Hepatitis C Virus NS5A Research Articles

Synthesis and Analysis of Properties of an Immunogenic Fragment from NS4A Polypeptide of Hepatitis C Virus

—The work is aimed at the synthesis and analysis from NS4A of hepatitis C virus (HCV) antigen peptide fragment that contains a conserved B-cell and T-helper epitopes. The 24-mer peptide VIVGRIILSGRPAVIPDREVLYRK-NH2, which contains the main immunogenic site 24–46 of HCV NS4A antigen (corresponding to the 1681–1703 amino acid residues of the HCV polypeptide), subtype 1b, has been prepared via solid-phase synthesis according to the Fmoc-protocol. Particles with diameters of 73 ± 10 nm (30%) and 236 ± 5 nm (70%) have been detected in the water solution of the highly purified peptide (0.5 mg/mL) by dynamic light scattering. The polydispersity index of 0.377 ± 0.012 implies the existence of heterogeneity because of the aggregation of the peptide molecules. The ζ-potential of the peptide aggregates has been determined as 7.0 ± 0.5 mV by means of electrophoretic light scattering. These data confirm the possibility for the development of a nanoscale liposome form of the peptide preparation. Immunoreactivity of the synthesized highly purified peptide has been studied with the use of blood sera of patients with chronic hepatitis C. Antipeptide immunoglobulins G have been detected in 41.7% of serum samples. Thus, this peptide has been shown to reproduce at least one B-epitope, to which antibodies are raised during natural HCV infection. The synthesized 24-mer peptide is a promising candidate for further research and for use as a potential immunogen for the design of a nanoscale therapeutic immunogenic liposomal peptide composition with synthetic lipids as an adjuvant.

Berberis lyceum and Fumaria indica: in vitro cytotoxicity, antioxidant activity, and in silico screening of their selected phytochemicals as novel hepatitis C virus nonstructural protein 5A inhibitors

Berberis lyceum and Fumaria indica are two Pakistani indigenous herbal medicines used to treat liver infections, including hepatitis C virus (HCV). This study aimed to evaluate the cytotoxicity, and antioxidant activity of these plant extracts and computationally screen their selected phytoconstituents as HCV NS5A inhibitors. The viability of HepG2 cells was assessed 24 h and 48 h post-treatment using colorimetric and dye exclusion methods. Antioxidant properties were examined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH), reducing power, and total antioxidant capacity assays. Seventeen known phytochemicals identified from each plant were docked into the active binding site of HCV NS5A protein. The top hit ligands were analyzed for their druglikeness properties and the indices of absorption, distribution, metabolism, elimination, and toxicity (ADMET). The results showed that both plant extracts were non-toxic (CC50 > 200 µg/ml). The IC50 values of DPPH-radical scavenging activity were 51.02 ± 0.94 and 62.91 ± 1.85 µg/ml for B. lyceum and F. indica, respectively. They also exhibited reducing power and total antioxidant capacity.The phytochemicals were identified as potent HCV NS5A inhibitors with good druglikeness and ADMET properties. Six of the docked phytochemicals exhibited higher binding scores (-17.9 to −19.2 kcal/mol) with HCV NS5A protein than the standard drug, daclatasvir (-17.2 kcal/mol). Molecular dynamics (MD) simulation confirmed the stability of two compounds, berbamine and paprafumine at 100 ns with active site of HCV NS5A protein. The identified compounds through molecular docking and MD simulation could have potential as HCV NS5A inhibitor after further validation. Communicated by Ramaswamy H. Sarma

Expression profiling of miRNA-196a biomarker in naïve hepatitis C virus-infected and Sofosbuvir plus Daclatasvir-treated patients.

Micro-RNA (miRNA) is a short stretch of nucleotides that can regulate many genes associated with the various stages of the hepatitis C virus (HCV) life cycle and disease progression. This study evaluates the expression profiling of miRNA-196a in naïve HCV-infected, and Sofosbuvir plus Daclatasvir-treated patients. MiRNA-196a can inhibit HCV replication by silencing the HCV NS5A protein or downregulating the human BACH-I mRNA. The expression level of miRNA-196a was determined by quantitative reverse transcription PCR (RT-qPCR) using the whole RNA extracted from the recruited participant's serum. Results showed a 0.83-fold decrease in the miRNA-196a level in naïve HCV-infected than controls. On the contrary, an increase in the expression level by 0.06-fold was observed in Sofosbuvir plus Daclatasvir-treated patients. A negative but significant correlation was recorded between the HCV-RNA load and miRNA-196a expression level in the naïve-infected patients. Serum miRNA-196a ROC curve analysis revealed an area under the curve of 0.8278 (95% CI 0.7033-0.9524, p < 0.0001) with 82.05% sensitivity and 76.19% specificity in discriminating the healthy controls from the HCV-infected samples. In conclusion, our study explored the comparative expression levels of miRNA-196a in HCV-infected and Sofosbuvir plus Daclatasvir patients. Further studies are needed to examine the possible role of miR-196a as a therapeutic agent for treating HCV-infected patients.

Amino Acid Substitutions Associated with Treatment Failure for Hepatitis C Virus Infection.

Despite the high virological response rates achieved with current directly acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral response. The identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultradeep sequencing (UDS) methods, for HCV characterization and patient management. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV, which are not bona fide resistance-associated substitutions (RAS), from 220 patients who failed therapy. They were present frequently in basal and posttreatment virus of patients who failed different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. They also have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value for treatment response, are discussed.

S2657 A Common Hepatitis C Treatment With an Uncommon Finding: Abdominal Perforation After Initiation of Mavyret

INTRODUCTION: Mavyret (glecaprevir/pibrentasvir) is a prescription medicine used to treat adults with chronic Hepatitis C Virus (HCV) typically over a course of 8–16 weeks. The mechanism of action involves inhibiting HCV NS3/4A and HCV NS5A proteases, both of which are essential for viral RNA replication. The most common severe gastrointestinal (GI) adverse effects reported include reactivation of Hepatitis B infection and in rare cases hepatic failure. We present a case of an adult female found to have an atypical GI adverse effect: a contained abdominal perforation seven days after the initiation of Mavyret. CASE DESCRIPTION/METHODS: A 60-year-old Caucasian female with past medical history of hypothyroidism and cholecystectomy was referred for Hepatitis C treatment in August 2018. She denied jaundice, edema, confusion or ascites. Labs revealed elevated AST/ALT to 118/106 IU/L, normal bilirubin, alkaline phosphatase and total protein. A hepatitis panel was positive for hepatitis C antibody and negative for hepatitis A and B. Additional labs revealed a positive RNA viral load, genotype 1b with FibroSURE fibrosis score of 0.84. An abdominal US showed no cirrhosis. A FibroScan showed cirrhosis with F4 disease. In April 2019, the patient began HCV treatment with Mavyret. One week later she developed severe, sharp abdominal pain localized to the lower abdomen. She presented to an outside emergency department and was admitted after an abdominal CT scan showed severe bowel wall thickening of a loop of the distal jejunum with associated pneumatosis and potential focal perforation. The Surgery team was consulted and recommended conservative management. Mavyret was discontinued and IV antibiotics were started. Within a few days she had marked improvement in abdominal pain and was discharged home to complete a seven day course of Ciprofloxacin/Flagyl. A repeat abdominal CT scan three weeks later showed a normal liver and no abnormal bowel wall thickening, obstruction or evidence of pneumatosis. DISCUSSION: This case identifies a female found to have abdominal pain early in the onset of taking Mavyret with an abdominal CT scan concerning for a contained perforation. No other cases of perforation have been reported with Mavyret use. Whether this was an actual adverse effect of Mavyret is remains uncertain; however this case documents a potentially new and life threatening side effect of Mavyret about which patients and providers should be aware.

Cortactin Interacts with Hepatitis C Virus Core and NS5A Proteins: Implications for Virion Assembly

Hepatitis C virus (HCV) exploits cellular proteins to facilitate viral propagation. To identify the cellular factors involved in the HCV life cycle, we previously performed protein microarray assays using either HCV nonstructural 5A (NS5A) protein or core protein as a probe. Interestingly, cellular cortactin strongly interacted with both NS5A and core. Cortactin is an actin-binding protein critically involved in tumor progression by regulating the migration and invasion of cancerous cells. Protein interaction between cortactin and NS5A or core was confirmed by coimmunoprecipitation and immunofluorescence assays. We showed that cortactin interacted with NS5A and core via the N-terminal acidic domain of cortactin. Cortactin expression levels were not altered by HCV infection. Small interfering RNA (siRNA)-mediated knockdown of cortactin dramatically decreased HCV protein expression and infectivity levels, whereas overexpression of cortactin increased viral propagation. Ectopic expression of the siRNA-resistant cortactin recovered the viral infectivity, suggesting that cortactin was specifically required for HCV propagation. We further showed that cortactin was involved in the assembly step without affecting viral entry, HCV internal ribosome entry site (IRES)-mediated translation, and the replication steps of the HCV life cycle. Of note, silencing of cortactin markedly reduced both NS5A and core protein levels on the lipid droplets (LDs), and this effect was reversed by the overexpression of cortactin. Importantly, NS5A and core promoted cell migration by activating the phosphorylation of cortactin at tyrosine residues 421 and 466. Taken together, these data suggest that cortactin is not only involved in HCV assembly but also plays an important role in the cell migration.IMPORTANCE Cortactin is a cytoskeletal protein that regulates cell migration in response to a number of extracellular stimuli. The functional involvement of cortactin in the virus life cycle is not yet fully understood. The most significant finding is that cortactin strongly interacted with both hepatitis C virus (HCV) core and NS5A. Cortactin is involved in HCV assembly by tethering core and NS5A on the lipid droplets (LDs) with no effect on LD biogenesis. It was noteworthy that HCV NS5A and core activated cortactin by phosphorylation at tyrosines 421 and 466 to regulate cell migration. Collectively, our study shows that cortactin is a novel host factor involved in viral production and HCV-associated pathogenesis.

Hepatitis C virus NS3-4A protease regulates the lipid environment for RNA replication by cleaving host enzyme 24-dehydrocholesterol reductase

Many RNA viruses create specialized membranes for genome replication by manipulating host lipid metabolism and trafficking, but in most cases, we do not know the molecular mechanisms responsible or how specific lipids may impact the associated membrane and viral process. For example, hepatitis C virus (HCV) causes a specific, large-fold increase in the steady-state abundance of intracellular desmosterol, an immediate precursor of cholesterol, resulting in increased fluidity of the membrane where HCV RNA replication occurs. Here, we establish the mechanism responsible for HCV's effect on intracellular desmosterol, whereby the HCV NS3-4A protease controls activity of 24-dehydrocholesterol reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol. Our cumulative evidence for the proposed mechanism includes immunofluorescence microscopy experiments showing co-occurrence of DHCR24 and HCV NS3-4A protease; formation of an additional, faster-migrating DHCR24 species (DHCR24*) in cells harboring a HCV subgenomic replicon RNA or ectopically expressing NS3-4A; and biochemical evidence that NS3-4A cleaves DHCR24 to produce DHCR24* in vitro and in vivo. We further demonstrate that NS3-4A cleaves DHCR24 between residues Cys91 and Thr92 and show that this reduces the intracellular conversion of desmosterol to cholesterol. Together, these studies demonstrate that NS3-4A directly cleaves DHCR24 and that this results in the enrichment of desmosterol in the membranes where NS3-4A and DHCR24 co-occur. Overall, this suggests a model in which HCV directly regulates the lipid environment for RNA replication through direct effects on the host lipid metabolism.

Effectiveness of direct-acting antivirals for hepatitis C virus infection in hepatitis C/HIV coinfected individuals: A multicenter study.

In a hepatitis C virus (HCV)/HIV-positive Brazilian cohort, evaluate the safety and efficacy of HCV DAAs, the frequency of resistance substitutions in the HCV NS5A and NS5B genes and identify predictors of treatment failure.Retrospective multicenter study of HCV/HIV patients treated with sofosbuvir (SOF)-based regimens at 10 reference centers in Brazil.Clinical and virological data were collected. Genetic diversity in the NS5A and NS5B genes was assessed by direct nucleotide sequencing. The primary outcome was sustained virological response (SVR) 12 weeks after DAA completion.Of 643 HCV/HIV patients analyzed, 74.7% were male, median CD4+ T cell count was 617 cells/mm3, 90% had an undetectable HIV viral load. HCV genotype 1 was detected in 80.2%, and 60% were taking at least 1 medication other than antiretroviral drugs during their DAA therapy. Cirrhosis was present in 42%. An SOF/daclatasvir (DCV) regimen was used in most patients (98%). The frequency of NS5A polymorphisms associated with clinically relevant resistance to DCV was 2%; no relevant NS5B variants were identified. The SVR12 rate was 92.8% in an intention to treat (ITT) analysis and 96% in a modified ITT (m-ITT) analysis. AE occurred in 1.6% of patients. By multivariate analysis, therapeutic failure was associated, in the m-ITT analysis, with concomitant use of anticonvulsant drugs (P = .001), age (P = .04), and female gender (P = .04).SOF/DCV regimens were associated with a high SVR rate in an HCV/HIV population. The use of concurrent anticonvulsant drugs and DAAs decreases the chances of achieving an SVR.

Hepatitis C virus exploits cyclophilin A to evade PKR.

Counteracting innate immunity is essential for successful viral replication. Host cyclophilins (Cyps) have been implicated in viral evasion of host antiviral responses, although the mechanisms are still unclear. Here, we show that hepatitis C virus (HCV) co-opts the host protein CypA to aid evasion of antiviral responses dependent on the effector protein kinase R (PKR). Pharmacological inhibition of CypA rescues PKR from antagonism by HCV NS5A, leading to activation of an interferon regulatory factor-1 (IRF1)-driven cell intrinsic antiviral program that inhibits viral replication. These findings further the understanding of the complexity of Cyp-virus interactions, provide mechanistic insight into the remarkably broad antiviral spectrum of Cyp inhibitors, and uncover novel aspects of PKR activity and regulation. Collectively, our study identifies a novel antiviral mechanism that harnesses cellular antiviral immunity to suppress viral replication.

HCV-2a NS5A downregulates viral translation predominantly through domain I

Hepatitis C virus (HCV) non-structural protein NS5A is a multifunctional protein with critical roles in viral replication and assembly. We previously showed that HCV-1b NS5A downregulates viral translation only in the presence of the poly(U/UC) tract in 3′UTR. As NS5A of different HCV genotypes may have different functions or carry out the same functions through genotype-specific mechanisms, we investigated the effect of HCV-2a NS5A on viral translation. We found that HCV-2a NS5A downregulates RNA translation of both HCV-2a and -1b, whereas the effect of HCV-1b NS5A is limited to HCV-1b only. In addition, individual regions of 3′UTR are not required for HCV-2a NS5A to downregulate viral RNA translation. We also found that HCV-2a NS5A inhibits capped mRNA translation. Mapping experiments showed that the translation downregulation by HCV-2a NS5A is predominantly mediated by domain I. Furthermore, we found that the integrity of serine-146 residue plays an important role in translation downregulation by NS5A. Our results increased our understanding on genotype-specific functions of HCV NS5A.

Hepatitis C Virus Proteins Core and NS5A Are Highly Sensitive to Oxidative Stress-Induced Degradation after eIF2α/ATF4 Pathway Activation.

Hepatitis C virus (HCV) infection is accompanied by increased oxidative stress and endoplasmic reticulum stress as a consequence of viral replication, production of viral proteins, and pro-inflammatory signals. To overcome the cellular stress, hepatocytes have developed several adaptive mechanisms like anti-oxidant response, activation of Unfolded Protein Response and autophagy to achieve cell survival. These adaptive mechanisms could both improve or inhibit viral replication, however, little is known in this regard. In this study, we investigate the mechanisms by which hepatocyte-like (Huh7) cells adapt to cellular stress in the context of HCV protein overexpression and oxidative stress. Huh7 cells stably expressing individual HCV (Core, NS3/4A and NS5A) proteins were treated with the superoxide anion donor menadione to induce oxidative stress. Production of reactive oxygen species and activation of caspase 3 were quantified. The activation of the eIF2α/ATF4 pathway and changes in the steady state levels of the autophagy-related proteins LC3 and p62 were determined either by quantitative polymerase chain reaction (qPCR) or Western blotting. Huh7 cells expressing Core or NS5A demonstrated reduced oxidative stress and apoptosis. In addition, phosphorylation of eIF2α and increased ATF4 and CHOP expression was observed with subsequent HCV Core and NS5A protein degradation. In line with these results, in liver biopsies from patients with hepatitis C, the expression of ATF4 and CHOP was confirmed. HCV Core and NS5A protein degradation was reversed by antioxidant treatment or silencing of the autophagy adaptor protein p62. We demonstrated that hepatocyte-like cells expressing HCV proteins and additionally exposed to oxidative stress adapt to cellular stress through eIF2a/ATF4 activation and selective degradation of HCV pro-oxidant proteins Core and NS5A. This selective degradation is dependent on p62 and results in increased resistance to apoptotic cell death induced by oxidative stress. This mechanism may provide a new key for the study of HCV pathology and lead to novel clinically applicable therapeutic interventions.

Real Life Egyptian Experience of Daclatasvir Plus Sofosbuvir with Ribavirin in Naïve Difficult to Treat HCV Patients.

Chronic infection with Hepatitis C virus (HCV) is considered as a major cause for developing liver cirrhosis and hepatocellular carcinoma. A new era in HCV treatment is ongoing using Direct Acting Antiviral activity (DAA). The first approved DAA drug was Sofosbuvir which has a high tolerability and preferable pharmacokinetic profile. Another recently developed drug is Daclatasvir a first-in-class HCV NS5A replication complex inhibitor. Both drugs are administered orally once daily and have potent antiviral activity with wide genotypic coverage. In the outpatient clinic, one hundred and fifty naïve difficult to treat chronic HCV patients were recruited from Tropical Medicine Department at Fayoum public hospital. A combination of Daclatasvir (60 mg) and Sofosbuvir (400 mg) (DCV/SOF) has been administered for those patients once daily with Ribavirin (1200 mg or 1000 mg based on patients' weight on two divided doses) over a period of 12 weeks. All patients have been followed up for clinical, laboratory assessment and HCV PCR to detect the efficacy and safety of the therapy. Sustained Virologic Response rate (SVR12) was achieved in the vast majority of patients (90.67%). Cirrhotic patients showed lower SVR compared to non-cirrhotic patients (88.89% vs 90.91%, respectively). Around half of the patients (49.33%) developed adverse events (AEs) during treatment. The most common AEs were headache, fatigue and abdominal pain. The available evidence seems to suggest that combination therapy of (DCV/SOF with RBV) in the treatment of chronic HCV genotype IV naïve difficult to treat patients either cirrhotic or non-cirrhotic is safe and effective. Monitoring for clinical and laboratory hepatic parameters was the basis for these findings.

The pattern of antiviral protein expression induced by interferon λ1 in peripheral blood mononuclear cells of patients with chronic hepatitis C virus infection.

Interferon lambda was discovered in recent years to be an antiviral agent, and research on different aspects of this antiviral factor in viral infection and investigations of its effectiveness are also progressing. The immunological effects of interferon lambda on different cell populations is not precisely known, which may be due to its use of a heterodimeric receptor consisting of IL-10R2 and IFN-λR1, which are not broadly expressed in all types of cells. In the present study, signaling by interferon lambda and its effect on the expression of hepatitis C virus (HCV) proteins were measured, and the expression pattern of some antiviral proteins and IL-10 levels were investigated in peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from 50 patients with chronic genotype 1a HCV infection and 10 healthy individuals as controls. The PBMCs were treated with various doses of interferon lambda at different times of cultivation. Real-time PCR was used for relative quantification of Mxa, PKR, OAS, ISG15 and HCV core mRNAs. Expression of the NS5A protein was measured by flow cytometry, and IL-10 production was assessed by ELISA. A significant increase in the expression of mRNA encoding antiviral proteins and a decrease in the expression of mRNAs encoding the HCV core protein were observed when cells were treated with interferon lambda in an intermittent manner. The expression of HCV NS5A protein and interleukin 10 levels were also lower than in the control group. It was shown that the maximum antiviral effect of interferon lambda in PBMCs is dependent on the dose and treatment time.

Prevalence of baseline HCV NS5A resistance associated substitutions in genotype 1a, 1b and 3 infection in Australia

BackgroundDirect-acting antivirals (DAA) have revolutionised hepatitis C virus (HCV) treatment, and most regimens include an NS5A inhibitor. Certain amino-acid substitutions confer resistance to NS5A inhibitors, termed resistance-associated substitutions (RAS). If present at baseline, they can reduce virological response rates. Population-based sequencing (PBS) is generally used for baseline sequencing, however next generation sequencing (NGS) reduces the threshold for detection of sequences encoding RAS from 20% to 5%. We determined the prevalence of NS5A RAS at baseline amongst Australian chronically infected with genotype (GT)1a, GT1b and GT3 HCV, using both PBS and NGS. MethodsSamples from DAA-naïve individuals were received at the Victorian Infectious Disease Reference Laboratory between June 2016 and December 2018. All samples were analysed for NS5A RAS using PBS. A subset of GT1 HCV samples were processed using NGS technology (Vela Diagnostics, Singapore) to determine the improvement in sensitivity. ResultsIn total, 672 samples were analysed using PBS. The baseline prevalence of NS5A RAS was 7.6% for GT1a (n = 25/329), 15.7% for GT1b (n = 8/51) and 15.1% for GT3 (n = 44/292). NGS only marginally increased sensitivity for NS5A RAS at baseline in GT1a (16% vs 17%) and GT1b (29% vs 36%). ConclusionThe prevalence of NS5A RAS in GT1a HCV in Australia was low compared with international data, and was similar to other reported international prevalence for GT1b and GT3 infection. NGS at baseline only marginally increased sensitivity for the detection of NS5A RAS in patients with GT1 HCV and cannot be recommended for routine use at baseline in clinical practice.

HCV NS5A hyperphosphorylation is involved in viral translation modulation

Hepatitis C virus (HCV) non-structural (NS) 5A protein is a multifunctional phosphoprotein. NS5A exists as hypo- and hyper-phosphorylated forms and the dynamic transitions between these two states are involved in the functions of NS5A. Hyperphosphorylation occurs primarily at six serine residues within the low complexity sequence I of NS5A. We previously showed that NS5A downregulates viral translation. In this study, we investigated the role of NS5A hyperphosphorylation in translation modulation. By analyzing the effects of phospho-ablative and phospho-mimetic mutants of the six serine residues on translation, we showed that NS5A hyperphospho-ablative mutation at all six serine residues can no longer downregulate viral translation. We then studied the effects of phospho-mutations at each of the six serine residues on translation. We found that phosphorylation of S222, S225, S235 is not involved in translation downregulation by NS5A. In contrast, NS5A with alanine mutations at S229 or S238 can no longer downregulate translation, whereas S229D or S238D mutations have no effect. Interestingly, S232D NS5A, but not S232A, abrogates translation downregulation by NS5A. Since dimerization of NS5A plays an important role in its functions, we also studied the effects of phospho-mutants of S229, S232, and S238 on dimerization in a protein-protein interaction assay. We showed that phopho-mimetic S229D or S238D mutations enhances NS5A dimerization, whereas the phospho-ablative mutations of these two residues have no effect. Neither phospho-ablative nor phopho-mimetic mutations of S232 affect dimerization. These results indicate that phosphorylation of NS5A at S229, S232, and S238 is involved in viral translation regulation and NS5A dimerization.

Anti-viral activity of compounds from Agrimonia pilosa and Galla rhois extract mixture

Hepatitis C virus (HCV) infection is a significant health problem, with a worldwide prevalence of about 170 million. Recently, the development of direct acting antiviral (DAA) as a therapeutic agent for HCV has been rapidly increasing. However, DAA has a side effect and is costly. Therefore, it is still necessary to develop a therapeutic agent to treat HCV infection using products.Agrimonia pilosa (AP) and Galla rhois (RG) are traditional medicines and are known to display therapeutic activity on various diseases. Notably, they have been reported to have an anti-viral effect on HBV and influenza virus infections. It is expected that anti-viral activity will increase when two extracts are mixed.To investigate their anti-viral activity, the expression level of HCV Core 1b and NS5A was measured. Remarkably, AP, RG, and their mixed compound (APRG64) strongly inhibited the expression of viral proteins, which led us to identify their metabolites. A total of 14 metabolites were identified using liquid chromatography mass spectrometry (LC-MS). These metabolites were evaluated for their anti-HCV activity to identify active ingredients.In conclusion, our results unveiled that anti-HCV activity of Agrimonia pilosa and Galla rhois extract mixture could lead to the development of a novel therapy for HCV infection.

Exploring the hepatitis C virus genome using single molecule real-time sequencing.

Single molecular real-time (SMRT) sequencing, also called third-generation sequencing, is a novel sequencing technique capable of generating extremely long contiguous sequence reads. While conventional short-read sequencing cannot evaluate the linkage of nucleotide substitutions distant from one another, SMRT sequencing can directly demonstrate linkage of nucleotide changes over a span of more than 20 kbp, and thus can be applied to directly examine the haplotypes of viruses or bacteria whose genome structures are changing in real time. In addition, an error correction method (circular consensus sequencing) has been established and repeated sequencing of a single-molecule DNA template can result in extremely high accuracy. The advantages of long read sequencing enable accurate determination of the haplotypes of individual viral clones. SMRT sequencing has been applied in various studies of viral genomes including determination of the full-length contiguous genome sequence of hepatitis C virus (HCV), targeted deep sequencing of the HCV NS5A gene, and assessment of heterogeneity among viral populations. Recently, the emergence of multi-drug resistant HCV viruses has become a significant clinical issue and has been also demonstrated using SMRT sequencing. In this review, we introduce the novel third-generation PacBio RSII/Sequel systems, compare them with conventional next-generation sequencers, and summarize previous studies in which SMRT sequencing technology has been applied for HCV genome analysis. We also refer to another long-read sequencing platform, nanopore sequencing technology, and discuss the advantages, limitations and future perspectives in using these third-generation sequencers for HCV genome analysis.

Frequency of baseline NS5A resistance-associated substitutions in patients infected with genotype 1 of hepatitis C virus in Croatia

The backbone of current treatment for chronic Hepatitis C virus (HCV) infection are direct-acting antivirals targeting viral nonstructural proteins (NS3, NS4A, NS5A, NS5B). To date, there are six NS5A inhibitors approved for treatment of chronic HCV infection. The presence of drug-associated resistance substitutions is mainly due to fast error-prone replication, showing differential frequency between genotypes and subtypes. The aim of this study was to determine the frequency of baseline resistance to NS5A protein inhibitors in patients with genotype 1 HCV in Croatia. Resistance-associated substitutions (RAS) were detected by Sanger sequencing of HCV NS5A region amplified from 84 patients followed by phylogenetic analysis and analysis with Geno2Pheno algorithm. The frequency of NS5A RAS was 14.3% and highly dependent on viral subtype. The overall frequency of NS5A RAS was higher in patients infected with HCV subtype 1b (24.2%) than in those infected with HCV subtype 1a (7.8%). Overall, three resistance-conferring mutations were detected (Q30R, M28T and Y93H) along with two mutations (M28V and L31I) that cause reduced susceptibility to NS5A inhibitors. Analysis of the sequences showed two distinct subtype 1a clades with RAS detected in 4.3% (1/23) clade I and 10.7% (3/28) clade II sequences. Only a few distinct NS5A RAS were detected suggesting a high degree of homogeneity of the viral population. High frequency of clinically relevant NS5A RAS in Croatia suggest that the analysis of frequency and patterns of resistance mutations in local populations and evaluation of their possible clinical impact could be beneficial.

High efficacy of resistance-guided retreatment of HCV patients failing NS5A inhibitors in the real world

Most hepatitis C virus (HCV)-infected patients failing NS5A inhibitors develop resistance-associated substitutions (RASs). Here we report the use of resistance-guided retreatment of patients who failed prior NS5A inhibitor-containing regimens in the GEHEP-004 cohort. This is the largest direct-acting antiviral (DAA)-resistance cohort study conducted in Spain. We aim to provide indications on how to use resistance information in settings where sofosbuvir/velpatasvir/voxilaprevir may not be available. GEHEP-004 is a prospective multicenter cohort enrolling HCV-infected patients treated with interferon (IFN)-free DAA regimens. Prior to retreatment, population-based sequencing of HCV NS3, NS5A and NS5B genes was performed. After receiving a comprehensive resistance interpretation report, the retreatment regimen was chosen and the sustained virological response (SVR) at 12 weeks after treatment completion (SVR12) was recorded. A total of 342 patients experiencing virological failure after treatment with sofosbuvir/ledipasvir±ribavirin (54%), sofosbuvir/daclatasvir±ribavirin (23%), or paritaprevir-ritonavir/ombitasvir±dasabuvir±ribavirin (20%) were studied. After a resistance report, 186 patients were retreated. An SVR12 was achieved for 88.1% of the patients who failed after sofosbuvir/ledipasvir±ribavirin, 83.3% of the patients who failed after sofosbuvir/daclatasvir±ribavirin, 93.7% of the patients who failed after paritaprevir-ritonavir+ombitasvir±dasabuvir±ribavirin. In our study, we show how resistance-guided retreatment in conjunction with an interpreted report allows patients to achieve SVR rates close to 90%. We hypothesize that SVR rates may even be improved if resistance data are discussed between experienced virologists and treating clinicians. We believe that our data may be relevant for countries where the access to new DAA combination regimens is limited. Hepatitis C infection can be cured with currently available antiviral agents. Only a small proportion of patients experience treatment failure, however, in absolute numbers, a high number of patients may require retreatment. Highly effective combinations of antivirals are also available for retreatment. However, these antivirals might not be available in resource-limited settings. Herein, we show how, by analyzing the cause of resistance, retreatment efficacy with old drugs can get very close to the efficacy of new drug combinations.

Glecaprevir/pibrentasvir for the treatment of chronic hepatitis C: design, development, and place in therapy.

Direct-acting antiviral (DAA) therapy has changed the landscape of hepatitis C virus (HCV) management and has changed the focus to the possibility of HCV elimination in the near future. Glecaprevir, an NS3/4A protease inhibitor, and pibrentasvir, an HCV NS5A inhibitor, have addressed many of the existing shortcomings in the DAA therapy spectrum. This combination has proven to be a highly efficacious pan-genotypic DAA with a high barrier to resistance as a once-daily, all-oral medication. This review explores the design and development of glecaprevir and pibrentasvir, its place in current HCV management in the midst of a myriad of DAA therapy options, and also remaining challenges.