Characterization of primary direct-acting antiviral (DAA) drugs resistance mutations in NS5A/NS5B regions of hepatitis C virus with genotype 1a and 1b from patients with chronic hepatitis

ABSTRACT The Hepatitis C virus (HCV) infection is a public health problem. The high level of HCV replication and its lack of post-transcriptional correction mechanisms results in the emergence of viral variants and the difficulty in determining polymorphisms and variants that contain the substitutions associated with resistance towards new antivirals. The main focus of this study was to map the NS5A and NS5B polymorphisms and resistance mutations to new antiviral drugs in HCV strains genotype 1 from patients with chronic hepatitis C infection. Serum samples were collected from patients who underwent routine viral load tests at the Instituto Adolfo Lutz, Sao Paulo city, Brazil. A total of 698 and 853 samples were used for the characterization of NS5A and NS5B regions, respectively, which comprise the HCV genotypes 1a and 1b. The prevalence of resistance mutations found in the NS5A region was 6.4%, with Y93H, L31M, Q30R, and Y93N as the main resistance-associated substitutions (RAS). No NS5B-associated RAS was observed for any of the analyzed drugs. These findings support that the RAS test should be offered to individuals with poor response to double combination regimens prior to treatment initiation, thereby assisting strain vigilance and selection of effective treatment or retreatment options using DAA regimens.

Natural phytochemicals: Potential anti-HCV targets in silico approach

Objective: To identify phytochemicals as NS5B inhibitors against viral NS5B polymerase in silico model. The NS5Bpolymerase is a hepatitis C virus (HCV) protein involved in the HCV replication. HCV infection can cause progressiveliver damage.Material and Methods: Molecular docking method is used to identify binding efficiency between the NS5B(PDB ID: 3UPI) and the ligands (phytochemicals), i.e., Gallic acid, Catechin, Resveratrol, Apigenin, and Silibinin.Molinspiration tool is also used to determine the druglikeness properties of ligands (Lipinski’s rules of five). Thedocking results were compared to the reference ligand, Dasabuvir.Results: The molecular docking study revealed that all phytochemicals were formed complex with the HCV NS5Bpolymerase via hydrogen bonding interactions. The phytochemicals showed good binding efficacy with the dockingscore: gallic acid (−5.47 kcal/mol), catechin (−7.31 kcal/mol), resveratrol (−8.14 kcal/mol), apigenin (−8.75 kcal/mol),and silibinin (−10.75 kcal/mol) compared to the reference drug, Dasabuvir (−11.43 kcal/mol).Conclusion: The docking results suggested that all phytochemicals showed good binding affinity against hepatitisNS5B polymerase which might be due their antiviral properties.

In vitro inhibitory analysis of consensus siRNAs against NS3 gene of hepatitis C virus 1a genotype

OBJECTIVE@#To explore inhibitory effects of genome-specific, chemically synthesized siRNAs (small interference RNA) against NS3 gene of hepatitis C virus (HCV) 1a genotype in stable Huh-7 (human hepatoma) cells as well as against viral replication in serum-inoculated Huh-7 cells.@*METHODS@#Stable Huh-7 cells persistently expressing NS3 gene were produced under antibiotic gentamycin (G418) selection. The cell clones resistant to 1000 μg antibiotic concentration (G418) were picked as stable cell clones. The NS3 gene expression in stable cell clone was confirmed by RT-PCR and Western blotting. siRNA cell cytotoxicity was determined by MTT cell proliferation assay. Stable cell lines were transfected with sequence specific siRNAs and their inhibitory effects were determined by RT-PCR, real-time PCR and Western blotting. The viral replication inhibition by siRNAs in serum inoculated Huh-7 cells was determined by real-time PCR.@*RESULTS@#RT-PCR and Western blot analysis confirmed NS3 gene and protein expression in stable cell lines on day 10, 20 and 30 post transfection. MTT cell proliferation assay revealed that at most concentrated dose tested (50 nmol/L), siRNA had no cytotoxic effects on Huh-7 cells and cell proliferation remained unaffected. As demonstrated by the siRNA time-dependent inhibitory analysis, siRNA NS3-is44 showed maximum inhibition of NS3 gene in stable Huh-7 cell clones at 24 (80%, P = 0.013) and 48 h (75%, P = 0.002) post transfection. The impact of siRNAs on virus replication in serum inoculated Huh-7 cells also demonstrated significant decrease in viral copy number, where siRNA NS3-is44 exhibited 70% (P < 0.05) viral RNA reduction as compared to NS3-is33, which showed a 64% (P < 0.05) decrease in viral copy number. siRNA synergism (NS3-is33 + NS3-is44) decreased viral load by 84% (P < 0.05) as compared to individual inhibition by each siRNA (i.e., 64%-70% (P < 0.05)) in serum-inoculated cells. Synthetic siRNAs mixture (NS5B-is88 + NS3-is33) targeting different region of HCV genome (NS5B and NS3) also decreased HCV viral load by 85% (P < 0.05) as compared to siRNA inhibitory effects alone (70% and 64% respectively, P < 0.05).@*CONCLUSIONS@#siRNAs directed against NS3 gene significantly decreased mRNA and protein expression in stable cell clones. Viral replication was also vividly decreased in serum infected Huh-7 cells. Stable Huh-7 cells expressing NS3 gene is helpful to develop anti-hepatitis C drug screening assays. siRNA therapeutic potential along with other anti-HCV agents can be considered against hepatitis C.

Design and synthesis of anti-HCV drug sofosbuvir:research advances / 国际药学研究杂志

Sofosbuvir is a new type of direct-acting antiviral(DAA)drugs against hepatitis C.By competitive combination to NS5B polymerase activity sites,sofosbuvir can terminate genome synthesis of newly born virus,and finally inhibit the replication of hepatitis C virus(HCV).The research and development process of sofosbuvir is based on the principles of nucleoside antiviral drug metabolism.The subtle structure modification improves the drug′s structure stability and absorption process,which makes sofosbuvir a liver targeted anti-HCV drug.Sofosbuvir has become a clinical fundamental drug for anti-HCV infection,and then used alone or in combination with other drugs,with higher recovery rate,better safety and anti-drug resistance.This article reviews the research back?ground,development process,clinical application and synthetic methods for sofosbuvir.

In vitro inhibitory analysis of consensus siRNAs against NS3 gene of hepatitis C virus 1a genotype

Objective To explore inhibitory effects of genome-specific, chemically synthesized siRNAs (small interference RNA) against NS3 gene of hepatitis C virus (HCV) 1a genotype in stable Huh-7 (human hepatoma) cells as well as against viral replication in serum-inoculated Huh-7 cells. Methods Stable Huh-7 cells persistently expressing NS3 gene were produced under antibiotic gentamycin (G418) selection. The cell clones resistant to 1 000 μg antibiotic concentration (G418) were picked as stable cell clones. The NS3 gene expression in stable cell clone was confirmed by RT-PCR and Western blotting. siRNA cell cytotoxicity was determined by MTT cell proliferation assay. Stable cell lines were transfected with sequence specific siRNAs and their inhibitory effects were determined by RT-PCR, real-time PCR and Western blotting. The viral replication inhibition by siRNAs in serum inoculated Huh-7 cells was determined by real-time PCR. Results RT-PCR and Western blot analysis confirmed NS3 gene and protein expression in stable cell lines on day 10, 20 and 30 post transfection. MTT cell proliferation assay revealed that at most concentrated dose tested (50 nmol/L), siRNA had no cytotoxic effects on Huh-7 cells and cell proliferation remained unaffected. As demonstrated by the siRNA time-dependent inhibitory analysis, siRNA NS3-is44 showed maximum inhibition of NS3 gene in stable Huh-7 cell clones at 24 (80%, P = 0.013) and 48 h (75%, P = 0.002) post transfection. The impact of siRNAs on virus replication in serum inoculated Huh-7 cells also demonstrated significant decrease in viral copy number, where siRNA NS3-is44 exhibited 70% (P < 0.05) viral RNA reduction as compared to NS3-is33, which showed a 64% (P < 0.05) decrease in viral copy number. siRNA synergism (NS3-is33 + NS3-is44) decreased viral load by 84% (P < 0.05) as compared to individual inhibition by each siRNA (i.e., 64%–70% (P < 0.05)) in serum-inoculated cells. Synthetic siRNAs mixture (NS5B-is88 + NS3-is33) targeting different region of HCV genome (NS5B and NS3) also decreased HCV viral load by 85% (P < 0.05) as compared to siRNA inhibitory effects alone (70% and 64% respectively, P < 0.05). Conclusions siRNAs directed against NS3 gene significantly decreased mRNA and protein expression in stable cell clones. Viral replication was also vividly decreased in serum infected Huh-7 cells. Stable Huh-7 cells expressing NS3 gene is helpful to develop anti-hepatitis C drug screening assays. siRNA therapeutic potential along with other anti-HCV agents can be considered against hepatitis C.

Caracterización molecular del virus de la Hepatitis C en pacientes remitidos al Laboratorio Regional de Salud Pública del Hospital Universitario “Antonio Patricio de Alcalá”, Cumaná, Venezuela / Molecular characterization of hepatitis C virus in patients referred to a reference laboratory of public health, University Hospital “Antonio Patricio de Alcalá”, Cumaná, Venezuela

La Organización Mundial de la Salud estima que aproximadamente 170 millones de personas están crónicamente infectadas por el virus de la hepatitis C (VHC). En este estudio se evaluó la presencia de anticuerpos contra el VHC en pacientes remitidos durante enero de 2010 a febrero de 2013, al Laboratorio Regional de Salud Pública del Hospital Universitario “Antonio Patricio de Alcalᔠen Cumaná, Venezuela. La presencia de anticuerpos se hizo mediante dos ensayos de ELISA y se determinaron los genotipos circulantes a través de análisis filogenéticos de fragmentos de genoma viral amplificados por la región 5’ no codificante (5’NC) y región no estructural 5b (NS5b) usando la transcripción reversa y reacción en cadena de la polimerasa en dos rondas (RT-PCR). Se encontró una prevalencia de anticuerpos contra el VHC del 0,57 % (17/3005), siendo el grupo etario mayor de 41 años el más afectado (0,9 %). Un total de 16 muestras resultaron positivas para la presencia del ARN viral por RT-PCR en la región 5´NC (16/17, 94 %). El análisis filogenético de la región 5´NC permitió identificar la circulación del genotipo 2 y 1 y de un genotipo 3 y uno 4. Mediante análisis filogenéticos de la región NS5b, se observó la presencia de diversos subtipos dentro del genotipo 2 (2a, 2j y 2s), lo que concuerda con estudios anteriores que muestran que este genotipo es relativamente diverso en nuestro país.

The World Health Organization estimates that approximately 170 million people are chronically infected with hepatitis C virus (HCV). This study evaluated the presence of antibodies against HCV by two immunoassays. HCV genotypes were analyzed by phylogenetic analysis of viral genome fragments amplified from the 5 ‘non-coding (5’NC) region and non-structural region 5b (NS5b), using reverse transcription and nested polymerase chain reaction (RT-PCR), in patients referred from January 2010 to February 2013 to the Reference Laboratory of Public Health, University Hospital “Antonio Patricio de Alcalá”. The prevalence of anti-HCV antibodies was 0.57% (17/3005), being the group of patients older than 41 years the most affected (0.9%). A total of 16 samples were found positive for HCV RNA by RT-PCR in the 5’NC region (16/17, 94%). Phylogenetic analysis of the 5´NC region allowed to identify the circulation of genotypes 2 and 1, and one genotype 3 and one 4. By phylogenetic analysis of the NS5b region, diverse subtypes of HCV genotype 2 were identified (2a, 2j and 2s). This finding is in accordance with previous studies that indicate that this genotype is relatively diverse in our country.

Impact of directly sequenced core and non-structrural protein 5B regions on hepatitis C virus genotyping / 中华传染病杂志

Objective To explore the sensitivity and accuracy of directly sequenced core and non-structrural protein (NS)5B regions for hepatitis C virus (HCV)genotyping.Methods Fifty-one serum samples from chronic hepatitis C patients were collected in the study.Reverse transcription-polymerase chain reaction was used to amplify core and NS5B regions.Genotypes or subtypes were determined by the phylogenetic analysis of directly sequenced core and NS5B regions.Results Among the 51 samples,49 (96.1 %)were successfully typed by phylogenetic analysis of directly sequenced core region.There were overall five genotypes determined in the area,including 1b (61 .2%,30/49 ),2a (20.4%,10/49 ),2b (2.0%,1/49),3a (4.1 %,2/49 )and 6a (12.2%,6/49 ).The positive rate of HCV genotying was 88.2% (45/51 )on the basis of NS5B region.HCV genotypes 1b,2a,2b,3a and 6a were found in 62.2% (28/45),20.0% (9/45 ),2.2% (1/45 ),4.4% (2/45 )and 11 .1 % (5/45 )of the patients, respectively.Conclusion The HCV genotyping based on core regions,compared with that based on NS5B,shows the advantages of primer design,amplification efficiency and accuracy,suggesting that it has the priority to be used in the epidemiological and clinical study of HCV genotyping.

Naturally Occurring Mutations of Hepatitis B virus and Hepatitis C Virus in Korean Chronic Patients by Distinct CD4 T Cell Responses

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are among the most common causes of chronic liver disease worldwide. The host immune pressure against hepatitis viruses during the chronic infection has led to mutations in their coding genes, which could play a pivotal role in the clinical outcomes of chronic patients. Our recent molecular epidemiologic studies regarding the HBV precore/core (preC/C) regions and HCV nonstructural 5B (NS5B) protein suggest the presence of distinct CD4 T cell immune pressure against HBV and HCV in Korean chronic patients. However, induced HBV and HCV mutations seem to exert an opposite effect on Korean chronic hepatitis B (CHB) and chronic hepatitis C (CHC) patients, respectively. On the basis of two of our recent papers, we focused in this review on the relationships between the mutation patterns of HBV preC/C and HCV NS5B, which were presumed to be caused by distinct CD4 T cell pressure in the Korean population and their effect on the clinical outcomes and liver disease progression of CHB and CHC patients.

Genotypes of hepatitis C virus in the Indian sub-continent: A decade-long experience from a tertiary care hospital in South India.

Background: Hepatitis C virus (HCV) is a leading cause of chronic liver disease (CLD) that can progress to cirrhosis and hepatocellular carcinoma. Genotypes of HCV can vary in pathogenicity and can impact on treatment outcome. Objectives: To study the different genotypes among patients with HCV related CLD attending a tertiary care hospital in south India during 2002-2012. Study Design: Study subjects were those referred to clinical virology from the liver clinic. Genotyping was performed using the genotype specifi c core primers in nested polymerase chain reaction (PCR), 5′ non-coding regions based PCR- restriction fragment length polymorphism and NS5B sequencing methods. With the latter method, obtained sequences were compared with published GenBank sequences to determine the genotype. Results: Of the 451 samples tested, HCV genotype 3 was found to be the most predominant (63.85%). Other genotypes detected were genotype 1 (25.72%), genotype 2 (0.002%), genotype 4 (7.5%) and genotype 6 (2.7%). Genotype 3 was the common genotype in patients from Eastern India while genotype 1 and 4 were mainly seen in South Indian patients. Genotype 6 was seen exclusively in patients from North-Eastern India. Two other patients were infected with recombinants of genotype 1 and 2. Conclusions: In this study spanning a decade, HCV genotype 3 and genotype 1 were found to be the predominant genotypes in the Indian sub-continent. Genotype 4 and genotype 6 appeared to show some geographic restriction. A continued monitoring of HCV genotypes is essential for the optimum management of these chronically infected patients. In addition, knowledge of circulating genotypes could impact on future vaccine formulations.

Avaliação da presença de mutações de resistência no gene da NS5B e do prognóstico da infecção pelo HCV através da IL-28B em pacientes monoinfectados com HCV no RJ / Evaluation of resistance mutations presence in the NS5B gene and prognosis of HCV infection throught IL-28B in HCV monoinfected patients of RJ

Estima-se que a prevalência global da população mundial com hepatite C é de 3%. Pouco se sabe sobre a resposta ao tratamento com respeito à resistência viral. Algumas mutações no fragmento de 109 aminoácidos da NS5B são associadas com resistência ao interferon (IFN) e ribavirina (RBV). Estudos moleculares e clínicos identificaram fatores associados com o hospedeiro e vírus relacionados associada com a resposta ao tratamento, tal como o gene que codifica a IL-28B. Este estudo foi dividido em duas fases, cujos objetivos foram caracterizar a frequência de mutações que conferem resistência ao HCV e avaliar a relevância das mutações em pacientes Respondedores (R) ou Não Respondedores (NR) ao tratamento e caracterizar geneticamente as populações sobre polimorfismos genéticos nos SNPs da IL-28B em relação ao prognóstico da resposta ao tratamento. As amostras dos pacientes foram submetidas a testes de genotipagem e carga viral. As sequências geradas foram comparadas no BLAST e no banco de dados Los Alamos HCV. Realizamos o alinhamento das sequências homólogas e as mutações identificadas. Com base no genótipo e carga viral determinamos a classificação dos pacientes de acordo com a resposta à terapia. O DNA genômico foi isolado a partir de sangue periférico para a realização da tipagem de SNPs de IL-28B. A metodologia utilizada foi de PCR em tempo real utilizando sondas TaqMan SNP específico. A análise dos dados foi realizada utilizando GraphPad Prism com qui-quadrado, risco relativo (RR), Odds Ratio (OR) e intervalo de confiança de 95%, com um nível de significância de P <0,05. Foi encontrado na primeira fase deste estudo uma taxa significativa mutações associadas ao tratamento nas amostras estudadas. A prevalência de mutações associadas à resistência ao IFN e RBV bem como a novos medicamentos antivirais localizados no fragmento de 109 aminoácidos da NS5B foi examinado em 69 indivíduos infectados naïve no Rio de Janeiro, Brasil. Na segunda fase, as mutações foram ...

It is estimated that the overall prevalence of the average world population with hepatitis C is 3%. Little is known about the treatment response with respect to viral resistance. Some mutations in the 109-aminoacid fragment of NS5B are associated to Interferon (IFN) and Ribavirin (RBV) resistance. Molecular and clinical studies have identified factors associated with the host and related viruses associated with response to treatment, as the gene encoding IL-28B. This study was divided into two phases whose objectives were to characterize the frequency of mutations conferring resistance to HCV viral evaluating the relevance of these in Responders (R) or Non-Responders (NR) patients to treatment and to characterize genetically the populations regarding genetic polymorphisms SNPs IL-28B in relation to prognosis of response to treatment for HCV. Patient samples were subjected to tests for genotyping and viral load. The sequences generated were compared in the BLAST and the Los Alamos database HCV. We conducted the alignment of homologous sequences and mutations identified. Based on virological parameters genotype and viral load determined the classification of patients according to response to therapy. Genomic DNA was isolated from peripheral blood for carrying out the typing of SNPs of IL-28B. The methodology used was real-time PCR using TaqMan probes specific SNPs. Data analysis was performed using GraphPad Prism with chi-square, relative risk (RR), Odds Ratio (OR) and confidence interval of 95% with a significance level of P <0.05. To study these biological parameters we associated the responsive patients, non-responders, the viral load, genotype, and IL-28B polymorphism to treatment outcome. We found in the first phase of this study a significant rate of treatment-associated mutations in the samples studied. The prevalence of mutations associated to resistance to interferon and ribavirin (IFN/RBV) as well new antiviral drugs located in the 109 aminoacid ...

Association of hepatitis C virus NS5B variants with resistance to new antiviral drugs among untreated patients

Mutations located in the 109-amino acid fragment of NS5B are typically associated with resistance to interferon (IFN) and ribavirin (RIB) and to new antiviral drugs. The prevalence of these mutations was examined in 69 drug-naïve individuals with hepatitis C virus (HCV) infections in Rio de Janeiro, Brazil. Mutations related to non-response to IFN/RIB were observed in all subtypes studied (1a, 1b, 2b, 3a and 4). The most common mutation was Q309R, present in all subtypes, except subtype 2b with frequency above 20 percent. D244N was detected only in subtype 3a and A333E was detected only in subtype 2b. We did not detect the S282T, S326G or T329I mutations in any of the samples analysed. Of note, the C316N mutation, previously related to a new non-nucleoside compound (HCV796 and AG-021541), was observed in only eight of 33 (24 percent) samples from subtype 1b. Site 316 was under positive selection in this HCV variant. Our data highlight the presence of previously described resistance mutations in HCV genotypes from drug-naïve patients.

Recent advance in antiviral drugs for hepatitis C / 中南大学学报(医学版)

Hepatitis C virus (HCV) infection is the leading cause of chronic liver diseases worldwide.There is no vaccine to prevent HCV infection.Current standard of care (SOC) for hepatitis C is pegylated interferon-α (pegIFN-α) in combination with ribavirin (RBV).However,the efficacy of pegIFN-α and RBV combination therapy is less than 50％ for genotype 1 HCV,which is the dominant virus in human.Additionally,IFN and RBV are highly toxic,causing severe side effects.Therefore,it is urgent to develop safer and more efficacious anti-HCV drugs.Over the last decade,a number of HCV-specific inhibitors have been discovered with many of them reached to late stages of clinical trials.Recently,2 HCV NS3 protease inhibitors,telaprevir and boceprevir,have been approved by the Unite States Food and Drug Administration (FDA).This opens up a new era for anti-HCV therapy.Several new classes of antiviral drugs targeting HCV NS3 protease,NS5A and NSSB RNA-dependence RNA polymerase (RdRp) are currently at various stages of preclinical and clinical studies.Upon approval of more NS3 protease,NS5A and NS5B polymerase inhibitors,future clinical studies will lead to optimal combination therapies which will have desirable parameters such as IFN-free,higher efficacy,safe,one daily dose and short duration.

Biochemical properties of full-length hepatitis C virus RNA-dependent RNA polymerase expressed in insect cells

The hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B protein, is the key viral enzyme responsible for replication of the HCV viral RNA genome. Although several full-length and truncated forms of the HCV NS5B proteins have been expressed previously in insect cells, contamination of host terminal transferase (TNTase) has hampered analysis of the RNA synthesis initiation mechanism using natural HCV RNA templates. We have expressed the HCV NS5B protein in insect cells using a recombinant baculovirus and purified it to near homogeneity without contaminated TNTase. The highly purified recombinant HCV NS5B was capable of copying 9.6-kb full-length HCV RNA template, and mini-HCV RNA carrying both 5'- and 3'-untranslated regions (UTRs) of the HCV genome. In the absence of a primer, and other cellular and viral factors, the NS5B could elongate over HCV RNA templates, but the synthesized products were primarily in the double stranded form, indicating that no cyclic replication occurred with NS5B alone. RNA synthesis using RNA templates representing the 3'-end region of HCV minus-strand RNA and the X-RNA at the 3'-end of HCV RNA genome was also initiated de novo. No formation of dimersize self-primed RNA products resulting from extension of the 3'-end hydroxyl group was observed. Despite the internal de novo initiation from the X-RNA, the NS5B could not initiate RNA synthesis from the internal region of oligouridylic acid (U)20, suggesting that HCV RNA polymerase initiates RNA synthesis from the selected region in the 3'-UTR of HCV genome.

CLONING AND EXPRESSION OF SOLUBLE HCV NS5B GENE IN EUKARYOCYTES / 解放军医学杂志

HCV NS5B, acting as a RNA dependent replication enzyme,has emerged as an attractive protein used as a target for screenig of drugs against HCV NS5B, and plays an important role in HCV replication. In the report the gene expression of NS5B in E.coli was investigated. PCR was performed to gain the gene of HCV NS5B from plasmid pBRTM/HCV 1 which contains whole sequence of HCV, and the truncated NS5B gene containing no hydrophobic domain was cloned into pGEM Teasy vector. The gene of the truncated NS5B was cut from pGEM Teasy vector and cloned into E.coli expression plasmid pET 21b, then pET 21b NS5B was transfected into E.coli cells. The protein E.coli lysates were purified and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blotting assay. The RdRp activity of NS5B was examined by scintillation proximity assay (SPA). The truncated NS5B gene was successfully cloned into pET 21b. The results of SDS PAGE and Western blotting assay showed: ①the molecular weight of the expressed product was about 68 000 D, ②The truncated NS5B protein was existed in media of E.coli cells, ③The activity of NS5BDCT21 His from HCV 1b amounted to 6 900 cpm (total incorporation of approximately). These findings suggest that soluble NS5B can be successfully expressed in E.coli and could secret into media.