ABSTRACT
Introdução e objetivos: Polimorfismos naturais de resistência aos agentes antivirais de atuação direta (DAAs) no vírus da hepatite C (HCV) podem representar um fator limitante para a eficácia dessa terapia. A análise de sequências virais de regiões geográficas distintas pode apresentar diferenças significativas nas frequências de mutações de resistência aos DAAs. Neste contexto, o objetivo deste estudo foi investigar as mutações associadas à resistência aos DAAs nos genes nãoestruturais (NS) do HCV em isolados virais que circulam no Brasil. Métodos: Foram estudados um total de 390 sequências do genótipo 1 do HCV de pacientes brasileiros virgens de tratamento cronicamente infectados. O RNA viral foi extraído, e as regiões abrangendo os genes NS3, NS4 e NS5 foram amplificadas por RT-PCR e sequenciadas. Resultados: No domínio NS3/4A protease, a variação V36L foi encontrada em 5,60 porcento dos isolados do subtipo 1b, e a substituição T54S em 4,16 porcento das sequências do subtipo 1a; na posição 55, 4,16 porcento dos isolados continham a variação V55A, responsável por causar constrição no sítio de ligação do inibidor de protease boceprevir. Em relação à proteína NS4B, um isolado do subtipo 1b apresentou a substituição F98L, responsável por conferir resistência aos inibidores AP80978, PTC725 e silibina. Na proteína NS5A, 3,84 porcento das sequências do subtipo 1a mostraram as mutações de resistência M28T ou Y93H, e 13,46 porcento, as mutações secundárias H58P e E62D. Dentre os isolados do subtipo 1b, 3,70 porcento continham a mutação Y93H, e 14,8 porcento, as mutações secundárias R30Q, L31M, P58S e I280VEm NS5B, 25 porcento das sequências do subtipo 1b brasileiras apresentaram a variação L159F na população viral dominante, mas nenhuma sequência do subtipo 1a exibiu tal substituição...
Background and aims: Natural resistance polymorphisms to direct antiviral agents (DAAs)in hepatitis C virus (HCV) may represent a limiting factor for therapy efficacy. Analysis of viralsequences from distinct geographical regions may show significant differences in the frequencies ofresistance mutations to DAAs. In this context, the aim of this study was to investigate mutationsassociated with resistance to DAAs of HCV non-structural genes (NS) in viral isolates circulating inBrazil. Methods: A total of 390 HCV genotype 1 sequences from therapy-naive Brazilian patientschronically infected. Viral RNA was extracted, and the region encompassing the non-structuralgenes NS3, NS4, and NS5 was RT-PCR amplified and sequenced. Results: In the NS3/4A proteasedomain, a V36L variation was found in 5,60 percent of subtype 1b isolates and a T54S substitution in4,16 percent of subtype 1a sequences; at position 55, 4,16 percent of strains contained the V55A variationwhich is responsible to cause constriction in the binding site of the protease inhibitor boceprevir.Concerning the NS4B protein, one subtype 1b isolate showed the F98L substitution, responsible forconferring resistance to inhibitors AP80978, PTC725 and Silybin. In the NS5A protein, 3,84 percent ofsubtype 1a sequences showed the resistance mutations M28T and Y93H, while 13,46 percent , thesecondary mutations H58P and E62D. Among subtype 1b isolates, 3,70 percent of patients showed theY93H mutation, while 14,8 percent the secondary mutations R30Q, L31M, P58S and I280V. For NS5B,25 percent of Brazilian subtype 1b sequences presented the L159F variation in the dominant viralpopulation, but none of subtype 1a sequence showed such substitution. Moreover, 15 subtype 1bisolates (29 percent ), were observed the C316N variant, responsible to confer resistance to non-nucleosideinhibitors, while only 2,12 percent of subtype 1a isolates showed the C316Y substitution...
Subject(s)
Humans , Antiviral Agents/antagonists & inhibitors , Hepacivirus/chemistry , Hepatitis C, Chronic/transmission , RNA, ViralABSTRACT
O objetivo deste estudo foi otimizar protocolos e padronizar métodos de diagnóstico molecular para infecção pelo HCV em amostras de sangue coletado empapel de filtro (SPF) para facilitar o acesso ao diagnóstico em áreas remotas ou com recursos limitados. Para isto, 99 indivíduos forneceram amostras pareadas de soro e SPF, dentre os quais 59eram anti-HCV reagente e 40 eram não reagentes em suas amostras de soro. As amostras anti-HCVreagentes foram submetidas à técnica de quantificação comercial. Para o desenvolvimento da RTPCRquantitativa (RT-qPCR) in house, uma curva padrão interna foi construída utilizando umplasmídeo contendo o inserto do HCV e iniciadores e sonda foram desenhados para a região 5´NCdo HCV. Para otimização da técnica, a concentração de cDNA, transcriptase reversa e númerosde ciclos de reação foram avaliados. Para a extração de RNA de HCV em amostras de SPF, setemétodos foram avaliados. A sensibilidade, especificidade, correlação, reprodutibilidade epresença de inibidores da RT-PCR quantitativa também foram determinados. O conjunto deQIAamp DNA Mini Kit foi o método mais eficiente para extração do RNA do HCV em SPF. A RTqPCRin house desenvolvida foi capaz de quantificar o HCV no soro de 44 amostras onde a medianade carga viral foi inferior aquela observada na técnica comercial (log10 4,94 e log10 6 cópias deHCV/mL, respectivamente)...
The aim of this studywas to optimize protocols and standardize molecular diagnostic methods for HCV infection in driedblood samples (DBS) to facilitate access to diagnosis in remote areas or with limited resources. Forthis, 99 individuals provided paired serum and DBS samples, where 59 of them were anti-HCVreactive and 40 were non-reactive in their serum samples. Anti-HCV positive samples were subjectedto commercial quantification technique. For the development of quantitative RT-PCR (RT-qPCR) inhouse, an internal standard curve was constructed using a plasmid containing the insert and HCVprimers and probe designed for the 5' non coding (NC) region of HCV. For optimization of thesetechniques, the concentration of cDNA, reverse transcriptase and numbers of cycles of reaction wereevaluated. For the extraction of HCV RNA in DBS samples, seven methods were evaluated. Thesensitivity, specificity, correlation, reproducibility and presence of inhibitors in quantitative RT-PCRwere also determined. QIAamp DNA Mini Kit was the most efficient method for HCV RNA extractionamong DBS samples. The in house RT-qPCR was able to quantify HCV among 44 serum sampleswhere the median viral load was lower than that observed in commercial technique (4.94 log10 and 6log10 copies of HCV / mL, respectively). The range of HCV detection using in house RT-qPCR was 10-109 copies of HCV per reaction...
Subject(s)
Humans , Blood Specimen Collection , Hepacivirus/chemistry , Hepatitis C/history , Molecular Diagnostic Techniques , Reverse Transcriptase Polymerase Chain Reaction , Serologic TestsABSTRACT
Aim: To know the seroprevalence of Hepatitis C Virus (HCV) in Multi-transfused Thalassemic Children attending a tertiary care hospital, Ahmedabad, Gujarat. Material and Methods: Serum sample were tested by Enzyme Linked Immuno Sorbent Assay (ELISA) test for Anti HCV antibody from the thalassemic children over a period of 4 years from January 2006 to December 2009. Result: A total of 163 thalassemic children were tested for antibody of HCV. Out of these HCV antibodies were positive in 38 (23.31 %) patients. Conclusion: Prevalence of HCV infection among the thalassemic cases is much higher than the routine blood donors. in the light of this result a nationwide survey is recommended to confirm this pattern in the other areas and more sophisticated diagnostic tool should be employed to rule out window period of these Transfusion Associated infections.
Subject(s)
Child , Hepacivirus/analysis , Hepacivirus/chemistry , Seroepidemiologic Studies , Thalassemia/blood , Blood Transfusion , India , Tertiary Care CentersABSTRACT
The present review article is an update on various features of hepatitis C virus [HCV] core protein including its molecular biology, role in HCV replication, involvement in HCV pathogenesis, etiological role in hepatocellular carcinogenesis, significance in diagnosis and vaccination against HCV infection. Core protein is a structural protein of HCV virus and has only recently been characterized. It was found to play a major role in HCV-induced viral hepatitis. Although published information shows a lot about the clinical significance of HCV core protein, several studies are still needed to demonstrate its exact significance in viral biology and underlying HCV pathogenesis
Subject(s)
Humans , Hepacivirus/chemistry , Hepacivirus/ultrastructure , Hepatitis C , Apoptosis , Carcinoma, Hepatocellular , Viral Core Proteins/ultrastructureABSTRACT
Nonstructural 3 (NS3) protein of hepatitis C virus (HCV) is one of the antigens commonly used in diagnostic assays for antibody to hepatitis C virus. However, immune response to the NS3 protein from one genotype may not cross-react with that from other genotypes. In the development of an anti-HCV assay, the NS3 genes from genotypes 1 and 3 commonly found in Thailand were amplified and cloned into a bacterial expression system. These recombinant NS3 proteins were immunogenic and reacted with plasma samples of Thai patients infected with various HCV genotypes. Interestingly, the NS3 proteins from the Thai genotypes could react with 3 plasma samples from HCV infected Thai blood donors, which could not bind to the NS3.1 protein in the commercial HCV immunoblot kit using antigen from HCV genotype 1. This finding supports our prior observation that the appropriate HCV antigens used in a diagnostic assay should be derived from the virus genotypes commonly found in that geographical region.
Subject(s)
Amino Acid Sequence , Base Sequence , Biomarkers/blood , DNA Primers/genetics , DNA, Viral/genetics , Electrophoresis, Polyacrylamide Gel , Genetic Vectors/genetics , Genotype , Hepacivirus/chemistry , Hepatitis C/diagnosis , Humans , Immunoblotting , Molecular Sequence Data , Nucleic Acid Amplification Techniques , Protein Isoforms/genetics , Recombinant Proteins/diagnosis , Sensitivity and Specificity , Thailand , Viral Core Proteins/genetics , Viral Nonstructural Proteins/diagnosisABSTRACT
Specific single-chain Fvs (scFvs) of human immunoglobulin that specifically recognized the recombinant hepatitis C virus (HCV) nucleocapsid protein were isolated from a large phage display antibody library. This universal library of genetically engineered filamentous phagemids displayed random pairings of the variable regions of both human heavy and light chain immunoglobulin in the scFv format. Specific clones were isolated by affinity selection with purified recombinant HCV protein fused to glutathione-S-transferase (GST). The GST-specific clones were excluded by blocking the phagemid library with GST prior to the selection. After 4 rounds of selection, the HCV-reactive clones were enriched by a factor of 100,000. About 4% and 9% of the clones from rounds 4 and 5, respectively, specifically reacted to the HCV portion of the fusion protein in an enzyme immunoassay. The specificity was confirmed by specific binding inhibition with plasma from an HCV-infected individual. Nucleotide sequence analysis of 3 HCV-specific clones indicated that all 3 clones contained an almost identical VH gene sequence which was derived from the VH3 germline gene family. These clones had different VL gene sequences of the lambda type. There were some differences between nucleotide and amino acid sequences of the HCV-specific scFv genes and those of the closest matched germline genes, indicating the presence of somatic mutation. This study illustrated the feasibility of using antibody engineering technology with the universal phage display library to isolate human antibodies with predefined specificity to important microbial pathogen which may be useful for future therapeutic purpose.
Subject(s)
Amino Acid Sequence , Bacteriophages/genetics , Cloning, Molecular/methods , Enzyme-Linked Immunosorbent Assay , Epitopes , Glutathione Transferase/genetics , Hepacivirus/chemistry , Hepatitis C Antibodies/genetics , Humans , Immunoglobulin Fragments/genetics , Molecular Sequence Data , Nucleocapsid Proteins/genetics , Recombinant Fusion Proteins/genetics , Sequence AlignmentABSTRACT
Con el objeto de evaluar la prevalencia de los anticuerpos contra el virus de la hepatitis C (antiHCV IgG) en diferentes hepatopatías crónicas hemos estudiado un grupo de 148 pacientes divididos en: Grupo I compuesto por 35 pacientes con hepatitis crónica con o sin cirrosis, con antecedentes definidos de contagio parenteral, Grupo II de 39 pacientes con hepatitis crónica con o sin cirrosis sin antecedentes de transmisión parenteral, Grupo III de 37 pacientes con el diagnóstico de hepatitis crónica autoinmune, Grupo IV compuesto de 31 pacientes con cirrosis biliar primaria y Grupo V de 6 pacientes con síndrome colestático intermedio entre CBP y HCA. En todos se descartó la etiología medicamentosa, por el virus B, acoholismo, afecciones metabólicas o colangiotis esclerosante primaria. Las determinaciones en por lo mesmo dos instancias en cada uno de antiHCV IgG se efectuaron con equipos de laboratorio Ortho-Chiron (ELISA). Se consideraron como positivos sólo aquellos con relación de positividad superior a 2.0. Los resultados se expresan en la siguiente tabla: Grupo I: 35 pacientes, anti HCV IgG 32, 94.2%; Grupo II: 39 pacientes, anti HCV IgG 15, 38.4%; Grupo III: 37 pacientes, antiHCV IgG 8, 21.6%; Grupo IV: 31 pacientes, anti HCV IgG 1, 3.22%; Grupo V: 6 pacientes, antiHCV IgG 2, 33.3%. En 2 de los pacientes con anti HCV del grupo 3 y en uno del Grupo 4 hubo antecedentes transfusionales que podrían justificar dicha positividad independientemente de la enfermedad de base. Conclusiones: Se considera útil la detección de los anticuerpos anti-HCV IgG para caracterizar a las hepatitis NANB con antecedentes transfusionales o parenterales. Parece ser frecuente la etiología por el virus C también en pacientes con hepatitis crónica sin antecedentes parenterales. El virus C parece hallarse excepcionalmente en la CBP, en cambio algunos casos con síndrome intermedio podrían obedecer a esta etiología. Cabe plantear la duda si en algunos de los pacientes con suspecha HCA se trata de HC por virus C con componente inmunológico o si la metodología serológica aún presenta un margen de error que deberá ser subsanado en el futuro