Pipeline for specific subtype amplification and drug resistance detection in hepatitis C virus.

Autor: Soria ME; Liver Unit, Internal Medicine Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain., Gregori J; Liver Unit, Internal Medicine Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain.; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain.; Roche Diagnostics, S.L, Sant Cugat del Vallés, Barcelona, Spain., Chen Q; Liver Unit, Internal Medicine Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain., García-Cehic D; Liver Unit, Internal Medicine Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain.; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain., Llorens M; Liver Unit, Internal Medicine Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain., de Ávila AI; Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, Madrid, Spain., Beach NM; Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, Madrid, Spain., Domingo E; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain.; Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, Madrid, Spain., Rodríguez-Frías F; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain.; Liver Pathology Unit, Department of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Barcelona, Spain.; Universitat Autónoma de Barcelona, Barcelona, Spain., Buti M; Liver Unit, Internal Medicine Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain.; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain.; Universitat Autónoma de Barcelona, Barcelona, Spain., Esteban R; Liver Unit, Internal Medicine Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain.; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain.; Universitat Autónoma de Barcelona, Barcelona, Spain., Esteban JI; Liver Unit, Internal Medicine Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain.; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain.; Universitat Autónoma de Barcelona, Barcelona, Spain., Quer J; Liver Unit, Internal Medicine Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain.; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain.; Universitat Autónoma de Barcelona, Barcelona, Spain., Perales C; Liver Unit, Internal Medicine Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Barcelona, Spain. celia.perales@vhir.org.; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain. celia.perales@vhir.org.; Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, Madrid, Spain. celia.perales@vhir.org.
Jazyk: angličtina
Zdroj: BMC infectious diseases [BMC Infect Dis] 2018 Sep 03; Vol. 18 (1), pp. 446. Date of Electronic Publication: 2018 Sep 03.
DOI: 10.1186/s12879-018-3356-6
Abstrakt: Background: Despite the high sustained virological response rates achieved with current directly-acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 5-10% of treated patients do not respond to current antiviral therapies, and basal resistance to DAAs is increasingly detected among treatment-naïve infected individuals. Identification of amino acid substitutions (including those in minority variants) associated with treatment failure requires analytical designs that take into account the high diversification of HCV in more than 86 subtypes according to the ICTV website (June 2017).
Methods: The methodology has involved five sequential steps: (i) to design 280 oligonucleotide primers (some including a maximum of three degenerate positions), and of which 120 were tested to amplify NS3, NS5A-, and NS5B-coding regions in a subtype-specific manner, (ii) to define a reference sequence for each subtype, (iii) to perform experimental controls to define a cut-off value for detection of minority amino acids, (iv) to establish bioinformatics' tools to quantify amino acid replacements, and (v) to validate the procedure with patient samples.
Results: A robust ultra-deep sequencing procedure to analyze HCV circulating in serum samples from patients infected with virus that belongs to the ten most prevalent subtypes worldwide: 1a, 1b, 2a, 2b, 2c, 2j, 3a, 4d, 4e, 4f has been developed. Oligonucleotide primers are subtype-specific. A cut-off value of 1% mutant frequency has been established for individual mutations and haplotypes.
Conclusion: The methodological pipeline described here is adequate to characterize in-depth mutant spectra of HCV populations, and it provides a tool to understand HCV diversification and treatment failures. The pipeline can be periodically extended in the event of HCV diversification into new genotypes or subtypes, and provides a framework applicable to other RNA viral pathogens, with potential to couple detection of drug-resistant mutations with treatment planning.
Databáze: MEDLINE
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