Independent acquisition of short insertions at the RIR1 site in the spike N-terminal domain of the SARS-CoV-2 BA.2 lineage

Although the SARS-CoV-2 variants BA.1 and BA.2 share over 30 non-synonymous substitutions in the spike glycoprotein, they show several unique mutations that were likely acquired after the split between these two major omicron lineages. One of the most intriguing mutations associated with BA.1 is the presence of the inserted tripeptide Glu-Pro-Glu within the N-terminal domain. While the functional implications of this insertion are still unclear, several other SARS-CoV-2 lineages had previously independently acquired similarly short insertions at the very same site, named RIR1. We have previously identified this site, located approximately between codon 212 and codon 216, as a hotspot of insertions, which usually involve small nucleotide sequences including three or four codons.

Here we show that similar insertion events have independently occurred at least 13 times in early 2022 within the BA.2 lineage, being occasionally associated with significant community transmission. One of these omicron sublineages, characterized by a Ser-Gly-Arg insertion in position 212, is responsible of over 2% of all SARS-CoV-2 cases recorded in Denmark, as of early April 2022. Molecular surveillance data highlight a slow but steady growth compared with the parental BA.2 lineage in all Danish regions, suggesting that the RIR1 insertion may confer a selective advantage. We report the identification of other currently circulating BA.2 sublineages showing similar insertions, whose spread should be therefore carefully monitored in the upcoming months.