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Whole-genome phylogenetic analysis, the most suitable strategy for subtyping bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2, is not feasible for many laboratories. Consequently, BVDV isolates/strains have been frequently subtyped based on analysis of single genomic regions, mainly the 5' untranslated region (UTR). This approach, however, may lead to inaccurate and/or poorly statistically supported viral classification. Herein, we describe novel primer sets whose amplicons may be easily sequenced and used for BVDV subtyping. Initially, genomic regions previously described as the most suitable targets for BVDV subtyping were analyzed for design of high-coverage primers. The putative amplicons were analyzed in silico for their suitability to reproduce the phylogenetic classification of 118 BVDV-1 and 88 BVDV-2 complete/near-complete genomes (CNCGs) (GenBank). This analysis was also performed considering the region amplifiable by primers HCV90-368, 324-326 and BP189-389 (5'UTR), which have been used for BVDV diagnosis and/or classification. After confirming the agreement between the analyses of our primers' amplicon versus the CNCGs, we optimized the RT-PCRs and evaluated their performance for amplification of BVDV isolates/strains (n = 35 for BVDV-1; n = 33 for BVDV-2). Among the potential targets for BVDV subtyping, we designed high-coverage primers for NS3-NS4A (BVDV-1) (526 bp amplicon) and NS5B (BVDV-2) (728 bp). The classification based on these regions fully reproduced the subtyping of all CNCGs. On the other hand, subtyping based on the putative amplicons from primers HCV90-368, 324-326 and BP189-389 showed disagreements in relation the CNCG analysis. The NS3-NS4A and NS5B primers also allowed the amplification of all BVDV isolates/strains tested. Finally, we suggest the use of these primers in future phylogenetic and epidemiological studies of BVDVs.
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Doença das Mucosas por Vírus da Diarreia Viral Bovina , Vírus da Diarreia Viral Bovina Tipo 1 , Vírus da Diarreia Viral Bovina Tipo 2 , Vírus da Diarreia Viral Bovina , Animais , Bovinos , Vírus da Diarreia Viral Bovina Tipo 1/genética , Vírus da Diarreia Viral Bovina Tipo 2/genética , Filogenia , Genômica , Regiões 5' não Traduzidas/genética , Vírus da Diarreia Viral Bovina/genéticaRESUMO
Hepatitis C virus (HCV) is responsible for more than 180 million infections worldwide, and about 80â% of infections are reported in Low and Middle-income countries (LMICs). Therapy is based on the administration of interferon (INF), ribavirin (RBV) or more recently Direct-Acting Antivirals (DAAs). However, amino acid substitutions associated with resistance (RAS) have been extensively described and can contribute to treatment failure, and diagnosis of RAS requires considerable infrastructure, not always locally available. Dried serum spots (DSS) sampling is an alternative specimen collection method, which embeds drops of serum onto filter paper to be transported by posting to a centralized laboratory. Here, we assessed feasibility of genotypic analysis of HCV from DSS in a cohort of 80 patients from São Paulo state Brazil. HCV RNA was detected on DSS specimens in 83â% of samples of HCV infected patients. HCV genotypes 1a, 1b, 2a, 2c and 3a were determined using the sequence of the palm domain of NS5B region, and RAS C316N/Y, Q309R and V321I were identified in HCV 1b samples. Concerning therapy outcome, 75â% of the patients who used INF +RBV as a previous protocol of treatment did not respond to DAAs, and 25â% were end-of-treatment responders. It suggests that therapy with INF plus RBV may contribute for non-response to a second therapeutic protocol with DAAs. One patient that presented RAS (V321I) was classified as non-responder, and combination of RAS C316N and Q309R does not necessarily imply in resistance to treatment in this cohort of patients. Data presented herein highlights the relevance of studying circulating variants for a better understanding of HCV variability and resistance to the therapy. Furthermore, the feasibility of carrying out genotyping and RAS phenotyping analysis by using DSS card for the potential of informing future treatment interventions could be relevant to overcome the limitations of processing samples in several location worldwide, especially in LMICs.
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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.
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PURPOSE: Globally, it is estimated that 71 million people are chronically infected with hepatitis C, and 10-20% of these will develop cirrhosis and hepatocellular carcinoma. The development of new direct-acting antiviral (DAA) drugs has contributed to sustained virological response (SVR), eliminating the infection and achieving cure of chronic hepatitis C. However, treated patients can develop HCV resistance to DAAs, which can contribute to the failure of treatment. Here, we aimed to evaluate the prevalence and specific pattern of NS5A and NS5B resistance-associated substitutions (RAS) in samples from patients chronically infected with HCV genotype 3a at a public health laboratory, Instituto Adolfo Lutz, São Paulo, Brazil. PATIENTS AND METHODS: Serum samples from the enrolled individuals were submitted to "in-house" polymerase chain reaction amplification of NS5A and NS5B non-structural protein genes, which were then sequenced by Sanger method. RESULTS: A total of 170 and 190 samples were amplified and analyzed for NS5A and NS5B, respectively. For NS5A, 20 (12.0%) samples showed some important RAS; 16 (9.0%) showed some type of substitution and 134 (79.0%) showed no polymorphism. No sample showed any RAS for NS5B. CONCLUSION: This study found important RAS in samples from naïve chronic HCV patients in some areas from São Paulo. The most prevalent were A62S, A30K, and Y93H, which could indicate an increase in resistance to some DAAs used in HCV treatment.
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Infections produced by hepaciviruses have been associated with liver disease in horses. Currently, at least three viruses belonging to the Flaviviridae family are capable of producing a chronic infection in equines: non-primate hepacivirus (NPHV), Theiler's disease-associated virus (TDAV), and equine pegivirus (EPgV). The RNA-dependent RNA polymerases of viruses (RdRp) (NS5 protein), from the flavivirus family, use de novo RNA synthesis to initiate synthesis. The two antiviral drugs currently used to treat hepatitis C (HCV), sofosbuvir and dasabuvir, act on the viral NS5B polymerase as nucleoside and non-nucleoside inhibitors, respectively. Both drugs have shown significant clinical inhibition of viral response. In this work, we aimed to model the NS5B polymerase of the equine hepacivirus (EHCV) subtypes 1 and 2, TDAV and EPgV, to assess whether current direct-acting antiviral drugs against HCV interact with these proteins. Crystal structures of HCV-NS5B were used as templates for modeling target sequences in both conformations (open and closed). Also, molecular docking of sofosbuvir and dasabuvir were performed to predict their possible binding modes at the modeled NS5B polymerase binding sites. We observed that the NS5B models of the EHCV and EPgV shared well-conserved 3D structures to HCV-NS5B and other RdRps, suggesting functional conservation. Interactions of EHCV subtypes 1, 2 and TDAV polymerases with sofosbuvir showed a similar molecular interaction pattern compared to HCV-NS5B, while interactions with dasabuvir were less conserved. In silico studies of molecular interactions between these modeled structures and sofosbuvir suggest that this compound could be efficient in combating equine pathogens, thus contributing to animal welfare.
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Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/química , Pegivirus/química , Proteínas não Estruturais Virais/química , Animais , Antivirais/química , Inibidores Enzimáticos/química , Hepacivirus/efeitos dos fármacos , Cavalos/virologia , Simulação de Acoplamento Molecular , Pegivirus/efeitos dos fármacos , Alinhamento de SequênciaRESUMO
Hepatitis C virus (HCV) infection affects an estimated 71 million people worldwide. HCV is classified into eight genotypes and >70 subtypes. Determination of HCV genotype is important for selection of type and duration of antiviral therapy, and genotype is also a predictor of treatment response. The most commonly used HCV genotyping method in clinical laboratories is a hybridization-based line probe assay (LiPA; Versant HCV Genotype 2.0). However, these methods have a lack of specificity in genotype identification and subtype assignment. Here, we compared the performance of Versant HCV Genotype 2.0 with the gold standard direct sequencing of the NS5B region, in 97 samples from Mexican patients. We found a genotypic concordance of 63.9% between these methods. While 68 samples (70%) were classified into HCV genotype 1 (GT1) by NS5B sequencing, it was not true for 17 samples (17.5%), which were not match HCV subtype by LiPA. Furthermore, nine of the 33 samples classified by NS5B sequencing as GT1a were not identified by LiPA. Use of direct sequencing could improve selection of the optimal therapy, avoid possible failures of therapy and avoid high costs resulting from incorrect genotyping tests in settings without broad access to pangenotypic regimens.
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Técnicas de Genotipagem/métodos , Hepacivirus/genética , Hepatite C/virologia , RNA Viral , Análise de Sequência de RNA/métodos , Proteínas não Estruturais Virais/genética , Estudos Transversais , Humanos , MéxicoRESUMO
OBJECTIVE: Hepatitis C virus (HCV) infection is a public health problem and a major cause of chronic hepatitis. This virus exhibits a great genetic variability, with 8 genotypes and numerous subtypes. The aim of this study was to evaluate the fluctuations of HCV subtypes during 2 decades in Venezuela. METHODS: HCV genotypes were determined by direct sequencing of the 5'-noncoding region in 392 isolates circulating in patients attended during the years 2014-2015. HCV subtype assignment was confirmed in a subset of samples (n = 24) by partial sequencing of the NS5B region. The genotype distribution was compared with the one observed in a previous study of patients followed up during the years 1994-1996 and 2005-2006. RESULTS: Some variation was observed in the HCV genotype distribution over these 20 years. HCV genotype 1b prevalence was reduced significantly from 1994-1995 to 2004-2005, as previously described, and then remained constant. During the last 10 years, a significant decrease of HCV subtype 2b (36/237 in 2005-2006 vs. 24/392 in 2014-2015, p < 0.001) was observed. Patients infected with HCV G2acj were significantly older than the ones infected with G1 (53 vs. 47 years, p = 0.004), and male sex was significantly more prevalent among G3a-infected patients compared to the other ones (71 vs. 47%, p = 0.047). CONCLUSIONS: Fluctuations in HCV subtype distribution have been observed over 2 decades in Venezuela. Different major mode of transmission and susceptibility to the available HCV treatment during each period might be playing a role in the observed fluctuations in HCV subtype distribution.
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Posttranslational modification (PTM) of proteins is critical to modulate protein function and to improve the functional diversity of polypeptides. In this report, we have analyzed the PTM of both hepatitis C virus NS3 and NS5B enzyme proteins, upon their individual expression in insect cells under the baculovirus expression system. Using mass spectrometry, we present evidence that these recombinant proteins exhibit diverse covalent modifications on certain amino acid side chains, such as phosphorylation, ubiquitination, and acetylation. Although the functional implications of these PTM must be further addressed, these data may prove useful toward the understanding of the complex regulation of these key viral enzymes and to uncover novel potential targets for antiviral design.
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Hepacivirus/genética , Hepatite C/virologia , Proteínas não Estruturais Virais/genética , Regulação Viral da Expressão Gênica/genética , Hepacivirus/patogenicidade , Hepatite C/genética , Humanos , Processamento de Proteína Pós-Traducional/genéticaRESUMO
Among the hepacivirus species recently described, the non-primate hepacivirus/hepacivirus A found in horses and donkeys is closely related to the human hepatitis C virus (HCV). Therefore, the equine is an attractive surrogate large animal model for the study of HCV therapy, pathogenesis and prophylaxis. Despite global efforts, epidemiological and genetic studies have not elucidated the risk factors, virus distribution or genetic variability of the hepacivirus A, which are also important issues for the equine welfare. Little information about this background scenery is available in Brazil. The aims of this study were to investigate potential risk factors associated with hepacivirus A infection among different horse cohorts throughout the state of Rio de Janeiro and to evaluate the diversity of the viral NS5B gene and protein. Hepacivirus A RNA was detected in horse cohorts from all geographical mesoregions, independent of horse activity or breed investigated. Statewide prevalence ranged from 4.0% to 27.5%. Potential risk factors such as geographical location and age of female horses were significantly associated with the presence of virus RNA. Phylogenetic analysis revealed the circulation of subtype 2 in all mesoregions. NS5B gene sequences clustered according to geographical origin, while the NS5B fragments did not allow discriminant analysis. The predicted NS5B protein showed marked conservation, especially in the thumb domain. In conclusion, the higher frequency of hepacivirus A RNA detection in horses bred for reproduction purposes as well as in young females suggests a direct link between reproduction practices and the virus's spread. Additional studies are necessary to understand the distribution of this genetically conserved hepacivirus.
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Hepacivirus/genética , Hepatite C , Doenças dos Cavalos , Proteínas não Estruturais Virais/genética , Animais , Brasil/epidemiologia , Feminino , Hepatite C/epidemiologia , Hepatite C/virologia , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/virologia , Cavalos/virologia , Masculino , Epidemiologia Molecular , Fatores de RiscoRESUMO
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 Alcalá en 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 (5NC) 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 (5NC) 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 5NC 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.
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Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Adulto Jovem , Hepacivirus/genética , Hepatite C Crônica/virologia , Filogenia , Venezuela , Saúde Pública , Genótipo , Hospitais UniversitáriosRESUMO
The RNA-dependent RNA polymerase (RdRP) of the Hepatitis C virus (HCV), named NS5B, is phosphorylated by the cellular protein kinase C-related kinase 2 (PRK2) at two serine residues (Ser29 and Ser42) of the finger subdomain (genotype 1b). Herein, using bioinformatics, we selected four potential phosphorylation residues (Ser46, Ser76, Ser96 and Ser112) of NS5B (genotype 2a) for study. Whereas the NS5B Ser46D and Ser76D substitutions seemed to improve polymerase activity, the Ser96D mutation decreased colony formation efficiency. Active WT NS5B was utilized in in vitro kinase assays, and phosphopeptides were analyzed by mass spectrometry. Interestingly, the data indicated that both the NS5B Ser29 and Ser76 residues resulted phosphorylated. Thus, as Ser76 is absolutely conserved across HCV genotypes, our results confirmed the relevance of these sites for both genotypes and suggested that Ser76 becomes phosphorylated by a cellular kinase different from PRK2. By molecular dynamic simulations, we show that new interactions between space-adjacent amino acid chains could be established by the presence of a di-anionic phosphate group on the analyzed serines to possibly modify RNA polymerase activity. Together, our data present novel evidence on the complex regulation at the finger subdomain of HCV NS5B via phosphorylation.
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Hepacivirus/enzimologia , Hepatite C/virologia , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular , Hepacivirus/genética , Hepacivirus/fisiologia , Humanos , Simulação de Dinâmica Molecular , Fosforilação , Mutação Puntual , Estrutura Terciária de Proteína , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Replicação ViralRESUMO
Since 2011, treatment of chronic hepatitis C virus (HCV) includes direct-acting antivirals (DAAs) in addition to pegylated interferon-α (peg-IFN) and ribavirin (RBV). IFN-based treatment induces strong cytotoxic T-lymphocyte activity directed to the protease- and polymerase-derived epitopes. This enhanced immunological pressure could favour the emergence of viral epitope variants able to evade immune surveillance and, when resistance-associated variants (RAVs) are implicated, could also be co-selected as a hitchhiking effect. This study analysed the dynamics of the frequency of protease and polymerase inhibitor RAVs that could affect future HCV treatment in human immunodeficiency virus (HIV) co-infected patients on stable antiretroviral therapy with previous IFN-based treatment failure. HCV genotype 1a RNA was extracted from plasma samples of 18 patients prior to and during (24h and 4, 12, 24 and 48 weeks) therapy with peg-IFN+RBV. Next-generation sequencing was performed on HCV-RNA populations using NS3 and NS5B PCR-amplified coding regions. Two measures of genetic diversity were used to compare virus populations: average pairwise nucleotide diversity (π) and Tajima's D statistic. Several protease and polymerase RAVs were detected in all subjects at very low frequencies (<5%), and in most cases their presence was not constant during follow-up. Only samples from two patients for each region exhibited Q80R/K/L and A421V as highly predominant variants. No significant differences were observed among sampling times for either π or D values. In conclusion, previous therapy and failure of peg-IFN+RBV were not associated with an increase in DAA-targeting NS3 or NS5B RAVs that naturally exist in HIV co-infected subjects.
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Antivirais/uso terapêutico , Coinfecção/virologia , Farmacorresistência Viral , Hepacivirus/genética , Hepatite C Crônica/virologia , Interferons/uso terapêutico , Adulto , Substituição de Aminoácidos , Coinfecção/tratamento farmacológico , Feminino , Frequência do Gene , Variação Genética , Genótipo , Infecções por HIV/complicações , Hepacivirus/classificação , Hepacivirus/isolamento & purificação , Hepatite C Crônica/complicações , Hepatite C Crônica/tratamento farmacológico , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Proteínas Mutantes/genética , Mutação de Sentido Incorreto , RNA Viral/genética , Estudos Retrospectivos , Análise de Sequência de DNA , Falha de Tratamento , Proteínas não Estruturais Virais/genéticaRESUMO
AIM: To study the subtype prevalence and the phylogenetic relatedness of hepatitis C virus (HCV) sequences obtained from the Argentine general population, a large cohort of individuals was analyzed. METHODS: Healthy Argentinian volunteers (n = 6251) from 12 provinces representing all geographical regions of the country were studied. All parents or legal guardians of individuals younger than 18 years provided informed written consent for participation. The corresponding written permission from all municipal authorities was obtained from each city or town where subjects were to be included. HCV RNA reverse transcription-polymerase chain reaction products were sequenced and phylogenetically analyzed. The 5' untranslated region (5'UTR) was used for RNA detection and initial genotype classification. The NS5B polymerase region, encompassing nt 8262-8610, was used for subtyping. RESULTS: An unexpectedly low prevalence of HCV infection in the general population (0.32%) was observed. Our data contrasted with previous studies that reported rates ranging from 1.5% to 2.5%, mainly performed in selected populations of blood donors or vulnerable groups. The latter values are in keeping with the prevalence reported by the 2007 Argentinian HCV Consensus (approximately 2%). HCV subtypes were distributed as follows: 1a (25%), 1b (25%), 2c (25%), 3a (5%), and 2j (5%). Two isolates ascribed either to genotype 1 (5%) or to genotype 3 (5%) by 5'UTR phylogenetic analysis could not be subtyped. Subtype 1a sequences comprised a highly homogeneous population and clustered with United States sequences. Genotype 1b sequences represented a heterogeneous population, suggesting that this genotype might have been introduced from different sources. Most subtype 2c sequences clustered close to the 2c reported from Italy and Southern France. CONCLUSION: HCV has a low prevalence of 0.32% in the studied general population of Argentina. The pattern of HCV introduction and transmission in Argentina appears to be a consequence of multiple events and different for each subtype.
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Hepacivirus/genética , Hepatite C/epidemiologia , Hepatite C/genética , Filogenia , Regiões 5' não Traduzidas , Adulto , Análise de Variância , Argentina/epidemiologia , Distribuição de Qui-Quadrado , Feminino , Genótipo , Voluntários Saudáveis , Hepacivirus/imunologia , Hepatite C/sangue , Anticorpos Anti-Hepatite C/sangue , Humanos , Masculino , Epidemiologia Molecular , Prevalência , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas não Estruturais Virais/genéticaRESUMO
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 ...
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Humanos , Farmacorresistência Viral/genética , Hepatite C/virologia , Mutação , Antivirais/administração & dosagem , Antivirais/farmacologia , Técnicas de Genotipagem , Hepacivirus/patogenicidade , Interferons/farmacologia , Polimorfismo Genético , Reação em Cadeia da Polimerase em Tempo Real/métodos , Ribavirina/farmacologia , Resultado do Tratamento , Carga ViralRESUMO
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.