Association of mutant spectra of SARS-CoV-2 with COVID-19 disease severity

Abstract

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.