Evidence for SARS-CoV-2 related coronaviruses circulating in bats and pangolins in Southeast Asia

Abstract

Among the many questions unanswered for the COVID-19 pandemic are the origin of SARS-CoV-2 and the potential role of intermediate animal host(s) in the early animal-to-human transmission. The discovery of RaTG13 bat coronavirus in China suggested a high probability of a bat origin. Here we report molecular and serological evidence of SARS-CoV-2 related coronaviruses (SC2r-CoVs) actively circulating in bats in Southeast Asia. Whole genome sequences were obtained from five independent bats (Rhinolophus acuminatus) in a Thai cave yielding a single isolate (named RacCS203) which is most related to the RmYN02 isolate found in Rhinolophus malayanus in Yunnan, China. SARS-CoV-2 neutralizing antibodies were also detected in bats of the same colony and in a pangolin at a wildlife checkpoint in Southern Thailand. Antisera raised against the receptor binding domain (RBD) of RmYN02 was able to cross-neutralize SARS-CoV-2 despite the fact that the RBD of RacCS203 or RmYN02 failed to bind ACE2. Although the origin of the virus remains unresolved, our study extended the geographic distribution of genetically diverse SC2r-CoVs from Japan and China to Thailand over a 4800-km range. Cross-border surveillance is urgently needed to find the immediate progenitor virus of SARS-CoV-2.

Fig. 1. Molecular detection of a SC2r-CoV in bats in Thailand.

a Map of Asia illustrating the SC2r-CoVs detected in this region to date. b The Acuminatus horseshoe bats from which the SC2r-CoV was detected. Photo taken by the Thai research team of this study group. c Similarity plot (SimPlot) of whole-genome sequences of 10 SARSr-CoVs using the RacCS203 as a reference genome. d Phylogenetic tree based on whole-genome sequences. e Phylogenetic tree based on the RdRp gene sequences. The trees in d and e were generated using PhyML with general-time-reversible (GTR) substitution model and 1000 bootstrap replicates. Numbers (>70) above or below the branches are percentage bootstrap values for the associated nodes. The scale bar represents the number of substitutions per site. RacCS203 was highlighted in red.

Fig. 2. Analysis of RBD sequences and function.

a Phylogenetic tree based on the S gene sequences. b Phylogenetic tree based on the nucleotide sequences of the RBD coding regions. The trees in a and b were generated using PhyML with substitution model GTR and 1000 bootstraps. Numbers (>70) above or below the branches are percentage bootstrap values for the associated nodes. The scale bar represents the number of substitutions per site. c Amino acid sequence comparison of the receptor binding motif (RBM) of seven SARSr-CoVs with the red dots indicating amino acid residues critical for ACE2 interaction. d Structural modeling of the RBD-hACE2 interaction for RacCS203 based on the known structure of the SARS-CoV-2 RBD. e Multiplex binding assay to measure affinity of different SARSr-CoV RBDs to PE-conjugated hACE2. Data presented were derived from three independent experiments. Error bars indicate standard deviation. Graph was plotted using GraphPad Prism 8.

Fig. 3. Serological investigation.

a SARS-CoV-2 sVNT results from bat sera in Thailand. b SARS-CoV-2 sVNT results from pangolin sera in Thailand. c SARS-CoV VNT and C-ELISA testing for pangolin sera sampled from animals originated from Southeast Asia. d Multiplex-RBD binding study to examine antigenic cross reactivity between human sera and different RBDs. e SARS-CoV-2 sVNT assay to examine cross-neutralization ability of different rabbit anti-RBD sera. Data indicate percentage of inhibition at 1:20 dilution. f SARS-CoV-2 PRNT assay to examine cross-neutralization ability of different rabbit anti-RBD sera. Data indicate percentage of inhibition at 1:10 dilution. Red dot line indicates cut-off at 20% inhibition. Pre indicates pre-immunized sera; First and Second indicates sera obtained post first immunization and second immunization, respectively. All graphs and heatmap presented were generated using GraphPad Prism 8.

Fig. 4. Location distribution of Rhinolophus species carrying SC1r-CoV and SC2r-CoV.

Distribution of a SC2r-CoV and b SC1r-CoV in Southeast Asia; Phylogeny trees of c SARSr-CoVs and d Rhinolophus species constructed using PhyML with substitution model GTR and 1000 bootstraps. Numbers (>70) above or below the branches are percentage bootstrap values for the associated nodes. The scale bar represents the number of substitutions per site.