Proyecto de Investigación: SAF2014-52400-R
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SAF2014-52400-R
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Amino acid substitutions associated with treatment failure for Hepatitis C virus infection
(American Society for Microbiology, 2020-12) Soria, María Eugenia; García Crespo, Carlos; Martínez González, Brenda; Vázquez Sirvent, L.; Lobo Vega, Rebeca; Ávila, Ana Isabel de; Gallego, Isabel; Chen, Qian; García Cehic, Damir; Llorens Revull, Meritxell; Briones, C.; Gómez, Jordi; Ferrer Orta, Cristina; Verdaguer, Nuria; Gregori, Josep; Rodríguez Frías, Francisco; Buti, María; Ignacio Esteban, Juan; Domingo, Esteban; Quer, Josep; Perales, Celia; Ministerio de Economía y Competitividad (MINECO); Agencia Estatal de Investigación (AEI); Instituto de Salud Carlos III (ISCIII); Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas, España; Centro para el Desarrollo Tecnológico Industrial (CDTI); Fundación Ramón Areces; Banco de Santander; CSIC-INTA - Centro de Astrobiología, CAB; Unidad de Excelencia Científica María de Maeztu Centro de Astrobiología del Instituto Nacional de Técnica Aeroespacial y CSIC, MDM-2017-0737
Despite the high virological response rates achieved with current directly acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral response. The identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultradeep sequencing (UDS) methods, for HCV characterization and patient management. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV, which are not bona fide resistance-associated substitutions (RAS), from 220 patients who failed therapy. They were present frequently in basal and posttreatment virus of patients who failed different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. They also have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value for treatment response, are discussed.
Dissimilar Conservation Pattern in Hepatitis C Virus Mutant Spectra, Consensus Sequences, and Data Banks
(Multidisciplinary Digital Publishing Institute (MDPI), 2020-10-27) García Crespo, Carlos; Eugenia Soria, M.; Gallego, Isabel; Isabel de Ávila, A.; Martínez González, B.; Vázquez Sirvent, L.; Gómez, Jordi; Briones, C.; Gregori, Josep; Quer, J.; Perales, C.; Domingo, Esteban; Ministerio de Economía y Competitividad (MINECO); Comunidad de Madrid; Agencia Estatal de Investigación (AEI); Instituto de Salud Carlos III (ISCIII); Fundación Ramón Areces; García Crespo, C. [0000-0001-6561-5389]; Soria Benito, M. E. [0000-0002-4719-3351]; Martínez González, B. [0000-0002-4482-5181]; Briones, C. [0000-0003-2213-8353]; Gregori, J. [0000-0002-4253-8015]; Quer, J. [0000-0003-0014-084X]; Perales, C. [0000-0003-1618-1937]; Unidad de Excelencia Científica María de Maeztu Centro de Astrobiología del Instituto Nacional de Técnica Aeroespacial y CSIC, MDM-2017-0737
The influence of quasispecies dynamics on long-term virus diversification in nature is a largely unexplored question. Specifically, whether intra-host nucleotide and amino acid variation in quasispecies fit the variation observed in consensus sequences or data bank alignments is unknown. Genome conservation and dynamics simulations are used for the computational design of universal vaccines, therapeutic antibodies and pan-genomic antiviral agents. The expectation is that selection of escape mutants will be limited when mutations at conserved residues are required. This strategy assumes long-term (epidemiologically relevant) conservation but, critically, does not consider short-term (quasispecies-dictated) residue conservation. We calculated mutant frequencies of individual loci from mutant spectra of hepatitis C virus (HCV) populations passaged in cell culture and from infected patients. Nucleotide or amino acid conservation in consensus sequences of the same populations, or in the Los Alamos HCV data bank did not match residue conservation in mutant spectra. The results relativize the concept of sequence conservation in viral genetics and suggest that residue invariance in data banks is an insufficient basis for the design of universal viral ligands for clinical purposes. Our calculations suggest relaxed mutational restrictions during quasispecies dynamics, which may contribute to higher calculated short-term than long-term viral evolutionary rates.
Population Disequilibrium as Promoter of Adaptive Explorations in Hepatitis C Virus
(Multidisciplinary Digital Publishing Institute (MDPI), 2021-04-03) García Crespo, Carlos; Gallego, Isabel; Eugenia Soria, M.; Isabel de Ávila, A.; Martínez González, B.; Vázquez Sirvent, L.; Lobo Vega, Rebeca; Moreno, E.; Gómez, Jordi; Briones, C.; Gregori, Josep; Quer, J.; Domingo, Esteban; Perales, C.; Banco Santander; Fundación Ramón Areces; Instituto de Salud Carlos III (ISCIII); Agencia Estatal de Investigación (AEI); Ministerio de Economía y Competitividad (MINECO); García Crespo, C. [0000-0001-6561-5389]; Martínez González, B. [0000-0002-4482-5181]; Moreno, E. [0000-0002-2301-4558]; Briones, C. [0000-0003-2213-8353]; Quer, J. [0000-0003-0014-084X]; Unidad de Excelencia Científica María de Maeztu Centro de Astrobiología del Instituto Nacional de Técnica Aeroespacial y CSIC, MDM-2017-0737
Replication of RNA viruses is characterized by exploration of sequence space which facilitates their adaptation to changing environments. It is generally accepted that such exploration takes place mainly in response to positive selection, and that further diversification is boosted by modifications of virus population size, particularly bottleneck events. Our recent results with hepatitis C virus (HCV) have shown that the expansion in sequence space of a viral clone continues despite prolonged replication in a stable cell culture environment. Diagnosis of the expansion was based on the quantification of diversity indices, the occurrence of intra-population mutational waves (variations in mutant frequencies), and greater individual residue variations in mutant spectra than those anticipated from sequence alignments in data banks. In the present report, we review our previous results, and show additionally that mutational waves in amplicons from the NS5A-NS5B-coding region are equally prominent during HCV passage in the absence or presence of the mutagenic nucleotide analogues favipiravir or ribavirin. In addition, by extending our previous analysis to amplicons of the NS3- and NS5A-coding region, we provide further evidence of the incongruence between amino acid conservation scores in mutant spectra from infected patients and in the Los Alamos National Laboratory HCV data banks. We hypothesize that these observations have as a common origin a permanent state of HCV population disequilibrium even upon extensive viral replication in the absence of external selective constraints or changes in population size. Such a persistent disequilibrium—revealed by the changing composition of the mutant spectrum—may facilitate finding alternative mutational pathways for HCV antiviral resistance. The possible significance of our model for other genetically variable viruses is discussed
