Association of mutant spectra of SARS-CoV-2 with COVID-19 disease severity
Authors:   Department of Clinical Microbiology, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain, Martínez-González Brenda ,   Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain, Eugenia Soria María ,   Department of Clinical Microbiology, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain,   Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC-UAM), Madrid, Spain, Vázquez-Sirvent Lucía ,   1Department of Clinical Microbiology, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain, Ferrer-Orta Cristina ,   Structural Biology Department, Institut de Biología Molecular de Barcelona CSIC, Barcelona, Spain, Mínguez Pablo ,   Department of Genetics & Genomics, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain,   Centre for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Madrid, Spain,   Bioinformatics Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain, de la Fuente Lorena ,   Department of Genetics & Genomics, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain,   Centre for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Madrid, Spain,   Bioinformatics Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain, Llorens Carlos ,   Biotechvana, “Scientific Park”, Universidad de Valencia, Valencia, Spain, Soriano Beatriz ,   Biotechvana, “Scientific Park”, Universidad de Valencia, Valencia, Spain, Ramos Ricardo ,   Unidad de Genómica, “Scientific Park of Madrid”, Campus de Cantoblanco, Madrid, Spain, Cortón Marta ,   Department of Genetics & Genomics, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain,   Centre for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Madrid, Spain, García-Crespo Carlos ,   Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC-UAM), Madrid, Spain, Gallego Isabel ,   Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC-UAM), Madrid, Spain,   Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain, de Ávila Ana Isabel ,   Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC-UAM), Madrid, Spain, Gómez Jordi ,   Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain,   Instituto de Parasitología y Biomedicina ‘López-Neyra’ (CSIC), Parque Tecnológico Ciencias de la Salud, Granada, Spain, Salar-Vidal Llanos ,   Department of Clinical Microbiology, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain, Esteban Jaime ,   Department of Clinical Microbiology, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain, Fernandez-Roblas Ricardo ,   Department of Clinical Microbiology, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain, Gadea Ignacio ,   Department of Clinical Microbiology, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain, Ayuso Carmen ,   Centre for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Madrid, Spain, Ruíz-Hornillos Javier ,   Allergy Unit, Hospital Infanta Elena, Valdemoro, Madrid, Spain,   Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain,   Faculty of Medicine, Universidad Francisco de Vitoria, Madrid, Spain, Verdaguer Nuria ,   Structural Biology Department, Institut de Biología Molecular de Barcelona CSIC, Barcelona, Spain, Domingo Esteban ,   Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC-UAM), Madrid, Spain,   Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain, Perales Celia ,   Department of Clinical Microbiology, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain,   Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain,   Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
Published: June 2022

Introduction: Replication of RNA virus, as SARS-CoV-2, leads to composition of sequences which are at different frequencies in the infected host. Minority variants present in mutant spectra can have an epidemiological importance generating more infectious variants, resistance to vaccines and drugs and affecting to the resolution of the disease. How different are the mutant spectra of virus isolated from patients with different disease outcome has not yet been studied. Material and Methods: To approach this question, 30 patients infected with SARS-CoV-2 during the first pandemic wave in Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain were classified as mild, moderate and exitus according to the disease severity of COVID-19. RNA presents in the nasopharyngeal swabs of these patients was extracted. Four amplicons of nsp12 (polymerase) and two amplicons of spike (S) coding regions were amplified. PCR products were sequenced by ultra-deep sequencing (UDS) using MiSeq platform (Illumina). Sequences were analyzed with two pipelines and differences between the mutant spectra of each virus were determined. Possible structural and functional alterations of mutations detected have been studied. Results: Most of the substitutions were found at frequencies between 0.5% to 30% in the mutant spectra. The number of mutations was significantly higher in patients with mild symptoms. The analysis of the mutant spectra resulted in a higher number of transitions and non-synonymous substitutions for in all COVID-19 categories. A major complexity of mutant spectra of virus isolated from mild patients were reflected in the study of several diversity indices, with significant statistical differences between COVID-19 categories. Structural analysis of nsp12 and spike substitutions, detected in the mutant spectra, showed possible alterations in the structure or function of the proteins. Conclusion: The study of mutant spectra shows a positive association between the number of point mutations and the complexity of mutant spectra with a mild disease outcome. Possible models for this association are discussed. Structural and functional alterations are under study right now.