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1. 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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2. 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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3. 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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4. 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

5. 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

6. 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

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

Author

Martinez-Gonzalez, Brenda, Vazquez-Sirvent, Lucia, Soria, Maria E., Minguez, Pablo, Salar-Vidal, Llanos, Garcia-Crespo, Carlos, Gallego, Isabel, de Avila, Ana I., Llorens, Carlos, Soriano, Beatriz, Ramos-Ruiz, Ricardo, Esteban, Jaime, Fernandez- Roblas, Ricardo, Gadea, Ignacio, Ayuso, Carmen, Ruiz-Hornillos, Javier, Perez-Jorge, Concepcion, Domingo, Esteban, and Perales, Celia

Subjects
Gene mutations -- Research, Immunological research, Health care industry
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., Introduction SARS-CoV-2 continues its diversification worldwide, and a new variant termed Omicron (B.l.1.529), carrying a large number of mutations, was recently described in South Africa and classified as a potential [...]

Published
2022
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11. 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

12. 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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14. 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

15. 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

16. Epidemiology and prognosis of candidaemia in elderly patients

Author

Ramos‐Martínez, Antonio, Vicente‐López, Natalia, Sánchez‐Romero, Isabel, Padilla, Belén, Merino‐Amador, Paloma, Garnacho‐Montero, José, Ruiz‐Camps, Isabel, Montejo, Miguel, Salavert, Miguel, Mensa, José, Cuenca‐Estrella, Manuel, Muñoz, Patricia, Guinea, Jesús, Paño Pardo, José Ramón, García‐Rodríguez, Julio, Cerrada, Carlos García, Fortún, Jesús, Martín, Pilar, Gómez, Elia, Ryan, Pablo, Campelo, Carolina, de los Santos Gil, Ignacio, Buendía, Ventura, Gorricho, Beatriz Perez, Alonso, Mercedes, Sanz, Francisca Sanz, Aguado, José María, Romo, Fernando González, Gorgolas, Miguel, Gadea, Ignacio, Losa, Juan Emilio, Delgado‐Iribarren, Alberto, Romero, Yolanda, Zaragoza, Oscar, Rodriguez‐Baño, Jesús, Suarez, Ana Isabel, Loza, Ana, Aller García, Ana Isabel, Martín‐Mazuelos, Estrella, Pérez de Pipaón, Maite Ruiz, Ortiz, Carlos, Chávez, Mónica, Maroto, Fernando L., Salavert, Miguel, Pemán, Javier, Blanquer, José, Navarro, David, Camarena, Juan José, Zaragoza, Rafael, Abril, Vicente, Gimeno, Concepción, Hernáez, Silvia, Ezpeleta, Guillermo, Bereciartua, Elena, Hernández Almaraz, José L., Montejo, Miguel, Rivas, Rosa Ana, Ayarza, Rafael, Planes, Ana Mª, Almirante, Benito, Almela, Manel, Gurgui, Mercè, Sánchez‐Reus, Ferran, Martinez‐Montauti, Joaquin, Sierra, Montserrat, Horcajada, Juan Pablo, Sorli, Luisa, Gómez, Julià, Gené, Amadeu, Urrea, Mireia, Valerio, Maricela, Díaz‐Martín, Ana, Puchades, Francesc, and Mularoni, Alessandra

Published
2017
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17. 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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18. 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

19. 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

20. 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

21. 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

22. 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

24. 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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25. 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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30. 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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31. 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), Agencia Estatal de Investigación (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Fundación Ramón Areces, Banco Santander, Comunidad de Madrid, Instituto de Investigación Sanitaria Fundación Jiménez Díaz, Soria, María Eugenia [0000-0002-4719-3351], Lobo-Vega, Rebeca [0000-0002-4882-6763], Perales, Celia [0000-0003-1618-1937], 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), Agencia Estatal de Investigación (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (España), Fundación Ramón Areces, Banco Santander, Comunidad de Madrid, Instituto de Investigación Sanitaria Fundación Jiménez Díaz, Soria, María Eugenia [0000-0002-4719-3351], Lobo-Vega, Rebeca [0000-0002-4882-6763], Perales, Celia [0000-0003-1618-1937], 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 is to determine a possible association between the 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 have 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 exitus. Here we report a statistically significant correlation between viral load and disease severity, being high viral load 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 the 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
2020

32. Synergistic Lethal Mutagenesis of Hepatitis C Virus

Author

Gallego, Isabel, primary, Soria, María Eugenia, additional, Gregori, Josep, additional, de Ávila, Ana I., additional, García-Crespo, Carlos, additional, Moreno, Elena, additional, Gadea, Ignacio, additional, Esteban, Jaime, additional, Fernández-Roblas, Ricardo, additional, Esteban, Juan Ignacio, additional, Gómez, Jordi, additional, Quer, Josep, additional, Domingo, Esteban, additional, and Perales, Celia, additional

Published
2019
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34. Biofilm development by potentially pathogenic non-pigmented rapidly growing mycobacteria

Author

Fernández-Roblas Ricardo, Ayala Guillermo, Kinnari Teemu J, Martín-de-Hijas Nieves Z, Esteban Jaime, and Gadea Ignacio

Subjects
Microbiology, QR1-502
Abstract

Abstract Background A study to evaluate the biofilm-development ability in three different media (Middlebrook 7H9, sterile tap water and PBS-5% glucose) was performed with 19 collection strains from 15 different species on non-pigmented rapidly growing mycobacteria (NPRGM). A microtiter plate assay was developed to evaluate the percentage of covered surface of the microtiter plate wells in different days from day 1 to day 69. Results All strains were able to develop biofilm in all the tested media. Middlebrook 7H9 showed the fastest growth, followed by sterile tap water and PBS-5% glucose. A sigmoid growth curve was detected in all the strains both in Middlebrook 7H9 and in sterile tap water. A difference could be detected for Mycobacterium abscessus in tap water, where it showed faster growth than all the other strains. Conclusion Biofilm development seems to be a property of all the species of NPRGM and it depends on the nutrients present in the medium. The microtiter plate assay described here is a useful tool to evaluate differences in biofilm development among the different species of rapidly growing mycobacteria.

Published
2008
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35. Synergistic Lethal Mutagenesis of Hepatitis C Virus

Author

Instituto de Salud Carlos III, European Commission, 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, Gallego, Isabel, Soria, María Eugenia, Gregori, Josep, de Ávila, Ana I., García-Crespo, Carlos, Moreno, Elena, Gadea, Ignacio, Esteban, Jaime, Fernández-Roblas, Ricardo, Esteban, Juan Ignacio, Gómez-Castilla, Jordi, Quer, Josep, Domingo, Esteban, Perales, Celia, Instituto de Salud Carlos III, European Commission, 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, Gallego, Isabel, Soria, María Eugenia, Gregori, Josep, de Ávila, Ana I., García-Crespo, Carlos, Moreno, Elena, Gadea, Ignacio, Esteban, Jaime, Fernández-Roblas, Ricardo, Esteban, Juan Ignacio, Gómez-Castilla, Jordi, Quer, Josep, Domingo, Esteban, and Perales, Celia

Abstract

Lethal mutagenesis is an antiviral approach that consists of extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagenic agent, often a nucleotide analogue. One of its advantages is its broad-spectrum nature, which renders the strategy potentially effective against emergent RNA viral infections. Here we describe the synergistic lethal mutagenesis of hepatitis C virus (HCV) by a combination of favipiravir (T-705) and ribavirin. Synergy has been documented over a broad range of analogue concentrations using the Chou-Talalay method implemented in CompuSyn graphics software, with the average dose reduction index (DRI) being above 1 (68.02 101.6 for favipiravir and 5.83 6.07 for ribavirin) and the average combination indices (CI) being below 1 (0.52 0.28). Furthermore, analogue concentrations that individually did not extinguish high-fitness HCV in 10 serial infections extinguished high-fitness HCV in 1 to 2 passages when used in combination. Although both analogues displayed a preference for G ¡ A and C ¡ U transitions, deep sequencing analysis of mutant spectra indicated a different preference of the two analogues for the mutation sites, thus unveiling a new possible synergy mechanism in lethal mutagenesis. The prospects for synergy among mutagenic nucleotides as a strategy to confront emerging viral infections are discussed

Published
2019

37. Evaluation of a commercial multiplex PCR (Unyvero i60 ® ) designed for the diagnosis of bone and joint infections using prosthetic-joint sonication

Author

Prieto-Borja, Laura, primary, Rodriguez-Sevilla, Graciela, additional, Auñon, Alvaro, additional, Pérez-Jorge, Concepción, additional, Sandoval, Enrique, additional, Garcia-Cañete, Joaquín, additional, Gadea, Ignacio, additional, Fernandez-Roblas, Ricardo, additional, Blanco, Antonio, additional, and Esteban, Jaime, additional

Published
2017
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41. Evidence of Muller's ratchet in herpes simplex virus type 1

Author

Jaramillo, Nacarí, Domingo, Esteban, Múñoz-Egea, María del Carmen, Tabarés, Enrique, and Gadea, Ignacio

Subjects
DNA polymerase, viruses, Herpesvirus 1, Human, medicine.disease_cause, Genome, Virus, Evolution, Molecular, Lethal Dose 50, Mice, Viral Proteins, chemistry.chemical_compound, Mutation Rate, Virology, medicine, Animals, Point Mutation, Serial Passage, Gene, biology, Genetic Variation, RNA, Herpes Simplex, Sequence Analysis, DNA, Survival Analysis, Disease Models, Animal, Herpes simplex virus, chemistry, Thymidine kinase, DNA, Viral, biology.protein, Female, DNA
Abstract

Population bottlenecks can have major effects in the evolution of RNA viruses, but their possible influence in the evolution of DNA viruses is largely unknown. Genetic and biological variation of herpes simplex virus type 1 (HSV-1) has been studied by subjecting 23 biological clones of the virus to 10 plaque-to-plaque transfers. In contrast to large population passages, plaque transfers led to a decrease in replicative capacity of HSV-1. Two out of a total of 23 clones did not survive to the last transfer in 143 TK-cells. DNA from three genomic regions (DNA polymerase, glycoprotein gD and thymidine kinase) from the initial and passaged clones was sequenced. Nucleotide substitutions were detected in the TK and gD genes, but not in the DNA polymerase gene. Assuming a uniform distribution of mutations along the genome, the average rate of fixation of mutations was about five mutations per viral genome and plaque transfer. This value is comparable to the range of values calculated for RNA viruses. Four plaque-transferred populations lost neurovirulence for mice, as compared with the corresponding initial clones. LD50 values obtained with the populations subjected to serial bottlenecks were 4-to 67-fold higher than for their parental clones. These results equate HSV-1 with RNA viruses regarding fitness decrease as a result of plaque-to-plaque transfers, and show that population bottlenecks can modify the pathogenic potential of HSV-1. Implications for the evolution of complex DNA viruses are discussed. © 2013 SGM., Comunidad Autonoma de Madrid (GR/SAL/0413/2004); BFU2008-02816/BMC and MCINN from BFU2011-23604; Fundación Ramón Areces; (CIBERehd) in funded by the Instituto de Salud Calos III

Published
2013

42. Expression and characterization of the gD protein of HSV-2 fused to the tetramerization domain of the transcription factor p53

Author

Pérez-Hernández, M., Gadea, Ignacio, Escribano, J. M., Tabarés, E., Gómez-Sebastián, S., Pérez-Hernández, M., Gadea, Ignacio, Escribano, J. M., Tabarés, E., and Gómez-Sebastián, S.

Abstract

The highly immunogenic glycoprotein D (gD) of herpes simplex virus type 2 (HSV-2) is a very important element for entry of this virus into host cells. These characteristics have made this protein a very interesting HSV-2 subunit vaccine candidate. Despite efforts to prevent genital herpes using gD-based subunit vaccines, to date, clinical trials using this antigen have failed. Therefore, using a small animal model, we sought to determine if a tetramerized truncated form of gD subunit vaccine, produced by recombinant baculovirus infected insect larvae, would elicit better protection against genital herpes than a monomeric gD-2 subunit vaccine. Three out of 5 mice immunized with the tetramerized antigen produced in a baculovirus expression vector system, survived a lethal challenge with a wild type HSV-2 strain (for more than 3 weeks after challenge). In contrast, all the mice (5) immunized with the truncated protein, produced by the same methodology, died within 2 weeks after challenge. These results suggest that multimerization (increasing the structural complexity) of the truncated gD antigen might be more likely protective than the monomer form. Also the use of an alternative cost-efficient eukaryotic expression system is described. © 2015 Elsevier Inc. All rights reserved.

Published
2015

44. Initial Use of Echinocandins Does Not Negatively Influence Outcome in Candida parapsilosis Bloodstream Infection: A Propensity Score Analysis

Author

Fernández-Ruiz, Mario, primary, Aguado, José María, additional, Almirante, Benito, additional, Lora-Pablos, David, additional, Padilla, Belén, additional, Puig-Asensio, Mireia, additional, Montejo, Miguel, additional, García-Rodríguez, Julio, additional, Pemán, Javier, additional, Ruiz Pérez de Pipaón, Maite, additional, Cuenca-Estrella, Manuel, additional, Muñoz, Patricia, additional, Guinea, Jesús, additional, Paño Pardo, José Ramón, additional, Cerrada, Carlos García, additional, Fortún, Jesús, additional, Martín, Pilar, additional, Gómez, Elia, additional, Ryan, Pablo, additional, Campelo, Carolina, additional, de los Santos Gil, Ignacio, additional, Buendía, Ventura, additional, Gorricho, Beatriz Perez, additional, Alonso, Mercedes, additional, Sanz, Francisca Sanz, additional, Merino, Paloma, additional, Romo, Fernando González, additional, Gorgolas, Miguel, additional, Gadea, Ignacio, additional, Losa, Juan Emilio, additional, Delgado-Iribarren, Alberto, additional, Ramos, Antonio, additional, Romero, Yolanda, additional, Romero, Isabel Sánchez, additional, Zaragoza, Oscar, additional, Rodriguez-Baño, Jesús, additional, Suarez, Ana Isabel, additional, Loza, Ana, additional, Aller García, Ana Isabel, additional, Martín-Mazuelos, Estrella, additional, de Pipaón, Maite Ruiz Pérez, additional, Garnacho, José, additional, Ortiz, Carlos, additional, Chávez, Mónica, additional, Maroto, Fernando L., additional, Salavert, Miguel, additional, Blanquer, José, additional, Navarro, David, additional, Camarena, Juan José, additional, Zaragoza, Rafael, additional, Abril, Vicente, additional, Gimeno, Concepción, additional, Hernáez, Silvia, additional, Ezpeleta, Guillermo, additional, Bereciartua, Elena, additional, Hernández Almaraz, José L., additional, Rivas, Rosa Ana, additional, Ayarza, Rafael, additional, Ma Planes, Ana, additional, Ruiz Camps, Isabel, additional, Mensa, José, additional, Almela, Manel, additional, Gurgui, Mercè, additional, Sánchez-Reus, Ferran, additional, Martinez-Montauti, Joaquin, additional, Sierra, Montserrat, additional, Horcajada, Juan Pablo, additional, Sorli, Luisa, additional, Gómez, Julià, additional, Gené, Amadeu, additional, Urrea, Mireia, additional, Valerio, Maricela, additional, Díaz-Martín, Ana, additional, Puchades, Francesc, additional, and Mularoni, Alessandra, additional

Published
2014
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