Evidence of bottleneck effect on hepatitis C virus transmission between a couple under interferon based therapy.

Issues on the correlation of viral genetic diversity and treatment response to the hepatitis C infection remain uncertain. The bottleneck effect dictates the characteristics of the viral population that will establish the infection in a new host and is related to how the immune system and treatment will be effective against the virus. Here we evaluated the phylogenetic characteristics of quasispecies population and the treatment response pattern of a HCV infected couple. We also analyzed whether the viral population of these patients indicated that they were exposed to the same source for primer infection. This study included two patients (P10 and P11) HCV genotype 1b infected. The couple presented horizontal transmission. Viral RNA was isolated from serum samples collected before, during and after treatment, at specific time points. The HCV NS5A gene sequence was amplified, cloned and sequenced. Genetic and evolutionary analyses were performed to compare the quasispecies population of these two patients and local control patients. Genetic distance and diversity were calculated. Phylogenetic analyses were performed by using maximum likelihood and Bayesian methodologies. The analysis of the baseline samples showed that the genetic distance of the viral populations of patients P10 and P11 was significantly lower than when these patients and the control group based on sequences from local patients were analyzed, supporting the horizontal transmission hypothesis. Phylogenetic analysis with sequences from all the time point samples also demonstrated two patterns of evolution depending on the treatment response. The Bayesian analysis showed that one isolate corresponding to the baseline sample of P10 was grouped into the P11 clade, suggesting a way of infection and a bottleneck effect. Our data suggests that the patient P11 viral population may be originated from variants from P10 patient and consequently showing that clinical differences between treatment responses can emerge from the bottleneck effect on viral populations.

Evidence of bottleneck effect on hepatitis C virus transmission between a couple under interferon based therapy

Issues on the correlation of viral genetic diversity and treatment response to the hepatitis C infection remain uncertain. The bottleneck effect dictates the characteristics of the viral population that will establish the infection in a new host and is related to how the immune system and treatment will be effective against the virus. Here we evaluated the phylogenetic characteristics of quasispecies population and the treatment response pattern of a HCV infected couple. We also analyzed whether the viral population of these patients indicated that they were exposed to the same source for primer infection. This study included two patients (P10 and P11) HCV genotype 1b infected. The couple presented horizontal transmission. Viral RNA was isolated from serum samples collected before, during and after treatment, at specific time points. The HCV NS5A gene sequence was amplified, cloned and sequenced. Genetic and evolutionary analyses were performed to compare the quasispecies population of these two patients and local control patients. Genetic distance and diversity were calculated. Phylogenetic analyses were performed by using maximum likelihood and Bayesian methodologies. The analysis of the baseline samples showed that the genetic distance of the viral populations of patients P10 and P11 was significantly lower than when these patients and the control group based on sequences from local patients were analyzed, supporting the horizontal transmission hypothesis. Phylogenetic analysis with sequences from all the time point samples also demonstrated two patterns of evolution depending on the treatment response. The Bayesian analysis showed that one isolate corresponding to the baseline sample of P10 was grouped into the P11 clade, suggesting a way of infection and a bottleneck effect. Our data suggests that the patient P11 viral population may be originated from variants from P10 patient and consequently showing that clinical differences between treatment responses can emerge from the bottleneck effect on viral populations.

On hepatitis C virus evolution: the interaction between virus and host towards treatment outcome.

BACKGROUND: Hepatitis C is a disease spread throughout the world. Hepatitis C virus (HCV), the etiological agent of this disease, is a single-stranded positive RNA virus. Its genome encodes a single precursor protein that yields ten proteins after processing. NS5A, one of the non-structural viral proteins, is most associated with interferon-based therapy response, the approved treatment for hepatitis C in Brazil. HCV has a high mutation rate and therefore high variability, which may be important for evading the immune system and response to therapy. The aim of this study was to analyze the evolution of NS5A quasispecies before, during, and after treatment in patients infected with HCV genotype 3a who presented different therapy responses. METHODS: Viral RNA was extracted, cDNA was synthesized, the NS5A region was amplified and cloned, and 15 clones from each time-point were sequenced. The sequences were analyzed for evolutionary history, genetic diversity and selection. RESULTS: This analysis shows that the viral population that persists after treatment for most non-responder patients is present in before-treatment samples, suggesting it is adapted to evade treatment. In contrast, the population found in before treatment samples from most end-of-treatment responder patients either are selected out or appears in low frequency after relapse, therefore changing the population structure. The exceptions illustrate the uniqueness of the evolutionary process, and therefore the treatment resistance process, in each patient. CONCLUSION: Although evolutionary behavior throughout treatment showed that each patient presented different population dynamics unrelated to therapy outcome, it seems that the viral population from non-responders that resists the treatment already had strains that could evade therapy before it started.

Gene mutations and polymorphisms of TP53 and FHIT in chronic esophagitis and esophageal carcinoma.

AIM: The aim of this study was to investigate genetic changes of the TP53 (tumor protein p53) and FHIT (fragile histidine triad) genes in precursor lesion such as chronic esophagitis (CE) and in esophageal squamous cell carcinoma (ESCC). MATERIALS AND METHODS: PCR-Single Strand Conformation Polymorphism (SSCP) analysis and DNA sequencing in 30 CE and 10 ESCC specimens were performed. RESULTS: DNA sequencing indicated two novel mutations in the TP53 exons 5 (codon 147) and 6 (codon 197) in 2/9 SSCP positive cases of ESCC, but no mutation was found in the CE. The SIFT (Sorting Intolerant From Tolerant) web-based program showed that missense variant at codon 197 of TP53 could affect the protein function. Additionally, polymorphisms of the TP53 exon 4 (codon 36 and 72) and of the FHIT exon 7 (codon 88) were observed in 4/11 (36%) cases of CE and 6/9 (67%) SSCP positive cases of ESCC after DNA sequencing. CONCLUSION: TP53 gene mutations are not common events in CE, but are frequent in ESCC, and as polymorphisms of TP53 and FHIT may confer a greater risk for the development of esophageal carcinoma, further studies are required.

Quasispecies of hepatitis C virus genotype 1 and treatment outcome with peginterferon and ribavirin.

The majority of patients with chronic hepatitis C fail to respond to antiviral therapy. The genetic basis of this resistance is unknown. The quasispecies nature of HCV may have an important implication concerning viral persistence and response to therapy. The HCV nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy. To evaluate whether the NS5A quasispecies pre-treatment composition of HCV 1a/1b is related to responsiveness to combined pegylated interferon (PEG-IFN) and Ribavirin therapy, detailed analyses of the complete NS5A were performed. Fifteen full-length NS5A clones were sequenced from 11 pre-treatment samples of patients infected with genotype 1 HCV (3 virological sustained responders, 4 non-responders, and 4 end-of-treatment responders). Our study could not show a significant correlation between the mean number of mutations in HCV NS5A before treatment and treatment outcome, and the phylogenetic construction of complete NS5A sequences obtained from all patients failed to show any clustering associated with a specific response pattern. No single amino acid position was associated with different responses to therapy in any of the NS5A regions analyzed, and mutations were clustered downstream the ISDR, primarily in the V3 region. We observed that the CRS and NLS regions of the NS5A protein were conflicting for some variables analyzed, although no significant differences were found. If these two regions can have antagonistic functions, it seems viable that they present different mutation profiles when compared with treatment response. The patient sample that presented the lowest genetic distance values also presented the smallest number of variants, and the most heterogeneous pattern was seen in the end-of-treatment patients. These results suggest that a detailed molecular analysis of the NS5A region on a larger sample size may be necessary for understanding its role in the therapy outcome of HCV 1a/1b infection.