A novel SARS-CoV-2 related coronavirus in bats from Cambodia

Abstract

Knowledge of the origin and reservoir of the coronavirus responsible for the ongoing COVID-19 pandemic is still fragmentary. To date, the closest relatives to SARS-CoV-2 have been detected in Rhinolophus bats sampled in the Yunnan province, China. Here we describe the identification of SARS-CoV-2 related coronaviruses in two Rhinolophus shameli bats sampled in Cambodia in 2010. Metagenomic sequencing identifies nearly identical viruses sharing 92.6% nucleotide identity with SARS-CoV-2. Most genomic regions are closely related to SARS-CoV-2, with the exception of a region of the spike, which is not compatible with human ACE2-mediated entry. The discovery of these viruses in a bat species not found in China indicates that SARS-CoV-2 related viruses have a much wider geographic distribution than previously reported, and suggests that Southeast Asia represents a key area to consider for future surveillance for coronaviruses.

Fig. 1. Phylogenetic analysis of SARS-CoV-2 and representative sarbecoviruses and geographical context.

a Maximum likelihood phylogeny of the subgenus Sarbecovirus (genus Betacoronavirus; n = 39) estimated from complete genome sequences using IQ-TREE and 1000 replicates. The coronaviruses of the SARS-CoV-2 lineage are color coded by country of sampling as on the map. In orange, Cambodia, light orange, Thailand and blue, China. Taxa names include the isolate name, country and province of sampling, and host. The scientific names of the hosts are abbreviated as follows: Bats: R. affinis, Rhinolophus affinis; R. sinicus, Rhinolophus sinicus; R. ferrumequinum, Rhinolophus ferrumequinum; R. malayanus, Rhinolophus malayanus; R. acuminatus, Rhinolophus acuminatus; C. plicata, Chaerephon plicata; R. pusillus, Rhinolophus pusillus; R. macrotis, Rhinolophus macrotis; R. monoceros, Rhinolophus monoceros; R. cornutus, Rhinolophus cornutus; Pangolin: M_javanica, Manis javanica and human: H. sapiens, Homo sapiens. A maximum clade credibility tree is available in Supplementary Fig. 3. b map of parts of China and Southeast Asia. Regions where viruses of the SARS-CoV-2 lineage were sampled are colored as in the tree. A black dot indicates a sampling site when known, and the red dot shows the location of Wuhan, where the first cases of SARS-CoV-2 infection were reported.

Fig. 2. Recombination analysis.

a Sliding window analysis of changing patterns of sequence similarity between SARS-CoV-2 and related coronaviruses from China and Cambodia. CoVZXC21 and CoVZC45 were merged for this analysis. Source data are provided as a Source Data file. b Phylogenetic tree of different genomic regions. From left to right, region spanning: nsp1-nsp3, nsp4-nsp8, rdrp-nsp16, spike N terminal domain (NTD), spike receptor binding domain (RBD), orf3-N. Branch support obtained from 1000 bootstrap replicates are shown. SARS-CoV and SARS-CoV-2 sequences are collapsed, and trees are midpoint rooted for clarity.

Fig. 3. Structural modeling.

a Homology modeling of the RBD structure. The three-dimensional structure of the RshSTT200 Spike RBD was modeled using the Swiss-Model program employing the structure of SARS-CoV-2 (PDB: 6yla.1) as a template. The core and external subdomains are colored orange, and gray for RshSTT200 and SARS-CoV-2, respectively. The shortening a loop near the receptor-binding site of RshSTT200 are indicated by a black rectangle. The cysteines involved in a conserved disulfide bond are indicated. b Alignment of the receptor binding motif amino acid sequences of selected betaCoVs. c RshSTT200 spike (deleted for the last 21 amino acids) pseudovirus entry into HEK293T cells transfected with either RshACE2, hACE2 or an empty vector (pLenti-puro). d SARS-CoV-2 spike pseudovirus entry into HEK293T cells transfected with either RshACE2, hACE2 or an empty vector (pLenti-puro). Data are represented as mean ± standard deviation of technical replicates (n = 4) and are representative of three independent experiments. Source data are provided as a Source Data file.