Insertions in SARS-CoV-2 genome caused by template switch and duplications give rise to new variants that merit monitoring

Abstract

The appearance of multiple new SARS-CoV-2 variants during the winter of 2020-2021 is a matter of grave concern. Some of these new variants, such as B.1.351 and B.1.1.17, manifest higher infectivity and virulence than the earlier SARS-CoV-2 variants, with potential dramatic effects on the course of the COVID-19 pandemic. So far, analysis of new SARS-CoV-2 variants focused primarily on point nucleotide substitutions and short deletions that are readily identifiable by comparison to consensus genome sequences. In contrast, insertions have largely escaped the attention of researchers although the furin site insert in the spike protein is thought to be a determinant of SARS-CoV-2 virulence and other inserts might have contributed to coronavirus pathogenicity as well. Here, we investigate insertions in SARS-CoV-2 genomes and identify 141 unique inserts of different lengths. We present evidence that these inserts reflect actual virus variance rather than sequencing errors. Two principal mechanisms appear to account for the inserts in the SARS-CoV-2 genomes, polymerase slippage and template switch that might be associated with the synthesis of subgenomic RNAs. We show that inserts in the Spike glycoprotein can affect its antigenic properties and thus have to be monitored. At least, two inserts in the N-terminal domain of the Spike (ins246DSWG and ins15ATLRI) that were first detected in January 2021 are predicted to lead to escape from neutralizing antibodies whereas other inserts might result in escape from T-cell immunity.

The Remarkable Evolutionary Plasticity of Coronaviruses by Mutation and Recombination: Insights for the COVID-19 Pandemic and the Future Evolutionary Paths of SARS-CoV-2

“…This FCS has not yet been found in other sarbecoviruses, but it is present in other Beta-CoV subgenera [4,5,90,91]. One hypothesis about the origin of the FCS is that it was generated naturally by a template switch, followed by substitutions that eventually erased its similarity to the original sequence [87]. The FCS sequences of several CoVs resemble polybasic low-complexity regions that tend to evolve fast [92].…”

“…Large-scale SARS-CoV-2 sequencing data (>1.7 million genomes) coupled with sophisticated bioinformatics analyses have revealed many such events, which are located more frequently at the 3 half of the genome. These occur especially at the spike region and have the potential to lead to escape from neutralizing antibodies or even T-cell immunity [87]. For example, the Omicron variant has three deletions and one insertion within the spike and three deletions in other genomic regions.…”

There is still no evidence of SARS‐CoV‐2 laboratory origin: Response to Segreto and Deigin (10.1002/bies.202100137)

“…This scenario seems plausible, since it has been shown that insertions are not infrequent in SARS-CoV-2 and that "although this insert has a high GC-content compared to the genomic average of SARS-CoV-2, it falls within the GC-content range of the long inserts'' and is located within 20 nucleotides of a template switch hotspot at position 22 582. [33] Furthermore, Holmes et al reported that "both CGG codons are more than 99.8% conserved among the > 1,800,000 near complete SARS-CoV-2 genomes sequenced to date," [10] suggesting that these codons are preserved by stabilizing selection and, therefore, may be associated with high fitness. In this regard, the FCS insertion structure appears to be well consistent with the hypothesis of natural origin and subsequent evolution-driven codon optimization.…”

“…This scenario seems plausible, since it has been shown that insertions are not infrequent in SARS‐CoV‐2 and that “although this insert has a high GC‐content compared to the genomic average of SARS‐CoV‐2, it falls within the GC‐content range of the long inserts'' and is located within 20 nucleotides of a template switch hotspot at position 22 582. [ 33 ]…”

“…Since the start of the pandemics, more than 20 long insertions in the coronavirus genome have been identified, some of which are even bigger than the 12‐nt FCS insertion and have also resulted in a codon split. [ 33 ]…”