RESUMO
BACKGROUND: The quasispecies composition of Hepatitis C virus (HCV) could have important implications with regard to viral persistence and response to interferon-based therapy. The complete NS5A was analyzed to evaluate whether the composition of NS5A quasispecies of HCV 1a/1b is related to responsiveness to combined interferon pegylated (PEG-IFN) and ribavirin therapy. METHODS: Viral RNA was isolated from serum samples collected before, during and after treatment from virological sustained responder (SVR), non-responder (NR) and the end-of-treatment responder patients (ETR). NS5A region was amplified, cloned and sequenced. Six hundred and ninety full-length NS5A sequences were analyzed. RESULTS: This study provides evidence that lower nucleotide diversity of the NS5A region pre-therapy is associated with viral clearance. Analysis of samples of NRs and the ETRs time points showed that genetic diversity of populations tend to decrease over time. Post-therapy population of ETRs presented higher genetic distance from baseline probably due to the bottleneck phenomenon observed for those patients in the end of treatment. The viral effective population of those patients also showed a strong decrease after therapy. Otherwise, NRs demonstrated a continuous variation or stability of effective populations and genetic diversity over time that did not seem to be related to therapy. Phylogenetic relationships concerning complete NS5A sequences obtained from patients did not demonstrate clustering associated with specific response patterns. However, distinctive clustering of pre/post-therapy sequences was observed. In addition, the evolution of quasispecies over time was subjected to purifying or relaxed purifying selection. Codons 157 (P03), 182 and 440 (P42), 62 and 404 (P44) were found to be under positive selective pressure but it failed to be related to the therapy. CONCLUSION: These results confirm the hypothesis that a relationship exists between NS5A heterogeneity and response to therapy in patients infected with chronic hepatitis C.
Assuntos
Antivirais/uso terapêutico , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/virologia , Adulto , Sequência de Aminoácidos , Análise por Conglomerados , Evolução Molecular , Feminino , Hepacivirus/classificação , Hepacivirus/genética , Hepatite C Crônica/sangue , Hepatite C Crônica/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Filogenia , RNA Viral/sangue , Alinhamento de Sequência , Análise de Sequência de RNA , Proteínas não Estruturais Virais/genéticaRESUMO
BACKGROUND: The quasispecies nature of HCV may have important implications for viral persistence, pathogenicity and resistance to antiviral agents. The variability of one of the viral proteins, NS5A, is believed to be related to the response to IFN therapy, the standard treatment for infection. In this study we analyzed the quasispecies composition of NS5A protein in patients infected with HCV genotype 3a, before IFN therapy. METHODS: Viral RNA was isolated from samples of 12 patients: four sustained virological responders (SVR), four non-responders (NR), and four end-of-treatment responders (ETR). cDNA was synthesized, the NS5A region was amplified and the fragments obtained were cloned. Fifteen clones from each patient were sequenced with eight primers, generating 179 contigs. RESULTS: Higher values for substitution (either synonymous or non-synonymous) and for distance were found in the SVR group. However, the NR group showed relatively more non-synonymous mutations than the other groups, owing to the higher values of dN/dS in complete NS5A and most specific regions. Overall, NS5A protein is undergoing purifying selection, since all dN/dS ratios values are below 0.5. CONCLUSIONS: Our study provides an overview of the genetic variability of complete NS5A protein in HCV genotype 3a.
Assuntos
Antivirais/uso terapêutico , Variação Genética , Hepacivirus/classificação , Hepacivirus/isolamento & purificação , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/virologia , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Análise por Conglomerados , Genótipo , Hepacivirus/genética , Humanos , Dados de Sequência Molecular , Filogenia , Mutação Puntual , RNA Viral/genética , Análise de Sequência de DNA , Resultado do TratamentoRESUMO
Globally, hepatitis C virus (HCV) infection affects approximately 130 million people and 3 million new infections occur annually. HCV is also recognized as an important cause of chronic liver disease in children. The absence of proofreading properties of the HCV RNA polymerase leads to a highly error prone replication process, allowing HCV to escape host immune response. The adaptive nature of HCV evolution dictates the outcome of the disease in many ways. Here, we investigated the molecular evolution of HCV in three unrelated children who acquired chronic HCV infection as a result of mother-to-child transmission, two of whom were also coinfected with HIV-1. The persistence of discrete HCV variants and their population structure were assessed using median joining network and Bayesian approaches. While patterns of viral evolution clearly differed between subjects, immune system dysfunction related to HIV coinfection or persistent HCV seronegativity stand as potential mechanisms to explain the lack of molecular evolution observed in these three cases. In contrast, treatment of HCV infection with PegIFN, which did not lead to sustained virologic responses in all 3 cases, was not associated with commensurate variations in the complexity of the variant spectrum. Finally, the differences in the degree of divergence suggest that the mode of transmission of the virus was not the main factor driving viral evolution.