John Levis

Correlation between pre-treatment quasispecies complexity and treatment outcome in chronic HCV genotype 3a.

Pre-treatment HCV quasispecies complexity and diversity may predict response to interferon based anti-viral therapy. The objective of this study was to retrospectively (1) examine temporal changes in quasispecies prior to the start of therapy and (2) investigate extensively quasispecies evolution in a group of 10 chronically infected patients with genotype 3a, treated with pegylated α2a-Interferon and ribavirin.The degree of sequence heterogeneity within the hypervariable region 1 was assessed by analyzing 20–30 individual clones in serial serum samples. Genetic parameters, including amino acid Shannon entropy, Hamming distance and genetic distance were calculated for each sample. Treatment outcome was divided into (1) sustained virological responders (SVR) and (2) treatment failure (TF).Our results indicate, (1) quasispecies complexity and diversity are lower in the SVR group, (2) quasispecies vary temporally and (3) genetic heterogeneity at baseline can be use to predict treatment outcome.We discuss the results from the perspective of replicative homeostasis.

Insertion and recombination events at hypervariable region 1 over 9.6 years of hepatitis C virus chronic infection.

Hepatitis C virus (HCV) exists as a quasispecies within an infected individual. We have previously reported an in-frame 3 bp insertion event at the N-terminal region of the E2 glycoprotein from a genotype 4a HCV isolate giving rise to an atypical 28 aa hypervariable region (HVR) 1. To further explore quasispecies evolution at the HVR1, serum samples collected over 9.6 years from the same chronically infected, treatment naïve individual were subjected to retrospective clonal analysis. Uniquely, we observed that isolates containing this atypical HVR1 not only persisted for 7.6 years, but dominated the quasispecies swarm. Just as striking was the collapse of this population of variants towards the end of the sampling period in synchrony with variants containing a classical HVR1 from the same lineage. The replication space was subsequently occupied by a second minor lineage, which itself was only intermittently detectable at earlier sampling points. In conjunction with the observed genetic shift, the coexistence of two distinct HVR1 populations facilitated the detection of putative intra-subtype recombinants, which included the identification of the likely ancestral parental donors. Juxtaposed to the considerable plasticity of the HVR1, we also document a degree of mutational inflexibility as each of the HVR1 subpopulations within our dataset exhibited overall genetic conservation and convergence. Finally, we raise the issue of genetic analysis in the context of mixed lineage infections.

Separation of Hepatitis C genotype 4a into IgG-depleted and IgG-enriched fractions reveals a unique quasispecies profile

BackgroundHepatitis C virus (HCV) circulates in an infected individual as a heterogeneous mixture of closely related viruses called quasispecies. The E1/E2 region of the HCV genome is hypervariable (HVR1) and is targeted by the humoral immune system. Hepatitis C virions are found in two forms: antibody associated or antibody free.The objective of this study was to investigate if separation of Hepatitis C virions into antibody enriched and antibody depleted fractions segregates quasispecies populations into distinctive swarms.ResultsA HCV genotype 4a specimen was fractionated into IgG-depleted and IgG-enriched fractions by use of Albumin/IgG depletion spin column. Clonal analysis of these two fractions was performed and then compared to an unfractionated sample. Following sequence analysis it was evident that the antibody depleted fraction was significantly more heterogeneous than the antibody enriched fraction, revealing a unique quasispecies profile. An in-frame 3 nt insertion was observed in 26% of clones in the unfractionated population and in 64% of clones in the IgG-depleted fraction. In addition, an in-frame 3 nt indel event was observed in 10% of clones in the unfractionated population and in 9% of clones in the IgG-depleted fraction. Neither of these latter events, which are rare occurrences in genotype 4a, was identified in the IgG-enriched fraction.ConclusionIn conclusion, the homogeneity of the IgG-enriched species is postulated to represent a sequence that was strongly recognised by the humoral immune system at the time the sample was obtained. The heterogeneous nature of the IgG-depleted fraction is discussed in the context of humoral escape.

Strategy for the maximization of clinically relevant information from hepatitis C virus, RT-PCR quantification.

BACKGROUND The increasing clinical application of viral load assays for monitoring viral infections has been an incentive for the development of standardized tests for the hepatitis C virus. OBJECTIVE To develop a simple model for the prediction of baseline viral load in individuals infected with the hepatitis C virus. METHODOLOGY Viral load quantification of each patients first sample was assessed by RT-PCR-ELISA using the Roche MONITOR assay in triplicate. Genotype of the infecting virus was identified by reverse line probe hybridization, using amplicons resulting from the qualitative HCV Roche AMPLICOR assay. RESULTS Retrospective evaluation of first quantitative values suggested that 82.4% (n=168/204) of individuals had a viral load between 4.3 and 6.7 log(10) viral copies per ml. A few patients (3.4%; n=7/204) have a serum viremia less than the lower limit of the linear range of the RT-PCR assay. Subsequent, prospective evaluation of hepatitis C viral load of all new patients using a model based on the dynamic range of viral load in the retrospective group correctly predicted the dynamic range in 75.9% (n=33/54). CONCLUSION The dynamic range of hepatitis C viremia extends beyond the linear range of the Roche MONITOR assay. Accurate determination of serum viremia is substantially improved by dilution of specimens prior to quantification.

The pan-genotype specificity of the hepatitis C virus anti-core monoclonal antibody C7-50 is contingent on the quasispecies profile of a population

The inter/intra-genotype quasispecies makeup of hepatitis C virus (HCV) has retarded the development of antibodies capable of pan-genotype reactivity. Mutations, even in conserved domains, are tolerated to a degree. In this report, we characterise the pan-genotype specificity of the commercially available monoclonal anti-HCV core antibody C7-50. We demonstrate the antibody’s ability to detect HCV core protein following infection of Huh7 cells with serum-derived HCV of genotypes 2-5 and that a single-site polymorphism in a genotype 3a core amino acid sequence is sufficient to disrupt antibody recognition of the epitope. This same polymorphism is a feature of genotype 3 viruses.

Nonlinear Mixed Effects Modelling Viral Load in Untreated Patients with Chronic Hepatitis C

It is well known that viral load of the hepatitis C virus (HCV) is related to the efficacy of interferon therapy. We have previously observed that viral load can fluctuate within an untreated patient population. The complex biological parameters that impact on viral load are essentially unknown. No mathematical model exists to describe HCV viral load dynamics in untreated patients. We carried out an empirical modelling to investigate whether different fluctuation patterns exist and how these patterns (if exist) are related to host-specific factors. Data was collected from 147 untreated patients chronically infected with hepatitis C, each contributing between 2 to 10 years of measurements. We propose to use a three parameter logistic model to describe the overall pattern of viral load fluctuation based on an exploratory analysis of the data. To incorporate the correlation feature of longitudinal data and patient to patient variation we introduced random effects components into the model. On the base of this nonlinear mixed effects modelling, we investigated effects of host-specific factors on viral load fluctuation by incorporating covariates into the model. The proposed model provided a good fit for describing fluctuations of viral load measured with varying frequency over different time intervals. The average viral load growth time was significantly different between infection sources. There was a large patient to patient variation in viral load asymptote.

Clinical relevance of detectable hepatitis C virus RNA in the context of direct-acting antivirals†

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