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1. 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

2. 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

3. SARS-CoV-2 mutant spectra at different depth levels reveal an overwhelming abundance of low frequency mutations

Author

Brenda Martínez-González, María Eugenia Soria, Lucía Vázquez-Sirvent, Cristina Ferrer-Orta, Rebeca Lobo-Vega, Pablo Mínguez, Lorena de la Fuente, Carlos Llorens, Beatriz Soriano, Ricardo Ramos-Ruíz, Marta Cortón, Rosario López-Rodríguez, Carlos García-Crespo, Pilar Somovilla, Antoni Durán-Pastor, Isabel Gallego, Ana Isabel de Ávila, Soledad Delgado, Federico Morán, Cecilio López-Galíndez, Jordi Gómez, Luis Enjuanes, Llanos Salar-Vidal, Mario Esteban-Muñoz, Jaime Esteban, Ricardo Fernández-Roblas, Ignacio Gadea, Carmen Ayuso, Javier Ruíz-Hornillos, Nuria Verdaguer, Esteban Domingo, Celia Perales, UAM. Departamento de Medicina Preventiva y Salud Pública y Microbiología, 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, Soria, María Eugenia, Lobo-Vega, Rebeca, Mínguez, Pablo, Llorens, Carlos, Ramos-Ruiz, Ricardo, Cortón, Marta, García-Crespo, Carlos, Durán-Pastor, Antoni, Delgado, Soledad, López-Galíndez, Cecilio, Enjuanes Sánchez, Luis, Salar-Vidal, Llanos, Esteban, Jaime, Ayuso, Carmen, Verdaguer, Núria, 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], and Verdaguer, Núria [0000-0001-8826-7129]

Subjects
Microbiology (medical), ultra-deep sequencing, Ultra-deep sequencing, RNA virus, COVID-19, viral quasispecies, mutation, deletion, nsp12 (polymerase), spike, General Immunology and Microbiology, Medicina, Spike, Deletion, Infectious Diseases, Mutation, Immunology and Allergy, Molecular Biology
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., This work was supported by Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (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) 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). 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 supported by predoctoral contract PRE2018-083422 from MCIU. P.S. is supported by postdoctoral contract “Margarita Salas” CA1/RSUE/2021 from MCIU. B.S. was supported by a predoctoral research fellowship (Doctorados Industriales, DI-17-09134) from Spanish MINECO.

Published
2022
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4. High SARS-CoV-2 viral load is associated with a worse clinical outcome of COVID-19 disease

Author

Ignacio Gadea, Javier Ruiz-Hornillos, Rebeca Lobo-Vega, Jaime Esteban, Carmen Ayuso, A. Herrero, Marta Corton, Juan Carlos Taracido, Pablo Minguez, Ricardo Fernández-Roblas, Rosario López-Rodríguez, Ignacio Mahillo, María Eugenia Soria, Alicia Macías-Valcayo, Berta Almoguera, Lucía Vázquez-Sirvent, Brenda Martínez-González, Celia Perales, 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], Banco Santander, Perales, Celia [0000-0003-1618-1937], Soria, María Eugenia, Lobo-Vega, Rebeca, Perales, Celia, Cortón, Marta, Martínez-González, Brenda, Vázquez-Sirvent, Lucía, Mínguez, Pablo, Macías-Valcayo, Alicia, Esteban, Jaime, Gadea, Ignacio, and Ayuso, Carmen

Subjects
medicine.medical_specialty, education.field_of_study, business.industry, viruses, Population, Short Communications, Outbreak, COVID-19, Disease, medicine.disease, viral load, Risk factors, Diabetes mellitus, Intensive care, Internal medicine, Cohort, medicine, risk factors, General Materials Science, Viral load, education, business, Asthma
Abstract

Publisher's version disponible en: http://hdl.handle.net/10261/257116, 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., This work was supported by Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (COVID-19 Research Call COV20/00181) 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 Cientificas (CSIC), BFU2017-91384-EXP from Ministerio de Ciencia, Innovacion y Universidades (MICIU), PI18/00210 from Instituto de Salud Carlos III. C.P., M.C. and P.M. are supported by the Miguel Servet program of the Instituto de Salud Carlos III (CPII19/00001, CPII17/00006 and CP16/00116, respectively) cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigacion en Red de Enfermedades Hepaticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundacion Ramon 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. R L-R is sponsored by the IIS-Fundacion Jimenez Diaz-UAM Genomic Medicine Chair.

Published
2021

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