCV) infects the human liver, and its chronic infection is one of the major causes of Hepatocellular carcinoma. Translation of HCV RNA is mediated by an Internal Ribosome Entry Site (IRES) element located in the 5'UTR of viral RNA. Several RNA Binding proteins of the host interact with the HCV IRES and modulate its function. Here, we demonstrate that PSPC1 (Paraspeckle Component 1), an essential paraspeckle component, upon HCV infection is relocalized and interacts with HCV IRES to prevent viral RNA translation. Competition UV-crosslinking experiments showed that PSPC1 interacts explicitly with the SLIV region of the HCV IRES, which is known to play a vital role in ribosomal loading to the HCV IRES via interaction with Ribosomal protein S5 (RPS5). Partial silencing of PSPC1 increased viral RNA translation and, consequently, HCV replication, suggesting a negative regulation by PSPC1. Interestingly, the silencing of PSPC1 protein leads to an increased interaction of RPS5 at the SLIV region, leading to an overall increase in the viral RNA in polysomes. Overall, our results showed how the host counters viral infection by relocalizing nuclear protein to the cytoplasm as a survival strategy.

Development of an effective single-chain variable fragment recognizing a novel epitope in the hepatitis C virus E2 protein that restricts virus entry into hepatocytes.

Previously, we reported a neutralizing monoclonal antibody, A8A11, raised against a novel conserved epitope within the hepatitis C virus (HCV) E2 protein, that could significantly reduce HCV replication. Here, we report the nucleotide sequence of A8A11 and demonstrate the efficacy of a single-chain variable fragment (scFv) protein that mimics the antibody, inhibits the binding of an HCV virus-like particle to hepatocytes, and reduces viral RNA replication in a cell culture system. More importantly, scFv A8A11 was found to effectively restrict the increase of viral RNA levels in the serum of HCV-infected chimeric mice harbouring human hepatocytes. These results suggest a promising approach to neutralizing-antibody-based therapeutic interventions against HCV infection.

Biomarkers in Detection of Hepatitis C Virus Infection.

The hepatitis C virus (HCV) infection affects 58 million people worldwide. In the United States, the incidence rate of acute hepatitis C has doubled since 2014; during 2021, this increased to 5% from 2020. Acute hepatitis C is defined by any symptom of acute viral hepatitis plus either jaundice or elevated serum alanine aminotransferase (ALT) activity with the detection of HCV RNA, the anti-HCV antibody, or hepatitis C virus antigen(s). However, most patients with acute infection are asymptomatic. In addition, ALT activity and HCV RNA levels can fluctuate, and a delayed detection of the anti-HCV antibody can occur among some immunocompromised persons with HCV infection. The detection of specific biomarkers can be of great value in the early detection of HCV infection at an asymptomatic stage. The high rate of HCV replication (which is approximately 1010 to 1012 virions per day) and the lack of proofreading by the viral RNA polymerase leads to enormous genetic diversity, creating a major challenge for the host immune response. This broad genetic diversity contributes to the likelihood of developing chronic infection, thus leading to the development of cirrhosis and liver cancer. Direct-acting antiviral (DAA) therapies for HCV infection are highly effective with a cure rate of up to 99%. At the same time, many patients with HCV infection are unaware of their infection status because of the mostly asymptomatic nature of hepatitis C, so they remain undiagnosed until the liver damage has advanced. Molecular mechanisms induced by HCV have been intensely investigated to find biomarkers for diagnosing the acute and chronic phases of the infection. However, there are no clinically verified biomarkers for patients with hepatitis C. In this review, we discuss the biomarkers that can differentiate acute from chronic hepatitis C, and we summarize the current state of the literature on the useful biomarkers that are detectable during acute and chronic HCV infection, liver fibrosis/cirrhosis, and hepatocellular carcinoma (HCC).

A Systematic Review of the Impact of Resveratrol on Viral Hepatitis and Chronic Viral Hepatitis-related Hepatocellular Carcinoma.

Resveratrol (RSV) is used for the treatment of various diseases due to their anti-inflammatory and antioxidant activities. However, its beneficial aspects on viral hepatitis have been less investigated. This report reviews the impact of resveratrol on viral hepatitis and chronic viral hepatitis-related hepatocellular carcinoma (HCC). The systematic review was performed and reported according to the PRISMA 2020 statement. Several core databases, such as Cochrane Library, PubMed, Web of Science, EMBASE, and Scopus, were used for search on September 6, 2023. After extraction of the data, the desired information of the full text of the studies was recorded in Excel, and the outcomes and mechanisms were reviewed. RSV inhibits viral replication through anti-HCV NS3 helicase activity, maintains redox homeostasis via glutathione (GSH) synthesis, improves T and B cell activity, and suppresses miR-155 expression. It also enhances viral replication by enhancing hepatitis C virus (HCV) RNA transcription, activating sirtuin-1 (SIRT1), which can increase peroxisome proliferator-activated receptor (PPAR), and SIRT1 activates the HBV X protein (HBx). Moreover, RSV is responsible for hepatitis-related HCC proliferation via suppression of mammalian target of rapamycin (mTOR), SIRT1 up-regulation, inhibiting expression of HBx, and reducing expression of cyclin D1. Despite the promising properties of RSV in inhibiting hepatitis-related HCC cell proliferation, its antiviral effects in viral hepatitis are controversial. The antihepatitis behaviors of RSV are mainly dose-dependent, and in some studies, activating some hepatoprotective pathways increases the transcription and replication of chronic HBV and HCV. Therefore, healthcare providers should be aware of viral hepatitis before using RSV supplements.

In Vitro Study of Garcinia dulcis (Roxb.) Kurz Leaves Extract against Hepatitis C Virus

Garcinia dulcis is a plant commonly used in traditional medicine in tropical regions. It has anti-bacterial, anti-inflammatory, anti-tumor, and anti-malaria properties. Despite its potential benefits, studies on its effectiveness as an antiviral are limited. This study investigated the antiviral properties of G. dulcis leaf extract against the JFH-1 strain genotype 2a as a treatment for Hepatitis C Virus (HCV). Huh7it-1 cells infected with the HCV genotype 2a strain JFH-1 were used to determine the antiviral effect of methanol extracts of G. dulcis leaves (GD-LE). GD-LE antiviral activity was investigated using a focus-forming assay for anti-HCV and 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay for cytotoxicity. A time-of-addition study of anti-HCV was also performed to determine the mode of action of GD-LE. The results showed that GD-LE inhibited HCV replication with an IC50 of 17.06 µg/mL without giving any toxicity to the cells. The mode-of-action of GD-LE was found to inhibit HCV replication in the co-addition and post-infection stages. These findings suggest that GD-LE could be a promising candidate for anti-HCV treatment. However, further study regarding GD-LE bioactive compound isolation needs to be done to prepare an effective antiviral.

Co-infection with hepatitis B and C viruses: current state of the problem

Co-infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) is a complex clinical disease with an estimated worldwide prevalence of 1-15%. The transmission routes for HCV and HBV are similar. During co-infection, four serological profiles are observed: codominant, HCV dominant, HBV dominant and non-replicative. Although both HBV and HCV replicate in hepatocytes, their life cycles are quite different. Viral replication in co-infected cells is characterized by the dominance of HCV replication over HBV replication. Three theories of interaction between HCV and HBV are discussed. There are no established recommendations for the treatment of HBV/HCV co-infection. Treatment of chronic hepatitis C without HBV suppression increases the risk of HBV reactivation. In the review, we evaluate studies of both direct-acting antivirals and interferon-based therapies. Screening and prevention of co-infection are important to prevent serious HBV reactivation.

Коморбидные состояния как внепеченочные проявления хронической HCV-инфекции

Chronic hepatitis C virus (HCV) infection is a serious public health problem. In 2022, more than 33.8 thousand cases of chronic hepatitis C virus (HCV) were registered in the Russian Federation, with a total economic burden of RUB 60.9 billion. In addition to liver damage, a significant number of patients with CHC have various comorbid conditions, the development of which may be due to extrahepatic replication of HCV. In clinical practice, it is difficult to prove the association of comorbid pathology with extrahepatic manifestations of HCV. This literature review presents the results of meta-analyses and systematic studies that demonstrate the development of extrahepatic manifestations in chronic HCV infection. The data on cardiovascular and endocrine system damage, development of mixed cryoglobulinemia, neurological disorders are discussed. The analysis of scientific studies on the influence of HCV therapy with direct antiviral drugs on the course of comorbid pathology is carried out, personalized approaches to antiviral therapy for HCV patients with comorbid pathology and the prospects of clinical research to study this problem are substantiated. Key words: chronic HCV infection, chronic hepatitis C, extrahepatic manifestations, comorbid conditions, direct antiviral drugs

Hydrogen Peroxide Inhibits Hepatitis C Virus Replication by Downregulating Hepatitis C Virus Core Levels through E6-Associated Protein-Mediated Proteasomal Degradation.

Hepatitis C virus (HCV) is constantly exposed to considerable oxidative stress, characterized by elevated levels of reactive oxygen species, including hydrogen peroxide (H2O2), during acute and chronic infection in the hepatocytes of patients. However, the effect of oxidative stress on HCV replication is largely unknown. In the present study, we demonstrated that H2O2 downregulated HCV Core levels to inhibit HCV replication. For this purpose, H2O2 upregulated p53 levels, resulting in the downregulation of both the protein and enzyme activity levels of DNA methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b, and activated the expression of E6-associated protein (E6AP) through promoter hypomethylation in the presence of HCV Core. E6AP, an E3 ligase, induced the ubiquitin-dependent proteasomal degradation of HCV Core in a p53-dependent manner. The inhibitory effect of H2O2 on HCV replication was almost completely nullified either by treatment with a representative antioxidant, N-acetyl-L-cysteine, or by knockdown of p53 or E6AP using a specific short hairpin RNA, confirming the roles of p53 and E6AP in the inhibition of HCV replication by H2O2. This study provides insights into the mechanisms that regulate HCV replication under conditions of oxidative stress in patients.

In silico docking studies of Ag nanoparticles and its derivatives against NS5B protein (HCV): Integrating nanobiotechnology and nanoinformatics

The present study focused on the prediction of interactions of three ligands, i.e., silver nanoparticles, tyrosine-capped silver nanoparticles, and silver oxide nanoparticles, with the nonstructural protein 5B (NS5B) protein of the hepatitis-C virus (HCV). ΑutoDock 4, Discovery Studio, ChemDraw Ultra, OpenBabel, and Chimera software were used. Computational docking helps to evaluate the conformations of small ligands attached to macromolecular proteins. NS5B plays a crucial role in HCV replication. It is an RNA-dependent RNA membrane-associated polymerase, weighing approximately 66-kDa . The results were obtained from AutoDock 4 and visualized in Discovery Studio and Chimera. Silver nanoparticles showed interactions with All these three ligands showed promising results to inhibit the NS5B enzyme preventing HCV replication. The most effective ligand was tyrosine-capped silver nanoparticles. Its relative highest binding energy i.e. -5.29 kcal/mol showed its intense binding with the protein molecule causing more damage to the integral residues forming the active site.

Ser235 phosphorylation of hepatitis C virus NS5A is required for NS5A dimerization and drug resistance

Hepatitis C virus (HCV) infection is recognized as a major causative agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV non-structural protein 5A (NS5A) is a dimeric phosphoprotein with a hyperphosphorylated form to act as a switch that regulates HCV replication and assembly. NS5A inhibitors have been utilized as the scaffold for combination therapy of direct-acting antiviral agents (DAA). However, the mode of action of NS5A inhibitors is still unclear due to the lack of mechanistic detail regarding NS5A phosphorylation and dimerization in the HCV life cycle. It has been demonstrated that phosphorylation of NS5A at Ser235 is essential for RNA replication of the JFH1 strain. In this report, we found that NS5A phosphomimetic Ser235 substitution (Ser-to-Asp mutation) formed a dimer that was resistant to disruption by NS5A inhibitors as was the NS5A resistance-associated substitution Y93H. Phosphorylation of NS5A at Ser235 residue was required for the interaction of two NS5A-WT molecules in JFH1-based cell culture system but not absolutely required for dimerization of the NS5A-Y93H mutant. Interestingly, HCV nonstructural proteins from the subgenomic replicon NS3-5A was required for NS5A-WT dimerization but not required for NS5A-Y93H dimerization. Our data suggest that spontaneous Ser235 phosphorylation of NS5A and ensuing dimerization account for resistance of the JFH1/NS5A-Y93H mutant to NS5A inhibitors.

Generation of hepatitis C virus–resistant liver cells by genome editing–mediated stable expression of RNA aptamer

Hepatitis C virus (HCV) infections frequently recur after liver transplantation in patients with HCV-related liver diseases. About 30% of these patients progress to cirrhosis within five years after surgery. In this study, we proposed an effective therapeutic strategy to overcome the recurrence of HCV. CRISPR/Cas9 was used to insert an expression cassette encoding an RNA aptamer targeting HCV NS5B replicase as an anti-HCV agent into adeno-associated virus integration site 1 (AAVS1), known as a “safe harbor”, in a hepatocellular carcinoma cell line to confer resistance to HCV. The RNA aptamer expression system based on a dihydrofolate reductase (DFHR) minigene was precisely knocked-in into AAVS1, leading to the stable expression of aptamer RNA in the developed cell line. HCV replication was effectively inhibited at both the RNA and protein levels in cells transfected with HCV RNA or infected with HCV. RNA immunoprecipitation and competition experiments strongly suggested that this HCV inhibition was due to the RNA aptamer-mediated sequestration of HCV NS5B. No off-target insertion of the RNA aptamer expression construct was observed. The findings suggest that HCV-resistant liver cells produced by genome editing technology could be utilized as a new alternative in the development of a treatment for HCV-induced liver diseases.

The Epigenetic Controller Lysine-Specific Demethylase 1 (LSD1) Regulates the Outcome of Hepatitis C Viral Infection.

Hepatitis C virus (HCV) alters gene expression epigenetically to rearrange the cellular microenvironment in a beneficial way for its life cycle. The host epigenetic changes induced by HCV lead to metabolic dysfunction and malignant transformation. Lysine-specific demethylase 1 (LSD1) is an epigenetic controller of critical cellular functions that are essential for HCV propagation. We investigated the putative role of LSD1 in the establishment of HCV infection using genetic engineering and pharmacological inhibition to alter endogenous LSD1 levels. We demonstrated for the first time that HCV replication was inhibited in LSD1-overexpressing cells, while specific HCV proteins differentially fine-tuned endogenous LSD1 expression levels. Electroporation of the full-length HCV genome and subgenomic replicons in LSD1 overexpression enhanced translation and partially restored HCV replication, suggesting that HCV might be inhibited by LSD1 during the early steps of infection. Conversely, the inhibition of LSD1, followed by HCV infection in vitro, increased viral replication. LSD1 was shown to participate in an intriguing antiviral mechanism, where it activates endolysosomal interferon-induced transmembrane protein 3 (IFITM3) via demethylation, leading endocytosed HCV virions to degradation. Our study proposes that HCV-mediated LSD1 oscillations over countless viral life cycles throughout chronic HCV infection may promote epigenetic changes related to HCV-induced hepatocarcinogenesis.

Direct-Acting Antiviral Drug Modulates the Mitochondrial Biogenesis in Different Tissues of Young Female Rats.

(1) Background: Hepatitis C virus (HCV) infection is endemic in Egypt, with the highest prevalence rate worldwide. Sofosbuvir (SOF) is a nucleos(t)ide analog that specifically inhibits HCV replication. This study aimed to explore the possible effects of the therapeutic dose of SOF on the mitochondrial biogenesis and functions of the liver, muscle, and ovarian tissues of young normal female rats. (2) Methods: This study was conducted on 20 female Wistar rats, classified into two groups, the control group and the exposed group; the latter was orally supplemented with 4 mg/kg/day of SOF for 3 months. (3) Results: The exposure to SOF impairs mitochondrial biogenesis via mitochondrial DNA copy number decline and suppressed mitochondrial biogenesis-regulated parameters at mRNA and protein levels. Also, SOF suppresses the DNA polymerase γ (POLG) expression, citrate synthase activity, and mitochondrial NADH dehydrogenase subunit-5 (ND5) content, which impairs mitochondrial functions. SOF increased lipid peroxidation and oxidative DNA damage markers and decreased tissue expression of nuclear factor erythroid 2-related factor 2 (Nfe2l2). (4) Conclusions: The present findings demonstrate the adverse effects of SOF on mitochondrial biogenesis and function in different tissues of young female rats, which mostly appeared in ovarian tissues.

A Synopsis of Hepatitis C Virus Treatments and Future Perspectives.

Hepatitis C virus (HCV) infection is a worldwide public health problem. Chronic infection with HCV can lead to liver cirrhosis or cancer. Although some immune-competent individuals can clear the virus, others develop chronic HCV disease due to viral mutations or an impaired immune response. IFNs type I and III and the signal transduction induced by them are essential for a proper antiviral effect. Research on the viral cycle and immune escape mechanisms has formed the basis of therapeutic strategies to achieve a sustained virological response (SVR). The first therapies were based on IFNα; then, IFNα plus ribavirin (IFN-RBV); and then, pegylated-IFNα-RBV (PEGIFNα-RIV) to improve cytokine pharmacokinetics. However, the maximum SVR was 60%, and several significant side effects were observed, decreasing patients' treatment adherence. The development of direct-acting antivirals (DAAs) significantly enhanced the SVR (>90%), and the compounds were able to inhibit HCV replication without significant side effects, even in paediatric populations. The management of coinfected HBV-HCV and HCV-HIV patients has also improved based on DAA and PEG-IFNα-RBV (HBV-HCV). CD4 cells are crucial for an effective antiviral response. The IFNλ3, IL28B, TNF-α, IL-10, TLR-3, and TLR-9 gene polymorphisms are involved in viral clearance, therapeutic responses, and hepatic pathologies. Future research should focus on searching for strategies to circumvent resistance-associated substitution (RAS) to DAAs, develop new therapeutic schemes for different medical conditions, including organ transplant, and develop vaccines for long-lasting cellular and humoral responses with cross-protection against different HCV genotypes. The goal is to minimise the probability of HCV infection, HCV chronicity and hepatic carcinoma.