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1. Reply to Qu et al., “Quasispecies are constantly selected through virus-encoded intracellular reproductive population bottlenecking”

Author

Ministerio de Ciencia e Innovación (España), Instituto de Salud Carlos III, Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Comunidad de Madrid, Ministerio de Ciencia, Innovación y Universidades (España), Domingo, Esteban, Martínez-González, Brenda, García-Crespo, Carlos, Somovilla, Pilar, Ávila, Ana Isabel de, Soria, María Eugenia, Durán-Pastor, Antoni, Perales, Celia, Ministerio de Ciencia e Innovación (España), Instituto de Salud Carlos III, Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Comunidad de Madrid, Ministerio de Ciencia, Innovación y Universidades (España), Domingo, Esteban, Martínez-González, Brenda, García-Crespo, Carlos, Somovilla, Pilar, Ávila, Ana Isabel de, Soria, María Eugenia, Durán-Pastor, Antoni, and Perales, Celia

Abstract

We appreciate the careful reading by F. Qu, K. Khemsom, C. Perdoncini Carvalho, and J. Han of our minireview on SARS-CoV-2 quasispecies recently published in the Journal of Virology (1). Qu and colleagues disagree with one of our assertions about viral quasispecies in general, namely that the mutant genome copies generated during viral RNA replication can collectively engage in evolution. Their main argument is that when a viral population enters a cell, it becomes strongly bottlenecked, and only one or a few of the viruses have a chance to replicate. The remaining copies are degraded, although they may contribute to the cellular modifications required for the formation of replication organelles. An advantage of such bottlenecking for virus survival is that genomes that, during prior replication, mutated to encode a different phenotype find an unchallenged cellular environment to feely multiply and express the deviant phenotype. This model is termed “Bottleneck, Isolate, Amplify, Select” (BIAS), and it is presented as contrary to the premise of quasispecies theory because “the BIAS model is unapologetically deterministic and predicts natural selection as the primary driver of virus evolution.”

Published
2024

2. Puzzles, challenges, and information reservoir of SARS-CoV-2 quasispecies

Author

Ministerio de Ciencia e Innovación (España), Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Instituto de Salud Carlos III, Fundació La Marató de TV3, Comunidad de Madrid, Fundación Ramón Areces, Fundación Banco Santander, Domingo, Esteban [0000-0002-0573-1676], Perales, Celia [0000-0003-1618-1937], Domingo, Esteban, Martínez-González, Brenda, García-Crespo, Carlos, Somovilla, Pilar, Ávila, Ana Isabel de, Soria, María Eugenia, Durán-Pastor, Antoni, Perales, Celia, Ministerio de Ciencia e Innovación (España), Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Instituto de Salud Carlos III, Fundació La Marató de TV3, Comunidad de Madrid, Fundación Ramón Areces, Fundación Banco Santander, Domingo, Esteban [0000-0002-0573-1676], Perales, Celia [0000-0003-1618-1937], Domingo, Esteban, Martínez-González, Brenda, García-Crespo, Carlos, Somovilla, Pilar, Ávila, Ana Isabel de, Soria, María Eugenia, Durán-Pastor, Antoni, and Perales, Celia

Abstract

Upon the emergence of SARS-CoV-2 in the human population, it was conjectured that for this coronavirus the dynamic intra-host heterogeneity typical of RNA viruses would be toned down. Nothing of this sort is observed. Here we review the main observations on the complexity and diverse composition of SARS-CoV-2 mutant spectra sampled from infected patients, within the framework of quasispecies dynamics. The analyses suggest that the information provided by myriads of genomic sequences within infected individuals may have a predictive value of the genomic sequences that acquire epidemiological relevance. Possibilities to reconcile the presence of broad mutant spectra in the large RNA coronavirus genome with its encoding a 3' to 5' exonuclease proofreading-repair activity are considered. Indeterminations in the behavior of individual viral genomes provide a benefit for the survival of the ensemble. We propose that this concept falls in the domain of "stochastic thinking," a notion that applies also to cellular processes, as a means for biological systems to face unexpected needs.

Published
2023

3. Atypical Mutational Spectrum of SARS-CoV-2 Replicating in the Presence of Ribavirin

Author

Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (España), Consejo Superior de Investigaciones Científicas (España), European Commission, Fundació La Marató de TV3, Agencia Estatal de Investigación (España), Ministerio de Ciencia, Innovación y Universidades (España), Comunidad de Madrid, Ministerio de Economía y Competitividad (España), Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Fundación Ramón Areces, Banco Santander, Somovilla, Pilar, García-Crespo, Carlos, Martínez-González, Brenda, Soria, María Eugenia, Ávila, Ana Isabel de, Gallego, Isabel, Mínguez, Pablo, Durán-Pastor, Antoni, Ferrer-Orta, Cristina, Salar-Vidal, Llanos, Esteban-Muñoz, Mario, Zúñiga Lucas, Sonia, Solá Gurpegui, Isabel, Enjuanes Sánchez, Luis, Esteban, Jaime, Fernandez-Roblas, Ricardo, Gadea, Ignacio, Gómez-Castilla, Jordi, Verdaguer, Núria, Domingo, Esteban, Perales, Celia, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (España), Consejo Superior de Investigaciones Científicas (España), European Commission, Fundació La Marató de TV3, Agencia Estatal de Investigación (España), Ministerio de Ciencia, Innovación y Universidades (España), Comunidad de Madrid, Ministerio de Economía y Competitividad (España), Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Fundación Ramón Areces, Banco Santander, Somovilla, Pilar, García-Crespo, Carlos, Martínez-González, Brenda, Soria, María Eugenia, Ávila, Ana Isabel de, Gallego, Isabel, Mínguez, Pablo, Durán-Pastor, Antoni, Ferrer-Orta, Cristina, Salar-Vidal, Llanos, Esteban-Muñoz, Mario, Zúñiga Lucas, Sonia, Solá Gurpegui, Isabel, Enjuanes Sánchez, Luis, Esteban, Jaime, Fernandez-Roblas, Ricardo, Gadea, Ignacio, Gómez-Castilla, Jordi, Verdaguer, Núria, Domingo, Esteban, and Perales, Celia

Abstract

We report that ribavirin exerts an inhibitory and mutagenic activity on SARS-CoV-2-infecting Vero cells, with a therapeutic index higher than 10. Deep sequencing analysis of the mutant spectrum of SARS-CoV-2 replicating in the absence or presence of ribavirin indicated an increase in the number of mutations, but not in deletions, and modification of diversity indices, expected from a mutagenic activity. Notably, the major mutation types enhanced by replication in the presence of ribavirin were A→G and U→C transitions, a pattern which is opposite to the dominance of G→A and C→U transitions previously described for most RNA viruses. Implications of the inhibitory activity of ribavirin, and the atypical mutational bias produced on SARS-CoV-2, for the search for synergistic anti-COVID-19 lethal mutagen combinations are discussed.

Published
2023

4. SARS-CoV-2 mutant spectra reveal differences between COVID-19 severity categories

Author

Martínez-González, Brenda, Soria, María Eugenia, Vázquez-Sirvent, Lucía, Ferrer-Orta, Cristina, Lobo-Vega, Rebeca, Mínguez, Pablo, Fuente, Lorena de la, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruíz, Ricardo, Cortón, Marta, López-Rodríguez, Rosario, García-Crespo, Carlos, Somovilla, Pilar, Durán-Pastor, Antoni, Gallego, Isabel, Ávila, Ana Isabel de, Delgado, Soledad, Morán, Federico, López-Galíndez, Cecilio, Gómez, Jordi, Enjuanes Sánchez, Luis, Salar-Vidal, Llanos, Esteban-Muñoz, Mario, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Verdaguer, Núria, Domingo, Esteban, Perales, Celia, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (España), Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Agencia Estatal de Investigación (España), Ministerio de Ciencia, Innovación y Universidades (España), Comunidad de Madrid, European Commission, Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Fundación Ramón Areces, Banco Santander, Ministerio de Sanidad y Consumo (España), and Ministerio de Economía y Competitividad (España)

Abstract

Trabajo presentado en el XVI Congreso Nacional de Virología, celebrado en Málaga (España) del 06 al 09 de septiembre de 2022., RNA virus populations are composed of complex mixtures of genomes that are termed mutant spectra. SARS-CoV-2 replicates as a viral quasispecies, and mutations that are detected at low frequencies in a host can be dominant in subsequent variants. We have studied mutant spectrum complexities of SARS-CoV-2 populations derived from thirty nasopharyngeal swabs of patients infected during the first wave (April 2020) in the Hospital Universitario Fundación Jiménez Díaz. The patients were classified according to the COVID-19 severity in mild (non-hospitalized), moderate (hospitalized) and exitus (hospitalized with ICU admission and who passed away due to COVID-19). Using ultra-deep sequencing technologies (MiSeq, Illumina), we have examined four amplicons of the nsp12 (polymerase)-coding region and two amplicons of the spike-coding region. Ultra-deep sequencing data were analyzed with different cut-off frequency for mutation detection. Average number of different point mutations, mutations per haplotype and several diversity indices were significantly higher in SARS-CoV-2 isolated from patients who developed mild disease. A feature that we noted in the SARS-CoV-2 mutant spectra from diagnostic samples is the remarkable absence of mutations at intermediate frequencies, and an overwhelming abundance of mutations at frequencies lower than 10%. Thus, the decrease of the cut-off frequency for mutation detection from 0.5% to 0.1% revealed an increasement (50- to 100 fold) in the number of different mutations. The significantly higher frequency of mutations in virus from patients displaying mild than moderate or severe disease was maintained with the 0.1% cut- off frequency. To evaluate whether the frequency repertoire of amino acid substitutions differed between SARS-CoV-2 and the well characterized hepatitis C virus (HCV), we performed a comparative study of mutant spectra from infected patients using the same bioinformatics pipelines. HCV did not show the deficit of intermediate frequency substitutions that was observed with SARS-CoV-2. This difference was maintained when two functionally equivalent proteins, the corresponding viral polymerases, were compared. In conclusion, SARS-CoV-2 mutant spectra are rich reservoirs of mutants, whose complexity is not uniform among clinical isolates. Virus from patients who developed mild disease may be a source of new variants that may acquire epidemiological relevance., This work was supported by Instituto de Salud Carlos III, Spanish Ministry of Science and In-novation (COVID-19 Research Call COV20/00181), and co-financed by European Development Regional Fund ‘A way to achieve Europe’. The work was also supported by grants CSIC-COV19-014 from Consejo Superior de Investigaciones Científicas (CSIC), project 525/C/2021 from Fundació La Marató de TV3, PID2020-113888RB-I00 from Ministerio de Ciencia e Innovación, BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MCIU), PI18/00210 and PI21/00139 from Instituto de Salud Carlos III, and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P., M.C., and P.M. are supported by the Miguel Servet programme of the Instituto de Salud Carlos III (CPII19/00001, CPII17/00006, and CP16/00116, respectively) co-financed by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investi-gación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo) cofinanced by Fondo Social Europeo (FSE). R.L.-V. is supported by predoctoral contract PEJD-2019-PRE/BMD-16414 from Comunidad de Madrid. C.G.-C. is sup-ported by predoctoral contract PRE2018-083422 from MCIU. BS was supported by a predoctoral research fellowship (Doctorados Industriales, DI-17-09134) from Spanish MINECO.

Published
2022

5. Amino acid substitutions associated with treatment failure of hepatitis C virus infection

Author

Soria, María Eugenia, García-Crespo, Carlos, Martínez-González, Brenda, Vázquez-Sirvent, Lucía, Lobo-Vega, Rebeca, Ávila, Ana Isabel de, Gallego, Isabel, Chen, Qian, García-Cehic, Damir, Llorens-Revull, Meritxell, Briones, Carlos, Gómez, Jordi, Ferrer-Orta, Cristina, Verdaguer, Núria, Gregori, Josep, Rodríguez-Frías, Francisco, Buti, María, Esteban, Juan Ignacio, Domingo, Esteban, Quer, Josep, Perales, Celia, Ministerio de Economía y Competitividad (España), Agencia Estatal de Investigación (España), Comunidad de Madrid, Instituto de Salud Carlos III, Fundación Ramón Areces, Banco Santander, Global Virus Network, Centro para el Desarrollo Tecnológico Industrial (España), and Ministerio de Ciencia, Innovación y Universidades (España)

Abstract

Trabajo presentado en el XVI Congreso Nacional de Virología, celebrado en Málaga (España) del 06 al 09 de septiembre de 2022., Despite the high sustained virological response rates achieved with current directly-acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of patients do not achieve such a response. Identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultra-deep sequencing (UDS) methods for HCV characterization and patient management. By deep sequencing analysis of 220 subtyped HCV samples from infected patients who failed therapy, collected from 39 Spanish hospitals, we determined amino acid sequences of the DAA-target proteins NS3, NS5A and NS5B, by UDS of HCV patient samples, in search of resistanceassociated substitutions (RAS). Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV, which are not bona fide RAS. They were present frequently in basal and post-treatment virus of patients who failed therapy to different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Coherently, 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. Also, they have limited predicted disruptive effects on the threedimensional structures of the proteins harboring them. The information on HRSs that will be gathered during sequencing should be relevant not only to help predict treatment outcomes and disease progression but also to further understand HCV population dynamics, which appears much more complex than thought prior to the introduction of deep sequencing., The work at CBMSO was supported by grants SAF2014-52400-R from MINECO, SAF2017-87846-R and BFU2017-91384-EXP MICIU, PI18/00210 from ISCIII, S2013/ABI-2906 (PLATESA) and S2018/BAA-4370 (PLATESA2) from Comunidad de Madrid/FEDER. C.P. is supported by the Miguel Servet program of the ISCIII (CP14/00121 and CPII19/00001), cofinanced by the European Regional Development Fund (ERDF). CIBERehd is funded by ISCIII. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). The work in Barcelona was supported by ISCIII, cofinanced by ERDF grant number PI19/00301 and by the Centro para el Desarrollo Tecnológico Industrial (CDTI) from the MICIU, grant number IDI20151125. Work at CAB was supported by MINECO grant BIO2016-79618R and PID2019-104903RB-I00 (funded by the EU under the FEDER program) and by the Spanish State research agency (AEI) through project number MDM-2017-0737 Unidad de Excelencia “María de Maeztu”-Centro de Astrobiología (CSIC-INTA). Work at IBMB was supported by MICIN grant BIO2017-83906-P (funded by the EU under the FEDER program). C.G.-C. is supported by predoctoral contract PRE2018-083422 from MICIU. B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo), cofinanced by Fondo Social Europeo (FSE).

Published
2022

6. Vaccine breakthrough infections with SARS-CoV-2 Alpha mirror mutations in Delta Plus, Iota, and Omicron

Author

Instituto de Salud Carlos III, Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Comunidad de Madrid, European Commission, Fundación Ramón Areces, Banco Santander, Ministerio de Sanidad y Consumo (España), Ministerio de Economía y Competitividad (España), Martínez-González, Brenda, Vázquez-Sirvent, Lucía, Soria, María Eugenia, Mínguez, Pablo, Salar-Vidal, Llanos, García-Crespo, Carlos, Gallego, Isabel, Ávila, Ana Isabel de, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Pérez-Jorge, Concepción, Domingo, Esteban, Perales, Celia, Instituto de Salud Carlos III, Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Comunidad de Madrid, European Commission, Fundación Ramón Areces, Banco Santander, Ministerio de Sanidad y Consumo (España), Ministerio de Economía y Competitividad (España), Martínez-González, Brenda, Vázquez-Sirvent, Lucía, Soria, María Eugenia, Mínguez, Pablo, Salar-Vidal, Llanos, García-Crespo, Carlos, Gallego, Isabel, Ávila, Ana Isabel de, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Pérez-Jorge, Concepción, Domingo, Esteban, and Perales, Celia

Abstract

Replication of SARS-CoV-2 in the human population is defined by distributions of mutants that are present at different frequencies within the infected host and can be detected by ultra-deep sequencing techniques. In this study, we examined the SARS-CoV-2 mutant spectra of amplicons from the spike-coding (S-coding) region of 5 nasopharyngeal isolates derived from patients with vaccine breakthrough. Interestingly, all patients became infected with the Alpha variant, but amino acid substitutions that correspond to the Delta Plus, Iota, and Omicron variants were present in the mutant spectra of the resident virus. Deep sequencing analysis of SARS-CoV-2 from patients with vaccine breakthrough revealed a rich reservoir of mutant types and may also identify tolerated substitutions that can be represented in epidemiologically dominant variants.

Published
2022

7. Efficacy decrease of antiviral agents when administered to ongoing hepatitis C virus infections in cell culture

Author

Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (España), Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Comunidad de Madrid, European Commission, Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Fundación Ramón Areces, Banco Santander, García-Crespo, Carlos, Vázquez-Sirvent, Lucía, Somovilla, Pilar, Soria, María Eugenia, Gallego, Isabel, Ávila, Ana Isabel de, Martínez-González, Brenda, Durán-Pastor, Antoni, Domingo, Esteban, Perales, Celia, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (España), Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Comunidad de Madrid, European Commission, Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Fundación Ramón Areces, Banco Santander, García-Crespo, Carlos, Vázquez-Sirvent, Lucía, Somovilla, Pilar, Soria, María Eugenia, Gallego, Isabel, Ávila, Ana Isabel de, Martínez-González, Brenda, Durán-Pastor, Antoni, Domingo, Esteban, and Perales, Celia

Abstract

We report a quantification of the decrease of effectiveness of antiviral agents directed to hepatitis C virus, when the agents are added during an ongoing infection in cell culture vs. when they are added at the beginning of the infection. Major determinants of the decrease of inhibitory activity are the time post-infection of inhibitor administration and viral replicative fitness. The efficacy decrease has been documented with antiviral assays involving the combination of the direct-acting antiviral agents, daclatasvir and sofosbuvir, and with the combination of the lethal mutagens, favipiravir and ribavirin. The results suggest that strict antiviral effectiveness assays in preclinical trials may involve the use of high fitness viral populations and the delayed administration of the agents, relative to infection onset.

Published
2022

8. Guanosine inhibits hepatitis C virus replication and increases indel frequencies, associated with altered intracellular nucleotide pools

Author

Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Fundació La Marató de TV3, Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Ministerio de Economía y Competitividad (España), Comunidad de Madrid, Junta de Comunidades de Castilla-La Mancha, Junta de Andalucía, Universidad de Málaga, Sabariegos, Rosario, Ortega-Prieto, Ana M., Díaz-Martínez, Luis, Grande-Pérez, Ana, García-Crespo, Carlos, Gallego, Isabel, Ávila, Ana Isabel de, Albentosa González, Laura, Soria, María Eugenia, Gastaminza, Pablo, Domingo, Esteban, Perales, Celia, Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Fundació La Marató de TV3, Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Ministerio de Economía y Competitividad (España), Comunidad de Madrid, Junta de Comunidades de Castilla-La Mancha, Junta de Andalucía, Universidad de Málaga, Sabariegos, Rosario, Ortega-Prieto, Ana M., Díaz-Martínez, Luis, Grande-Pérez, Ana, García-Crespo, Carlos, Gallego, Isabel, Ávila, Ana Isabel de, Albentosa González, Laura, Soria, María Eugenia, Gastaminza, Pablo, Domingo, Esteban, and Perales, Celia

Abstract

In the course of experiments aimed at deciphering the inhibition mechanism of mycophenolic acid and ribavirin in hepatitis C virus (HCV) infection, we observed an inhibitory effect of the nucleoside guanosine (Gua). Here, we report that Gua, and not the other standard nucleosides, inhibits HCV replication in human hepatoma cells. Gua did not directly inhibit the in vitro polymerase activity of NS5B, but it modified the intracellular levels of nucleoside di- and tri-phosphates (NDPs and NTPs), leading to deficient HCV RNA replication and reduction of infectious progeny virus production. Changes in the concentrations of NTPs or NDPs modified NS5B RNA polymerase activity in vitro, in particular de novo RNA synthesis and template switching. Furthermore, the Gua-mediated changes were associated with a significant increase in the number of indels in viral RNA, which may account for the reduction of the specific infectivity of the viral progeny, suggesting the presence of defective genomes. Thus, a proper NTP:NDP balance appears to be critical to ensure HCV polymerase fidelity and minimal production of defective genomes.

Published
2022

9. Amino acid substitutions associated with treatment failure of hepatitis C virus infection

Author

Ministerio de Economía y Competitividad (España), Agencia Estatal de Investigación (España), Comunidad de Madrid, Instituto de Salud Carlos III, Fundación Ramón Areces, Banco Santander, Global Virus Network, Centro para el Desarrollo Tecnológico Industrial (España), Ministerio de Ciencia, Innovación y Universidades (España), Soria, María Eugenia, García-Crespo, Carlos, Martínez-González, Brenda, Vázquez-Sirvent, Lucía, Lobo-Vega, Rebeca, Ávila, Ana Isabel de, Gallego, Isabel, Chen, Qian, García-Cehic, Damir, Llorens-Revull, Meritxell, Briones, Carlos, Gómez-Castilla, Jordi, Ferrer-Orta, Cristina, Verdaguer, Núria, Gregori, Josep, Rodríguez-Frías, Francisco, Buti, María, Esteban, Juan Ignacio, Domingo, Esteban, Quer, Josep, Perales, Celia, Ministerio de Economía y Competitividad (España), Agencia Estatal de Investigación (España), Comunidad de Madrid, Instituto de Salud Carlos III, Fundación Ramón Areces, Banco Santander, Global Virus Network, Centro para el Desarrollo Tecnológico Industrial (España), Ministerio de Ciencia, Innovación y Universidades (España), Soria, María Eugenia, García-Crespo, Carlos, Martínez-González, Brenda, Vázquez-Sirvent, Lucía, Lobo-Vega, Rebeca, Ávila, Ana Isabel de, Gallego, Isabel, Chen, Qian, García-Cehic, Damir, Llorens-Revull, Meritxell, Briones, Carlos, Gómez-Castilla, Jordi, Ferrer-Orta, Cristina, Verdaguer, Núria, Gregori, Josep, Rodríguez-Frías, Francisco, Buti, María, Esteban, Juan Ignacio, Domingo, Esteban, Quer, Josep, and Perales, Celia

Abstract

Despite the high sustained virological response rates achieved with current directly-acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of patients do not achieve such a response. Identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultra-deep sequencing (UDS) methods for HCV characterization and patient management. By deep sequencing analysis of 220 subtyped HCV samples from infected patients who failed therapy, collected from 39 Spanish hospitals, we determined amino acid sequences of the DAA-target proteins NS3, NS5A and NS5B, by UDS of HCV patient samples, in search of resistanceassociated substitutions (RAS). Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV, which are not bona fide RAS. They were present frequently in basal and post-treatment virus of patients who failed therapy to different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Coherently, 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. Also, they have limited predicted disruptive effects on the threedimensional structures of the proteins harboring them. The information on HRSs that will be gathered during sequencing should be relevant not only to help predict treatment outcomes and disease progression but also to further understand HCV population dynamics, which appears much more complex than thought prior to the introduction of deep sequencing.

Published
2022

10. SARS-CoV-2 mutant spectra reveal differences between COVID-19 severity categories

Author

Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (España), Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Agencia Estatal de Investigación (España), Ministerio de Ciencia, Innovación y Universidades (España), Comunidad de Madrid, European Commission, Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Fundación Ramón Areces, Banco Santander, Ministerio de Sanidad y Consumo (España), Ministerio de Economía y Competitividad (España), Martínez-González, Brenda, Soria, María Eugenia, Vázquez-Sirvent, Lucía, Ferrer-Orta, Cristina, Lobo-Vega, Rebeca, Mínguez, Pablo, Fuente, Lorena de la, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Cortón, Marta, López-Rodríguez, Rosario, García-Crespo, Carlos, Somovilla, Pilar, Durán-Pastor, Antoni, Gallego, Isabel, Ávila, Ana Isabel de, Delgado, Soledad, Morán, Federico, López-Galíndez, Cecilio, Gómez-Castilla, Jordi, Enjuanes Sánchez, Luis, Salar-Vidal, Llanos, Esteban-Muñoz, Mario, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Verdaguer, Núria, Domingo, Esteban, Perales, Celia, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (España), Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Agencia Estatal de Investigación (España), Ministerio de Ciencia, Innovación y Universidades (España), Comunidad de Madrid, European Commission, Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Fundación Ramón Areces, Banco Santander, Ministerio de Sanidad y Consumo (España), Ministerio de Economía y Competitividad (España), Martínez-González, Brenda, Soria, María Eugenia, Vázquez-Sirvent, Lucía, Ferrer-Orta, Cristina, Lobo-Vega, Rebeca, Mínguez, Pablo, Fuente, Lorena de la, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Cortón, Marta, López-Rodríguez, Rosario, García-Crespo, Carlos, Somovilla, Pilar, Durán-Pastor, Antoni, Gallego, Isabel, Ávila, Ana Isabel de, Delgado, Soledad, Morán, Federico, López-Galíndez, Cecilio, Gómez-Castilla, Jordi, Enjuanes Sánchez, Luis, Salar-Vidal, Llanos, Esteban-Muñoz, Mario, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Verdaguer, Núria, Domingo, Esteban, and Perales, Celia

Abstract

RNA virus populations are composed of complex mixtures of genomes that are termed mutant spectra. SARS-CoV-2 replicates as a viral quasispecies, and mutations that are detected at low frequencies in a host can be dominant in subsequent variants. We have studied mutant spectrum complexities of SARS-CoV-2 populations derived from thirty nasopharyngeal swabs of patients infected during the first wave (April 2020) in the Hospital Universitario Fundación Jiménez Díaz. The patients were classified according to the COVID-19 severity in mild (non-hospitalized), moderate (hospitalized) and exitus (hospitalized with ICU admission and who passed away due to COVID-19). Using ultra-deep sequencing technologies (MiSeq, Illumina), we have examined four amplicons of the nsp12 (polymerase)-coding region and two amplicons of the spike-coding region. Ultra-deep sequencing data were analyzed with different cut-off frequency for mutation detection. Average number of different point mutations, mutations per haplotype and several diversity indices were significantly higher in SARS-CoV-2 isolated from patients who developed mild disease. A feature that we noted in the SARS-CoV-2 mutant spectra from diagnostic samples is the remarkable absence of mutations at intermediate frequencies, and an overwhelming abundance of mutations at frequencies lower than 10%. Thus, the decrease of the cut-off frequency for mutation detection from 0.5% to 0.1% revealed an increasement (50- to 100 fold) in the number of different mutations. The significantly higher frequency of mutations in virus from patients displaying mild than moderate or severe disease was maintained with the 0.1% cut- off frequency. To evaluate whether the frequency repertoire of amino acid substitutions differed between SARS-CoV-2 and the well characterized hepatitis C virus (HCV), we performed a comparative study of mutant spectra from infected patients using the same bioinformatics pipelines. HCV did not show the deficit of intermediat

Published
2022

11. SARS-CoV-2 Mutant Spectra at Different Depth Levels Reveal an Overwhelming Abundance of Low Frequency Mutations

Author

Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación y Universidades (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Agencia Estatal de Investigación (España), Comunidad de Madrid, Fundación Ramón Areces, Banco Santander, Ministerio de Sanidad y Consumo (España), Ministerio de Economía y Competitividad (España), Martínez-González, Brenda [0000-0002-4482-5181], Soria, María Eugenia [0000-0002-4719-3351], Lobo-Vega, Rebeca [0000-0002-4882-6763], Mínguez, Pablo [0000-0003-4099-9421], Llorens, Carlos [0000-0003-1402-4743], Ramos-Ruiz, Ricardo [0000-0002-6331-9786], Cortón, Marta [0000-0003-0087-1626], García-Crespo, Carlos [0000-0001-6561-5389], Durán-Pastor, Antoni [0000-0002-4248-0554], Delgado, Soledad [0000-0003-4868-3712], López-Galíndez, Cecilio [0000-0002-2324-9584], Enjuanes Sánchez, Luis [0000-0002-0854-0226], Salar-Vidal, Llanos [0000-0002-3052-0176], Esteban, Jaime [0000-0002-8971-3167], Ayuso, Carmen [0000-0002-9242-7065], Verdaguer, Núria [0000-0001-8826-7129], Martínez-González, Brenda, Soria, María Eugenia, Vázquez-Sirvent, Lucía, Ferrer-Orta, Cristina, Lobo-Vega, Rebeca, Mínguez, Pablo, Fuente, Lorena de la, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Cortón, Marta, López-Rodríguez, Rosario, García-Crespo, Carlos, Somovilla, Pilar, Durán-Pastor, Antoni, Gallego, Isabel, Ávila, Ana Isabel de, Delgado, Soledad, Morán, Federico, López-Galíndez, Cecilio, Gómez-Castilla, Jordi, Enjuanes Sánchez, Luis, Salar-Vidal, Llanos, Esteban-Muñoz, Mario, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Verdaguer, Núria, Domingo, Esteban, Perales, Celia, Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación y Universidades (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Agencia Estatal de Investigación (España), Comunidad de Madrid, Fundación Ramón Areces, Banco Santander, Ministerio de Sanidad y Consumo (España), Ministerio de Economía y Competitividad (España), Martínez-González, Brenda [0000-0002-4482-5181], Soria, María Eugenia [0000-0002-4719-3351], Lobo-Vega, Rebeca [0000-0002-4882-6763], Mínguez, Pablo [0000-0003-4099-9421], Llorens, Carlos [0000-0003-1402-4743], Ramos-Ruiz, Ricardo [0000-0002-6331-9786], Cortón, Marta [0000-0003-0087-1626], García-Crespo, Carlos [0000-0001-6561-5389], Durán-Pastor, Antoni [0000-0002-4248-0554], Delgado, Soledad [0000-0003-4868-3712], López-Galíndez, Cecilio [0000-0002-2324-9584], Enjuanes Sánchez, Luis [0000-0002-0854-0226], Salar-Vidal, Llanos [0000-0002-3052-0176], Esteban, Jaime [0000-0002-8971-3167], Ayuso, Carmen [0000-0002-9242-7065], Verdaguer, Núria [0000-0001-8826-7129], Martínez-González, Brenda, Soria, María Eugenia, Vázquez-Sirvent, Lucía, Ferrer-Orta, Cristina, Lobo-Vega, Rebeca, Mínguez, Pablo, Fuente, Lorena de la, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Cortón, Marta, López-Rodríguez, Rosario, García-Crespo, Carlos, Somovilla, Pilar, Durán-Pastor, Antoni, Gallego, Isabel, Ávila, Ana Isabel de, Delgado, Soledad, Morán, Federico, López-Galíndez, Cecilio, Gómez-Castilla, Jordi, Enjuanes Sánchez, Luis, Salar-Vidal, Llanos, Esteban-Muñoz, Mario, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Verdaguer, Núria, Domingo, Esteban, and Perales, Celia

Abstract

Populations of RNA viruses are composed of complex and dynamic mixtures of variant genomes that are termed mutant spectra or mutant clouds. This applies also to SARS-CoV-2, and mutations that are detected at low frequency in an infected individual can be dominant (represented in the consensus sequence) in subsequent variants of interest or variants of concern. Here we briefly review the main conclusions of our work on mutant spectrum characterization of hepatitis C virus (HCV) and SARS-CoV-2 at the nucleotide and amino acid levels and address the following two new questions derived from previous results: (i) how is the SARS-CoV-2 mutant and deletion spectrum composition in diagnostic samples, when examined at progressively lower cut-off mutant frequency values in ultra-deep sequencing; (ii) how the frequency distribution of minority amino acid substitutions in SARS-CoV-2 compares with that of HCV sampled also from infected patients. The main conclusions are the following: (i) the number of different mutations found at low frequency in SARS-CoV-2 mutant spectra increases dramatically (50- to 100-fold) as the cut-off frequency for mutation detection is lowered from 0.5% to 0.1%, and (ii) that, contrary to HCV, SARS-CoV-2 mutant spectra exhibit a deficit of intermediate frequency amino acid substitutions. The possible origin and implications of mutant spectrum differences among RNA viruses are discussed.

Published
2022

12. SARS-CoV-2 Point Mutation and Deletion Spectra and Their Association with Different Disease Outcomes

Author

Ramos-Ruiz, Ricardo [0000-0002-6331-9786], Enjuanes Sánchez, Luis [0000-0002-0854-0226], Esteban, Jaime [0000-0002-8971-3167], Verdaguer, Núria [0000-0001-8826-7129], Domingo, Esteban [0000-0002-0573-1676], Perales, Celia [0000-0003-1618-1937], Martínez-González, Brenda, Soria, María Eugenia, Vázquez-Sirvent, Lucía, Ferrer-Orta, Cristina, Lobo-Vega, Rebeca, Mínguez, Pablo, Fuente, Lorena de la, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Cortón, Marta, López-Rodríguez, Rosario, García-Crespo, Carlos, Gallego, Isabel, Ávila, Ana Isabel de, Gómez-Castilla, Jordi, Enjuanes Sánchez, Luis, Salar-Vidal, Llanos, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Verdaguer, Núria, Domingo, Esteban, Perales, Celia, Ramos-Ruiz, Ricardo [0000-0002-6331-9786], Enjuanes Sánchez, Luis [0000-0002-0854-0226], Esteban, Jaime [0000-0002-8971-3167], Verdaguer, Núria [0000-0001-8826-7129], Domingo, Esteban [0000-0002-0573-1676], Perales, Celia [0000-0003-1618-1937], Martínez-González, Brenda, Soria, María Eugenia, Vázquez-Sirvent, Lucía, Ferrer-Orta, Cristina, Lobo-Vega, Rebeca, Mínguez, Pablo, Fuente, Lorena de la, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Cortón, Marta, López-Rodríguez, Rosario, García-Crespo, Carlos, Gallego, Isabel, Ávila, Ana Isabel de, Gómez-Castilla, Jordi, Enjuanes Sánchez, Luis, Salar-Vidal, Llanos, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Verdaguer, Núria, Domingo, Esteban, and Perales, Celia

Abstract

Mutant spectra of RNA viruses are important to understand viral pathogenesis and response to selective pressures. There is a need to characterize the complexity of mutant spectra in coronaviruses sampled from infected patients. In particular, the possible relationship between SARS-CoV-2 mutant spectrum complexity and disease associations has not been established. In the present study, we report an ultradeep sequencing (UDS) analysis of the mutant spectrum of amplicons from the nsp12 (polymerase)- and spike (S)-coding regions of 30 nasopharyngeal isolates (diagnostic samples) of SARS-CoV-2 of the first COVID-19 pandemic wave (Madrid, Spain, April 2020) classified according to the severity of ensuing COVID-19. Low-frequency mutations and deletions, counted relative to the consensus sequence of the corresponding isolate, were overwhelmingly abundant. We show that the average number of different point mutations, mutations per haplotype, and several diversity indices was significantly higher in SARS-CoV-2 isolated from patients who developed mild disease than in those associated with moderate or severe disease (exitus). No such bias was observed with RNA deletions. Location of amino acid substitutions in the three-dimensional structures of nsp12 (polymerase) and S suggest significant structural or functional effects. Thus, patients who develop mild symptoms may be a richer source of genetic variants of SARS-CoV-2 than patients with moderate or severe COVID-19.

Published
2022

13. SARS-CoV-2 Point Mutation and Deletion Spectra, and Their Association with Different Disease Outcome

Author

Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Instituto de Salud Carlos III, Comunidad de Madrid, Fundación Ramón Areces, Banco Santander, Ministerio de Sanidad y Consumo (España), Ministerio de Economía y Competitividad (España), Ramos-Ruiz, Ricardo [0000-0002-6331-9786], Enjuanes Sánchez, Luis [0000-0002-0854-0226], Esteban, Jaime [0000-0002-8971-3167], Verdaguer, Núria [0000-0001-8826-7129], Domingo, Esteban [0000-0002-0573-1676], Perales, Celia [0000-0003-1618-1937], Martínez-González, Brenda, Soria, María Eugenia, Vázquez-Sirvent, Lucía, Ferrer-Orta, Cristina, Lobo-Vega, Rebeca, Mínguez, Pablo, Fuente, Lorena de la, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Cortón, Marta, López-Rodríguez, Rosario, García-Crespo, Carlos, Gallego, Isabel, Ávila, Ana Isabel de, Gómez-Castilla, Jordi, Enjuanes Sánchez, Luis, Salar-Vidal, Llanos, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Verdaguer, Núria, Domingo, Esteban, Perales, Celia, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Fundació La Marató de TV3, Instituto de Salud Carlos III, Comunidad de Madrid, Fundación Ramón Areces, Banco Santander, Ministerio de Sanidad y Consumo (España), Ministerio de Economía y Competitividad (España), Ramos-Ruiz, Ricardo [0000-0002-6331-9786], Enjuanes Sánchez, Luis [0000-0002-0854-0226], Esteban, Jaime [0000-0002-8971-3167], Verdaguer, Núria [0000-0001-8826-7129], Domingo, Esteban [0000-0002-0573-1676], Perales, Celia [0000-0003-1618-1937], Martínez-González, Brenda, Soria, María Eugenia, Vázquez-Sirvent, Lucía, Ferrer-Orta, Cristina, Lobo-Vega, Rebeca, Mínguez, Pablo, Fuente, Lorena de la, Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Cortón, Marta, López-Rodríguez, Rosario, García-Crespo, Carlos, Gallego, Isabel, Ávila, Ana Isabel de, Gómez-Castilla, Jordi, Enjuanes Sánchez, Luis, Salar-Vidal, Llanos, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruíz-Hornillos, Javier, Verdaguer, Núria, Domingo, Esteban, and Perales, Celia

Abstract

Mutant spectra of RNA viruses are important to understand viral pathogenesis, and response to selective pressures. There is a need to characterize the complexity of mutant spectra in coronaviruses sampled from infected patients. In particular, the possible relationship between SARS-CoV-2 mutant spectrum complexity and disease associations has not been established. In the present study, we report an ultra-deep sequencing (UDS) analysis of the mutant spectrum of amplicons from the nsp12 (polymerase)- and spike (S)-coding regions of thirty nasopharyngeal isolates (diagnostic samples) of SARS-CoV-2 of the first COVID-19 pandemic wave (Madrid, Spain, April 2020) classified according to the severity of ensuing COVID-19. Low frequency mutations and deletions, counted relative to the consensus sequence of the corresponding isolate, were overwhelmingly abundant. We show that the average number of different point mutations, mutations per haplotype and several diversity indices was significantly higher in SARS-CoV-2 isolated from patients who developed mild disease than in those associated with moderate or severe disease (exitus). No such bias was observed with RNA deletions. Location of amino acid substitutions in the three dimensional structures of nsp12 (polymerase) and S suggest significant structural or functional effects. Thus, patients who develop mild symptoms may be a richer source of genetic variants of SARS-CoV-2 than patients with moderate or severe COVID-19.

Published
2022

14. Population Disequilibrium as Promoter of Adaptive Explorations in Hepatitis C Virus

Author

García-Crespo, Carlos, Gallego, Isabel, Soria, María Eugenia, Ávila, Ana Isabel de, Martínez-González, Brenda, Vázquez-Sirvent, Lucía, Lobo-Vega, Rebeca, Moreno, Elena, Gómez, Jordi, Briones, Carlos, Gregori, Josep, Quer, Josep, Domingo, Esteban, Perales, Celia, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Instituto de Salud Carlos III, European Commission, Global Virus Network, CSIC-INTA - Centro de Astrobiología (CAB), Fundación Ramón Areces, Fundación Banco Santander, and Agencia Estatal de Investigación (España)

Subjects
hepatitis C virus, universal vaccines, sequence space, mutational waves, residue conservation, COVID-19, Antiviral drug resistance, viral quasispecies
Abstract

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. The work at CBMSO was supported by grants SAF2014-52400-R from Ministerio de Economía y Competitividad (MINECO), SAF2017-87846-R and BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MCIU), PI18/00210 from Instituto de Salud Carlos III, S2013/ABI-2906 (PLATESA from Comunidad de Madrid/FEDER), and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CPII19/00001), cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). The work in Barcelona was supported by Instituto de Salud Carlos III, cofinanced by the European Regional Development Fund (ERDF) Grant No. PI19/00301 and by the Centro para el Desarrollo Tecnológico Industrial (CDTI) from the MICIU, Grant No. IDI-20200297. Work at CAB was supported by MINECO grant BIO2016-79618R and PID2019-104903RB-I00 (funded by the EU under the FEDER program) and by the Spanish State research agency (AEI) through project number MDM-2017-0737 Unidad de Excelencia “María de Maeztu”-Centro de Astrobiología (CSIC-INTA). C.G.-C. is supported by predoctoral contract PRE2018-083422 from MCIU. B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo), cofinanced by Fondo Social Europeo (FSE).

Published
2021

15. Population Disequilibrium as Promoter of Adaptive Explorations in Hepatitis C Virus

Author

Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Instituto de Salud Carlos III, European Commission, Global Virus Network, CSIC-INTA - Centro de Astrobiología (CAB), Fundación Ramón Areces, Fundación Banco Santander, Agencia Estatal de Investigación (España), García-Crespo, Carlos, Gallego, Isabel, Soria, María Eugenia, Ávila, Ana Isabel de, Martínez-González, Brenda, Vázquez-Sirvent, Lucía, Lobo-Vega, Rebeca, Moreno, Elena, Gómez-Castilla, Jordi, Briones, Carlos, Gregori, Josep, Quer, Josep, Domingo, Esteban, Perales, Celia, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Instituto de Salud Carlos III, European Commission, Global Virus Network, CSIC-INTA - Centro de Astrobiología (CAB), Fundación Ramón Areces, Fundación Banco Santander, Agencia Estatal de Investigación (España), García-Crespo, Carlos, Gallego, Isabel, Soria, María Eugenia, Ávila, Ana Isabel de, Martínez-González, Brenda, Vázquez-Sirvent, Lucía, Lobo-Vega, Rebeca, Moreno, Elena, Gómez-Castilla, Jordi, Briones, Carlos, Gregori, Josep, Quer, Josep, Domingo, Esteban, and Perales, Celia

Abstract

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.

Published
2021

16. Dissimilar Conservation Pattern in Hepatitis C Virus Mutant Spectra, Consensus Sequences, and Data Banks

Author

García-Crespo, Carlos, primary, Soria, María Eugenia, additional, Gallego, Isabel, additional, Ávila, Ana Isabel de, additional, Martínez-González, Brenda, additional, Vázquez-Sirvent, Lucía, additional, Gómez, Jordi, additional, Briones, Carlos, additional, Gregori, Josep, additional, Quer, Josep, additional, Perales, Celia, additional, and Domingo, Esteban, additional

Published
2020
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17. A new implication of quasispecies dynamics: Broad virus diversification in absence of external perturbations

Author

Domingo, Esteban, Soria, María Eugenia, Gallego, I., Ávila, Ana Isabel de, García-Crespo, Carlos, Martínez-González, Brenda, Gómez, Jordi, Briones, Carlos, Gregori, J., Quer, Josep, Perales, Celia, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), and Instituto de Salud Carlos III

Subjects
Mutational waves, RNA viruses, Foot-and-mouth disease virus, Hepatitis C virus, Selective forces, Adaptive strategies
Abstract

RNA genetic elements include many important animal and plant pathogens. They share high mutability, a trait that has multiple implications for the interactions with their host organisms. Here we review evidence of a new adaptive feature of RNA viruses that we term “broadly diversifying selection”. It constitutes a new type of positive selection without participation of any external selective agent, and which is built upon a progressive increase of the number of different genomes that dominate the population. The evidence was provided by analyses of mutant spectrum composition of two important viral pathogens, foot-and-mouth disease virus (FMDV) and hepatitis C virus (HCV) after prolonged replication in their respective cell culture environment. Despite being fueled by mutations that arise randomly and in absence of an external guiding selective force, this type of selection prepares the viral population for a response to selective forces still to occur. Since current evidence suggests that broadly diversifying selection is favored by elevated mutation rates and population sizes, it may constitute a more general behavior, relevant also to the adaptive dynamics of microbial populations and cancer cells. We are indebted to many staff scientists and students who have work at Centro de Biología Molecular Severo Ochoa (CBMSO), and have contributed to the understanding of viral quasispecies. The work at CBMSO was supported by grants SAF2014-52400-R from Ministerio de Economía y Competitividad (MINECO), SAF2017-87846-R, BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MICIU), PI18/00210 from Instituto de Salud Carlos III, S2013/ABI-2906, (PLATESA from Comunidad de Madrid/FEDER) and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/00121) cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). The work in Barcelona was supported by Instituto de Salud Carlos III, cofinanced by the European Regional Development Fund (ERDF) grant number PI19/00301 and by the Centro para el Desarrollo Tecnológico Industrial (CDTI) from the MICIU, grant number IDI-20151125. Work at CAB was supported by MINECO grant BIO2016-79618R (funded by EU under the FEDER program) and by the Spanish State research agency (AEI) through Project number MDM-2017-0737 (Unidad de Excelencia “María de Maeztu”-Centro de Astrobiología (CSIC-INTA). C. G.-C. is supported by predoctoral contract PRE2018-083422 from MICIU. B. M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III.

Published
2020

18. Dissimilar conservation pattern in hepatitis C virus mutant spectra, consensus sequences, and data banks

Author

García-Crespo, Carlos, Soria, María Eugenia, Gallego, Isabel, Ávila, Ana Isabel de, Martínez-González, Brenda, Vázquez-Sirvent, Lucía, Gómez-Castilla, Jordi, Briones, Carlos, Gregori, Josep, Quer, Josep, Perales, Celia, Domingo, Esteban, García-Crespo, Carlos, Soria, María Eugenia, Gallego, Isabel, Ávila, Ana Isabel de, Martínez-González, Brenda, Vázquez-Sirvent, Lucía, Gómez-Castilla, Jordi, Briones, Carlos, Gregori, Josep, Quer, Josep, Perales, Celia, and Domingo, Esteban

Published
2020

19. Amino acid substitutions associated with treatment failure for Hepatitis C virus infection

Author

Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Centro para el Desarrollo Tecnológico Industrial (España), Fundación Ramón Areces, Banco Santander, CSIC-INTA - Centro de Astrobiología (CAB), Soria, María Eugenia, García-Crespo, Carlos, Martínez-González, Brenda, Vázquez-Sirvent, Lucía, Lobo-Vega, Rebeca, Ávila, Ana Isabel de, Gallego, Isabel, Chen, Qian, García-Cehic, Damir, Llorens-Revull, Meritxell, Briones, Carlos, Gómez-Castilla, Jordi, Ferrer-Orta, Cristina, Verdaguer, Núria, Gregori, Josep, Rodríguez-Frías, Francisco, Buti, María, Esteban, Juan Ignacio, Domingo, Esteban, Quer, Josep, Perales, Celia, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Centro para el Desarrollo Tecnológico Industrial (España), Fundación Ramón Areces, Banco Santander, CSIC-INTA - Centro de Astrobiología (CAB), Soria, María Eugenia, García-Crespo, Carlos, Martínez-González, Brenda, Vázquez-Sirvent, Lucía, Lobo-Vega, Rebeca, Ávila, Ana Isabel de, Gallego, Isabel, Chen, Qian, García-Cehic, Damir, Llorens-Revull, Meritxell, Briones, Carlos, Gómez-Castilla, Jordi, Ferrer-Orta, Cristina, Verdaguer, Núria, Gregori, Josep, Rodríguez-Frías, Francisco, Buti, María, Esteban, Juan Ignacio, Domingo, Esteban, Quer, Josep, and Perales, Celia

Abstract

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.

Published
2020

20. Broad and dynamic diversification of infectious hepatitis c virus in a cell culture environment

Author

Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Comunidad de Madrid, Instituto de Salud Carlos III, Fundación Ramón Areces, Banco Santander, European Commission, Agencia Estatal de Investigación (España), Ministerio de Economía y Empresa (España), Gallego, Isabel, Soria, María Eugenia, García-Crespo, Carlos, Chen, Q., Martínez-Barragán, P., Khalfaoui, S., Martínez-González, Brenda, Sanchez-Martin, I., Palacios-Blanco, Inés, Ávila, Ana Isabel de, García-Cehic, Damir, Esteban, Juan Ignacio, Gómez-Castilla, Jordi, Briones, Carlos, Gregori, Josep, Quer, Josep, Perales, Celia, Domingo, Esteban, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Comunidad de Madrid, Instituto de Salud Carlos III, Fundación Ramón Areces, Banco Santander, European Commission, Agencia Estatal de Investigación (España), Ministerio de Economía y Empresa (España), Gallego, Isabel, Soria, María Eugenia, García-Crespo, Carlos, Chen, Q., Martínez-Barragán, P., Khalfaoui, S., Martínez-González, Brenda, Sanchez-Martin, I., Palacios-Blanco, Inés, Ávila, Ana Isabel de, García-Cehic, Damir, Esteban, Juan Ignacio, Gómez-Castilla, Jordi, Briones, Carlos, Gregori, Josep, Quer, Josep, Perales, Celia, and Domingo, Esteban

Abstract

Previous studies documented that long-term hepatitis C virus (HCV) replication in human hepatoma Huh-7.5 cells resulted in viral fitness gain, expansion of the mutant spectrum, and several phenotypic alterations. In the present work, we show that mutational waves (changes in frequency of individual mutations) occurred continuously and became more prominent as the virus gained fitness. They were accompanied by an increasing proportion of heterogeneous genomic sites that affected 1 position in the initial HCV population and 19 and 69 positions at passages 100 and 200, respectively. Analysis of biological clones of HCV showed that these dynamic events affected infectious genomes, since part of the fluctuating mutations became incorporated into viable genomes. While 17 mutations were scored in 3 biological clones isolated from the initial population, the number reached 72 in 3 biological clones from the population at passage 200. Biological clones differed in their responses to antiviral inhibitors, indicating a phenotypic impact of viral dynamics. Thus, HCV adaptation to a specific constant environment (cell culture without external influences) broadens the mutant repertoire and does not focus the population toward a limited number of dominant genomes. A retrospective examination of mutant spectra of foot-and-mouth disease virus passaged in cell cultures suggests a parallel behavior here described for HCV. We propose that virus diversification in a constant environment has its basis in the availability of multiple alternative mutational pathways for fitness gain. This mechanism of broad diversification should also apply to other replicative systems characterized by high mutation rates and large population sizes. IMPORTANCE The study shows that extensive replication of an RNA virus in a constant biological environment does not limit exploration of sequence space and adaptive options. There was no convergence toward a restricted set of adapted genomes. Mutational waves a

Published
2020

21. A new implication of quasispecies dynamics: Broad virus diversification in absence of external perturbations

Author

Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Instituto de Salud Carlos III, Domingo, Esteban, Soria, María Eugenia, Gallego, I., Ávila, Ana Isabel de, García-Crespo, Carlos, Martínez-González, Brenda, Gómez-Castilla, Jordi, Briones, Carlos, Gregori, J., Quer, Josep, Perales, Celia, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Instituto de Salud Carlos III, Domingo, Esteban, Soria, María Eugenia, Gallego, I., Ávila, Ana Isabel de, García-Crespo, Carlos, Martínez-González, Brenda, Gómez-Castilla, Jordi, Briones, Carlos, Gregori, J., Quer, Josep, and Perales, Celia

Abstract

RNA genetic elements include many important animal and plant pathogens. They share high mutability, a trait that has multiple implications for the interactions with their host organisms. Here we review evidence of a new adaptive feature of RNA viruses that we term “broadly diversifying selection”. It constitutes a new type of positive selection without participation of any external selective agent, and which is built upon a progressive increase of the number of different genomes that dominate the population. The evidence was provided by analyses of mutant spectrum composition of two important viral pathogens, foot-and-mouth disease virus (FMDV) and hepatitis C virus (HCV) after prolonged replication in their respective cell culture environment. Despite being fueled by mutations that arise randomly and in absence of an external guiding selective force, this type of selection prepares the viral population for a response to selective forces still to occur. Since current evidence suggests that broadly diversifying selection is favored by elevated mutation rates and population sizes, it may constitute a more general behavior, relevant also to the adaptive dynamics of microbial populations and cancer cells.

Published
2020

22. The increasing impact of lethal mutagenesis of viruses

Author

Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Comunidad de Madrid, Centro para el Desarrollo Tecnológico Industrial (España), Fundación Ramón Areces, Banco Santander, Perales, Celia, Gallego, Isabel, Ávila, Ana Isabel de, Soria, María Eugenia, Gregori, Josep, Quer, Josep, Domingo, Esteban, Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Comunidad de Madrid, Centro para el Desarrollo Tecnológico Industrial (España), Fundación Ramón Areces, Banco Santander, Perales, Celia, Gallego, Isabel, Ávila, Ana Isabel de, Soria, María Eugenia, Gregori, Josep, Quer, Josep, and Domingo, Esteban

Abstract

Selection of viral mutants resistant to compounds used in therapy is a major determinant of treatment failure, a problem akin to antibiotic resistance in bacteria. In this scenario, mutagenic base and nucleoside analogs have entered the picture because they increase the mutation rate of viral populations to levels incompatible with their survival. This antiviral strategy is termed lethal mutagenesis. It has found a major impulse with the observation that some antiviral agents, which initially were considered only inhibitors of virus multiplication, may in effect exert part of their antiviral activity through mutagenesis. Here, we review the conceptual basis of lethal mutagenesis, the evidence of virus extinction through mutagenic nucleotide analogs and prospects for application in antiviral designs.

Published
2019

23. Viral fitness: history and relevance for viral pathogenesis and antiviral interventions

Author

Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Comunidad de Madrid, Domingo, Esteban, Ávila, Ana Isabel de, Gallego, Isabel, Sheldon, Julie, Perales, Celia, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Comunidad de Madrid, Domingo, Esteban, Ávila, Ana Isabel de, Gallego, Isabel, Sheldon, Julie, and Perales, Celia

Abstract

The quasispecies dynamics of viral populations (continuous generation of variant genomes and competition among them) has as one of its frequent consequences variations in overall multiplication capacity, a major component of viral fitness. This parameter has multiple implications for viral pathogenesis and viral disease control, some of them unveiled thanks to deep sequencing of viral populations. Darwinian fitness is an old concept whose quantification dates back to the early developments of population genetics. It was later applied to viruses (mainly to RNA viruses) to quantify relative multiplication capacities of individual mutant clones or complex populations. The present article reviews the fitness concept and its relevance for the understanding of the adaptive dynamics of viruses in constant and changing environments. Many studies have addressed the fitness cost of escape mutations (to antibodies, cytotoxic T cells or inhibitors) as an influence on the efficacy of antiviral interventions. Here, we summarize the evidence that the basal fitness level can be a determinant of inhibitor resistance.

Published
2019

24. Molecular and functional bases of selection against a mutation bias in an RNA virus

Author

Ministerio de Economía y Competitividad (España), European Commission, Comunidad de Madrid, Fundación Ramón Areces, Instituto de Salud Carlos III, ALBA Synchrotron, National Institutes of Health (US), Higuera, Ignacio de la, Ferrer-Orta, Cristina, Ávila, Ana Isabel de, Perales, Celia, Sierra, Macarena, Singh, Kamalendra, Sarafianos, Stefan G., Dehouck, Yves, Bastolla, Ugo, Verdaguer, Núria, Domingo, Esteban, Ministerio de Economía y Competitividad (España), European Commission, Comunidad de Madrid, Fundación Ramón Areces, Instituto de Salud Carlos III, ALBA Synchrotron, National Institutes of Health (US), Higuera, Ignacio de la, Ferrer-Orta, Cristina, Ávila, Ana Isabel de, Perales, Celia, Sierra, Macarena, Singh, Kamalendra, Sarafianos, Stefan G., Dehouck, Yves, Bastolla, Ugo, Verdaguer, Núria, and Domingo, Esteban

Abstract

The selective pressures acting on viruses that replicate under enhanced mutation rates are largely unknown. Here, we describe resistance of foot-and-mouth disease virus to the mutagen 5-fluorouracil (FU) through a single polymerase substitution that prevents an excess of A to G and U to C transitions evoked by FU on the wild-type foot-and-mouth disease virus, while maintaining the same level of mutant spectrum complexity. The polymerase substitution inflicts upon the virus a fitness loss during replication in absence of FU but confers a fitness gain in presence of FU. The compensation of mutational bias was documented by in vitro nucleotide incorporation assays, and it was associated with structural modifications at the N-terminal region and motif B of the viral polymerase. Predictions of the effect of mutations that increase the frequency of G and C in the viral genome and encoded polymerase suggest multiple points in the virus life cycle where the mutational bias in favor of G and C may be detrimental. Application of predictive algorithms suggests adverse effects of the FU-directed mutational bias on protein stability. The results reinforce modulation of nucleotide incorporation as a lethal mutagenesis-escape mechanism (that permits eluding virus extinction despite replication in the presence of a mutagenic agent) and suggest that mutational bias can be a target of selection during virus replication

Published
2017

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