Genetic Recombination Sites Away from the Insertion/Deletion Hotspots in SARS-Related Coronaviruses

The genome sequences of severe acute respiratory syndrome (SARS)-related coronaviruses (sarbecoviruses) have been reported to include many long and complex insertions/deletions (indels) in specific genomic regions, including open reading frame 1a (ORF1a), S1 domain of the spike, and ORF8 genes. These indel hotspots incorporate various non-classical, long, and complex indels with uncertain developmental processes. A possible explanation for these complex and diversified indels at the hotspots is genetic recombination. To determine the possible association between recombination events and development of indel hotspots, this study investigated the genome sequences of many sarbecoviruses from different countries and hosts and compared the distributions of the indel hotspots and recombination sites by performing multiple sequence alignments and recombination analyses. The genomes of 19 SARS-related coronaviruses (15 coronaviruses that infect bats, two that infect humans, one that infects pangolins, and one that infects civets), including human-infecting SARS-CoV and SARS-CoV-2, were evaluated. Hotspots of complex indels with diverse RNA sequences around gaps were clustered in non-structural protein 2 (Nsp2) and Nsp3 of ORF1a, S1, and ORF8. Phylogenetic reconstructions revealed different structures of the inferred phylogenetic trees between genomic regions, and recombination analyses identified multiple recombination sites across ORF1ab and S genes. However, the nucleotide positions of the indel hotspots were not identical with the identified recombination sites in the recombinant viruses, suggesting the involvement of different developmental processes of indel hotspots and genetic recombination. Further research is required to elucidate the developmental mechanisms underpinning clustered complex indels and recombination events in the evolutionary history of sarbecoviruses.