A novel SARS-CoV-2 related coronavirus with complex recombination isolated from bats in Yunnan province, China

Abstract

At the end of 2019, A new type of beta-CoV, SARS-CoV-2 emerged and triggered the COVID-19 pandemic, which spread overwhelmingly around the world in less than a year. However, the origin and direct ancestral viruses of SARS-CoV-2 remain unknown. RaTG13, a novel coronavirus found in bats in China's Yunnan Province, is the closest relative virus of the SARS-CoV-2 identified so far. In this study, a new SARS-CoV-2 related virus, provisionally named PrC31, was discovered in Yunnan province by retrospectively analyse bat next generation sequencing (NGS) data of intestinal samples collected in 2018. PrC31 shared 90.7% and 92.0% nucleotide identities to the genomes of SARS-CoV-2 and the bat SARSr-CoV ZC45, respectively. Sequence alignment of PrC31 showed that several genomic regions, especially orf1a and orf8 had the highest homology with those corresponding genomic regions of SARS-CoV-2 than any other related viruses. Phylogenetic analysis indicated that PrC31 shared a common ancestor with SARS-CoV-2 in evolutionary history. The differences between the PrC31 and SARS-CoV-2 genomes were mainly manifested in the spike genes. The amino acid homology between the receptor binding domains of PrC31 and SARS-CoV-2 was only 64.2%. Importantly, recombination analysis revealed that PrC31 underwent multiple complex recombination events (including three recombination breakpoints) involving the SARS-CoV and SARS-CoV-2 sub-lineages, indicating that PrC31 evolved from yet-to-be-identified intermediate recombination strains. Combined with previous studies, it is revealed that the beta-CoVs may possess a more complex recombination mechanism than we thought.

Keywords: SARS-CoV-2; bats; coronaviruses; evolution; recombination.

Figure 1.

Homology modelling structures and Characterization of Receptor binding domain (RBD) of PrC31 and representative Beta-CoVs. (A-D) Homology modelling structures of PrC31 and representative Beta-CoVs. The three-dimensional structures of PrC31, ZC45 and SARS-CoV-2 RBDs were modelled using the Swiss-Model programme, using the SARS-CoV-2 RBD structure (PDB: 7a91.1) as a template. The two deletion loops in PrC31 and ZC45 are marked with a circle. (E) Characterization of the RBDs of PrC31 and representative beta-CoVs. The six critial amino acid residues for ACE2 interaction were marked using red star.

Figure 2.

Phylogenetic trees of SARS-CoV-2 and representative sarbecoviruses. (A)Complete genome; (B)RdRp gene (C)S gene (D)RBD region. SARS-CoV lineage and SARS-CoV-2-related lineages are shown in orange and purple shadow, respectively. Viruses that originated in bats are labelled in blue, human viruses are labelled in red and pangolin viruses are labelled in green. The PrC31 identified in this study is highlighted in yellow shadow. Phylogenetic analyses were performed with RaxML software (v8.2.11) using the GTR nucleotide substitution model, GAMMA distribution of rates among sites, and 1000 bootstrap replicates

Figure 3.

Genome organization and Recombination analysis. A. Genome organization of PrC31. (B) Similarity plot and (C) Bootstrap plot of full-length genome of human SARS-CoV-2, pangolin- and bat beta-CoVs using PrC31as the query. Slide window was set to 1000 bp with 100 bp steps.

Figure 4.

Phylogenetic trees of various regions of the PrC31 genome. SARS-CoV and SARS-CoV-2-related lineages are shown as orange and purple shadow, respectively. The PrC31 virus identified in this study is indicated with yellow shadow. Viral taxonomy is labelled in colour that originated in bats are labelled in blue, humans in red, and pangolins in green. Phylogenetic analyses were performed with RaxML software (v8.2.11) using the GTR nucleotide substitution model, GAMMA distribution of rates among sites, and 1000 bootstrap replicates. Region 1:1-12,719 bp; Region 2: 12,720-20,244; Region 3: 20,244-27142; Region 4: 27,143-29,749.