Study population and setting
To investigate the evolutionary origins of SARS-CoV-2 in animals, researchers sampled 100 Rhinolophus acuminatus bats from a roost in an irrigation pipe in Chachoengsao, Thailand, in June 2020. Coronavirus RNA was detected in rectal swabs using PCR targeting the RNA-dependent RNA polymerase gene (RdRp). Samples with high viral RNA levels were chosen for next-generation sequencing to assemble a complete virus genome. Phylogenetic analysis was performed on the RdRp gene, the whole genome, the full-length spike protein, and the spike receptor-binding domain (RBD). The ability of the RBD to bind to the human angiotensin-converting enzyme 2 (ACE2) and enter human cells was evaluated with a binding assay and via infection of cell cultures with vesicular stomatitis virus (VSV) pseudoviruses expressing coronavirus spike protein. In addition to bats, the researchers sampled ten Malayan pangolins (Manis javanica) confiscated at three checkpoint stations central and southern Thailand between February and July 2020; an additional seven Malayan pangolin samples from imported animals confiscated in Guangdong, China, in May 2003 were also included. Serum samples from R. acuminatus and M. javanica were tested for the presence of neutralizing antibodies against SARS-CoV-2 or related viruses.
Summary of Main Findings
Thirteen of the 100 rectal swabs from R. acuminatus were positive for coronavirus RNA, all with an identical RdRp sequence that had 95.86% sequence identity to SARS-CoV-2. Next generation sequencing of five samples with high viral RNA produced nearly identical genomes, the highest quality of which was named RacCS203. The novel coronavirus RacCS203 fits within the phylogenetic group of betacoronaviruses closely related to SARS-CoV-2 found in Rhinolophus bats and trafficked Malayan pangolins. The closest relative of RacCS203 is the RmYN02 virus isolated from R. malayanus in Yunnan province, China, sharing 93.7% sequence identity across the whole genome. Phylogenetic analysis of the RBD indicated that RacCS203 did not cluster closely to SARS-CoV-2 or other related viruses capable of binding human ACE2, which was confirmed through cell experiments, demonstrating that VSV pseudovirus expressing RacCS203 spike was unable to infect cells expressing human ACE2. Serological analysis showed that 4/98 serum samples from R. acuminatus and 1/10 pangolins from Thailand had neutralizing antibodies against SARS-CoV-2 (all four bats had PCR-positive rectal swabs); 1/7 pangolins sampled in China in 2003 had antibodies against SARS-CoV.
This is this first study to report detection of SARS-CoV-2-related viruses in bats and pangolins sampled in 2020 during the COVID-19 pandemic. The study not only produced a full genome of the novel virus, but also assessed the potential for the virus to enter human cells.
Due to the very limited sample size from bats and pangolins, the prevalence and geographic distribution of SARS-CoV-2-related viruses in these animals in Thailand is unclear. While the researchers performed some analysis of the cross-reactivity of SARS-CoV-2 antibodies to other related viruses, it is not clear from this work whether the neutralizing antibodies detected in Thai bats and pangolins were against SARS-CoV-2, RacCS203, or another related but undetected coronavirus. Finally, none of the pangolins were positive for viral RNA, so it is unknown which virus or viruses these animals may have been exposed to and whether this differed from the infections in bats.
This study expands the observed geographic distribution of SARS-CoV-2 related viruses in bats, which now covers Thailand, Cambodia, southern China, and Japan. In addition, the RacCS203 virus had similar amino acid insertions to RmYN02 in the furin cleavage site of the spike protein. While these insertions had been thought to be unique to SARS-CoV-2, the RacCS203 genome independently confirms similar insertions in a bat virus, providing further evidence that this unique feature in SARS-CoV-2 likely derives from variation in viruses circulating naturally in animal reservoirs.
This review was posted on: 12 March 2021