Study population and setting
To investigate the animal origins of SARS-CoV-2, research teams retrospectively analyzed a set of archived samples collected from six families of bats in Cambodia in 2010, and between 2012 and 2018. The samples included 162 oral swabs and 268 rectal swabs that were tested for the presence of coronaviruses using real-time PCR (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. Samples positive for viruses related to SARS-CoV-2 at RdRP were subjected to metagenomic sequencing to obtain full-length genome sequences. Phylogenetic analyses, identification of recombinant regions, and comparison of the spike receptor binding domain (RBD) between SARS-CoV-2 and the novel viruses were performed using the obtained sequences.
Summary of Main Findings
Out of the 430 swab samples tested, 16 (3.72%) were positive for coronaviruses, 5 of which were betacoronaviruses. Samples positive for betacoronaviruses were tested using another RT-PCR targeting RdRp of sarbecoviruses, the group of coronaviruses that includes SARS-CoV, SARS-CoV-2, and related bat coronaviruses. Two of the 5 samples were positive, collected from Rhinolophus shameli bats in December 2010 in Steung Treng province in northeastern Cambodia. Sequencing produced nearly full-length genomes (RshSTT182 and RshSTT200) that were nearly identical to one another in sequence identity and genome structure. Phylogenetic analysis shows that these two viruses form a new sublineage of SARS-CoV-2-related viruses more closely related to SARS-CoV-2 than coronaviruses identified in pangolins but more distant than two viruses identified in Rhinolophus species in Yunnan province, China (RaTG13 and RmYN02). At the whole genome level, the two new viruses share 92.6% nucleotide identity with SARS-CoV-2. One divergent region of the spike N terminal domain clusters more closely with SARS-related coronaviruses, indicating some history of recombination with other sarbecoviruses in this new lineage. The RBD of RshSTT182 and RshSTT200 are highly similar to SARS-CoV-2, sharing 5/6 key amino acids involved in binding to the ACE2 receptor for entry into human cells. However, the unique polybasic cleavage site within the spike protein of SARS-CoV-2 is absent in the two new viruses.
The study benefits from a moderately large sample size of bats from multiple species in Cambodia, a region with a high diversity of Rhinolophus species. The sequencing methods used were appropriate for indicating the unique features of RshSTT182 and RshSTT200 compared to SARS-CoV-2 and other viruses.
While the viruses detected in Cambodian bats show important similarities with SARS-CoV-2, the viruses are still quite distantly related to SARS-CoV-2 and do not represent the direct evolutionary progenitor of SARS-CoV-2. The study was a retrospective analysis of samples, so the results may not represent the diversity of coronaviruses circulating currently in this region. Analysis of the RBD amino acids is not sufficient to establish that these viruses may enter human or bat cells; additional experiments using cell cultures must be performed to ascertain this. Data on the other bat species or the sample types (oral or fecal) that were positive were reported in the Supplementary Materials, which are not currently available with the preprint.
Although the new viruses do not represent the closest relative of SARS-CoV-2 in bats, this paper does expand the species range and geographic area where SARS-CoV-2-related viruses may be circulating. Similar to SARS-related coronaviruses, it appears that these viruses may be widespread in Rhinolophus bats in East and Southeast Asia and that recombination among virus lineages is facilitated by the overlapping geographic ranges of Rhinolophus species in this region.
This review was posted on: 12 February 2021