Key Points
Background
The Delta variant (Pango lineage B.1.617.2) spread rapidly around the world in 2021 and was associated with major waves of COVID-19 infections. Scientists have extensively studied the mutations that enhanced its transmissibility, but less attention has been paid to the types of genetic changes that generated those mutations. The SARS-CoV-2 spike protein is responsible for viral entry into host cells and plays a central role in transmission and immune evasion. As the virus evolved, mutations in the spike protein helped successive variants spread despite immunity acquired through infection or vaccination.
The Delta variant carries seven defining amino acid substitutions in its spike protein in addition to the widely shared D614G mutation. Two of these substitutions, T19R and P681R, originated through C→G transversions. Because C→G transversions are among the rarest mutation types observed in SARS-CoV-2, the researcher investigated whether this pattern represented an unusual evolutionary event.
Research Methods
The study analyzed publicly available SARS-CoV-2 genome sequences deposited in the NCBI GenBank and GISAID databases. Mutation patterns were compared across major SARS-CoV-2 lineage transitions and within individual viral lineages.
Researchers examined all 12 possible nucleotide substitution types and evaluated them separately in the spike-coding region and in the remainder of the viral genome. Particular attention was given to C→G transversions because previous studies have consistently shown them to be exceptionally rare in SARS-CoV-2 and other coronaviruses.
Results
C→G transversions were nearly absent during the evolution of other major SARS-CoV-2 variants. Excluding Delta, only 3 of 342 substitutions (0.88%) detected during major lineage transitions were C→G transversions. Importantly, none of the 165 spike-region substitutions observed during these transitions involved a C→G event. This remained true even when major evolutionary transitions leading to Omicron sublineages were included in the analysis.
A similarly low frequency was observed during evolution within individual viral lineages. In lineage B.1.1, only 1 of 179 spike-region substitutions (0.56%) was a C→G transversion. In the Alpha variant lineage (B.1.1.7), only 1 of 865 spike-region substitutions (0.12%) involved a C→G change.
In contrast, the Delta lineage contained two C→G transversions among 32 characteristic substitutions across its genome, and both were located in the spike gene. As a result, 2 of the 7 defining spike substitutions (28.6%) were C→G transversions. Statistical analyses demonstrated that this proportion was significantly higher than those observed during non-Delta lineage evolution and during evolution within the B.1.1 and Alpha lineages, even after correction for multiple comparisons.
The study also found that four of the seven defining amino acid substitutions in the Delta spike protein introduced positively charged basic amino acids. Two of these substitutions, T19R and P681R, resulted directly from C→G transversions. Previous studies have shown that P681R enhances furin-mediated cleavage of the spike protein and is thought to contribute to increased infectivity and transmissibility.
Significance
Why the Delta variant acquired this distinctive mutation pattern remains uncertain. To investigate possible explanations, the researcher examined viral genomes collected before the emergence of Delta. No viral sequences carrying four or more Delta-defining spike mutations simultaneously were identified before the first recorded Delta sequence, suggesting that the observed mutation pattern is not readily explained by simple recombination among previously circulating lineages.
One possible explanation is that the Delta lineage evolved, at least temporarily, in a biological environment where C→G transversions occur more frequently than they do in typical human SARS-CoV-2 transmission. Previous studies have reported elevated frequencies of C→G substitutions in some non-human environments, including murine systems. However, no direct evidence currently identifies a specific host species or environment responsible for the emergence of Delta. The author emphasizes that this hypothesis remains speculative and requires further investigation.
The findings highlight a previously underappreciated feature of Delta variant evolution and may help improve scientific understanding of the evolutionary mechanisms that contribute to the emergence of highly transmissible viral variants.
Glossary
C→G Transversion
A nucleotide substitution in which cytosine (C) is replaced by guanine (G). Among the twelve possible single-nucleotide substitution types, C→G transversions are among the rarest observed during SARS-CoV-2 evolution.
NCBI GenBank
A publicly accessible nucleotide sequence database maintained by the U.S. National Center for Biotechnology Information (NCBI).
GISAID
An international platform that enables rapid sharing of genomic sequence data for influenza viruses, SARS-CoV-2, and other pathogens.
T19R
A spike-protein amino acid substitution in which threonine (T) at position 19 is replaced by arginine (R).
P681R
A spike-protein amino acid substitution in which proline (P) at position 681 is replaced by arginine (R). The substitution has been linked to enhanced furin-mediated spike cleavage and increased transmissibility.
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This research was funded by the Transdisciplinary Review Association, Japan.
Title of original paper:
Unusual Enrichment of Rare C-to-G Transversions in the SARS-CoV-2 Delta Spike
Journal:
Infection, Genetics and Evolution
DOI:
10.1016/j.meegid.2026.105989
Associate Professor KAKEYA, Hideki
Institute of Systems and Information Engineering, University of Tsukuba
Institute of Systems and Information Engineering
Unusual enrichment of rare C-to-G transversions in the SARS-CoV-2 delta spike
11-Jul-2026