A comprehensive evolutionary and epidemiological characterization of insertion and deletion mutations in SARS-CoV-2 genomes.

SARS-CoV-2, which causes the current pandemic of respiratory illness, is evolving continuously and generating new variants. Nevertheless, most of the sequence analyses thus far focused on nucleotide substitutions despite the fact that insertions and deletions (indels) are equally important in the evolution of SARS-CoV-2. In this study, we analyzed 1,099,664 high-quality sequences of SARS-CoV-2 genomes to re-construct the evolutionary and epidemiological histories of indels. Our analysis revealed 289 circulating indel types (237 deletion and 52 insertion types, each represented by more than ten genomic sequences), among which eighteen were recurrent indel types, each represented by more than 500 genome sequences. Although indels were identified across the entire genome, most of them were identified in nsp6, S, ORF8, and N genes, among which ORF8 indel types had the highest frequencies of frameshift. Geographical and temporal analyses of these variants revealed a few alterations of dominant indel types, each accompanied by geographic expansion to different countries and continents, which resulted in the fixation of several types of indels in the field, including the current variants of concern. Evolutionary and structural analyses revealed that indels involving S N-terminal domain regions were linked to the 3/4 variants of concern, resulting in significantly altered S protein that might contribute to the selective advantage of the corresponding variant. In sum, our study highlights the important role of insertions and deletions in the evolution and spread of SARS-CoV-2.

A comprehensive evolutionary and epidemiological characterization of insertion and deletion mutations in SARS-CoV-2 genomes

SARS-CoV-2, which causes the current pandemic of respiratory illness, is evolving continuously and generating new variants. Nevertheless, most of the sequence analyses thus far focused on nucleotide substitutions despite the fact that insertions and deletions (indels) are equally important in the evolution of SARS-CoV-2. In this study, we analyzed 1,099,664 high-quality sequences of SARS-CoV-2 genomes to re-construct the evolutionary and epidemiological histories of indels. Our analysis revealed 289 circulating indel types (237 deletion and 52 insertion types, each represented by more than ten genomic sequences), among which eighteen were recurrent indel types, each represented by more than 500 genome sequences. Although indels were identified across the entire genome, most of them were identified in nsp6, S, ORF8, and N genes, among which ORF8 indel types had the highest frequencies of frameshift. Geographical and temporal analyses of these variants revealed a few alterations of dominant indel types, each accompanied by geographic expansion to different countries and continents, which resulted in the fixation of several types of indels in the field, including the current variants of concern. Evolutionary and structural analyses revealed that indels involving S N-terminal domain regions were linked to the 3/4 variants of concern, resulting in significantly altered S protein that might contribute to the selective advantage of the corresponding variant. In sum, our study highlights the important role of insertions and deletions in the evolution and spread of SARS-CoV-2.

A Convenient and Biosafe Replicon with Accessory Genes of SARS-CoV-2 and Its Potential Application in Antiviral Drug Discovery.

SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far. Due to the high infectivity and pathogenicity of this virus, all studies on the live virus are strictly confined in the biosafety level 3 (BSL3) laboratory but this would hinder the basic research and antiviral drug development of SARS-CoV-2 because the BSL3 facility is not commonly available and the work in the containment is costly and laborious. In this study, we constructed a reverse genetics system of SARS-CoV-2 by assembling the viral cDNA in a bacterial artificial chromosome (BAC) vector with deletion of the spike (S) gene. Transfection of the cDNA into cells results in the production of an RNA replicon that keeps the capability of genome or subgenome replication but is deficient in virion assembly and infection due to the absence of S protein. Therefore, such a replicon system is not infectious and can be used in ordinary biological laboratories. We confirmed the efficient replication of the replicon by demonstrating the expression of the subgenomic RNAs which have similar profiles to the wild-type virus. By mutational analysis of nsp12 and nsp14, we showed that the RNA polymerase, exonuclease, and cap N7 methyltransferase play essential roles in genome replication and sgRNA production. We also created a SARS-CoV-2 replicon carrying a luciferase reporter gene and this system was validated by the inhibition assays with known anti-SARS-CoV-2 inhibitors. Thus, such a one-plasmid system is biosafe and convenient to use, which will benefit both fundamental research and development of antiviral drugs.