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Master Thesis

Étude du génome complet du virus de l'hépatite C par séquençage de nouvelle génération : mise au point et applications

Abstract : Directly-acting antivirals brought considerable progress to hepatitis C treatment, but their activity varies depending on viral genotypes and subtypes. Therefore, accurate genotyping is still of major importance, as well as the detection of resistance-associated mutations. To answer these goals, an approach to amplify hepatitis C virus near-complete genome with a single long-range PCR and sequence it with a next-generation technique was developed. The overall amplification success rate was of 73% for usual genotypes (i.e. HCV1a, 1b, 3a and 4a, n = 16/22) and 45% for recombinant forms RF_2k/1b (n = 5/11). After pyrosequencing and de novo assembly, a near-full-length genomic consensus sequence was obtained for 19/21 samples. Genotype and subtype were confirmed by phylogenetic analysis for every sample, including the suspected recombinant forms. Circulation of this particular strain, difficult to identify, is probably underestimated. Ten cases have been detected in the Rhône-Alpes region between January and August 2014 in patients from the Caucasus area. All viral types considered, resistance-associated mutations were detected in 7/13 patients at baseline, in NS3 (n = 3) or NS5A region (n = 4). One of them was treated with daclatasvir and experienced a relapse. Viral sequences from pre- and post-treatment samples of four patients who had relapsed after a sofosbuvir-based therapy were compared: the selected variants seem too far from NS5B active site to be held responsible. Although tested on a limited set of samples and with technical improvements still needing to be made, this assay has proved to be successful both for genotyping and mutation detection on several HCV types.
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Master Thesis
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  • HAL Id : dumas-01315019, version 1


Pauline Trémeaux. Étude du génome complet du virus de l'hépatite C par séquençage de nouvelle génération : mise au point et applications. Sciences pharmaceutiques. 2015. ⟨dumas-01315019⟩