SARS-CoV-2 biological clones are genetically heterogeneous, and include clade-discordant residues
Defective genomes are part of SARS-CoV-2 quasispecies. High resolution, ultra-deep sequencing of bulk RNA from viral populations does not distinguish RNA mutations, insertions and deletions in viable genomes from those in defective genomes. To quantify SARS-CoV-2 infectious variant progeny, virus from four individual plaques (biological clones) of a preparation of isolate USA-WA1/2020, formed on Vero E6 cell monolayers, was subjected to further biological cloning, to yield 9 second-generation and 15 third-generation sub-clones. Consensus genomic sequences of the biological clones and sub-clones included an average of 2.7 variations per viable genome, relative to the consensus sequence of the parental USA-WA1/2020 virus. This value is 8.4-fold lower than the estimates for biological clones of other RNA viruses such as bacteriophage Qß, foot-and-mouth disease virus or hepatitis C virus in cell culture. The mutant spectrum complexity of the nsp12 (polymerase)- and spike (S)-coding region was unique in the progeny of each of 10 third-generation sub-clones; they shared 2.4% of the total of 164 different mutations and deletions scored. The presence of minority out-of-frame deletions revealed the ease of defective genome production from an individual infectious genome. Several low-frequency point mutations and deletions were clade-discordant in that they were not typical of USA-WA1/2020, but served to define the consensus sequences of future SARS-CoV-2 clades. Implications for SARS-CoV-2 adaptability and COVID-19 control of the viable genome heterogeneity and the generation of complex mutant spectra from individual genomes are discussed.