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 f...
| Autores: | , , , , , , , , , , , , , |
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| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2025 |
| País: | España |
| Institución: | Consejo Superior de Investigaciones Científicas (CSIC) |
| Repositorio: | DIGITAL.CSIC. Repositorio Institucional del CSIC |
| OAI Identifier: | oai:digital.csic.es:10261/390102 |
| Acceso en línea: | http://hdl.handle.net/10261/390102 |
| Access Level: | acceso abierto |
| Palabra clave: | RNA virus defective viral genome deletion diversity indices mutant spectrum point mutation population complexity quasispecies Ultra-deep sequencing viral clade |
| Sumario: | 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 2.8 variations per viable genome, relative to the consensus sequence of the parental USA-WA1/2020 virus. This value is 6.5-fold lower than the estimates for biological clones of other RNA viruses such as bacteriophage Qβ, 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 in the 3,719 genomic residues that were screened. 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.IMPORTANCESequencing of biological clones is a means to identify mutations, insertions, and deletions located in viable genomes. This distinction is particularly important for viral populations, such as those of SARS-CoV-2, that contain large proportions of defective genomes. By sequencing biological clones and sub-clones, we quantified the heterogeneity of the viable complement of USA-WA1/2020 to be lower than exhibited by other RNA viruses. This difference may be due to a reduced mutation rate or to limited tolerance of the large coronavirus genome to incorporate mutations and deletions and remain functional or a combination of both influences. The presence of clade-discordant residues in the progeny of individual biological sub-clones suggests limitations in the occupation of sequence space by SARS-CoV-2. However, the complex and unique mutant spectra that are rapidly generated from individual genomes suggest an aptness to confront selective constraints. |
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