Incipient functional SARS-CoV-2 diversification identified through neural network haplotype maps. A SARS-CoV-2 ultra-deep sequencing analysis of the mutant spectrum and consensus sequences of nasopharyngeal swabs from patients that became infected in Spain in April 2020

Since its introduction in the human population, SARS-CoV-2 has evolved into multiple clades, but the events in its intra-host 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. The analysis has identified mutations in SARS-CoV-2 quasispecies that occurred during an initial stage of the COVID-19 pandemics within a brief time period, and which are functionally relevant in viral genome replication.