A comprehensive survey of bat sarbecoviruses across China for the origin tracing of SARS-CoV and SARS-CoV-2

doi:10.21203/rs.3.rs-885194/v1

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A comprehensive survey of bat sarbecoviruses across China for the origin tracing of SARS-CoV and SARS-CoV-2

https://doi.org/10.21203/rs.3.rs-885194/v1

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published 10 Oct, 2022

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Severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 have been thought to originate from bat, but whether the cross-species transmission occurred directly from bat to human or through an intermediate host remains elusive. In this study, we performed CoV screening of 102 samples collected from animal-selling stalls of Wuhan Huanan Market (WHM) and pharyngeal and anal swabs from13,064 bats collected at 703 locations across China, covering almost all known southern hotspots for sarbecovirus, between 2016 and 2021. This is the first systematic survey of bat CoV in China during the outbreak of Corona Virus Disease 2019. We found four non-sarbeco CoVs in samples of WHM, and 142 SARS-CoV related CoVs (SARSr-CoV) and 4 recombinant CoVs in bats, of which YN2020B-G share the highest sequence identity with SARS-CoV among all known bat CoVs, suggesting endemic SARSr-CoVs in bats in China. However, we did not find any SARS-CoV-2 related CoVs (SC2r-CoV) in any samples, including specimens collected from the only two domestic places where RaTG13 and RmYN02 were previously reported (the Tongguan caves and the karst caves around the Xishuangbanna Tropical Botanical Garden), indicating that SC2r-CoVs might not actively circulate among bats in China. Phylogenetic analysis showed that there are three different lineages of sarbecoviruses, L1 (SARSr-CoV), L2 (SC2r-CoV), and L-R (a novel CoV lineage from L1 and L2 recombination), in China. Of note, L-R CoVs are only found in R. pusillus. Further macroscopical analysis of the genetic diversity, host specificity for colonization and accidental infection, and geographical characteristics of available CoVs in database revealed the presence of a general geographical distribution pattern for bat sarbecoviruses, with the highest genetic diversity and sequence homology to SARS-CoV or SARS-CoV-2 along the southwest border of China, the least in the northwest of China. Considering the receptor binding motifs for spike gene of sarbecoviruses in Indochina Peninsula show the greatest diversity, our data provide the rationale that extensive surveys in further south and southwest to or of China might be needed for finding closer ancestors of SARS-CoV and SARS-CoV-2.

General Microbiology

Virology

SARS-CoV

SARS-CoV-2

bats

China

origin tracing

Coronaviruses (CoVs) are a group of enveloped viruses with a large positive single-stranded RNA genome within the subfamily Orthocoronavirinae that infect varieties of mammals and birds¹. Phylogenetically, CoVs are classified into four genera, Alphacoronavirus (a-CoV), Betacoronavirus (b-CoV), Deltacoronavirus (D-CoV), and Gammacoronavirus (g-CoV) (ICTV; https://talk.ictvonline.org/). Prior to 2019, there are only six known human CoVs (hCoVs), including 229E, NL63, OC43, HKU1, SARS-CoV, and MERS-CoV. Among them, 229E and NL63 belong to a-CoVs, and the remaining four are b-CoVs. While infection by 229E, NL63, OC43 and HKU1 leads to common cold, SARS-CoV and MERS-CoV can cause severe pneumonia, even death². HKU1 and OC43 are thought to originate from rodent, and 229E, NL63, SARS-CoV, and MERS-CoV are considered to be evolved from different bat CoVs^2-6. SARS-related CoVs (SARSr-CoVs) were found in several horseshoe bats (Rhinolophidae) and a few of other bat species, and MERS-related CoVs (MERSr-CoVs) were found in members of the families Vespertilionidae and Nycteridae^5,7-14. However, the immediate precursor, SARS-CoV or MERS-CoV identical virus, has never been detected in bats during the past seventeen years.

In late 2019, a new hCoV, named SARS-CoV-2, was first detected in Wuhan China. Subsequently, as of August 31, 2021, it has spread across the globe and results in over 216 million confirmed cases and more than 4.5 million deaths, posing great threat to global health and economy (https://covid19.who.int/). Genome sequence of SARS-CoV-2 shares about 78.9% homology with that of SARS-CoV. Similar to SARS-CoV, SARS-CoV-2 also belong to Sarbecovirus, a subgenus of b-CoV, and SARS-CoV-2 also uses human angiotensin-converting enzyme 2 (hACE2) as the entry receptor. Several SARS-CoV-2 related CoVs (SC2r-CoVs), including strains RshSTT182, RshSTT200, RacCS203, Rc-o139, RaTG13, RmYN02, and RpYN06, have been identified in several Rhinolophus species (Rhinolophus affinis, R. malayanus, R. pusillus, R. shameli, and R. acuminatus) from Cambodia, Thailand, Japan, and the southern border area of Yunnan separately^15-20, suggesting that SARS-CoV-2 might also be originated from bat. Moreover, several SC2r-CoVs were also found in Malayan pangolins (Manis javanica) seized during anti-smuggling operations^21,22. However, whether these bat and pangolin species could serve as the direct natural reservoirs and intermediate hosts for SARS-CoV-2, respectively, remains unknown because of significant remaining genome differences²³. Discovery of bat CoVs with higher homology than previously reported are warranted for further confirmation of bats as the direct natural hosts of SARS-CoV and SARS-CoV-2.

To develop the databases of the potential zoonotic viral pathogens and their reservoir hosts, we have conducted a series of viral surveys on rodents and bats across China and Southeast Asia (SEA) in the past ten years^24-26, and established two online virome databases (DBatVir and DRodVir)^27,28. In this study, we conducted retrospective CoV tests of environmental samples collected from Wuhan Huanan Market (WHM) and performed virome analysis of samples from over 13 thousand bats collected prior to and during Corona Virus Disease 2019 (COVID-19) pandemic from 14 provinces of China. We discovered a new recombinant lineage of sarbecoviruses and found divergence trend for sarbecoviruses present in China with increase of diversity and homology with SARS-CoV and SARS-CoV-2 in southern China. These data indicate that SC2r-CoV is extremely rare in bats in China, and the more related ancestors of SARS-CoV and SARS-CoV-2 may have been circulating in Indochina Peninsula or even further south.

No SARS-CoV-2 sequences in environmental samples of WHM animal-selling stalls.

Since most of early cases of COVID-19 had connections with WHM, WHM was considered as where the initial outbreak of SARS-CoV-2 occurred. According to WHO-convened global study of origins of SARS-CoV-2, this virus most likely originated from animal spillover to human²⁹. With these in mind, we collected 22 environmental samples from cold storages for animal products and 80 samples from environment around animal-selling stalls of WHM, once we had brief access to WHM in February, 2020. These samples included swabs taken from grounds, walls, sewers, door handles, chopping blocks, knives, and scissors, and were combined into 11 pools, M1 to M11, and subjected for CoV screening and virome analysis (Table 1, Figure 2A). Sequence analysis revealed that there are four animal CoVs present in M1, M6, and M9 pools, indicating that the techniques and methods used are feasible. The M1 pool contained the sequences of hedgehog HKU31-related CoV under the genus Merbecovirus and rabbit HKU14-related CoV under the genus Embecovirus; M6 contained the sequences of canine CoV under the genus Tegacovirus; M9 contained the sequences of rat CoV of the genus Embecovirus. However, none of any sequence of SARS-CoV-2 or SC2r-CoVs was found in any samples, suggesting that the spillover event might not happen in WHM.

Hotspots delineation and bat sampling.

To date, a total of more than 36,000 bat individuals of 100 species across China have been sampled for extensive CoV survey previously. Sixteen bat species, including R. sinicus, R. pusillus, R. ferrumequinum, R. affinis, R. pearsonii, R. macrotis, R. malayanus, R. thomasi, R. rex, R. stheno, Chaerephon plicata, Hipposideros armiger, H. pratti, H. Pomona, Aselliscus stoliczkanus, and Myotis daubentonii, are reported to carry sarbecoviruses in China (Table S1). Among them, R. rex is an endemic species of China, R. ferrumequinum and M. daubentonii are widely distributed in Europa and Asia, while all other species are live in Asia and mainly in South Asia, SEA and southern China (Figure 1). However, only about 7,000 bat individuals of 60 species in Asia except China, and about 9,500 bat individuals of 108 species in Europa and Africa have been sampled for CoVs as of 2020, seven species (R. shameli, R. blasii, R. euryale, R. clivosus, R. acuminatus, R. ferrumequinum and H. galeritus) were reported to be sarbecovirus-positive (Figure 1 and Table S1).

Subsequently, based on region and species hotspots deduced from previous studies, including our first survey between 2010-2013 ²⁶, the sample collection was continuously proceeded in seven provinces of southern China between 2016-2019 (Figure 2B, Figure 2C, Table 2, and Table S2). After the outbreak of coronavirus disease 2019 (COVID-19), in order to seek for origin clues of SARS-CoV-2 and further investigate the diversity of bat CoVs in China, we adapted a much deeper sample collection strategy across Chinese mainland and the project restarted on Jan. 4, 2020. The newly added locations included a series of suspected hotspots of the area around Wuhan city in Hubei province, Zhoushan city in Zhejiang province, Liaoning province, and all the nine southern provinces and related border regions. It is worth noting that Rhinolophus species in the abandoned mine of Tongguan town, where RaTG13 was found, and the karst caves around the Xishuangbanna Tropical Botanical Garden in the southern border of Yunnan province, close to where RmYN02 and RpYN06 were found, were also sampled. In total, our survey covered 703 sampling sites in urban, rural, and wild areas, including 416 old and 287 new sites, where the bat species suspected or confirmed to carry sarbecoviruses are found. Pharyngeal and anal swabs from 13,064 individuals of 56 bat species were collected. The most commonly sampled species were members of the families Rhinolophidae, Hipposideridae, and Vespertilionidae. Twelve species, including R. lepidus, R. marshalli, M. ikonnikovi, M. brandti, M. montivagus, M. indochinensis, M. nipalensis, Scotophilus heathi, Hypsugo cadornae, Nyctalus velutinus, Megaderma spasma, and Pipistrellus ceylonicus, were sampled for the first time.

CoV screening and virome analysis

All pharyngeal and anal swab samples of bats were combined into 372 pools according to the collection date, sampling point and host species (numbered as P001-P372) (Table S3). Partial RNA-dependent RNA polymerase (RdRp) based PCR results showed that 113 pools were a-CoV-positive, 64 pools were b-CoV-positive, and 22 pools were both a-CoV and b-CoV positive (Figure 3A). Meanwhile, a total of 760.3 GB of nucleotide clean data with 1,718,361,529 valid reads was obtained from next generation sequencing. Among them, 569,389,447 reads (~33.14% of the total sequence reads) were matched with CoV proteins available in the NCBI NR database, and 199 out of 372 pools were found to be CoV-positive. The proportion of CoV-related reads in each pool varied from 0.0022% to 98.67% (Figure 3C). All CoV-related reads could be classified into 5 subgenera (Minunacovirus, Decacovirus, Pedacovirus, Rhinacovirus, and Nyctacovirus) of a-CoV and 4 subgenera (Nobecovirus, Merbecovirus, Sarbecovirus, and Embecovirus) of b-CoV.

b-CoVs were mainly found in R. sinicus, Eonycteris spelaea, Tylonycteris pachypus, P. abramus, and Eonycteris spelaea from Guangdong, Yunnan, Guangxi, Jiangxi, and Fujian. In details, nobecoviruses were mainly detected in E. spelaea and Rousettus leschenaulti captured in Yunnan, with 75.7%-99.0% nucleotide (nt) identities to each other within the 440nt fragment of RdRp; merbecoviruses were mainly presented in T. pachypus and P. abramus captured in Guangxi, Guangdong, Jiangxi, and Yunnan with 79.0%-100.0% nt identities to each other within the 440nt fragment of RdRp; sarbecoviruses were mainly found in R. sinicus, R. ferrumequinum and R. affinis captured in Guangdong, Yunnan, Fujian, Guangxi, Hubei, and Liaoning with 83.2%-100% nt identities to each other within the 440nt fragment of RdRp.

To further delineate the prevalence and positive rate of sarbecoviruses, all 1068 individual samples in 44 sarbecovirus-positive pools were selected for sample-by-sample RT-PCR screening, and 146 samples were sarbecovirus-positive (Figure 3B and Table 3). These sarbecoviruses were identified from R. ferrumequinum, R. sinicus, R. pusillus, R. affinis, R. rex, R. luctus, and R. siamensis captured in Guangdong, Yunnan, Fujian, Liaoning, Hunan, Hubei, Jiangxi, Anhui, and Guangxi Provinces, with 88.8%-100% nt identities to known SARSr-CoVs in the 440 nt fragment of RdRp. Among sarbecovirus positive samples, 69 out of the 146 strains were selected for genomic sequencing as quasi-species and their whole-genome sequence was used for subsequent analysis.

Evolution trend derivation for sarbecoviruses

By using the newly identified bat sarbecoviruses here and 123 CoVs available in GenBank or GISAID (list of GenBank and GISAID accession numbers available in Table S1), the phylogenetic reconstruction based on the 440nt RdRp sequences was conducted. As shown in Figure 4, Except one outgroup CoV identified from H. galeritus in Malaysia, all bat sarbecoviruses could be divided into three main lineages, lineage 1 (L1) as SARSr-CoVs, lineage 2 (L2) as SC2r-CoVs, and lineage 3 (L3) as sarbecoviruses found in Europe, Africa, and Taiwan province. An evolutionary origin within a single host family, Rhinolophidae, was found. All sarbecoviruses identified here (labeled by red boxes in Figure 4) were clustered into L1. L1 can be further divided into two sublineages, Lineage 1.1 (L1.1) and Lineage 1.2 (L1.2). L1.1 was mainly found in three bat rhinolophus species (R. sinicus, R. ferrumequinum, and R. pusillus) and occasionally can also be found in other bat species, including R. macrotis, R. thomasi, M daubentonii, and H. armiger, with a wide geographical distribution including China and South Korea. Of note, L1.1 can be further separated into two groups, central-to-northeast and central-to-southeast, showing different geographical distribution in China. L1.2 was mainly presented in four hosts (R.sinicus, R. ferrumequinum, R. pusillus, and R. affinis) and rarely found in other species (R. pearsonii, R. rex, R. siamensis, R. luctus, R. stheno, A. stoliczkanus, H. armiger, H. pratti, and H. Pomona) in southern China. To date, L2 was found in R. pusillus, R. malayanus, R. shameli, R. acuminatus, R. affinis, and R. stheno that scattered among the Indochina Peninsula, the southern border region of Yunnan province, and Japan. L3 contained CoVs found in Rhinolophidae species (R. ferrumequinum, R. blasii, R. euryale, R. hipposideros, R. pusillus, and R. clivosus) from regions outside Chinese mainland, including Taiwan province, Italy, Bulgaria, France, Russia, Luxembourg, Rwanda, Uganda, and Kenya.

The addition of newly identified CoVs here makes the maximum clade credibility (MCC) tree more stable and shows a more obvious clustering trend. Based on the most recent common ancestor (TMRCA) calculation, the subgenus Sarbecovirus might share a common ancestor about 737 years ago (95% highest probability density (HPD): 434–1056), and L3, L2, and L1 were further diverged in order from outgroup CoV (Figure 4). L3 might be diverged from L1 and L2 at 1821 (95% HPD: 1747-1881), and separation between L1 and L2 occurred approximately at 1867 (95% HPD: 1809-1917). The TMRCA of L2 was estimated in 1877 (95% HPD: 1821–1921). The two sublineages of L2, Japan sublineage (L2-JP) and Indochina Peninsula sublineage (L2-IP), may come from a divergence event happening between 1821-1921 (mean of 1874). L1.1 and L1.2 likely shared a common ancestor between 1879-1947 (mean of 1918), and L1.1 was further divided into two directions as central-to-northeast and central-to-southeast around 1927 (95% HPD: 1895–1955) (Figure 4). L1.2 was stem from Yunnan and evolved into different sublineages from southwest to central of China initiated in 1940 (95% HPD: 1909–1969). The differentiation trend of L3 was uncertain because nodes in this lineage had low posterior value due to limited sample size. The closer root distance of the outgroup of Sarbecovirus revealed that some undiscovered evolution events may have happened outside Chinese mainland between 1305 to 1821.

Identification of recombinant lineage between SARS-CoV and SARS-CoV-2 in Sarbecovirus through the whole genome sequence analysis

The nucleotide identities of all nonstructural proteins (NSPs) in ORF1ab, structure proteins, and accessory ORFs among sarbecovirus strains were analyzed (Table S4, S5, and Figure 5). All sarbecoviruses found in this study were related to each other with 78.9%–100% nt identities in ORF1ab and 78.0%–100% nt identities in whole genome level, and they also shared 73.2% to 98.9% nt identities in ORF1ab and 71.6% to 98.6% nt identities at the whole genome level with all available references, respectively.

When SARS-CoV and SARS-CoV-2 genomes are used as the query separately to conduct the homology analysis, in accordance with the phylogenetic tree based on the complete genome sequences, all known sarbecoviruses can be mainly divided into SARS-CoV related branches (L1 in the MCC tree) and SARS-COV-2 related branch (L2 in the MCC tree). Six newly identified isolates (YN2020B-G) collected from the southern border region of Honghe Prefecture in Yunnan share 95.8% nt identity with SARS-CoV at the whole genome level, the highest homology with SARS-CoV among all known bat CoVs. The S genes of YN2020B-G also highly similar to SARS-CoV with 93.3% nt identity.

Surprisingly, we did not detect any bat SC2r-CoVs (L2-IP) in this study despite such a large scale of sampling were conducted, indicating that bat SC2r-CoV might not be widely present in bats in China. Currently, L2-IP was only detected in the Indochina Peninsula and the southern border region of Xishuangbanna Prefecture of Yunnan province, where only limited survey has been conducted, more sampling should be done in these regions.

Of note, four strains, HN2021A, HN2021B, HN2021G, and YN2021, identified from R. pusillus in Hunan and Yunnan provinces, together with previously discovered PrC31, ZXC21, and ZC45 ^30,31, formed a new lineage, in which part of viral genome, from NSP7 or NSP9 to NSP15, shows higher similarity to SARS-CoV with 89.8%-97.8% nt identities, whereas the rest of genome is more homologous to that of SARS-CoV-2 with 87.9%-92.5% nt identities. These results indicate that multiple recombination events occurred in these strains during the evolution, therefore, we named the new lineage as L-R (R stands for recombination). The S genes of L-R are least conserved and showed only ~73.0% nt identity with SARS-CoV and ~75% nt identity with SARS-CoV-2, respectively.

The presence of L-R was long neglected within the subgenus Sarbecovirus, because of usage of RdRp for classification. The extent of L-R recombination history can be illustrated by three phylogenetic trees inferred from two main breakpoints (Figure 6A and 6B). Ancestral recombination events divided the genome of L-R strains into SARSr-CoV related region 2 and SC2r-CoV related regions 1 and 3. When the first 11 kb and last 10 kb of the genomes were used to construct the tree, seven L-Rs could be phylogenetically clustered with L2-IP. In contrast, when the middle part of genomes between NSP9 to NSP15 was inspected, the seven L-Rs were clustered in L1, consistent with 440 nt RdRp based evolution analysis (labeled with gray frame in Figure 4). Because all L-R bat CoVs were only found in R. pusillus, the recombinant events likely happened in R. pusillus. Interestingly, the isolate Rc-o319, belonging to L2-JP when 440 nt RdRp sequences were used to perform the analysis (Figure 4), was also detected in R. pusillus and also have an ambivalent homology character, suggesting that Rc-o319 might also be a member of L-R and R. pusillus might be the primary host for L-R bat CoVs.

Increase of diversity and prevailance of sarbecoviruses in bat along with direction from northeast to southwest in China

By combining the genome homology and the divergent results of MCC tree, the different genome homology distributed in each province were illustrated in Figure 7. A gross association of gradual reduction in viral genome diversity and homology of bat SARSr-CoVs with SARS-CoV and the geographic direction from southwest China to northeast can be found, when we use SARS-CoV genome as the query. The tendency grossly matches the possible spread directions of L1 we predicted in Figure 4. YN2020B-G shares 95.8% genome sequence identity with SARS-CoV, the highest among known bat CoVs. YN2020B-G was found in the southern border region of Honghe Prefecture of Yunnan province, raising the question whether there is any bat SARSr-CoV sharing even higher sequence identity with SARS-CoV in further south of Yunnan or not. More bat sampling and CoV survey should be conducted to see whether it is true.

Similarly, the L-R strains of R. pusillus also showed a trend with decrease in genome homology to SARS-CoV-2 from southwest to northeast (reduced from 91.0%, 90.1%, 88.1%, 88%, 87.8%, to 87.7% nt identities and finally linked R. pusillus SC2r-CoVs in L2-IP (94.7% nt identity) and L2-JP (79.3% nt identity)) along limited survey sites when SARS-CoV-2 genome was used as the query (Figure 7).

Characterization of S gene and receptor binding motif (RBM)

The CoV S proteins bind the host receptors and play essential roles in virus entry. SARS-CoV, SARS-CoV-2, and several SARSr-CoVs and SC2r-CoVs use hACE2 as the entry receptor^32,33, We then determined whether there is any way that can predict the usage of ACE2 by CoV S protein. The phylogenetic analysis of CoV S genes and sequence alignment of RBMs of different bat CoVs were performed. All of CoVs in RdRp based L1 lineage were regrouped (Figure 8). The presence of Upper-group was consistent with the foregoing homology heatmap which showed obvious gene exchange between L1.1 and L1.2. Most members in this group have broad host species and lack ability to use hACE2 because of the deletions in the two key regions of RBM, consistent with previous reports (Labeled with #)³⁴. The hACE2-usage-group-1 with intact RBM have a very narrow host and location ranges in Yunnan province, and all were found in R. sinicus with the exception of one in R. affinis. Their capability of using hACE2 as the entry receptor had been experimentally verified among six CoVs (Labeled with *)^10,33-35, indicated with * in Figure 8. The seven strains of L-R-group also failed to bind hACE2 because of presence of two deletions in the key regions of RBMs.

All Lower-group came from L2 and showed a more diverse deletion pattern in RBM than the three groups described above. The SARS-CoV-2 and SC2r-CoV (RaTG13 and several pangolin CoVs,) related hACE2-usage-group-2 had intact RBM. It's worth noting that MP789 from Malayan pangolin had an almost identical RBM with that of SARS-CoV2, except for residue 498 (Q498 in SARS-CoV-2, H498 in MP789). While the RBMs of RpYN06 and RmYN02 from the southwest border region of Yunnan and RaCS203 from Thailand also had deletions in the two key regions, the RBMs of RShSTT182 and RshSTT200 from Cambodian R. shameli only had a 4 aa deletion in region 1, and the RBMs of Rc-o319 from Japanese R. pussillus only had a 9 aa deletion in region 2. However, all of them failed to bind hACE2. Of note, although the S protein of RsYN04 also has a deletion in region 1 of RBD, it can still weakly bind to hACE2²⁰.

Bats are the second most diverse mammalians behind rodents with a wide geographical distribution, unique behaviors, and intimate interactions with humans and livestock³⁶. A large variety of viruses treat bats as natural reservoirs or evolutionary origins, including many important zoonotic viruses such as Nipa virus, Hendra virus, Ebola virus, and CoVs^3,14,37. Pteropus fruit bats were confirmed to be the natural reservoirs of Nipa virus and Hendra virus, and horses and pigs are intermediate hosts. Nipa virus could also be transmitted directly from bats to humans via fruits contaminated by bat excrements^3,38,39. The situation for CoVs is more complicated.

CoVs are the most diverse viruses carried by bats⁴⁰, and have high frequencies of recombination throughout the genome due to their unique mechanisms of viral replication and RNA synthesis, making their cross-species transmission relatively easy and adaptation to new hosts rapid^41,42. Since the emergences of SARS-CoV in 2002-2003, significant efforts have been devoted to identify the origins and the cross-species chains of this pathogen by examing wild and domestic animals. The discovery of bat SARSr-CoVs^7,9,11,43,44, particularly those using hACE2 as the entry receptor¹⁰, highlights bats as the origin of this highly transmissible and pathogenic CoV. Recently, several SC2r-CoVs were also detected in bats and some of them could also use hACE2 as the entry receptor^15-17,20. Therefore, bats were also considered as the origin of SARS-CoV-2. However, the hypothesis that bats act as the direct natural reservoir of SARS-CoV and SARS-CoV-2 is challenged by the remaining genetic gaps between them and related bat CoVs. To date, the bat CoVs sharing the highest nt identities with SARS-CoV and SARS-CoV-2 are YN2020B and RaTG13, and they only show 95.8% and 96.2% nucleotide sequence identity with SARS-CoV and SARS-CoV-2, respectively^10,15. The lineages composed by different CoVs started independent evolution since the estimated divergence events happened about 60-70 years ago between SARS-CoV and its closely relate bat SARSr-CoVs, and about 70-40 years ago between SARS-CoV-2 and RaTG13, indicating that none of the present known bat CoV could serve as the immediate precursors for these two HCoVs²³. The potential immediate ancestors of SARS-CoV and SARS-CoV-2 have not been found yet in any bat species so far, even in China.

The sampling criteria of this research focused on the following points: (1) Retrospectively sample the environment in the animal stalls in WHM to look for the traces of SC2r-CoVs and determine whether the source of cross-species spillover was presented or not; (2) Conduct bat sampling in mountain areas of Hubei province and around Wuhan city where the first COVID-19 outbreak was reported place; (3) Add significant amount of new sampling sites and neglected bat species in southern China, as well as conduct the routine survey; (4) Conduct bat sampling in possible hotspots where SC2r-CoVs were reported, such as Zhoushan City of Zhejiang province and the border regions of Yunnan province, to investigate if SC2r-CoVs are actively circulating in these places.

Animal spillover hypothesis was proposed as the most likely scenario of the origin of SARS-CoV-2, and WHM was the initial epicenter of COVID-19. Retrospectively, we collected and analyzed some environmental samples from WHM during the outbreak in Wuhan. Detection of CoVs under the genera Embecovirus and Tegacovirus in WHM samples is consistent with the animal species (hedgehog, rabbit, and bamboo rat) once sold in the market ²⁹. However, lacking of any SARS-CoV-2 and SC2r-CoV sequences in any environmental samples from WHM animal stalls suggest that the trace of cross-species spillover might not present in this market. Of note, since our samples were collected 40 days after WHM was closed down, and samples collected in this study might not be the representatives of animal species sold prior to COVID-19 pandemic, the exact role of WHM in the origin of SARS-CoV-2 remains elusive.

By the collection of more than seventeen thousand samples from 29 provinces between 2010-2021, we expanded the sample size of bat to about fifty thousand in China (by combining with published data). We not only covered most known geographic hotspots for bat CoVs, but also expanded to some new areas in China never being explored before, such as Hunan, Jiangxi, Fujian, Liaoning provinces, etc. A large number of new CoVs were detected, and some bat species, such as R. luctus and R. siamensis, were discovered to contain sarbecovirus for the first time. Sarbecoviruses were primarily found in Rhinolophid bats, and colonization of diverse SARSr-CoVs was revealed in four Rhinolophus species, R. sinicus, R. ferrumequinum, R. pusillus, and R. affinis. These four species tend to gregariously roost in group in cave with a relatively large population, which might facilitate active circulation and frequent recombination of such CoVs. Other accidentally related Rhinolophus species, such as R. macrotis, R. siamensis, R. luctus, etc., always exist in small population or even in pairs or alone, which might make it difficult to keep SARSr-CoV intraspecies circulating. Although Hipposideridae bats, such as H. armiger and H. pratti, have a large population size and extensive habitat area in China, they are rarely detected for SARSr-CoV. We speculate that these species may be contaminated or transiently infected by SARSr-CoV because many of them often share roosts with Rhinolophus species in the same cave, and they may not have the funcational receptor(s) or required protease(s) for various sarbecoviruses, which requires further investigation.

After analyzing 146 new sarbecoviruses in this study and 471 representative strains from database, we find that the distribution pattern of the bat sarbecoviruses in China appears to have a geographic trend from southwest to northeast/east of China, with a gradual decrease in virus diversity across L3, L2, and L1 lineages and their sub-lineages. Once a new lineage was established after differentiation, it might only spread in its own limited area, whether this phenomenon results from limited migration range of the hosts remains to be determine. Of note, this trend is also closely correlated with the degree of homology of genome sequences of SARSr-CoVs with SARS-CoV, higher genome homology in the southwest and lower genome homology in the northeast/east of China. If this trend were also true outside China, the fact that YN2020B-G sharing 95.8%, the highest homology among bat SARSr-CoVs, with SARS-CoV was found near China southwestern border raises another important question whether there is any SARSr-CoVs sharing even higher homology with SARS-CoV in further south, even outside of China or not.

Several SC2r-CoVs, including RaTG13, RmYN02, and RpYN06, were found in bats from Tongguan and Xishuangbanna, Yunnan Province^15,16,20. Surprisingly, we failed to find any SC2r-CoVs in this study, despite the fact that we collected 17,504 samples in 66 different bat species throughout significant parts of China between 2010-2021, including the border places where RaTG13, RmYN02, and RpYN06 were found or nearby, the suspected R. affinis and R. pusillus with large sampling sizes, and the mountainous areas around Wuhan city. This result suggests that such SC2r-CoVs are extremely rare in bats in China, and they might not have established the active circulation in large scale among bats in China. Recently, SC2r-CoVs were found in R. shameli and R. acuminatus, and they inhabit in SEA^17,19. R. malayanus, the host bat for RmYN02, is also an endemic species in SEA and was firstly recorded as a northward migrating species in southwestern frontier of China in 2015⁴⁵. Moreover, R. pusillus and R. affinis, in which RaTG13 and RpYN06 were found, are also commonly found in SEA, and south Asia. Whether there are more SC2r-CoVs actively circulating in these regions or other under sampling areas warrants further investigation to understand the exact evolutionary pathways from ancestors to SARS-CoV-2.

Even with such size of sampling and analysis, we still did not find the presences of SARS-CoV and SARS-CoV-2 or their proximal ancestors in bat populations of China. If the direct spillover pathway from bat to human is possible, there is only one possibility that, similar to described above, the exact lineages of SARS-CoV and SARS-CoV-2 may circulate in unreached bat population in extremely imperceptible ways. Since bat virome have been investigated very extensively in China, to better address this question, the CoV survey may be expanded to regions where suspectable Rhinolophus species is present, especially places with under sampling, such as SEA and South Asia, and other locations.

Another animal spillover theory, in which the virus spill over from bats to animal intermediate host, then to human, has been favored by many¹. Candidate intermediate hosts may include civets, Malayan pangolins, rabbits, ferrets, foxes, etc., because their ACE2s could bind to S of SARS-CoV and SARS-CoV-2 and facilitate the virus entry^33,46,47. However, the nationwide animal testing of over 90,000 animal samples for SARS-CoV-2 were all negative²⁹, suggesting that the spillover evens from bat to certain intermediate host might not necessarily occur frequently in China. Although the existence of diverse S genes within Rhinolophus species could maximumly facilitate the cross-species transmission, an S gene identical to that of SARS-CoV or SARS-CoV-2 has not yet been found in any bat species. The S based phylogenetic analysis showed that the S genes of L2 and L1.2 are more diverse than those of L1.1. Further focusing on RBM reveals that RBMs of L2 in the southwestern border of China and Indochina Peninsula are the most diverse and might have more flexibility to adapt new host via recombination. Considering the almost identical RBMs of SARS-CoV-2 were found in SC2r-CoV of smuggled imported Malayan pangolins, the diversity of S gene in L2 and Malayan pangolin related regions should be further studied to find the closest relative of SARS-CoV-2 S gene. This hypothesis was further supported partially by the detection of SARS-CoV-2 neutralizing antibodies in bats and Malayan pangolin in SEA recently¹⁷.

The detection of L-R only in R. pusillus reveals that complicated recombinant events have happened between SARSr-CoV related L1 and SC2r-CoV related L2-IP during virus-host co-evolution in this bat species. The ancestor of L-R may come from a L2-IP strain that exchanged the middle genome fragment with SARSr-CoV, this recombinant lineage firstly colonized in R. pusillus when entering the southwest border of China and then spread to the northeast with an independent evolution clade along the gradual migration or population symbiosis of R. pusillus. Further sequence analysis of viral genomes of RmYN02, RpYN06, L-R strains, and Rc-o319 reveals that the ancestor of L-R might originate from a L2-IP recombining with SARSr-CoV, then evolve to L2-JP in Japan. Although the risk of L-R strains to human remains low because of their hACE2 unusable feature until now, we should not underestimate their cross-species abilities through recombination to obtain new S genes.

In conclusion, our study provides a macroscopic view for bat CoVs in China. However, failure of finding any SC2r-CoV in such a broad and in-depth bat virome study in China, indicating the difficulty and complexity of probing the origin of SARS-CoV-2. Investigation through global collaboration in places with known bat species susceptible to SARS-CoV-2 infection and under sampled should be considered.

Sample collection

For WHM, to collect environment samples, swabs were applied to wipe repeatedly on the floors, walls, or surfaces of objects and then immersed in virus sampling tubes. For bat sampling, pharyngeal and anal swab samples were collected from live bat individuals and then immersed in virus sampling tubes (Yocon, Beijing, China) containing maintenance medium and temporarily stored at −20°C. Samples were then transported to the laboratory and stored at −80 °C. The bat species were initially determined morphologically and subsequently confirmed by barcoding of mitochondrial cytochrome b using patagium. The accurate locations of sampling sites were recorded by place names and GPS coordinates with latitude and longitude.

Library Construction and Next generation sequencing

Samples from the same bat species and from the adjacent same sites were pooled by adding 1 ml from each maintenance medium sample into one new sample tube. The pooled samples were processed with a virus-particle-protected, nucleic acid purification method as described previously⁴⁸. The samples were homogenized in virus maintenance medium and subsequently filtered through a 0.45 μm polyvinylidene difluoride filter (Millipore, Germany), the filtered samples were then centrifuged at 150,000 × g for 3 h. The pellet was digested in a cocktail of DNase and RNase enzymes. The viral DNA and RNA were simultaneously isolated using a QIAmp MinElute Virus Spin Kit (Qiagen, USA). First-strand viral cDNA was synthesized using the primer K-8N and a Superscript IV system (Invitrogen, USA). The cDNA was converted into dsDNA by Klenow fragment (NEB, USA). Sequence-independent PCR amplification was conducted using primer K. The PCR products which are from 300 to 2000 bps were purified by magnetic beads (Beckman Coulter, USA). The purified products were then subjected to Illumina HiSeq X Ten sequencer, for a paired-end read of 150 bp. The sequence reads were filtered using previously described criteria⁴⁹. Clean data were generated after adaptor sequence, primer K sequence, and low quality reads were removed.

Taxonomic Assignment and Genome assembly

Sequence-similarity-based taxonomic assignments were processed as described in our previous study²⁵. Reads in clean data were aligned to sequences in the NCBI non-redundant nucleotide database and non-redundant protein database using BLASTx. The taxonomies of the aligned reads with the best BLAST scores (E score <10−5) were parsed and extracted by MEGAN6⁵⁰. Extracted coronavirus reads were assembled by megahit v1.2.9 with default parameters. Assembled contigs were used as reference sequences during PCR screening and sequencing.

PCR screening and genome sequencing

CoV screening of individual samples of coronavirus positive pool was performed by amplifying a 440-bp fragment of the RNA-dependent RNA polymerase (RdRp) gene of all a-CoV and b-CoV using conserved primer pairs as described previously^7,51-53. Specific primers were designed from assembled contigs of Sarbecovirus and their closest reference sequence identified by Blast from Genbank. Each open reading frame (ORF) in viral genome was amplified with nested specific primers whose PCR product are nearly 1,500 bp and then sequenced with ABI3500 DNA analyzer (Applied Biosystems, USA). PCR products with low concentration or generating heterogeneity in the sequencing chromatograms were cloned into pMD-18T Vector (Takara) for sequencing.

Phylogenetic and recombination analysis

MEGA7.0 was used to align nucleotide sequences and deduced amino acid sequences using the MUSCLE package and default parameters⁵⁴. The best substitution model was then evaluated by the Model Selection package. Finally, we constructed a maximum-likelihood method using an appropriate model to process the phylogenetic analyses with 1,000 bootstrap replicates. Recombination among CoVs was detected with SimPlot software. All analyses were performed with a Kimura model, a window size of 200 bp, and a step size of 20 bp.

Bayesian divergence time estimation

Partial RdRp sequences (approximately 440 nt) were used to conduct a time-measured phylogenetic reconstruction analysis. It was performed using a Bayesian method implemented in BEAST v.1.10.4. A single GTR+Gamma substitution model, a strict clock model, and the constant size model as a coalescent tree prior were also selected for the analyses, which were run for 100 million steps with sampling at every 10,000 states. The BEAGLE parallel computation library was used to enhance the speed of the likelihood calculations. Finally, the resulting log file was checked using TRACER version 1.5 (http://tree.bio.ed.ac.uk/software/tracer) to confirm that all effective sample sizes were >200. TreeAnnotator was used to summarize posterior tree distributions and annotated the estimated values to a maximum clade credibility tree with a burn-in of 10%, which was visualized using FigTree v1.4.4.

Homology analysis of all NSP and structure protein

Separate alignments were generated for the main open reading frame (ORFs) and non-structure protein (NSP) gene of all complete genome of Sarbecovirus by MAFTT v.7.475. Sequence distance of 15 NSP genes belong to ORF1a and ORF1ab, structure protein genes, and several accessory proteins were analyzed by MegAlign (DNA Star package). Homology detection between each part of these complete genome strain was virtualized by heatmap made by TBtools v1.082⁵⁵.

Dataset

All genome sequences were submitted to GenBank. Accession numbers for the viruses are OK017594 to OK017860.

Description of Supplementary Information (SI)

Supplementary Tables are available with the online version of this paper.

Ethics approval and consent to participate

Animals were treated according to the guidelines of the Regulations for the Administration of Laboratory Animals (Decree No. 2 of the State Science and Technology Commission of the People’s Republic of China, 1988). Sampling procedures were approved by the Ethics Committee of the Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College (Approval number: IPB EC20100415).

Consent for publication

Not applicable.

Availability of data and materials

Datasets generated and analyzed during the current study are available in this published article (and its supplementary information files).

Competing interests

The authors declare that they have no competing interests

Funding

This work was supported by the CAMS Innovation Fund for Medical Sciences (2020-12M-CoV19-010), Beijing Natural Science Foundation (M21002), the National Natural Science Foundation of China (Grant No. 81772228 and 32070407), the National Key R&D Program of China (2020YFC0840800), the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2019PT310029), and the National S&T Major Project “China Mega-Project for Infectious Disease” (Grant No.2018ZX10101001 and 2018ZX10711001).

1 Cui, J., Li, F. & Shi, Z. L. Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol 17, 181-192, doi:10.1038/s41579-018-0118-9 (2019).

2 Forni, D., Cagliani, R., Clerici, M. & Sironi, M. Molecular Evolution of Human Coronavirus Genomes. Trends Microbiol 25, 35-48, doi:10.1016/j.tim.2016.09.001 (2017).

3 Smith, I. & Wang, L. F. Bats and their virome: an important source of emerging viruses capable of infecting humans. Curr Opin Virol 3, 84-91, doi:10.1016/j.coviro.2012.11.006 (2013).

4 Chinese, S. M. E. C. Molecular evolution of the SARS coronavirus during the course of the SARS epidemic in China. Science 303, 1666-1669, doi:10.1126/science.1092002 (2004).

5 Gao, H., Yao, H., Yang, S. & Li, L. From SARS to MERS: evidence and speculation. Front Med, doi:10.1007/s11684-016-0466-7 (2016).

6 Zaki, A. M., van Boheemen, S., Bestebroer, T. M., Osterhaus, A. D. & Fouchier, R. A. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med 367, 1814-1820, doi:10.1056/NEJMoa1211721 (2012).

7 Latinne, A. et al. Origin and cross-species transmission of bat coronaviruses in China. Nat Commun 11, 4235, doi:10.1038/s41467-020-17687-3 (2020).

8 Yang, L. et al. MERS-related betacoronavirus in Vespertilio superans bats, China. Emerg Infect Dis 20, 1260-1262, doi:10.3201/eid2007.140318 (2014).

9 Yang, L. et al. Novel SARS-like betacoronaviruses in bats, China, 2011. Emerg Infect Dis 19, 989-991, doi:10.3201/eid1906.121648 (2013).

10 Ge, X. Y. et al. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature 503, 535-538, doi:10.1038/nature12711 (2013).

11 Li, W. et al. Bats are natural reservoirs of SARS-like coronaviruses. Science 310, 676-679, doi:10.1126/science.1118391 (2005).

12 Han, Y. et al. Identification of Diverse Bat Alphacoronaviruses and Betacoronaviruses in China Provides New Insights Into the Evolution and Origin of Coronavirus-Related Diseases. Front Microbiol 10, 1900, doi:10.3389/fmicb.2019.01900 (2019).

13 Woo, P. C. et al. Comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features. J Virol 81, 1574-1585, doi:10.1128/JVI.02182-06 (2007).

14 Letko, M., Seifert, S. N., Olival, K. J., Plowright, R. K. & Munster, V. J. Bat-borne virus diversity, spillover and emergence. Nat Rev Microbiol 18, 461-471, doi:10.1038/s41579-020-0394-z (2020).

15 Zhou, P. et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270-273, doi:10.1038/s41586-020-2012-7 (2020).

16 Zhou, H. et al. A Novel Bat Coronavirus Closely Related to SARS-CoV-2 Contains Natural Insertions at the S1/S2 Cleavage Site of the Spike Protein. Curr Biol 30, 2196-2203 e2193, doi:10.1016/j.cub.2020.05.023 (2020).

17 Wacharapluesadee, S. et al. Evidence for SARS-CoV-2 related coronaviruses circulating in bats and pangolins in Southeast Asia. Nat Commun 12, 972, doi:10.1038/s41467-021-21240-1 (2021).

18 Murakami, S. et al. Detection and Characterization of Bat Sarbecovirus Phylogenetically Related to SARS-CoV-2, Japan. Emerg Infect Dis 26, 3025-3029, doi:10.3201/eid2612.203386 (2020).

19 Hul, V. et al. A novel SARS-CoV-2 related coronavirus in bats from Cambodia. bioRxiv, 2021.2001.2026.428212, doi:10.1101/2021.01.26.428212 (2021).

20 Zhou, H. et al. Identification of novel bat coronaviruses sheds light on the evolutionary origins of SARS-CoV-2 and related viruses. bioRxiv, 2021.2003.2008.434390, doi:10.1101/2021.03.08.434390 (2021).

21 Lam, T. T. et al. Identifying SARS-CoV-2 related coronaviruses in Malayan pangolins. Nature, doi:10.1038/s41586-020-2169-0 (2020).

22 Xiao, K. et al. Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins. Nature 583, 286-289, doi:10.1038/s41586-020-2313-x (2020).

23 Boni, M. F. et al. Evolutionary origins of the SARS-CoV-2 sarbecovirus lineage responsible for the COVID-19 pandemic. Nat Microbiol, doi:10.1038/s41564-020-0771-4 (2020).

24 Wu, Z. et al. Decoding the RNA viromes in rodent lungs provides new insight into the origin and evolutionary patterns of rodent-borne pathogens in Mainland Southeast Asia. Microbiome 9, 18, doi:10.1186/s40168-020-00965-z (2021).

25 Wu, Z. et al. Comparative analysis of rodent and small mammal viromes to better understand the wildlife origin of emerging infectious diseases. Microbiome 6, 178, doi:10.1186/s40168-018-0554-9 (2018).

26 Wu, Z. et al. Deciphering the bat virome catalog to better understand the ecological diversity of bat viruses and the bat origin of emerging infectious diseases. ISME J 10, 609-620, doi:10.1038/ismej.2015.138 (2016).

27 Chen, L., Liu, B., Wu, Z., Jin, Q. & Yang, J. DRodVir: A resource for exploring the virome diversity in rodents. J Genet Genomics 44, 259-264, doi:10.1016/j.jgg.2017.04.004 (2017).

28 Chen, L., Liu, B., Yang, J. & Jin, Q. DBatVir: the database of bat-associated viruses. Database (Oxford) 2014, bau021, doi:10.1093/database/bau021 (2014).

29 120 (World Health Organization, 2021).

30 Li, L.-l. et al. A novel SARS-CoV-2 related virus with complex recombination isolated from bats in Yunnan province, China. bioRxiv, 2021.2003.2017.435823, doi:10.1101/2021.03.17.435823 (2021).

31 Hu, D. et al. Genomic characterization and infectivity of a novel SARS-like coronavirus in Chinese bats. Emerg Microbes Infect 7, 154, doi:10.1038/s41426-018-0155-5 (2018).

32 Zhao, X. et al. Broad and differential animal ACE2 receptor usage by SARS-CoV-2. J Virol, doi:10.1128/JVI.00940-20 (2020).

33 Li, P. et al. The Rhinolophus affinis bat ACE2 and multiple animal orthologs are functional receptors for bat coronavirus RaTG13 and SARS-CoV-2. Sci Bull (Beijing), doi:10.1016/j.scib.2021.01.011 (2021).

34 Letko, M., Marzi, A. & Munster, V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat Microbiol 5, 562-569, doi:10.1038/s41564-020-0688-y (2020).

35 Shang, J. et al. Structural basis of receptor recognition by SARS-CoV-2. Nature 581, 221-224, doi:10.1038/s41586-020-2179-y (2020).

36 Calisher, C. H., Childs, J. E., Field, H. E., Holmes, K. V. & Schountz, T. Bats: important reservoir hosts of emerging viruses. Clin Microbiol Rev 19, 531-545, doi:10.1128/CMR.00017-06 (2006).

37 Leroy, E. M. et al. Fruit bats as reservoirs of Ebola virus. Nature 438, 575-576, doi:10.1038/438575a (2005).

38 Drexler, J. F. et al. Bats host major mammalian paramyxoviruses. Nat Commun 3, 796, doi:10.1038/ncomms1796 (2012).

39 Halpin, K. et al. Pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission. Am J Trop Med Hyg 85, 946-951, doi:10.4269/ajtmh.2011.10-0567 (2011).

40 Frutos, R., Serra-Cobo, J., Pinault, L., Lopez Roig, M. & Devaux, C. A. Emergence of Bat-Related Betacoronaviruses: Hazard and Risks. Front Microbiol 12, 591535, doi:10.3389/fmicb.2021.591535 (2021).

41 Latif, A. A. & Mukaratirwa, S. Zoonotic origins and animal hosts of coronaviruses causing human disease pandemics: A review. Onderstepoort J Vet Res 87, e1-e9, doi:10.4102/ojvr.v87i1.1895 (2020).

42 Ye, Z. W. et al. Zoonotic origins of human coronaviruses. Int J Biol Sci 16, 1686-1697, doi:10.7150/ijbs.45472 (2020).

43 Woo, P. C. et al. Molecular diversity of coronaviruses in bats. Virology 351, 180-187, doi:10.1016/j.virol.2006.02.041 (2006).

44 Tang, X. C. et al. Prevalence and genetic diversity of coronaviruses in bats from China. J Virol 80, 7481-7490, doi:10.1128/JVI.00697-06 (2006).

45 Liang, J., He, X., Peng, X., Xie, H. & Zhang, L. First record of existence of Rhinolophus malayanus (Chiroptera, Rhinolophidae) in China. Mammalia 84, 362-365, doi:10.1515/mammalia-2019-0062 (2020).

46 Wu, L. et al. Broad host range of SARS-CoV-2 and the molecular basis for SARS-CoV-2 binding to cat ACE2. Cell Discov 6, 68, doi:10.1038/s41421-020-00210-9 (2020).

47 Guan, Y. et al. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 302, 276-278, doi:10.1126/science.1087139 (2003).

48 Wu, Z. et al. Virome analysis for identification of novel mammalian viruses in bat species from Chinese provinces. J Virol 86, 10999-11012, doi:10.1128/JVI.01394-12 (2012).

49 Yang, J. et al. Unbiased parallel detection of viral pathogens in clinical samples by use of a metagenomic approach. J Clin Microbiol 49, 3463-3469, doi:10.1128/JCM.00273-11 (2011).

50 Huson, D. H. et al. MEGAN Community Edition - Interactive Exploration and Analysis of Large-Scale Microbiome Sequencing Data. PLoS Comput Biol 12, e1004957, doi:10.1371/journal.pcbi.1004957 (2016).

51 Watanabe, S. et al. Bat coronaviruses and experimental infection of bats, the Philippines. Emerg Infect Dis 16, 1217-1223, doi:10.3201/eid1608.100208 (2010).

52 Du, J. et al. Genetic diversity of coronaviruses in Miniopterus fuliginosus bats. Sci China Life Sci 59, 604-614, doi:10.1007/s11427-016-5039-0 (2016).

53 Vlasova, A. N. et al. Novel Canine Coronavirus Isolated from a Hospitalized Pneumonia Patient, East Malaysia. Clin Infect Dis, doi:10.1093/cid/ciab456 (2021).

54 Kumar, S., Stecher, G. & Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol 33, 1870-1874, doi:10.1093/molbev/msw054 (2016).

55 Chen, C. et al. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol Plant 13, 1194-1202, doi:10.1016/j.molp.2020.06.009 (2020).

Table 1. Sampling information in WHM.

Pool ID	Sampling area	Sample source	Sample Size	Coronavirus Detection Result	Accession number	Most related strain
M1	Cold storages	Chopping blocks and knives	4	Embecovirus, Merbecovirus	OK017789 OK017790	JN874561.1 rabbit CoV HKU14, MK907287.1 hedgehog CoV HKU31
M2	Cold storages	Grounds, walls and door handles	7
M3	Cold storages	Chopping blocks and knives	11
M4	Animal-selling stalls	Grounds	10
M5	Animal-selling stalls	Chopping blocks and knives	12
M6	Animal-selling stalls	Grounds	8	Tegacovirus	OK017788	JQ404409.1 Canine CoV strain 1-71
M7	Animal-selling stalls	Grounds and walls	12
M8	Animal-selling stalls	Chopping blocks, and knives	10
M9	Animal-selling stalls	Grounds	15	Embecovirus	OK017791	KF294371.1 rat CoV Longquan-370
M10	Animal-selling stalls	Scissors	3
M11	Animal-selling stalls	Grounds and sewers	10

Table 2. Bat Species and Sampling Size in each year.

Species	2016	2017	2018	2019	2020	2021	Total
Rhinolophus sinicus	129	192	45	67	584	398	1415
Rhinolophus pusillus	174	136	118	33	509	96	1066
Rhinolophus ferrumequinum	0	0	0	0	441	3	444
Rhinolophus affinis	59	113	78	149	160	121	680
Rhinolophus pearsonii	10	46	6	29	116	119	326
Rhinolophus macrotis	40	42	10	2	23	52	169
Rhinolophus rex	6	10	4	3	0	4	27
Rhinolophus marshalli	0	4	3	12	0	0	19
Rhinolophus malayanus	0	0	0	0	42	10	52
Rhinolophus luctus	1	5	0	0	7	0	13
Rhinolophus siamensis	0	5	3	3	2	33	46
Rhinolophus paradoxolophus	0	0	0	0	2	0	2
Rhinolophus spp.	0	0	0	0	0	11	11
Hipposideros larvatus	106	329	45	145	333	0	958
Hipposideros armiger	94	174	99	58	177	0	602
Hipposideros pratti	82	55	10	0	38	0	185
Aselliscus stoliczkanus	40	103	0	0	162	1	306
Hipposideros pomona	21	51	0	71	56	4	203
Hipposideros cineraceus	0	40	0	0	8	0	48
Myotis ricketti	11	233	96	83	70	0	493
Myotis chinensis	359	35	51	15	44	0	504
Myotis daubentonii	9	115	0	0	42	0	166
Myotis siligorensis	76	96	0	2	22	0	196
Myotis altarium	0	0	153	4	60	7	224
Myotis adversus	5	0	2	0	83	0	90
Myotis laniger	0	8	6	26	40	0	80
Myotis longipes	5	3	2	13	14	0	37
Myotis fimbriatus	4	5	0	0	3	0	12
Myotis montivagus	4	5	0	0	0	0	9
Myotis indochinensis	0	0	0	6	0	1	7
Myotis rufoniger	0	0	0	0	1	0	1
Myotis nipalensis	0	0	1	0	0	0	1
Myotis spp.	82	9	1	25	11	0	128
Chaerephon plicata	8	0	0	0	68	0	76
Megaderma spasma	0	15	0	0	4	0	19
Megaderma lyra	0	0	3	2	2	0	7
Eonycteris spelaea	34	87	0	1	98	0	220
Cynopterus sphinx	1	86	0	8	0	1	96
Rousettus leschenaulti	4	47	0	6	24	0	81
Eptesicus serotinus	0	0	0	0	71	0	71
Ia io	1	10	0	0	16	0	27
Murina spp.	0	0	0	0	4	0	4
Nyctalus velutinus	2	1	0	0	5	0	8
Hypsugo pulveratus	5	0	2	0	0	0	7
Hypsugo cadornae	0	0	1	3	2	0	6
Pipistrellus abramus	19	89	50	92	312	0	562
Pipistrellus pipistrellus	26	21	7	71	5	0	130
Pipistrellus ceylonicus	1	0	0	0	0	0	1
Pipistrellus tenuis	5	0	0	4	0	0	9
Pipistrellus spp.	1	5	0	29	47	0	82
Scotophilus kuhlii	0	497	141	407	0	0	1045
Scotophilus heathi	5	0	0	19	0	0	24
Taphozous melanopogon	70	145	6	20	73	0	314
Myotis formosus	0	0	0	0	0	1	1
Tylonycteris pachypus	486	259	55	214	0	0	1014
Tylonycteris robustula	75	55	4	53	0	0	187
Miniopterus pusillus	93	38	0	25	6	0	162
Miniopterus schreibersii	126	1	112	8	136	8	391
Total	2279	3170	1114	1708	3923	870	13064

Table 3. 146 Sarbecoviruses obtained in this study, include 69 complete genome and 77 partial RdRp sequence.

Pool	Species	Year	Location	Strain Name	Upload ID	Fragment	Accession
83	R. sinicus	2016	GD	GD2016A	BtRs-BetaCoV/GD2016A	Partial RdRp	OK017595
84	R. sinicus	2016	GD	GD2016B	BtRs-BetaCoV/GD2016B	Complete Genome	OK017812
93	R. sinicus	2016	YN	YN2016A	BtRs-BetaCoV/YN2016A	Complete Genome	OK017847
93	R. sinicus	2016	YN	YN2016B	BtRs-BetaCoV/YN2016B	Complete Genome	OK017848
93	R. sinicus	2016	YN	YN2016C	BtRs-BetaCoV/YN2016C	Complete Genome	OK017849
93	R. sinicus	2016	YN	YN2016D	BtRs-BetaCoV/YN2016D	Complete Genome	OK017850
93	R. sinicus	2016	YN	YN2016E	BtRs-BetaCoV/YN2016E	Complete Genome	OK017851
96	R. affinis	2017	GD	GD2017A	BtRa-BetaCoV/GD2017A	Partial RdRp	OK017610
96	R. affinis	2017	GD	GD2017B	BtRa-BetaCoV/GD2017B	Partial RdRp	OK017611
97	R. affinis	2017	GD	GD2017C	BtRa-BetaCoV/GD2017C	Partial RdRp	OK017612
97	R. affinis	2017	GD	GD2017D	BtRa-BetaCoV/GD2017D	Partial RdRp	OK017613
97	R. affinis	2017	GD	GD2017E	BtRa-BetaCoV/GD2017E	Partial RdRp	OK017614
97	R. affinis	2017	GD	GD2017F	BtRa-BetaCoV/GD2017F	Complete Genome	OK017792
87	R. sinicus	2017	GD	GD2017G	BtRs-BetaCoV/GD2017G	Complete Genome	OK017813
87	R. sinicus	2017	GD	GD2017H	BtRs-BetaCoV/GD2017H	Complete Genome	OK017814
87	R. sinicus	2017	GD	GD2017I	BtRs-BetaCoV/GD2017I	Complete Genome	OK017815
87	R. sinicus	2017	GD	GD2017J	BtRs-BetaCoV/GD2017J	Complete Genome	OK017816
87	R. sinicus	2017	GD	GD2017K	BtRs-BetaCoV/GD2017K	Complete Genome	OK017817
87	R. sinicus	2017	GD	GD2017L	BtRs-BetaCoV/GD2017L	Complete Genome	OK017818
87	R. sinicus	2017	GD	GD2017M	BtRs-BetaCoV/GD2017M	Complete Genome	OK017819
87	R. sinicus	2017	GD	GD2017N	BtRs-BetaCoV/GD2017N	Complete Genome	OK017820
87	R. sinicus	2017	GD	GD2017O	BtRs-BetaCoV/GD2017O	Complete Genome	OK017821
88	R. sinicus	2017	GD	GD2017P	BtRs-BetaCoV/GD2017P	Complete Genome	OK017822
88	R. sinicus	2017	GD	GD2017Q	BtRs-BetaCoV/GD2017Q	Complete Genome	OK017823
86	R. sinicus	2017	GD	GD2017R	BtRs-BetaCoV/GD2017R	Partial RdRp	OK017596
86	R. sinicus	2017	GD	GD2017S	BtRs-BetaCoV/GD2017S	Partial RdRp	OK017597
86	R. sinicus	2017	GD	GD2017T	BtRs-BetaCoV/GD2017T	Partial RdRp	OK017598
86	R. sinicus	2017	GD	GD2017U	BtRs-BetaCoV/GD2017U	Partial RdRp	OK017599
86	R. sinicus	2017	GD	GD2017V	BtRs-BetaCoV/GD2017V	Partial RdRp	OK017600
86	R. sinicus	2017	GD	GD2017W	BtRs-BetaCoV/GD2017W	Complete Genome	OK017824
86	R. sinicus	2017	GD	GD2017X	BtRs-BetaCoV/GD2017X	Partial RdRp	OK017601
86	R. sinicus	2017	GD	GD2017Y	BtRs-BetaCoV/GD2017Y	Partial RdRp	OK017602
86	R. sinicus	2017	GD	GD2017Z	BtRs-BetaCoV/GD2017Z	Partial RdRp	OK017603
112	R. rex	2017	GX	GX2017A	BtRr-BetaCoV/GX2017A	Partial RdRp	OK017615
112	R. luctus	2017	GX	GX2017B	BtRl-BetaCoV/GX2017B	Partial RdRp	OK017616
94	R. sinicus	2017	YN	YN2017A	BtRs-BetaCoV/YN2017A	Partial RdRp	OK017605
94	R. sinicus	2017	YN	YN2017B	BtRs-BetaCoV/YN2017B	Partial RdRp	OK017606
94	R. sinicus	2017	YN	YN2017C	BtRs-BetaCoV/YN2017C	Partial RdRp	OK017607
94	R. sinicus	2017	YN	YN2017D	BtRs-BetaCoV/YN2017D	Partial RdRp	OK017608
94	R. sinicus	2017	YN	YN2017E	BtRs-BetaCoV/YN2017E	Partial RdRp	OK017609
1	R. sinicus	2018	SC	SC2018B	BtRs-BetaCOV/SC2018B	Complete Genome	OK017846
89	R. sinicus	2019	GD	GD2019A	BtRs-BetaCoV/GD2019A	Complete Genome	OK017825
89	R. sinicus	2019	GD	GD2019B	BtRs-BetaCoV/GD2019B	Complete Genome	OK017826
89	R. sinicus	2019	GD	GD2019C	BtRs-BetaCoV/GD2019C	Partial RdRp	OK017604
90	R. sinicus	2019	GD	GD2019D	BtRs-BetaCoV/GD2019D	Complete Genome	OK017827
90	R. sinicus	2019	GD	GD2019E	BtRs-BetaCoV/GD2019E	Complete Genome	OK017828
112	R. siamensis	2019	GX	GX2019A	BtRsi-BetaCoV/GX2019A	Complete Genome	OK017617
112	R. rex	2019	GX	GX2019B	BtRr-BetaCoV/GX2019B	Partial RdRp	OK017859
118	R. sinicus	2020	GD	GD2020A	BtRs-BetaCoV/GD2020A	Partial RdRp	OK017618
118	R. sinicus	2020	GD	GD2020B	BtRs-BetaCoV/GD2020B	Partial RdRp	OK017619
118	R. sinicus	2020	GD	GD2020C	BtRs-BetaCoV/GD2020C	Partial RdRp	OK017620
118	R. sinicus	2020	GD	GD2020D	BtRs-BetaCoV/GD2020D	Partial RdRp	OK017621
118	R. sinicus	2020	GD	GD2020E	BtRs-BetaCoV/GD2020E	Partial RdRp	OK017622
118	R. sinicus	2020	GD	GD2020F	BtRs-BetaCoV/GD2020F	Partial RdRp	OK017623
118	R. sinicus	2020	GD	GD2020G	BtRs-BetaCoV/GD2020G	Partial RdRp	OK017624
119	R. sinicus	2020	GD	GD2020H	BtRs-BetaCoV/GD2020H	Partial RdRp	OK017625
119	R. sinicus	2020	GD	GD2020I	BtRs-BetaCoV/GD2020I	Partial RdRp	OK017626
119	R. sinicus	2020	GD	GD2020J	BtRs-BetaCoV/GD2020J	Partial RdRp	OK017627
119	R. sinicus	2020	GD	GD2020K	BtRs-BetaCoV/GD2020K	Partial RdRp	OK017628
123	R. sinicus	2020	GX	GX2020	BtRs-BetaCoV/GX2020	Partial RdRp	OK017629
303	R. ferrumequinum	2020	HB	HB2020A	BtRf-BetaCoV/HB2020A	Partial RdRp	OK017630
303	R. ferrumequinum	2020	HB	HB2020B	BtRf-BetaCoV/HB2020B	Partial RdRp	OK017631
303	R. ferrumequinum	2020	HB	HB2020C	BtRf-BetaCoV/HB2020C	Partial RdRp	OK017632
18	Rhinolophus sp.	2020	HB	HB2020D	BtRh-BetaCOV/HB2020D	Complete Genome	OK017594
20	Rhinolophus sp.	2020	HB	HB2020E	BtRh-BetaCOV/HB2020E	Complete Genome	OK017801
21	R. sinicus	2020	HB	HB2020F	BtRs-BetaCOV/HB2020F	Partial RdRp	OK017802
327	R. ferrumequinum	2020	LN	LN2020A	BtRf-BetaCoV/LN2020A	Complete Genome	OK017794
327	R. ferrumequinum	2020	LN	LN2020B	BtRf-BetaCoV/LN2020B	Complete Genome	OK017795
327	R. ferrumequinum	2020	LN	LN2020C	BtRf-BetaCoV/LN2020C	Complete Genome	OK017796
327	R. ferrumequinum	2020	LN	LN2020D	BtRf-BetaCoV/LN2020D	Partial RdRp	OK017633
327	R. ferrumequinum	2020	LN	LN2020E	BtRf-BetaCoV/LN2020E	Complete Genome	OK017797
329	R. ferrumequinum	2020	LN	LN2020F	BtRf-BetaCoV/LN2020F	Complete Genome	OK017798
329	R. ferrumequinum	2020	LN	LN2020G	BtRf-BetaCoV/LN2020G	Complete Genome	OK017799
329	R. ferrumequinum	2020	LN	LN2020H	BtRf-BetaCoV/LN2020H	Complete Genome	OK017800
330	R. ferrumequinum	2020	LN	LN2020I	BtRf-BetaCoV/LN2020I	Partial RdRp	OK017634
310	R. affinis	2020	YN	YN2020A	BtRa-BetaCoV/YN2020A	Complete Genome	OK017793
60	R. sinicus	2020	YN	YN2020B	BtRs-BetaCoV/YN2020B	Complete Genome	OK017852
60	R. sinicus	2020	YN	YN2020C	BtRs-BetaCoV/YN2020C	Complete Genome	OK017853
60	R. sinicus	2020	YN	YN2020D	BtRs-BetaCoV/YN2020D	Complete Genome	OK017854
60	R. sinicus	2020	YN	YN2020E	BtRs-BetaCoV/YN2020E	Complete Genome	OK017855
60	R. sinicus	2020	YN	YN2020F	BtRs-BetaCoV/YN2020F	Complete Genome	OK017856
60	R. sinicus	2020	YN	YN2020G	BtRs-BetaCoV/YN2020G	Complete Genome	OK017857
60	R. sinicus	2020	YN	YN2020H	BtRs-BetaCoV/YN2020H	Complete Genome	OK017858
356	R. sinicus	2021	FJ	FJ2021A	BtRs-BetaCoV/FJ2021A	Complete Genome	OK017807
357	R. sinicus	2021	FJ	FJ2021B	BtRs-BetaCoV/FJ2021B	Partial RdRp	OK017668
360	R. affinis	2021	FJ	FJ2021C	BtRa-BetaCoV/FJ2021C	Partial RdRp	OK017650
357	R. sinicus	2021	FJ	FJ2021D	BtRs-BetaCoV/FJ2021D	Complete Genome	OK017651
357	R. sinicus	2021	FJ	FJ2021E	BtRs-BetaCoV/FJ2021E	Complete Genome	OK017808
357	R. sinicus	2021	FJ	FJ2021F	BtRs-BetaCoV/FJ2021F	Partial RdRp	OK017641
358	R. sinicus	2021	FJ	FJ2021G	BtRs-BetaCoV/FJ2021G	Partial RdRp	OK017809
358	R. sinicus	2021	FJ	FJ2021H	BtRs-BetaCoV/FJ2021H	Partial RdRp	OK017810
358	R. sinicus	2021	FJ	FJ2021I	BtRs-BetaCoV/FJ2021I	Partial RdRp	OK017642
358	R. sinicus	2021	FJ	FJ2021J	BtRs-BetaCoV/FJ2021J	Partial RdRp	OK017643
358	R. sinicus	2021	FJ	FJ2021K	BtRs-BetaCoV/FJ2021K	Partial RdRp	OK017644
358	R. sinicus	2021	FJ	FJ2021L	BtRs-BetaCoV/FJ2021L	Partial RdRp	OK017645
358	R. sinicus	2021	FJ	FJ2021M	BtRs-BetaCoV/FJ2021M	Complete Genome	OK017646
358	R. sinicus	2021	FJ	FJ2021N	BtRs-BetaCoV/FJ2021N	Partial RdRp	OK017647
361	R. affinis	2021	FJ	FJ2021O	BtRa-BetaCoV/FJ2021O	Partial RdRp	OK017648
340	R. sinicus	2021	GZ	GZ2021A	BtRs-BetaCoV/GZ2021A	Partial RdRp	OK017811
340	R. sinicus	2021	GZ	GZ2021B	BtRs-BetaCoV/GZ2021B	Partial RdRp	OK017649
340	R. sinicus	2021	GZ	GZ2021C	BtRs-BetaCoV/GZ2021C	Complete Genome	OK017640
341	R. affinis	2021	GZ	GZ2021D	BtRa-BetaCoV/GZ2021D	Partial RdRp	OK017635
340	R. sinicus	2021	GZ	GZ2021E	BtRs-BetaCoV/GZ2021E	Partial RdRp	OK017636
340	R. sinicus	2021	GZ	GZ2021F	BtRs-BetaCoV/GZ2021F	Partial RdRp	OK017829
340	R. sinicus	2021	GZ	GZ2021G	BtRs-BetaCoV/GZ2021G	Partial RdRp	OK017637
340	R. sinicus	2021	GZ	GZ2021H	BtRs-BetaCoV/GZ2021H	Complete Genome	OK017638
340	R. sinicus	2021	GZ	GZ2021I	BtRs-BetaCoV/GZ2021I	Complete Genome	OK017639
333	R. pusillus	2021	HN	HN2021A	BtRp-BetaCoV/HN2021A	Complete Genome	OK017830
333	R. pusillus	2021	HN	HN2021B	BtRp-BetaCoV/HN2021B	Complete Genome	OK017831
336	R. sinicus	2021	HN	HN2021C	BtRs-BetaCoV/HN2021C	Complete Genome	OK017803
336	R. sinicus	2021	HN	HN2021D	BtRs-BetaCoV/HN2021D	Complete Genome	OK017804
336	R. sinicus	2021	HN	HN2021E	BtRs-BetaCoV/HN2021E	Complete Genome	OK017805
336	R. sinicus	2021	HN	HN2021F	BtRs-BetaCoV/HN2021F	Complete Genome	OK017832
333	R. pusillus	2021	HN	HN2021G	BtRp-BetaCoV/HN2021G	Complete Genome	OK017833
350	R. pusillus	2021	YN	YN2021	BtRp-BetaCoV/YN2021	Complete Genome	OK017834
364	R. sinicus	2021	JX	JX2021A	BtRs-BetaCoV/JX2021A	Partial RdRp	OK017835
364	R. sinicus	2021	JX	JX2021B	BtRs-BetaCoV/JX2021B	Partial RdRp	OK017654
364	R. sinicus	2021	JX	JX2021C	BtRs-BetaCoV/JX2021C	Complete Genome	OK017860
365	R. siamensis	2021	JX	JX2021D	BtRsi-BetaCoV/JX2021D	Complete Genome	OK017669
367	R. sinicus	2021	JX	JX2021E	BtRs-BetaCoV/JX2021E	Partial RdRp	OK017670
367	R. sinicus	2021	JX	JX2021F	BtRs-BetaCoV/JX2021F	Partial RdRp	OK017652
367	R. sinicus	2021	JX	JX2021G	BtRs-BetaCoV/JX2021G	Complete Genome	OK017836
366	R. affinis	2021	JX	JX2021H	BtRa-BetaCoV/JX2021H	Partial RdRp	OK017653
367	R. sinicus	2021	JX	JX2021I	BtRs-BetaCoV/JX2021I	Partial RdRp	OK017837
367	R. sinicus	2021	JX	JX2021J	BtRs-BetaCoV/JX2021J	Complete Genome	OK017655
367	R. sinicus	2021	JX	JX2021K	BtRs-BetaCoV/JX2021K	Complete Genome	OK017656
367	R. sinicus	2021	JX	JX2021L	BtRs-BetaCoV/JX2021L	Complete Genome	OK017838
367	R. sinicus	2021	JX	JX2021M	BtRs-BetaCoV/JX2021M	Complete Genome	OK017657
367	R. sinicus	2021	JX	JX2021N	BtRs-BetaCoV/JX2021N	Complete Genome	OK017839
368	R. sinicus	2021	JX	JX2021O	BtRs-BetaCoV/JX2021O	Complete Genome	OK017840
368	R. sinicus	2021	JX	JX2021P	BtRs-BetaCoV/JX2021P	Complete Genome	OK017841
368	R. sinicus	2021	JX	JX2021Q	BtRs-BetaCoV/JX2021Q	Partial RdRp	OK017842
368	R. sinicus	2021	JX	JX2021R	BtRs-BetaCoV/JX2021R	Partial RdRp	OK017843
368	R. sinicus	2021	JX	JX2021S	BtRs-BetaCoV/JX2021S	Partial RdRp	OK017844
368	R. sinicus	2021	JX	JX2021T	BtRs-BetaCoV/JX2021T	Partial RdRp	OK017845
368	R. sinicus	2021	JX	JX2021U	BtRs-BetaCoV/JX2021U	Partial RdRp	OK017658
368	R. sinicus	2021	JX	JX2021V	BtRs-BetaCoV/JX2021V	Partial RdRp	OK017659
369	R. sinicus	2021	JX	JX2021W	BtRs-BetaCoV/JX2021W	Partial RdRp	OK017660
369	R. sinicus	2021	JX	JX2021X	BtRs-BetaCoV/JX2021X	Partial RdRp	OK017661
369	R. sinicus	2021	JX	JX2021Y	BtRs-BetaCoV/JX2021Y	Partial RdRp	OK017662
369	R. sinicus	2021	JX	JX2021Z	BtRs-BetaCoV/JX2021Z	Partial RdRp	OK017663
370	R. sinicus	2021	AH	AH2021A	BtRs-BetaCoV/AH2021A	Complete Genome	OK017664
370	R. sinicus	2021	AH	AH2021B	BtRs-BetaCoV/AH2021B	Partial RdRp	OK017665
372	R. sinicus	2021	JX	JX2021AA	BtRs-BetaCoV/JX2021AA	Partial RdRp	OK017666
372	R. sinicus	2021	JX	JX2021AB	BtRs-BetaCoV/JX2021AB	Partial RdRp	OK017667
372	R. sinicus	2021	JX	JX2021AC	BtRs-BetaCoV/JX2021AC	Complete Genome	OK017806

There is NO Competing Interest.

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A comprehensive survey of bat sarbecoviruses across China for the origin tracing of SARS-CoV and SARS-CoV-2

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