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 sarbecovirus species 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 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. To estimate five gene phylogenetic maximum likelihood (ML) trees were inferred for different subdomains in ORF1ab (Nsp1–2, indel hotspot domain and subsequent regions of Nsp3, and Nsp4–16) and spike genes ( N terminal domain, receptor-binding domain, and S2) using the Tamura-Nei model (Tamura and Nei 1993). Phylogenetic reconstruction was performed for the membrane ( M ), ORF8 , and nucleocapsid ( N ) genes. Phylogenetic trees were constructed using the MEGA11 software with 100 bootstraps resamplings. The nearest-neighbor interchange method was used for the ML heuristic search process. Branch lengths were the genetic distances measured by the number of substitutions per site. The present study has several limitations. First, the structures of the inferred phylogenetic ML trees in some genomic regions were unstable, with relatively low reproducibility in 100 bootstrap resamplings, especially for the S1 and peri- ORF8 genes, both of which are indel hotspots with clustered long and complex indels. In these regions, virus species belonging to the SARS-clade and COVID-19-clade could not be clearly distinguished. Extra caution is required when interpreting the inferred phylogenetic trees in these regions, which may not be suitable for multiple sequence alignment or phylogenetic reconstructions. Second, this study could not determine the molecular mechanisms of genetic recombination, such as whether the recombination events were based on replicative or non-replicative mechanisms. The former replicative mechanisms include the copy-choice recombination based on RdRp template switching with homologous templates from co-infected different viral species (Chrisman et al. 2021; Francisco Junior et al. 2022); however, most of the mechanisms and processes underpinning copy-choice recombination remain unclear. Future studies are needed to elucidate the possible role of genetic recombination in the evolutionary history of SARS-related coronaviruses. trees between genomic of the distributions of recombination sites and indel hotspots were not identical in most recombinant viruses. Further studies are needed to delineate the roles and mechanisms of genetic recombination and clustered complex indels in the evolutionary history of SARS-related coronaviruses.