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1. Surgical Considerations for Minimally Invasive Gynecologic Surgery in Patient With Skeletal Dysplasia

Author: Borrayo-Garza, Viviane, Martínez-González, Brenda, Escarcega-Bordagaray, Jaime A., and García-Rodríguez, Luis Fernando
Published: 2024
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2. Orphan quality control shapes network dynamics and gene expression

Author: Mark, Kevin G, Kolla, SriDurgaDevi, Garshott, Danielle M, Martínez-González, Brenda, Xu, Christina, Akopian, David, Haakonsen, Diane L, See, Stephanie K, and Rapé, Michael
Subjects: Biochemistry and Cell Biology, Bioinformatics and Computational Biology, Biological Sciences, Genetics, Biotechnology, Generic health relevance
Abstract: All eukaryotes require intricate protein networks to translate developmental signals into accurate cell fate decisions. Mutations that disturb crucial interactions between network components often result in disease, but how the composition and dynamics of complex networks are established is unknown. Here, we identify the tumor suppressor E3 ligase UBR5 as a quality control enzyme that helps degrade unpaired subunits of multiple transcription factors that operate within a single network. By constantly turning over orphan subunits, UBR5 forces cells to continuously replenish network components through new protein synthesis. The resulting cycles of transcription factor synthesis and degradation allow cells to effectively execute the gene expression program, while remaining susceptible to environmental signals. We conclude that orphan quality control plays an essential role in establishing the dynamics of protein networks, which may explain the conserved need for protein degradation in transcription and offers unique opportunities to modulate gene expression in disease.
Published: 2022

3. Structural basis and regulation of the reductive stress response

Author: Manford, Andrew G, Mena, Elijah L, Shih, Karen Y, Gee, Christine L, McMinimy, Rachael, Martínez-González, Brenda, Sherriff, Rumi, Lew, Brandon, Zoltek, Madeline, Rodríguez-Pérez, Fernando, Woldesenbet, Makda, Kuriyan, John, and Rape, Michael
Subjects: Biochemistry and Cell Biology, Biological Sciences, 1.1 Normal biological development and functioning, Generic health relevance, Amino Acids, Animals, Carrier Proteins, Cell Cycle Proteins, Cell Line, Female, Humans, Ions, Mice, Mutant Proteins, Mutation, Protein Binding, Protein Stability, Reactive Oxygen Species, Stress, Physiological, Structure-Activity Relationship, Substrate Specificity, Ubiquitin-Protein Ligase Complexes, Ubiquitination, Zinc, BEX2, BEX3, CUL2, FEM1B, mitochondria, oxidative phosphorylation, reactive oxygen species, reductive stress, ubiquitin, Medical and Health Sciences, Developmental Biology, Biological sciences, Biomedical and clinical sciences
Abstract: Although oxidative phosphorylation is best known for producing ATP, it also yields reactive oxygen species (ROS) as invariant byproducts. Depletion of ROS below their physiological levels, a phenomenon known as reductive stress, impedes cellular signaling and has been linked to cancer, diabetes, and cardiomyopathy. Cells alleviate reductive stress by ubiquitylating and degrading the mitochondrial gatekeeper FNIP1, yet it is unknown how the responsible E3 ligase CUL2FEM1B can bind its target based on redox state and how this is adjusted to changing cellular environments. Here, we show that CUL2FEM1B relies on zinc as a molecular glue to selectively recruit reduced FNIP1 during reductive stress. FNIP1 ubiquitylation is gated by pseudosubstrate inhibitors of the BEX family, which prevent premature FNIP1 degradation to protect cells from unwarranted ROS accumulation. FEM1B gain-of-function mutation and BEX deletion elicit similar developmental syndromes, showing that the zinc-dependent reductive stress response must be tightly regulated to maintain cellular and organismal homeostasis.
Published: 2021

4. Orphan quality control shapes network dynamics and gene expression

Author: Mark, Kevin G., Kolla, SriDurgaDevi, Aguirre, Jacob D., Garshott, Danielle M., Schmitt, Stefan, Haakonsen, Diane L., Xu, Christina, Kater, Lukas, Kempf, Georg, Martínez-González, Brenda, Akopian, David, See, Stephanie K., Thomä, Nicolas H., and Rapé, Michael
Published: 2023
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5. Ubiquitin-dependent remodeling of the actin cytoskeleton drives cell fusion

Author: Rodríguez-Pérez, Fernando, Manford, Andrew G., Pogson, Angela, Ingersoll, Andrew J., Martínez-González, Brenda, and Rape, Michael
Published: 2021
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6. Synergism between remdesivir and ribavirin leads to SARS‐CoV‐2 extinction in cell culture.

Author: García‐Crespo, Carlos, de Ávila, Ana Isabel, Gallego, Isabel, Soria, María Eugenia, Durán‐Pastor, Antoni, Somovilla, Pilar, Martínez‐González, Brenda, Muñoz‐Flores, Javier, Mínguez, Pablo, Salar‐Vidal, Llanos, Esteban‐Muñoz, Mario, Cañar‐Camacho, Elizabeth, Ferrer‐Orta, Cristina, Zuñiga, Sonia, Sola, Isabel, Enjuanes, Luis, Esteban, Jaime, Fernández‐Roblas, Ricardo, Gadea, Ignacio, and Gómez, Jordi
Subjects: RIBAVIRIN, SARS-CoV-2, REMDESIVIR, COVID-19, VIRAL mutation, CELL culture
Abstract: Background and Purpose: There is a need for effective anti‐COVID‐19 treatments, mainly for individuals at risk of severe disease such as the elderly and the immunosuppressed. Drug repositioning has proved effective in identifying drugs that can find a new application for the control of coronavirus disease, in particular COVID‐19. The purpose of the present study was to find synergistic antiviral combinations for COVID‐19 based on lethal mutagenesis. Experimental Approach: The effect of combinations of remdesivir and ribavirin on the infectivity of SARS‐CoV‐2 in cell culture has been tested. Viral populations were monitored by ultra‐deep sequencing, and the decrease of infectivity as a result of the treatment was measured. Key Results: Remdesivir and ribavirin exerted a synergistic inhibitory activity against SARS‐CoV‐2, quantified both by CompuSyn (Chou‐Talalay method) and Synergy Finder (ZIP‐score model). In serial passage experiments, virus extinction was readily achieved with remdesivir‐ribavirin combinations at concentrations well below their cytotoxic 50 value, but not with the drugs used individually. Deep sequencing of treated viral populations showed that remdesivir, ribavirin, and their combinations evoked significant increases of the number of viral mutations and haplotypes, as well as modification of diversity indices that characterize viral quasi‐species. Conclusion and Implications: SARS‐CoV‐2 extinction can be achieved by synergistic combination treatments based on lethal mutagenesis. In addition, the results offer prospects of triple drug treatments for effective SARS‐CoV‐2 suppression. [ABSTRACT FROM AUTHOR]
Published: 2024
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7. A new implication of quasispecies dynamics: Broad virus diversification in absence of external perturbations

Author: Domingo, Esteban, Soria, María Eugenia, Gallego, Isabel, de Ávila, Ana Isabel, García-Crespo, Carlos, Martínez-González, Brenda, Gómez, Jordi, Briones, Carlos, Gregori, Josep, Quer, Josep, and Perales, Celia
Published: 2020
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8. Reply to Qu et al., “Quasispecies are constantly selected through virus-encoded intracellular reproductive population bottlenecking”

Author: Domingo, Esteban, primary, Martínez-González, Brenda, additional, García-Crespo, Carlos, additional, Somovilla, Pilar, additional, de Ávila, Ana Isabel, additional, Soria, María Eugenia, additional, Durán-Pastor, Antoni, additional, and Perales, Celia, additional
Published: 2024
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9. SARS-CoV-2 mutant spectra as variant of concern nurseries: endless variation?

Author: Martínez-González, Brenda, primary, Soria, María Eugenia, additional, Mínguez, Pablo, additional, Lorenzo-Redondo, Ramón, additional, Salar-Vidal, Llanos, additional, López-García, Alberto, additional, Esteban-Muñoz, Mario, additional, Durán-Pastor, Antoni, additional, Somovilla, Pilar, additional, García-Crespo, Carlos, additional, de Ávila, Ana Isabel, additional, Gómez, Jordi, additional, Esteban, Jaime, additional, Fernández-Roblas, Ricardo, additional, Gadea, Ignacio, additional, Domingo, Esteban, additional, and Perales, Celia, additional
Published: 2024
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10. Incipient functional SARS-CoV-2 diversification identified through neural network haplotype maps

Author: Delgado, Soledad, primary, Somovilla, Pilar, additional, Ferrer-Orta, Cristina, additional, Martínez-González, Brenda, additional, Vázquez-Monteagudo, Sergi, additional, Muñoz-Flores, Javier, additional, Soria, María Eugenia, additional, García-Crespo, Carlos, additional, de Ávila, Ana Isabel, additional, Durán-Pastor, Antoni, additional, Gadea, Ignacio, additional, López-Galíndez, Cecilio, additional, Moran, Federico, additional, Lorenzo-Redondo, Ramon, additional, Verdaguer, Nuria, additional, Perales, Celia, additional, and Domingo, Esteban, additional
Published: 2024
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11. Incipient functional SARS-CoV-2 diversification identified through neural network haplotype maps

Author: Agencia Estatal de Investigación (España), Ministerio de Ciencia, Innovación y Universidades (España), Ministerio de Ciencia e Innovación (España), European Commission, Instituto de Salud Carlos III, Consejo Superior de Investigaciones Científicas (España), Comunidad de Madrid, CSIC - Plataforma Temática Interdisciplinar del CSIC Salud Global (PTI Salud Global), Fundació La Marató de TV3, Banco Santander, Fundación Ramón Areces, CSIC-UAM - Centro de Biología Molecular Severo Ochoa (CBM), Ministerio de Economía y Competitividad (España), Ferrer-Orta, Cristina [0000-0002-1072-8463], Verdaguer, Núria [0000-0001-8826-7129], Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72], Delgado, Soledad, Somovilla, Pilar, Ferrer-Orta, Cristina, Martínez-González, Brenda, Vázquez-Monteagudo, Sergi, Muñoz-Flores, Javier, Soria, María Eugenia, García-Crespo, Carlos, de Ávila, Ana Isabel, Durán-Pastor, Antoni, Gadea, Ignacio, López-Galíndez, Cecilio, Moran, Federico, Lorenzo-Redondo, Ramon, Verdaguer, Núria, Perales, Celia, Domingo, Esteban, Agencia Estatal de Investigación (España), Ministerio de Ciencia, Innovación y Universidades (España), Ministerio de Ciencia e Innovación (España), European Commission, Instituto de Salud Carlos III, Consejo Superior de Investigaciones Científicas (España), Comunidad de Madrid, CSIC - Plataforma Temática Interdisciplinar del CSIC Salud Global (PTI Salud Global), Fundació La Marató de TV3, Banco Santander, Fundación Ramón Areces, CSIC-UAM - Centro de Biología Molecular Severo Ochoa (CBM), Ministerio de Economía y Competitividad (España), Ferrer-Orta, Cristina [0000-0002-1072-8463], Verdaguer, Núria [0000-0001-8826-7129], Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72], Delgado, Soledad, Somovilla, Pilar, Ferrer-Orta, Cristina, Martínez-González, Brenda, Vázquez-Monteagudo, Sergi, Muñoz-Flores, Javier, Soria, María Eugenia, García-Crespo, Carlos, de Ávila, Ana Isabel, Durán-Pastor, Antoni, Gadea, Ignacio, López-Galíndez, Cecilio, Moran, Federico, Lorenzo-Redondo, Ramon, Verdaguer, Núria, Perales, Celia, and Domingo, Esteban
Abstract: Since its introduction in the human population, SARS-CoV-2 has evolved into multiple clades, but the events in its intrahost diversification are not well understood. Here, we compare three-dimensional (3D) self-organized neural haplotype maps (SOMs) of SARS-CoV-2 from thirty individual nasopharyngeal diagnostic samples obtained within a 19-day interval in Madrid (Spain), at the time of transition between clades 19 and 20. SOMs have been trained with the haplotype repertoire present in the mutant spectra of the nsp12- and spike (S)-coding regions. Each SOM consisted of a dominant neuron (displaying the maximum frequency), surrounded by a low-frequency neuron cloud. The sequence of the master (dominant) neuron was either identical to that of the reference Wuhan-Hu-1 genome or differed from it at one nucleotide position. Six different deviant haplotype sequences were identified among the master neurons. Some of the substitutions in the neural clouds affected critical sites of the nsp12-nsp8-nsp7 polymerase complex and resulted in altered kinetics of RNA synthesis in an in vitro primer extension assay. Thus, the analysis has identified mutations that are relevant to modification of viral RNA synthesis, present in the mutant clouds of SARS-CoV-2 quasispecies. These mutations most likely occurred during intrahost diversification in several COVID-19 patients, during an initial stage of the pandemic, and within a brief time period.
Published: 2024

12. Synergism between remdesivir and ribavirin leads to SARS-CoV-2 extinction in cell culture

Author: Ministerio de Ciencia e Innovación (España), Ministerio de Ciencia, Innovación y Universidades (España), Instituto de Salud Carlos III, Fundació La Marató de TV3, García-Crespo, Carlos, de Ávila, Ana Isabel, Gallego, Isabel, Soria, María Eugenia, Durán-Pastor, Antoni, Somovilla, Pilar, Martínez-González, Brenda, Muñoz-Flores, Javier, Mínguez, Pablo, Salar-Vidal, Llanos, Esteban-Muñoz, Mario, Cañar-Camacho, Elizabeth, Ferrer-Orta, Cristina, Zuñiga, Sonia, Solá Gurpegui, Isabel, Enjuanes, Luis, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Gómez, Jordi, Verdaguer, Núria, Domingo, Esteban, Perales, Celia, Ministerio de Ciencia e Innovación (España), Ministerio de Ciencia, Innovación y Universidades (España), Instituto de Salud Carlos III, Fundació La Marató de TV3, García-Crespo, Carlos, de Ávila, Ana Isabel, Gallego, Isabel, Soria, María Eugenia, Durán-Pastor, Antoni, Somovilla, Pilar, Martínez-González, Brenda, Muñoz-Flores, Javier, Mínguez, Pablo, Salar-Vidal, Llanos, Esteban-Muñoz, Mario, Cañar-Camacho, Elizabeth, Ferrer-Orta, Cristina, Zuñiga, Sonia, Solá Gurpegui, Isabel, Enjuanes, Luis, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Gómez, Jordi, Verdaguer, Núria, Domingo, Esteban, and Perales, Celia
Abstract: There is a need for effective anti-COVID-19 treatments, mainly for individuals at risk of severe disease such as the elderly and the immunosuppressed. Drug repositioning has proved effective in identifying drugs that can find a new application for the control of coronavirus disease, in particular COVID-19. The purpose of the present study was to find synergistic antiviral combinations for COVID-19 based on lethal mutagenesis.
Published: 2024

13. SARS-CoV-2 mutant spectra as variant of concern nurseries: endless variation?

Author: Ministerio de Ciencia e Innovación (España), Instituto de Salud Carlos III, Fundació La Marató de TV3, Comunidad de Madrid, #NODATA#, Martínez-González, Brenda, Soria, María Eugenia, Mínguez, Pablo, Lorenzo-Redondo, Ramón, Salar-Vidal, Llanos, López-García, Alberto, Esteban-Muñoz, Mario, Durán-Pastor, Antoni, Somovilla, Pilar, García-Crespo, Carlos, de Ávila, Ana Isabel, Gómez, Jordi, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Domingo, Esteban, Perales, Celia, Ministerio de Ciencia e Innovación (España), Instituto de Salud Carlos III, Fundació La Marató de TV3, Comunidad de Madrid, #NODATA#, Martínez-González, Brenda, Soria, María Eugenia, Mínguez, Pablo, Lorenzo-Redondo, Ramón, Salar-Vidal, Llanos, López-García, Alberto, Esteban-Muñoz, Mario, Durán-Pastor, Antoni, Somovilla, Pilar, García-Crespo, Carlos, de Ávila, Ana Isabel, Gómez, Jordi, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Domingo, Esteban, and Perales, Celia
Abstract: SARS-CoV-2 isolates of a given clade may contain low frequency genomes that encode amino acids or deletions which are typical of a different clade.
Published: 2024

14. 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

15. Point mutations at specific sites of the nsp12-nsp8 interface dramatically affect the RNA polymerization activity of SARS-CoV-2.

Author: Ferrer-Orta, Cristina, Vázquez-Monteagudo, Sergi, Ferrero, Diego S., Martínez-González, Brenda, Perales, Celia, Domingo, Esteban, and Verdaguer, Nuria
Subjects: SARS-CoV-2, RNA replicase, RNA synthesis, COVID-19 pandemic, RNA regulation
Abstract: In a recent characterization of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variability present in 30 diagnostic samples from patients of the first COVID-19 pandemic wave, 41 amino acid substitutions were documented in the RNA-dependent RNA polymerase (RdRp) nsp12. Eight substitutions were selected in this work to determine whether they had an impact on the RdRp activity of the SARS-CoV-2 nsp12-nsp8-nsp7 replication complex. Three of these substitutions were found around the polymerase central cavity, in the template entry channel (D499G and M668V), and within the motif B (V560A), and they showed polymerization rates similar to the wild type RdRp. The remaining five mutations (P323L, L372F, L372P, V373A, and L527H) were placed near the nsp12-nsp8F contact surface; residues L372, V373, and L527 participated in a large hydrophobic cluster involving contacts between two helices in the nsp12 fingers and the long a-helix of nsp8F. The presence of any of these five amino acid substitutions resulted in important alterations in the RNA polymerization activity. Comparative primer elongation assays showed different behavior depending on the hydrophobicity of their side chains. The substitution of L by the bulkier F side chain at position 372 slightly promoted RdRp activity. However, this activity was dramatically reduced with the L372P, and L527H mutations, and to a lesser extent with V373A, all of which weaken the hydrophobic interactions within the cluster. Additional mutations, specifically designed to disrupt the nsp12-nsp8F interactions (nsp12-V330S, nsp12-V341S, and nsp8-R111A/D112A), also resulted in an impaired RdRp activity, further illustrating the importance of this contact interface in the regulation of RNA synthesis. [ABSTRACT FROM AUTHOR]
Published: 2024
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16. Puzzles, challenges, and information reservoir of SARS-CoV-2 quasispecies

Author: Domingo, Esteban, primary, Martínez-González, Brenda, additional, García-Crespo, Carlos, additional, Somovilla, Pilar, additional, de Ávila, Ana Isabel, additional, Soria, María Eugenia, additional, Durán-Pastor, Antoni, additional, and Perales, Celia, additional
Published: 2023
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17. Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing

Author: Llorens-Revull, Meritxell, primary, Martínez-González, Brenda, additional, Quer, Josep, additional, Esteban, Juan Ignacio, additional, Núñez-Moreno, Gonzalo, additional, Mínguez, Pablo, additional, Burgui, Idoia, additional, Ramos-Ruíz, Ricardo, additional, Soria, María Eugenia, additional, Rico, Angie, additional, Riveiro-Barciela, Mar, additional, Sauleda, Silvia, additional, Piron, María, additional, Corrales, Irene, additional, Borràs, Francesc E., additional, Rodríguez-Frías, Francisco, additional, Rando, Ariadna, additional, Ramírez-Serra, Clara, additional, Camós, Silvia, additional, Domingo, Esteban, additional, Bes, Marta, additional, Perales, Celia, additional, and Costafreda, Maria Isabel, additional
Published: 2023
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18. Fitness-Dependent, Mild Mutagenic Activity of Sofosbuvir for Hepatitis C Virus

Author: Martínez-González, Brenda, primary, Gallego, Isabel, additional, Gregori, Josep, additional, Soria, María Eugenia, additional, Somovilla, Pilar, additional, de Ávila, Ana Isabel, additional, García-Crespo, Carlos, additional, Durán-Pastor, Antoni, additional, Briones, Carlos, additional, Gómez, Jordi, additional, Quer, Josep, additional, Domingo, Esteban, additional, and Perales, Celia, additional
Published: 2023
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19. 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

20. Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing

Author: Instituto de Salud Carlos III, European Commission, Ministerio de Economía y Empresa (España), Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Fundació La Marató de TV3, Ministerio de Ciencia e Innovación (España), Comunidad de Madrid, CSIC - Plataforma Temática Interdisciplinar del CSIC Salud Global (PTI Salud Global), Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Fundación Ramón Areces, Banco Santander, Llorens-Revull, Meritxell, Martínez-González, Brenda, Quer, Josep, Esteban, Juan Ignacio, Núñez-Moreno, Gonzalo, Mínguez, Pablo, Burgui, Idoia, Ramos-Ruiz, Ricardo, Soria, María Eugenia, Rico, Angie, Riveiro-Barciela, Mar, Sauleda, Silvia, Piron, María, Corrales, Irene, Borràs, Francesc E., Rodríguez-Frías, Francisco, Rando, Ariadna, Ramírez-Serra, Clara, Camós, Silvia, Domingo, Esteban, Bes, Marta, Perales, Celia, Costafreda, María Isabel, Instituto de Salud Carlos III, European Commission, Ministerio de Economía y Empresa (España), Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Fundació La Marató de TV3, Ministerio de Ciencia e Innovación (España), Comunidad de Madrid, CSIC - Plataforma Temática Interdisciplinar del CSIC Salud Global (PTI Salud Global), Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Fundación Ramón Areces, Banco Santander, Llorens-Revull, Meritxell, Martínez-González, Brenda, Quer, Josep, Esteban, Juan Ignacio, Núñez-Moreno, Gonzalo, Mínguez, Pablo, Burgui, Idoia, Ramos-Ruiz, Ricardo, Soria, María Eugenia, Rico, Angie, Riveiro-Barciela, Mar, Sauleda, Silvia, Piron, María, Corrales, Irene, Borràs, Francesc E., Rodríguez-Frías, Francisco, Rando, Ariadna, Ramírez-Serra, Clara, Camós, Silvia, Domingo, Esteban, Bes, Marta, Perales, Celia, and Costafreda, María Isabel
Abstract: MicroRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) are potential diagnostic and prognostic biomarkers. However, discrepancies in miRNA patterns and their validation are still frequent due to differences in sample origin, EV isolation, and miRNA sequencing methods. The aim of the present study is to find a reliable EV isolation method for miRNA sequencing, adequate for clinical application. To this aim, two comparative studies were performed in parallel with the same human plasma sample: (i) isolation and characterization of EVs obtained using three procedures: size exclusion chromatography (SEC), iodixanol gradient (GRAD), and its combination (SEC+GRAD) and (ii) evaluation of the yield of miRNA sequences obtained using NextSeq 500 (Illumina) and three miRNA library preparation protocols: NEBNext, NEXTFlex, and SMARTer smRNA-seq. The conclusion of comparison (i) is that recovery of the largest amount of EVs and reproducibility were attained with SEC, but GRAD and SEC+GRAD yielded purer EV preparations. The conclusion of (ii) is that the NEBNext library showed the highest reproducibility in the number of miRNAs recovered and the highest diversity of miRNAs. These results render the combination of GRAD EV isolation and NEBNext library preparation for miRNA retrieval as adequate for clinical applications using plasma samples.
Published: 2023

21. 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

22. Fitness-Dependent, Mild Mutagenic Activity of Sofosbuvir for Hepatitis C Virus

Author: Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Fundació La Marató de TV3, Ministerio de Ciencia e Innovación (España), Comunidad de Madrid, Instituto de Salud Carlos III, European Commission, Fundación Ramón Areces, Banco Santander, Global Virus Network, Martínez-González, Brenda, Gallego, Isabel, Gregori, Josep, Soria, María Eugenia, Somovilla, Pilar, de Ávila, Ana Isabel, García-Crespo, Carlos, Durán-Pastor, Antoni, Briones, Carlos, Gómez, Jordi, Quer, Josep, Domingo, Esteban, Perales, Celia, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Fundació La Marató de TV3, Ministerio de Ciencia e Innovación (España), Comunidad de Madrid, Instituto de Salud Carlos III, European Commission, Fundación Ramón Areces, Banco Santander, Global Virus Network, Martínez-González, Brenda, Gallego, Isabel, Gregori, Josep, Soria, María Eugenia, Somovilla, Pilar, de Ávila, Ana Isabel, García-Crespo, Carlos, Durán-Pastor, Antoni, Briones, Carlos, Gómez, Jordi, Quer, Josep, Domingo, Esteban, and Perales, Celia
Abstract: The concept of a mild mutagen was coined to describe a minor mutagenic activity exhibited by some nucleoside analogues that potentiated their efficacy as antiretroviral agents. In the present study, we report the mild mutagen activity of sofosbuvir (SOF) for hepatitis C virus (HCV). Serial passages of HCV in human hepatoma cells, in the presence of SOF at a concentration well below its cytotoxic concentration 50 (CC) led to pre-extinction populations whose mutant spectra exhibited a significant increase of C!U transitions, relative to populations passaged in the absence of SOF. This was reflected in an increase in several diversity indices that were used to characterize viral quasispecies. The mild mutagenic activity of SOF was largely absent when it was tested with isogenic HCV populations that displayed high replicative fitness. Thus, SOF can act as a mild mutagen for HCV, depending on HCV fitness. Possible mechanisms by which the SOF mutagenic activity may contribute to its antiviral efficacy are discussed.
Published: 2023

23. 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

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

Author: Somovilla, Pilar, primary, García-Crespo, Carlos, additional, Martínez-González, Brenda, additional, Soria, María Eugenia, additional, de Ávila, Ana Isabel, additional, Gallego, Isabel, additional, Mínguez, Pablo, additional, Durán-Pastor, Antoni, additional, Ferrer-Orta, Cristina, additional, Salar-Vidal, Llanos, additional, Esteban-Muñoz, Mario, additional, Zuñiga, Sonia, additional, Sola, Isabel, additional, Enjuanes, Luis, additional, Esteban, Jaime, additional, Fernandez-Roblas, Ricardo, additional, Gadea, Ignacio, additional, Gómez, Jordi, additional, Verdaguer, Nuria, additional, Domingo, Esteban, additional, and Perales, Celia, additional
Published: 2023
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25. Orphan quality control shapes network dynamics and gene expression

Author: Mark, Kevin G., primary, Kolla, SriDurgaDevi, additional, Garshott, Danielle M., additional, Martínez-González, Brenda, additional, Xu, Christina, additional, Akopian, David, additional, Haakonsen, Diane L., additional, See, Stephanie K., additional, and Rapé, Michael, additional
Published: 2022
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26. Characterization of the in vitro polymerization activities of different SARS-CoV-2 nsp12 variants from patients' isolates

Author: Ferrer-Orta, Cristina, Vázquez-Monteagudo, Sergi, Ferrero, Diego, Martínez-González, Brenda, Guerra, Pablo, Perales, Celia, Domingo, Esteban, Verdaguer, Núria, Ministerio de Ciencia e Innovación (España), European Commission, CSIC - Instituto de Biología Molecular de Barcelona (IBMB), and Fundació La Marató de TV3
Abstract: Trabajo presentado en las II Jornadas Científicas PTI+ Salud Global, celebradas en Valencia (España) del 05 al 06 de octubre de 2022.
Published: 2022

27. 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

28. 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

29. High SARS-CoV-2 viral load is associated with a worse clinical outcome of COVID-19 disease

Author: Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación y Universidades (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Fundación Ramón Areces, Fundación Banco Santander, Agencia Estatal de Investigación (España), Comunidad de Madrid, Instituto de Investigación Sanitaria Fundación Jiménez Díaz, Soria, María Eugenia [0000-0002-4719-3351], Cortón, Marta [0000-0003-0087-1626], Martínez-González, Brenda [0000-0002-4482-5181], Lobo-Vega, Rebeca [0000-0002-4882-6763], Vázquez-Sirvent, Lucía [0000-0002-0396-7781], Mínguez, Pablo [0000-0003-4099-9421], Macías-Valcayo, Alicia [0000-0003-3879-0493], Esteban, Jaime [0000-0002-8971-3167], Gadea, Ignacio [0000-0003-4684-7816], Ayuso, Carmen [0000-0002-9242-7065], Perales, Celia [000-0003-1618-1937], Soria, María Eugenia, Cortón, Marta, Martínez-González, Brenda, Lobo-Vega, Rebeca, Vázquez-Sirvent, Lucía, López-Rodríguez, Rosario, Almoguera, Berta, Mahillo-Fernández, Ignacio, Mínguez, Pablo, Herrero, Antonio, Taracido, Juan Carlos, Macías-Valcayo, Alicia, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ruíz-Hornillos, Javier, Ayuso, Carmen, Perales, Celia, Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación y Universidades (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Fundación Ramón Areces, Fundación Banco Santander, Agencia Estatal de Investigación (España), Comunidad de Madrid, Instituto de Investigación Sanitaria Fundación Jiménez Díaz, Soria, María Eugenia [0000-0002-4719-3351], Cortón, Marta [0000-0003-0087-1626], Martínez-González, Brenda [0000-0002-4482-5181], Lobo-Vega, Rebeca [0000-0002-4882-6763], Vázquez-Sirvent, Lucía [0000-0002-0396-7781], Mínguez, Pablo [0000-0003-4099-9421], Macías-Valcayo, Alicia [0000-0003-3879-0493], Esteban, Jaime [0000-0002-8971-3167], Gadea, Ignacio [0000-0003-4684-7816], Ayuso, Carmen [0000-0002-9242-7065], Perales, Celia [000-0003-1618-1937], Soria, María Eugenia, Cortón, Marta, Martínez-González, Brenda, Lobo-Vega, Rebeca, Vázquez-Sirvent, Lucía, López-Rodríguez, Rosario, Almoguera, Berta, Mahillo-Fernández, Ignacio, Mínguez, Pablo, Herrero, Antonio, Taracido, Juan Carlos, Macías-Valcayo, Alicia, Esteban, Jaime, Fernández-Roblas, Ricardo, Gadea, Ignacio, Ruíz-Hornillos, Javier, Ayuso, Carmen, and Perales, Celia
Abstract: COVID-19 severity and progression are determined by several host and virological factors that may influence the final outcome of SARS-CoV-2-infected patients. The objective of this work was to determine a possible association between viral load, obtained from nasopharyngeal swabs, and the severity of the infection in a cohort of 448 SARS-CoV-2-infected patients from a hospital in Madrid during the first outbreak of the pandemic in Spain. To perform this, we clinically classified patients as mild, moderate and severe COVID-19 according to a number of clinical parameters such as hospitalization requirement, need of oxygen therapy, admission to intensive care units and/or death. Also, Ct values were determined using SARS-CoV-2-specific oligonucleotides directed to ORF1ab. Here we report a statistically significant association between viral load and disease severity, a high viral load being associated with worse clinical prognosis, independently of several previously identified risk factors such as age, sex, hypertension, cardiovascular disease, diabetes, obesity and lung disease (asthma and chronic obstructive pulmonary disease). The data presented here reinforce viral load as a potential biomarker for predicting disease severity in SARS-CoV-2-infected patients. It is also an important parameter in viral evolution since it relates to the numbers and types of variant genomes present in a viral population, a potential determinant of disease progression.
Published: 2021

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

Author: García-Crespo, Carlos, primary, Vázquez-Sirvent, Lucía, additional, Somovilla, Pilar, additional, Soria, María Eugenia, additional, Gallego, Isabel, additional, de Ávila, Ana Isabel, additional, Martínez-González, Brenda, additional, Durán-Pastor, Antoni, additional, Domingo, Esteban, additional, and Perales, Celia, additional
Published: 2022
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31. Inhibition of high fitness viruses by different antiviral strategies when the infection has started

Author: García-Crespo, Carlos, primary, Vázquez-Sirvent, Lucía, additional, Soria, Maria Eugenia, additional, Gallego, Isabel, additional, de Ávila, Ana Isabel, additional, Martínez-González, Brenda, additional, Domingo, Esteban, additional, and Perales, Celia, additional
Published: 2022
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32. SARS-CoV-2 Mutant Spectra at Different Depth Levels Reveal an Overwhelming Abundance of Low Frequency Mutations

Author: Martínez-González, Brenda, primary, Soria, María Eugenia, additional, Vázquez-Sirvent, Lucía, additional, Ferrer-Orta, Cristina, additional, Lobo-Vega, Rebeca, additional, Mínguez, Pablo, additional, de la Fuente, Lorena, additional, Llorens, Carlos, additional, Soriano, Beatriz, additional, Ramos-Ruíz, Ricardo, additional, Cortón, Marta, additional, López-Rodríguez, Rosario, additional, García-Crespo, Carlos, additional, Somovilla, Pilar, additional, Durán-Pastor, Antoni, additional, Gallego, Isabel, additional, de Ávila, Ana Isabel, additional, Delgado, Soledad, additional, Morán, Federico, additional, López-Galíndez, Cecilio, additional, Gómez, Jordi, additional, Enjuanes, Luis, additional, Salar-Vidal, Llanos, additional, Esteban-Muñoz, Mario, additional, Esteban, Jaime, additional, Fernández-Roblas, Ricardo, additional, Gadea, Ignacio, additional, Ayuso, Carmen, additional, Ruíz-Hornillos, Javier, additional, Verdaguer, Nuria, additional, Domingo, Esteban, additional, and Perales, Celia, additional
Published: 2022
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33. Vaccine breakthrough infections with SARS-CoV-2 Alpha mirror mutations in Delta Plus, Iota, and Omicron

Author: Martínez-González, Brenda, primary, Vázquez-Sirvent, Lucía, additional, Soria, María E., additional, Mínguez, Pablo, additional, Salar-Vidal, Llanos, additional, García-Crespo, Carlos, additional, Gallego, Isabel, additional, de Ávila, Ana I., additional, Llorens, Carlos, additional, Soriano, Beatriz, additional, Ramos-Ruiz, Ricardo, additional, Esteban, Jaime, additional, Fernandez-Roblas, Ricardo, additional, Gadea, Ignacio, additional, Ayuso, Carmen, additional, Ruíz-Hornillos, Javier, additional, Pérez-Jorge, Concepción, additional, Domingo, Esteban, additional, and Perales, Celia, additional
Published: 2022
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34. 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

35. 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

36. 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

37. 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

38. 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

39. Characterization of the in vitro polymerization activities of different SARS-CoV-2 nsp12 variants from patients' isolates

Author: Ministerio de Ciencia e Innovación (España), European Commission, CSIC - Instituto de Biología Molecular de Barcelona (IBMB), Fundació La Marató de TV3, Ferrer-Orta, Cristina, Vázquez-Monteagudo, Sergi, Ferrero, Diego, Martínez-González, Brenda, Guerra, Pablo, Perales, Celia, Domingo, Esteban, Verdaguer, Núria, Ministerio de Ciencia e Innovación (España), European Commission, CSIC - Instituto de Biología Molecular de Barcelona (IBMB), Fundació La Marató de TV3, Ferrer-Orta, Cristina, Vázquez-Monteagudo, Sergi, Ferrero, Diego, Martínez-González, Brenda, Guerra, Pablo, Perales, Celia, Domingo, Esteban, and Verdaguer, Núria
Published: 2022

40. 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

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

Author: Martínez-González, Brenda, primary, Soria, María Eugenia, additional, Vázquez-Sirvent, Lucía, additional, Ferrer-Orta, Cristina, additional, Lobo-Vega, Rebeca, additional, Mínguez, Pablo, additional, de la Fuente, Lorena, additional, Llorens, Carlos, additional, Soriano, Beatriz, additional, Ramos, Ricardo, additional, Cortón, Marta, additional, López-Rodríguez, Rosario, additional, García-Crespo, Carlos, additional, Gallego, Isabel, additional, de Ávila, Ana Isabel, additional, Gómez, Jordi, additional, Enjuanes, Luis, additional, Salar-Vidal, Llanos, additional, Esteban, Jaime, additional, Fernandez-Roblas, Ricardo, additional, Gadea, Ignacio, additional, Ayuso, Carmen, additional, Ruíz-Hornillos, Javier, additional, Verdaguer, Nuria, additional, Domingo, Esteban, additional, and Perales, Celia, additional
Published: 2022
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42. A Two-Level, Intramutant Spectrum Haplotype Profile of Hepatitis C Virus Revealed by Self-Organized Maps

Author: Delgado, Soledad, primary, Perales, Celia, additional, García-Crespo, Carlos, additional, Soria, María Eugenia, additional, Gallego, Isabel, additional, de Ávila, Ana Isabel, additional, Martínez-González, Brenda, additional, Vázquez-Sirvent, Lucía, additional, López-Galíndez, Cecilio, additional, Morán, Federico, additional, and Domingo, Esteban, additional
Published: 2021
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43. High SARS-CoV-2 viral load is associated with a worse clinical outcome of COVID-19 disease

Author: Soria, María Eugenia, primary, Cortón, Marta, additional, Martínez-González, Brenda, additional, Lobo-Vega, Rebeca, additional, Vázquez-Sirvent, Lucía, additional, López-Rodríguez, Rosario, additional, Almoguera, Berta, additional, Mahillo, Ignacio, additional, Mínguez, Pablo, additional, Herrero, Antonio, additional, Taracido, Juan Carlos, additional, Macías-Valcayo, Alicia, additional, Esteban, Jaime, additional, Fernandez-Roblas, Ricardo, additional, Gadea, Ignacio, additional, Ruíz-Hornillos, Javier, additional, Ayuso, Carmen, additional, and Perales, Celia, additional
Published: 2021
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44. 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

45. 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

46. A two-level, dynamic fitness landscape of hepatitis C virus revealed by self-organized haplotype maps

Author: Delgado, Soledad, primary, Perales, Celia, additional, García-Crespo, Carlos, additional, Soria, María Eugenia, additional, Gallego, Isabel, additional, de Ávila, Ana Isabel, additional, Martínez-González, Brenda, additional, Vázquez-Sirvent, Lucía, additional, López-Galíndez, Cecilio, additional, Morán, Federico, additional, and Domingo, Esteban, additional
Published: 2021
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47. Population Disequilibrium as Promoter of Adaptive Explorations in Hepatitis C Virus

Author: García-Crespo, Carlos, primary, Gallego, Isabel, additional, Soria, María Eugenia, additional, de Ávila, Ana Isabel, additional, Martínez-González, Brenda, additional, Vázquez-Sirvent, Lucía, additional, Lobo-Vega, Rebeca, additional, Moreno, Elena, additional, Gómez, Jordi, additional, Briones, Carlos, additional, Gregori, Josep, additional, Quer, Josep, additional, Domingo, Esteban, additional, and Perales, Celia, additional
Published: 2021
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48. Amino Acid Substitutions Associated with Treatment Failure for Hepatitis C Virus Infection

Author: Soria, María Eugenia, primary, García-Crespo, Carlos, additional, Martínez-González, Brenda, additional, Vázquez-Sirvent, Lucía, additional, Lobo-Vega, Rebeca, additional, de Ávila, Ana Isabel, additional, Gallego, Isabel, additional, Chen, Qian, additional, García-Cehic, Damir, additional, Llorens-Revull, Meritxell, additional, Briones, Carlos, additional, Gómez, Jordi, additional, Ferrer-Orta, Cristina, additional, Verdaguer, Nuria, additional, Gregori, Josep, additional, Rodríguez-Frías, Francisco, additional, Buti, María, additional, Esteban, Juan Ignacio, additional, Domingo, Esteban, additional, Quer, Josep, additional, and Perales, Celia, additional
Published: 2020
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49. High SARS-CoV-2 viral load is associated with a worse clinical outcome of COVID-19 disease

Author: Soria, María Eugenia, primary, Cortón, Marta, additional, Martínez-González, Brenda, additional, Lobo-Vega, Rebeca, additional, Vázquez-Sirvent, Lucía, additional, López-Rodríguez, Rosario, additional, Almoguera, Berta, additional, Mahillo, Ignacio, additional, Mínguez, Pablo, additional, Herrero, Antonio, additional, Taracido, Juan Carlos, additional, Macías-Valcayo, Alicia, additional, Esteban, Jaime, additional, Fernandez-Roblas, Ricardo, additional, Gadea, Ignacio, additional, Ruíz-Hornillos, Javier, additional, Ayuso, Carmen, additional, and Perales, Celia, additional
Published: 2020
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50. 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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