Journal Pre-proof

A novel bat coronavirus closely related to SARS-CoV-2 contains natural insertions at

the S1/S2 cleavage site of the spike protein

Hong Zhou, Xing Chen, Tao Hu, Juan Li, Hao Song, Yanran Liu, Peihan Wang, Di
Liu, Jing Yang, Edward C. Holmes, Alice C. Hughes, Yuhai Bi, Weifeng Shi
PII: S0960-9822(20)30662-X
DOI: https://doi.org/10.1016/j.cub.2020.05.023
Reference: CURBIO 16489

To appear in: Current Biology

Received Date: 16 April 2020

Revised Date: 1 May 2020
Accepted Date: 6 May 2020

Please cite this article as: Zhou H, Chen X, Hu T, Li J, Song H, Liu Y, Wang P, Liu D, Yang J, Holmes
EC, Hughes AC, Bi Y, Shi W, A novel bat coronavirus closely related to SARS-CoV-2 contains
natural insertions at the S1/S2 cleavage site of the spike protein, Current Biology (2020), doi: https://
doi.org/10.1016/j.cub.2020.05.023.

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 1 A novel bat coronavirus closely related to SARS-
SARS-CoV-
CoV-2
2 contains natural insertions at the S1/S2 cleavage site of
3 the spike protein

5 Hong Zhou1,8, Xing Chen2,8, Tao Hu1,8, Juan Li1,8, Hao Song3, Yanran Liu1, Peihan Wang1,

6 Di Liu4, Jing Yang5, Edward C. Holmes6, Alice C. Hughes2,*, Yuhai Bi5,*, Weifeng Shi1,7,9,*

8 1Key Laboratory of Etiology and Epidemiology of Emerging Infectious Diseases in

9 Universities of Shandong, Shandong First Medical University & Shandong Academy of

10 Medical Sciences, Taian 271000, China

11 2Landscape Ecology Group, Center for Integrative Conservation, Xishuangbanna Tropical

12 Botanical Garden, Chinese Academy of Sciences, Menglun, Mengla, Yunnan 666303,

13 China

14 3Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science,

15 Chinese Academy of Sciences, Beijing 100101, China

16 4Computational Virology Group, Center for Bacteria and Virus Resources and

17 Bioinformation, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan

18 430071, China

19 5CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of

20 Microbiology, CAS Center for Influenza Research and Early-warning (CASCIRE),

1
21 CAS-TWAS Center of Excellence for Emerging Infectious Diseases (CEEID), Chinese

22 Academy of Sciences, Beijing 100101, China

23 6Marie Bashir Institute for Infectious Diseases and Biosecurity, School of Life and

24 Environmental Sciences and School of Medical Sciences, The University of Sydney,

25 Sydney, New South Wales 2006, Australia

26 7The First Affiliated Hospital of Shandong First Medical University (Shandong Provincial

27 Qianfoshan Hospital), Ji’nan 250014, China

28 8These authors contributed equally

29 9Lead Contact

30 *Correspondence: shiwf@ioz.ac.cn (W.S.), beeyh@im.ac.cn (Y.B.) ，

31 ach_conservation2@hotmail.com (A.C.H.)

32

2
33 Summary

34 The unprecedented pandemic of pneumonia caused by a novel coronavirus,

35 SARS-CoV-2, in China and beyond has had major public health impacts on a global scale

36 [1,2]. Although bats are regarded as the most likely natural hosts for SARS-CoV-2 [3], the

37 origins of the virus remain unclear. Here, we report a novel bat-derived coronavirus,

38 denoted RmYN02, identified from a metagenomics analysis of samples from 227 bats

39 collected from Yunnan Province in China between May and October, 2019. Notably,

40 RmYN02 shares 93.3% nucleotide identity with SARS-CoV-2 at the scale of the complete

41 virus genome and 97.2% identity in the 1ab gene, in which it is the closest relative of

42 SARS-CoV-2 reported to date. In contrast, RmYN02 showed low sequence identity

43 (61.3%) to SARS-CoV-2 in the receptor binding domain (RBD) and might not bind to

44 angiotensin-converting enzyme 2 (ACE2). Critically, and in a similar manner to

45 SARS-CoV-2, RmYN02 was characterized by the insertion of multiple amino acids at the

46 junction site of the S1 and S2 subunits of the spike (S) protein. This provides strong

47 evidence that such insertion events can occur naturally in animal betacoronaviruses.

48 Keywords: SARS-CoV-2, bat coronavirus, COVID-19, spike protein, S1/S2 cleavage

49 site

3
50 Results and Discussion

51 Coronaviruses (CoVs) are common viral respiratory pathogens that primarily cause

52 symptoms in the upper respiratory and gastrointestinal tracts. In 1960s, two CoVs, 229E

53 and OC43, were identified in clinical samples from patients experiencing the common cold

54 [4]. More recently, four additional human CoVs have been successively identified: severe

55 acute respiratory syndrome coronavirus (SARS-CoV) in 2002, NL63 in late 2004, HKU1 in

56 January 2005, and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012.

57 However, two betacoronaviruses (beta-CoVs), SARS-CoV and MERS-CoV are notable as

58 they have caused severe and fatal infections, leading to 774 and 858 deaths to date,

59 respectively, suggesting that beta-CoVs may be of particular concern to human health. In

60 December 2019, viral pneumonia caused by an unidentified microbial agent was reported,

61 which was soon identified to be a novel coronavirus [1-3], now termed SARS-CoV-2 [5].

62 The number of patients infected with SARS-CoV-2 has increased sharply since January

63 21, 2020, and confirmed SARS-CoV-2 cases were present in all the Chinese provinces

64 and municipalities by the end of January. The virus also spread rapidly outside of China in

65 March and the World Health Organization had to declare a coronavirus disease 2019

66 (COVID-19) pandemic on March 11, 2020. As of April 15, 2020, more than two million

67 confirmed SARS-CoV-2 cases have been reported, with >130,000 deaths worldwide.

68

69 An epidemiological survey of several SARS-CoV-2 cases at an early stage of the outbreak

70 revealed that most had visited the Huanan seafood market in Wuhan city prior to illness,

4
71 where various wild animals were on sale before it was closed on January 1, 2020 due to

72 the outbreak. Phylogenetic analysis has revealed that SARS-CoV-2 is a novel beta-CoV

73 distinct from SARS-CoV and MERS-CoV [1-3]. To date, the most closely related virus to

74 SARS-CoV-2 is RaTG13, identified from a Rhinolophus affinis bat sampled in Yunnan

75 province in 2013 [3]. This virus shared 96.1% nucleotide identity and 92.9% identity in the

76 S gene, again suggesting that bats play a key role as coronavirus reservoirs [3]. Notably,

77 however, several novel beta-CoVs related to SARS-CoV-2 have also been identified in

78 Malayan pangolins (Manis javanica) that were illegally imported into Guangxi (GX) and

79 Guangdong (GD) provinces, southern China [6-8]. Although these pangolins CoVs are

80 more distant to SARS-CoV-2 than RaTG13 across the virus genome as a whole, they are

81 very similar to SARS-CoV-2 in the receptor binding domain (RBD) of the S protein,

82 including at the amino acid residues thought to mediate binding to ACE2 [7]. It is therefore

83 possible that pangolins play an important role in the ecology and evolution of CoVs.

84 Indeed, the discovery of viruses in pangolins suggests that there is a wide diversity of

85 CoVs still to be sampled in wildlife, some of which may be directly involved in the

86 emergence of SARS-CoV-2.

87

88 Between May and October, 2019, we collected a total of 302 samples from 227 bats from

89 Mengla County, Yunnan Province in southern China (Table S1). These bats belonged to

90 20 different species, with the majority of samples from Rhinolophus malayanus (n=48,

91 21.1%), Hipposideros larvatus (n=41, 18.1%) and Rhinolophus stheno (n=39, 17.2%).

92 The samples comprised multiple tissues, including patagium (n=219), lung (n=2) and liver

5
 93 (n=3), and feces (n=78). All but three bats were sampled alive and subsequently released.

94 Based on the bat species primarily identified according to morphological criteria and

95 confirmed through DNA barcoding, the 224 tissues and 78 feces were merged into 38 and

96 18 pools, respectively, with each pool including 1 to 11 samples of the same type (Table

97 S1). These pooled samples were then used for next generation sequencing (NGS).

98

99 Using next-generation metagenomic sequencing we successfully obtained 11,954 and

100 64,224 reads from pool no. 39 (from a total of 78,477,464 clean reads) that mapped to a

101 previously identified SARS-like bat coronavirus, Cp/Yunnan2011 [9] (JX993988), and to

102 SARS-CoV-2. From this, we generated two preliminary consensus sequences. After a

103 series of verification steps, including re-mapping, one partial (23395 bp) and one

104 complete (29671 bp) beta-CoV genome sequences were obtained and named

105 BetaCoV/Rm/Yunnan/YN01/2019 (RmYN01) and BetaCoV/Rm/Yunnan/YN02/2019

106 (RmYN02), respectively. Pool 39 comprised 11 fecal samples from Rhinolophus

107 malayanus collected between May 6 and July 30, 2019. RNAs from eight samples

108 remained for further analysis. Based on the assembled sequence of RmYN02,

109 TaqMan-based qPCR was performed to detect the existence of RmYN02 in the eight

110 additional samples (see primers and probe in Table S2): the virus was only detected in

111 sample no. 123 collected on June 25, 2019 from Rhinolophus malayanus with a cycle

112 threshold (Ct) value of 34.76 (Figure S1). RmYN01 and RmYN02 were also identified in

113 sample 123 using Tip green supermix (TransGen), with the Ct values 32.21 for RmYN02

114 and 35.54 for RmYN01. Sample 123 was therefore used to verify the RmYN02 genome

6
115 sequence using the RmYN02 specific primers (Table S2). The partially amplified gene

116 fragments were ~10000 bp in length covering the spike (Figure S2A) and 1b genes of

117 RmYN02, and results from Sanger sequencing were consistent with those from

118 metagenomic sequencing. Notably, 20 positions in the RmYN02 genome displayed

119 nucleotide polymorphisms in the NGS data, although these did not include the S1/S2

120 cleavage site (Figure S2B). Only a few reads in the remaining 55 pools could be mapped

121 to the reference CoV genomes. The sequence identity between RmYN01 and

122 Cp/Yunnan2011 across the aligned regions was 96.9%, whereas that between RmYN01

123 and SARS-CoV-2 was only 79.7% across the aligned regions and 70.4% in the spike

124 gene.

125

126 In contrast, RmYN02 was closely related to SARS-CoV-2, exhibiting 93.3% nucleotide

127 sequence identity, although it was less similar to SARS-CoV-2 than RaTG13 (96.1%)

128 across the genome as a whole (Table 1). RmYN02 and SARS-CoV-2 were extremely

129 similar (>96% sequence identity) in most genomic regions (e.g. 1ab, 3a, E, 6, 7a, N and

130 10) (Table 1). In particular, RmYN02 was 97.2% identical to SARS-CoV-2 in the longest

131 encoding gene region, 1ab (21,285 nucleotides). However, RmYN02 exhibited far lower

132 sequence identity to SARS-CoV-2 in the S gene (nucleotide 71.8%, amino acid 72.9%),

133 compared to 97.4% amino acid identity between RaTG13 and SARS-CoV-2 (Table 1).

134 Strikingly, RmYN02 only possessed 62.4% amino acid identity to SARS-CoV-2 in the

135 RBD, whereas the pangolin beta-CoV from Guangdong had amino acid identity of 97.4%

136 [6], and was the closest relative of SARS-CoV-2 in this region. A similarity plot estimated

7
137 using Simplot [10] also revealed that RmYN02 was more similar to SARS-CoV-2 than

138 RaTG13 in most genome regions (Figure 1A). Again, in the RBD, the

139 pangolin/MP789/2019 virus shared the highest sequence identity to SARS-CoV-2 (Figure

140 1B).

141

142 Results from both homology modelling [2], in vitro assays [3] and resolved

143 three-dimensional structure of the S protein [11] have revealed that like SARS-CoV,

144 SARS-CoV-2 could also use ACE2 as a cell receptor. We analyzed the RBD of RmYN02,

145 RaTG13, and the two pangolin beta-CoVs using homology modelling (Figure 2A-2F and

146 Figure S3 for sequence alignment). The amino acid deletions in RmYN02 RBD made two

147 loops near the receptor binding site that are shorter than those in SARS-CoV-2 RBD

148 (Figure 2A and 2F). Importantly, the conserved disulfide bond in the external subdomain

149 of SARS-CoV (PDB: 2DD8) [12], SARS-CoV-2 (PDB: 6LZG), RaTG13 (Figure 2B),

150 pangolin/MP789/2019 (Figure 2C) and pangolin/GX/P5L/2017 (Figure 2D) was missing in

151 RmYN02 (Figure 2F). We speculate that these deletions may cause conformational

152 variations and consequently reduce the binding of RmYN02 RBD with ACE2 or even

153 cause non-binding. It is possible that the bat SARS-related CoVs with loop deletions,

154 including RmYN02, ZXC21 and ZC45, use a currently unknown receptor. In contrast,

155 RaTG13 (Figure 2B), pangolin/ MP789/2019 (Figure 2C) and pangolin/P5L/2017 (Figure

156 2D) did not have the deletions, and had similar conformations at their external domains,

157 indicating that they may also use ACE2 as cell receptor although, with the exception of

158 pangolin/MP789/2019 (see below), all exhibited amino acid variation to SARS-CoV-2.

8
159 Indeed, the pangolin/MP789/2019 virus showed highly structural homology with

160 SARS-CoV-2 (Figure 2E).

161

162 Six amino acid residues at the RBD (L455, F486, Q493, S494, N501 and Y505) have

163 been reported to be major determinants of efficient receptor binding of SARS-CoV-2 to

164 ACE2 [13]. As noted above, and consistent with the homology modelling,

165 pangolin/MP789/2019 possessed the identical amino acid residues to SARS-CoV-2 at all

166 six positions [6]. In contrast, both RaTG13, RmYN02 and RmYN01 possessed the same

167 amino acid residue as SARS-CoV-2 at only one of the six positions each (RaTG13, L455;

168 RmYN02, Y505; RmYN01, Y505) (Figure 2G), despite RaTG13 being the closest relative

169 in the spike protein. Such an evolutionary pattern is indicative of a complex combination of

170 recombination and natural selection [6, 14].

171 The S protein of CoVs is functionally cleaved into two subunits, S1 and S2 [15] in a similar

172 manner to the haemagglutinin (HA) protein of avian influenza viruses (AIVs). The insertion

173 of polybasic amino acids at the cleavage site in the HAs of some AIV subtypes is

174 associated with enhanced pathogenicity [16, 17]. Notably, SARS-CoV-2 is characterized

175 by a four-amino-acid-insertion at the junction of S1 and S2, not observed in other lineage

176 B beta-CoVs [18]. This insertion, which represents a poly-basic (furin) cleavage site, is

177 unique to SARS-CoV-2 and is present in all SARS-CoV-2 sequenced so far. The insertion

178 of three residues, PAA, at the junction of S1 and S2 in RmYN02 (Figure 2H, and Figure

179 S2A for results from Sanger sequencing) is therefore of major importance. Although the

180 inserted residues (and hence nucleotides) are not the same as those in RmYN02, and

9
181 hence are indicative of an independent insertion event, that they are presented in wildlife

182 (bats) strongly suggests that they are of natural origin and have likely acquired by

183 recombination. As such, these data are strongly suggestive of a natural zoonotic origin of

184 SARS-CoV-2.

185 We next performed a phylogenetic analysis of RmYN02, RaTG13, SARS-CoV-2 and the

186 pangolin beta-CoVs. Consistent with a previous research [6], the pangolin beta-CoVs

187 formed two well-supported sub-lineages, representing animal seized by anti-smuggling

188 authorities in Guangxi (Pangolin-CoV/GX) and Guangdong (Pangolin-CoV/GD) provinces

189 (Figure 3A and Figure S4A). However, whether pangolins are natural reservoirs for these

190 viruses, or they acquired these viruses independently from bats or other wildlife, requires

191 further sampling [6]. More notable was that RmYN02 was the closest relative of

192 SARS-CoV-2 in most of the virus genome, although these two viruses were still separated

193 from each other by a relatively long branch length (Figure 3A and Figure S4A). In the

194 spike gene tree, SARS-CoV-2 clustered with RaTG13 and was distant from RmYN02,

195 suggesting that the latter virus has experienced recombination in this gene (Figure 3B and

196 Figure S4B). In phylogeny of the RBD, SARS-CoV-2 was most closely related to

197 pangolin-CoV/GD, with the bat viruses falling in more divergent positions, again indicative

198 of recombination (Figure 3C and Figure S4C). Finally, phylogenetic analysis of the

199 complete RNA dependent RNA polymerase (RdRp) gene, which is often used in the

200 phylogenetic analysis of RNA viruses, revealed that RmYN02, RaTG13 and SARS-CoV-2

201 formed a well-supported sub-cluster distinct from the pangolin viruses (Figure 3D and

202 Figure S4D).

10
203 We confirmed the bat host of RmYN02, Rhinolophus malayanus, by analyzing the

204 sequence of the cytochrome b (Cytb) gene from the next generation sequencing data; this

205 revealed 100% sequence identity to a Rhinolophus malayanus isolate (GenBank

206 accession MK900703). Both Rhinolophus malayanus and Rhinolophus affinis are widely

207 distributed in southwest China and southeast Asia. Generally, they do not migrate over

208 long distances and are highly gregarious such that they are likely to live in the same caves,

209 which might facilitate the exchange of viruses between them and the occurrence of

210 recombination. Notably, RaTG13 was identified from anal swabs and RmYN02 was

211 identified from feces, which is a simple, but feasible way for bats to spread the virus to

212 other animals, especially species that can utilize cave environments.

213 Our study reaffirms that bats, particularly those of the genus Rhinolophus, are important

214 natural reservoirs for coronaviruses and currently harbor the closest relatives of

215 SARS-CoV-2, although this picture may change with increased wildlife sampling. In this

216 context it is striking that the RmYN02 virus identified here in Rhinolophus malayanus is

217 the closest relative of SARS-CoV-2 in the long 1ab replicase gene, although the virus

218 itself has a complex history of recombination. Finally, the observation that RmYN02

219 contains a polybasic insertion at the S1/S2 cleavage site in the spike protein clearly

220 indicates that events of this kind are a natural and expected component of coronavirus

221 evolution [19].

222 Acknowledgments

223 This work was supported by the Academic Promotion Programme of Shandong First

11
224 Medical University (2019QL006 and 2019PT008), the Strategic Priority Research

225 Programme of the Chinese Academy of Sciences (XDB29010102 and XDA20050202),

226 the Chinese National Natural Science Foundation (U1602265), the National Major Project

227 for Control and Prevention of Infectious Disease in China (2017ZX10104001-006), and

228 the High-End Foreign Experts Program of Yunnan Province (Y9YN021B01). W.S. was

229 supported by the Taishan Scholars Programme of Shandong Province (ts201511056).

230 Y.B. is supported by the NSFC Outstanding Young Scholars (31822055) and Youth

231 Innovation Promotion Association of CAS (2017122). E.C.H. is supported by an ARC

232 Australian Laureate Fellowship (FL170100022). We thank all the scientists, especially

233 Professor Wuchun Cao and Professor Yi Guan, who kindly shared their genomic

234 sequences of the coronaviruses used in this study.

235 Author contributions

236 W.S., Y.B. and A.C.H. designed and supervised research. X.C. and A.C.H. collected the

237 samples. H.Z. and Y.L. processed the samples. H.T. and J.L. performed genome

238 assembly and annotation. H.Z., J.L. and T.H. performed the genome analysis and

239 interpretation. W.S., Y.B. and A.C.H. wrote the paper. X.C., P.W., D.L., J.Y. and E.C.H.

240 assisted in data interpretation and edited the paper.

241 Declaration of Interests

242 The authors declare no competing interests.

12
243 Figure Legends

244 Figure 1.
1. Patterns of sequence identity between
between the consensus sequences of

245 SARS-
SARS-CoV-
CoV-2 and representative beta-
beta-CoVs. (A) Whole genome similarity plot between

246 SARS-CoV-2 and representative viruses listed in Table 1. The analysis was performed

247 using Simplot, with a window size of 1000bp and a step size of 100bp. (B) Similarity plot in

248 the spike gene (positions 1-1658) between SARS-CoV-2 and representative viruses listed

249 in Table 1. The analysis was performed using Simplot, with a window size of 150bp and a

250 step size of 5bp. See also Table S3.

251 Figure 2. Homology modelling of the RBD structures and molecular characterizations of

252 beta-CoVs. (A-D) Homology

the S1/S2 cleavage site of RmYN02 and representative beta-

253 modelling and structural comparison of the RBD structures of RmYN02 and

254 representative beta-CoVs, including (A) RmYN02, (B) RaTG13, (C) pangolin/MP789/2019

255 and (D) pangolin/GX/P5L/2017. The three-dimensional structures of the RBD from

256 Bat-SL-CoV RmYN02, RaTG13, pangolin/MP789/2019 and pangolin/GX/P5L/2017 were

257 modelled using the Swiss-Model program [20] employing the RBD of SARS-CoV (PDB:

258 2DD8) as a template. All the core subdomains are colored magenta, and the external

259 subdomains of RmYN02, RaTG13, pangolin/MP789/2019 and pangolin/GX/P5L/2017 are

260 colored cyan, green, orange and yellow, respectively. The conserved disulfide bond in

261 RaTG13, pangolin/GD and pangolin/GX is highlighted, while it is missing in RmYN02 due

262 to a sequence deletion. (E-F) Superimposition of the RBD structure of

263 pangolin/MP789/2019 (E) and RmYN02 (F) with that of SARS-CoV-2. The two deletions

13
264 located in respective loops in RmYN02 are highlighted using dotted cycles. (G) Molecular

265 characterizations of the RBD of RmYN02 and the representative beta-CoVs. (H)

266 Molecular characterizations of the cleavage site of RmYN02 and the representative

267 beta-CoVs. See also Figures S2, S3 and Table S2.

268 Figure 3.
3. Phylogenetic analysis of SARS-
SARS-CoV-
CoV-2 and representative viruses from the

269 subgenus Sarbecoronavirus. (A) Phylogenetic tree of the full-length virus genome. (B) the

270 S gene. (C) the RBD. (D) the RdRp. Phylogenetic analysis was performed using RAxML

271 [21] with 1000 bootstrap replicates, employing the GTR nucleotide substitution model.

272 RBD is delimited as the gene region 991-1572 of the spike gene according to the

273 reference [6]. All the trees are midpoint rooted for clarity. See also Figure S4.

14
274 STAR Methods
ethods

275 RESOURCE AVAILABILITY

276 Lead Contact

277 Further information and requests for resources and reagents should be directed to and will

278 be fulfilled by the Lead Contact, Weifeng Shi (shiwf@ioz.ac.cn).

279 Materials Availability

280 This study did not generate new unique reagents.

281 Data and Code Availability

282 Sequence data that support the findings of this study have been deposited in the China

283 National Microbiological Data Center (project accession number NMDC1001304 and

284 sequence accession numbers: NMDC60013004-01, NMDC60013004-02, and

285 NMDCN0000001-NMDCN0000003). The sequences of RmYN01 and RmYN02

286 generated during this study are also available at the GISAID with accession numbers:

287 EPI_ISL_412976 and EPI_ISL_412977.

288 EXPERIMENTAL MODEL AND SUBJECT DETAILS

289 Twenty different species of bats were tested in this study (Table S1). Samples were

290 collected between May and October, 2019 from Mengla County, Yunnan Province in

291 southern China (101.27156323E, 21.91889683N and 21.5932019N, 101.2200914E).

292 Xishuangbanna Tropical Botanical Garden has an ethics committee which provided

293 permission for trapping and bat surveys within this study.

15
294 METHOD DETAILS

295 Sample collection

296 Between May and October, 2019, a total of 302 samples from 227 bats were collected

297 from Mengla County, Yunnan Province in southern China (Table S1). These bats

298 belonged to 20 different species, with the majority of samples taken from Rhinolophus

299 malayanus (n=48, 21.1%), Hipposideros larvatus (n=41, 18.1%) and Rhinolophus stheno

300 (n=39, 17.2%). The samples included patagium (n=219), lung (n=2) and liver (n=3), and

301 feces (n=78). All but three bats were sampled alive and subsequently released. All

302 samples were first stored in RNAlater and then kept at -80°C until use.

303 Next generation sequencing

304 Based on the bat species primarily identified according to morphological criteria and

305 confirmed through DNA barcoding, the 224 tissue and 78 fecal samples were merged into

306 38 and 18 pools, respectively, with each pool containing 1 to 11 samples of the same type

307 (Table S1). Samples were transferred into the RNAiso Plus reagent (TAKARA) for

308 homogenization with steel beads. Total RNA was extracted and subsequently purified

309 using EZNA Total RNA Kit (OMEGA). Libraries were constructed using the NEB Next

310 Ultra RNA Library Prep Kit (NEB). rRNA of feces or tissues was removed using the

311 TransNGS rRNA Depletion (Bacteria) Kit and TransNGS rRNA Depletion

312 (Human/Mouse/Rat) Kit (TransGen), respectively. Paired-end (150 bp) sequencing of

313 each RNA library was performed on the NovaSeq 6000 platform (Illumina) carried out by

16
314 Novogene Bioinformatics Technology (Beijing, China).

315 Genome assembly and annotation

316 Raw reads were obtained from the 56 pools and were then adaptor- and quality- trimmed

317 with the Fastp program [22]. The clean reads were then mapped to reference genomes of

318 representative CoV genomes using Bowtie 2 [23], including SARS-CoV-2

319 (MDC60013002-01), SARS-CoV (AY508724, AY485277, AY390556 and AY278489),

320 SARS-like-CoV (DQ084200, DQ648857, GQ153542, GQ153547, JX993987, JX993988,

321 KF294455, KF294457, KJ473814, KJ473815, KJ473816, KT444582, KY417142,

322 KY417145, KY417146, KY417148, KY417151, KY417152, KY770859, MK211374,

323 MK211376, and MK211377), ZC45 (MG772933), ZXC21 (MG772934), MERS-CoV

324 (JX869059), other representative beta-CoV genomes (AY391777, EF065505, EF065509,

325 EF065513, FJ647223, KC545386, KF636752, KM349744, KU762338, and MK167038)

326 and alphacoronavirus genomes (NC_002645, AY567487). A total of 11,954 and 64,224

327 reads in pool no. 39 (a total of 78,477,464 clean reads) were mapped to both a bat

328 coronavirus Cp/Yunnan2011 (JX993988) [9] and SARS-CoV-2, generating two

329 preliminary consensus sequences, named BetaCoV/Rm/Yunnan/YN01/2019 (RmYN01)

330 and BetaCoV/Rm/Yunnan/YN02/2019 (RmYN02), respectively. However, there were only

331 few reads in the remaining 55 pools that could be mapped to these reference CoV

332 genomes. Pool 39 comprised 11 fecal samples from Rhinolophus malayanus collected

333 between May 6 and July 30, 2019.

334 To validate the two novel CoV genomes, the clean reads of pool 39 were then de novo

17
335 assembled using Trinity [24] with default settings. The assembled contigs were compared

336 with the consensuses obtained in the previous step and merged using Geneious (version

337 11.1.5) (https://www.geneious.com). We found that contigs with high and low abundance

338 corresponded to RmYN02 and RmYN01, respectively, with the abundance of RmYN02

339 5-10 times greater than that of RmYN01. The gaps between contigs of RmYN02 were

340 complemented by re-mapping the reads to the ends of the contigs, which produced the

341 full-length genome sequence of RmYN02. However, due to the limited number of reads

342 available, only a partial genome sequence of RmYN01 was obtained (23395 bp). Reads

343 were then mapped to the full-length genome sequence of RmYN02 using Bowtie 2 to

344 check base consistency at each nucleotide site. Moreover, to perform de novo assembly

345 for pool 39 and to further distinguish RmYN01 and RmYN02, we used PeHaplo [25] with

346 various overlap parameters (70, 80, 100, and 120). The consensus of each run of

347 PeHaplo was then compared, generating the final full-length genome of RmYN02 (29671

348 bp). The sequence identity between RmYN01 and Cp/Yunnan2011 across the aligned

349 regions was 96.9%, whereas that between RmYN01 and SARS-CoV-2 was only 79.7%.

350 Bioinformatics analyses

351 Reference virus genomes were obtained from NCBI/GenBank

352 (https://www.ncbi.nlm.nih.gov/) using Blastn with SARS-CoV-2 as a query. The beta-CoVs

353 from pangolins (Table S3) were retrieved from GISAID (www.gisaid.org/). The open

354 reading frames (ORFs) of the verified genome sequences were predicted using Geneious

355 (version 11.1.5). Pairwise sequence identities were also calculated using Geneious.

18
356 Potential recombination events were investigated using Simplot (version 3.5.1) [10].

357 The three-dimensional structures of RBD from RmYN02, RaTG13, pangolin/GD and

358 pangolin/GX were modeled using Swiss-Model program [20] using SARS CoV RBD

359 structure (PDB: 2DD8) [12] as a template.

360 Multiple sequence alignment of SARS-CoV-2 and the reference sequences was

361 performed using Mafft [26]. Phylogenetic analyses of the complete genome and major

362 encoding regions were performed using RAxML [21] with 1000 bootstrap replicates,

363 employing the GTR nucleotide substitution model (Figure 3). Phylogenetic analysis was

364 also performed using MrBayes [27], employing the GTR nucleotide substitution model

365 (Figure S4). Ten million steps were run, with trees and parameters sampled every 1,000

366 steps.

367 Sanger sequencing

sequencing

368 Based on the spike gene sequence of RmYN02, a TaqMan-based qPCR was performed

369 to test the feces of pool 39 (Table S2). Pool 39 comprised 11 feces from Rhinolophus

370 malayanus collected between May 6 and July 30, 2019. However, only eight original

371 samples remained after NGS. The results indicated that the fecal sample no. 123 from R.

372 malayanus, collected on June 25, 2019, was positive for RmYN02 (Figure S1). To further

373 confirm the S1/S2 cleavage site and the 1b (RdRp) gene sequence of RmYN02, five pair

374 primers, F1/R1-F4/R4 and F6/R6, were designed for Sanger sequencing (Table S2). The

375 consensus gene sequence of Sanger sequencing of the amplified products was

376 consistent with those from NGS (Figure S2).

19
377 QUANTIFICATION AND STATISTICAL
STATISTICAL ANALYSIS

378 No statistical analyses were conducted as part of this study.

379

380 References

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23
eTOC Blurb

Zhou et al. report a bat-derived coronavirus, RmYN02, which is the closest relative of

SARS-CoV-2 in most of the virus genome reported to date. RmYN02 contains an insertion

at the S1/S2 cleavage site in the spike protein in a similar manner to SARS-CoV-2. This

suggests that such insertion events can occur naturally in animal betacoronaviruses.

Highlights

Metagenomic analysis identified a novel coronavirus, RmYN02, from R. malayanus.

RmYN02 was the closest relative of SARS-CoV-2 in most of the virus genome.

Two loop deletions in RBD may reduce the binding of RmYN02 with ACE2.

RmYN02 contains an insertion at the S1/S2 cleavage site in the spike protein.
 A Simplot-Query: SARS-CoV-2
SARS-CoV-2
Consensus
6 7b
RmYN02
1ab S 3a E M 7a 8 N 10
1.0

0.9

0.8
Similarity

0.7

0.6

0.5

0.4
RmYN02 RaTG13 ZC45 ZXC21 pangolin/GX/P5L/2017 SARS-CoV GZ02
0.3

Window:1000bp, Step: 100bp, GapStrip: On, Kimura (2-parameter), T/t: 2.0

0.2
posi�on 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000 13000 14000 15000 16000 17000 18000 19000 20000 21000 22000 23000 24000 25000 26000 27000 28000 29000

B Simplot-Query: SARS-CoV-2 Spike gene (1-1658)

1.0 991 RBD 1572

[
0.9

0.8

0.7
Similarity

0.6

0.5

0.4

0.3

0.2

0.1

Posi�on 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600

RmYN02 RaTG13 ZC45 ZXC21 pangolin/MP789/2019 pangolin/GX/P5L/2017 SARS-CoV GZ02

Window:150bp, Step: 5bp, GapStrip: On, Kimura (2-parameter), T/t: 2.0
 A RmYN02 B RaTG13 C pangolin/MP789/2019

Cys480 Cys476
Cys488 Cys484

external
subdomain

core subdomain

D pangolin/GX/P5L/2017 E SARS-CoV-2 / pangolin/MP789/2019 F SARS-CoV-2 / RmYN02

dele�on
Cys478
Cys486
dele�on

G
Consensus SARS-CoV-2 Y L Y R L F R K S N L K P F E R D I S T E I Y Q A G S T P C N G V E G F N C Y F P L Q S Y G F Q P T N G V G Y Q P Y R
RmYN02 Y F Y R S H R A V K L K P F E R D L S S D E - - - - - - - - N G V R - - - - - - T L S T Y D F N P N V P L D Y Q A T R
RaTG13 Y L Y R L F R K A N L K P F E R D I S T E I Y Q A G S K P C N G Q T G L N C Y Y P L Y R Y G F Y P T D G V G H Q P Y R
ZC45 Y F Y R S H R S T K L K P F E R D L S S D E - - - - - - - - N G V R - - - - - - T L S T Y D F N P N V P L E Y Q A T R
ZXC21 Y F Y R S H R S T K L K P F E R D L S S D E - - - - - - - - N G V R - - - - - - T L S T Y D F N P N V P L E Y Q A T R
pangolin/MP789/2019 Y L Y R L F R K S N L K P F E R D I S T E I Y Q A G S T P C N G V E G F N C Y F P L Q S Y G F H P T N G V G Y Q P Y R
pangolin/GX/P5L/2017 Y L Y R L F R K S K L K P F E R D I S T E I Y Q A G S T P C N G Q V G L N C Y Y P L E R Y G F H P T T G V N Y Q P F R
SARS-CoV GZ02 Y K Y R Y L R H G K L R P F E R D I S N V P F S P D G K P C T P - P A L N C Y W P L N D Y G F Y T T T G I G Y Q P Y R
RmYN01 Y Y Y R S S R K T K L K P F E R D L S S D E - - - - - - - - N G V R - - - - - - T L S T Y D F Y P S V P L E Y Q A T R
SARS-CoV-2 Numbering 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509

Receptor binding domain

（685/686, SARS-CoV-2 Numbering）

S1 S2
Consensus
Iden�ty
Consensus SARS-CoV-2
RmYN02
RaTG13
ZC45
ZXC21
pangolin/MP789/2019
pangolin/GX/P5L/2017
SARS-CoV GZ02
RmYN02

H Polybasic cleavage site

SARS-CoV-2 Numbering 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693
Consensus SARS-CoV-2 G A G I C A S Y Q T Q T N S P R R A R S V A S Q S I I
RmYN02 G A G V C A S Y - - - - N S P - A A R - V G T N S I I
RaTG13 G A G I C A S Y Q T Q T N S - - - - R S V A S Q S I I
ZC45 G A G I C A S Y H T A S I L - - - - R S T S Q K A I V
ZXC21 G A G I C A S Y H T A S I L - - - - R S T G Q K A I V
pangolin/MP789/2019 G A G I C A S Y Q T Q T N S - - - - R S V S S X A I I
pangolin/GX/P5L/2017 G A G I C A S Y H S M S S F - - - - R S V N Q R S I I
SARS-CoV GZ02 G A G I C A S Y H T V S L L - - - - R S T S Q K S I V
RmYN01 G A G I C A S Y H T A S L L - - - - R N T G Q K S I V
O-linked glycan residues
 A 100
100
SARS-CoV B 100
100
SARS-CoV
YN2018B|Yunnan|R. affinis 100 LYRa11
100
Rs3367|Yunnan|R. sinicus 96 LYRa3
Rf4092|Yunnan|R. ferrumequinum 100 BtKY72
100
BM48-31/BGR
BtRs-BetaCoV/YN2013|Yunnan|R. sinicus HKU3-13
99
Rs672|Yunnan|R. sinicus 100 HKU3-3
100 100 Yunnan2011|C. plicata 100 HKU3-12
BetaCoV/LYRa11|China|R. affinis
100
Longquan_140
BtRl-BetaCoV/SC2018|China|Rhinolophus sp. BtRs-BetaCoV_GX2013
96 BtCoV/279/2005
100 279|Hubei|R. macrotis 85 Rp3
87 Rm1|Hubei|R. macrotis Rs672
BtRs-BetaCoV/HuB2013|R. sinicus 100 Rs4237
Shaanxi2011|R. pusillus As6526
99
85
YN2018A
Rf1|R. ferrumequinum Rs4247
HKU3-3|Hongkong|R. sinicus Jiyuan_84
HKU3-13|Hongkong|R. sinicus BtRf-BetaCoV/HeB2013
HKU3-12|Hongkong|R. sinicus BtRf-BetaCoV/HuB2013
99 BtCoV/273/2005
HKU3-7|Guangdong|R. sinicus 100
Rf1
Longquan-140|China|R. monoceros 85 100 BtRf-BetaCoV/JL2012
Wuhan/WH-01/2019 JTMC15
100 Wuhan/WH-03/2020 SARS-CoV-2 86
Yunnan2011
100 87
Wuhan/WH-04/2020 100 BtRl-BetaCoV/SC2018
100 Rp/Shaanxi2011
RmYN02|R. malayanus 100 Anlong_112
100 RaTG13|R. affinis BtRs-BetaCoV/YN2013
MP789 Guangxi/P2V
100
Pangolin-CoV/GD Guangxi/P5L
Guangdong/P2S Guangxi/P5E Pangolin-CoV/GX
100 Guangxi/P1E 100 Guangxi/P1E
Guangxi/P5L Guangxi/P3B
Guangxi/P2V Guangxi/P4L
Guangxi/P5E Pangolin-CoV/GX 92
Wuhan/WH-03/2020
100
100 100 Wuhan/WH-04/2020 SARS-CoV-2
Guangxi/P4L 100 100 Wuhan/WH-01/2019
Guangxi/P3B RaTG13
Bat-SL-CoVZXC21|Zhejiang|R. sinicus 100 MP789
Guangdong/P2S Pangolin-CoV/GD
100 100
Bat-SL-CoVZC45|Zhejiang|R. sinicus 100 100 Bat-SL-CoVZXC21
100 BtKY72|Kenya|Rhinolophus sp. Bat-SL-CoVZC45
BM48-31/BGR|Bulgaria|R. blasii RmYN02
0.05 0.07

C 70
99
SARS-CoV D 83
98
SARS-CoV
100 BtKY72 YNLF_31C
BM48-31/BGR BtRl-BetaCoV/SC2018
Wuhan/WH-01/2019 WIV1
100 Wuhan/WH-04/2020 SARS-CoV-2 Rf4092
100 83 Wuhan/WH-03/2020 Rs672
MP789 WIV16
80 100
Guangdong/P2S Pangolin-CoV/GD 100
BtRs-BetaCoV/YN2018B
RaTG13 BtRs-BetaCoV/GX2013
Guangxi/P4L 94 HKU3-7
87
Guangxi/P5L HKU3-3
Guangxi/P2V Pangolin-CoV/GX 100 HKU3-12
Guangxi/P1E 100
bat-SL-CoVZC45
100 Guangxi/P5E 100 Longquan-140
Guangxi/P3B 73 bat-SL-CoVZXC21
Rs4081 99 BtRs-BetaCoV/HuB2013
Rs4087-2 BtCoV/279/2005
Rs4096 100 JTMC15
YN2018D BtRf-BetaCoV/JL2012
Rs672 100
100 BtRf-BetaCoV/HuB2013
Rs4237 Jiyuan_84
Rs4255 100
BtRf_BetaCoV_SX2013
88
Rs4108 Rp/Shaanxi2011
100 As6526 MLHJC35
YN2018C 100 BM48-31/BGR
Rp3 BtKY72
Rs4085 Wuhan/WH-04/2020
98 Rs4247 100 Wuhan/WH-01/2019 SARS-CoV-2
98
Rs3267-2 97 Wuhan/WH-03/2020
Rs3262-2 RaTG13
BtRs-BetaCoV/YN2018A 100 RmYN02
BtRl-BetaCoV/SC2018 Guangdong/P2S
BtRs-BetaCoV/HuB2013 100
MP789 Pangolin-CoV/GD
100 100 bat-SL-CoVZXC21 Guangxi/P5E
bat-SL-CoVZC45 100
Guangxi/P1E
100 YNLF-31C Guangxi/P4L
YNLF-34C Guangxi/P5L Pangolin-CoV/GX
100 Rs4092 100 Guangxi/P3B
Rf4092 Guangxi/P2V
0.08 RmYN02 0.02
Table 1. Sequence identity for SARS-CoV-2 compared with RmYN02 and representative beta-CoVs genomes.

Complete Gene region

Strain
genome 1ab S RBD 3a E M 6 7a 7b 8 N 10
RmYN02 93.3% a 97.2% 71.9% 61.3% 96.4% 98.7% 94.8% 96.8% 96.2% 92.4% 45.8% 97.3% 99.1%
RaTG13 96.1% 96.5% 92.9% 85.3% 96.3% 99.6% 95.4% 98.4% 95.6% 99.2% 97.0% 96.9% 99.1%
ZC45 87.6% 89.0% 75.1% 62.1% 87.8% 98.7% 93.4% 95.2% 88.8% 94.7% 88.5% 91.1% 99.1%
Nucleotide
ZXC21 87.4% 88.7% 74.6% 60.6% 88.9% 98.7% 93.4% 95.2% 89.1% 95.5% 88.5% 91.2% /b
sequences
Pangolin/GD/2019 1 - 90.8% 89.3% - 93.4% 98.3% 93.1% 94.6% 93.4% - 92.1% 96.1% -
Pangolin/GX/P5L/2017 85.2% 84.7% 83.2% 79.9% 87.0% 97.4% 91.3% 90.9% 86.6% 81.8% 80.6% 91.0% 94.0%
SARS-CoV GZ02 78.9% 79.6% 72.3% 73.8% 75.6% 93.5% 85.1% 74.5% 82.1% 83.0% 45.3% 88.1% /
RmYN02 NA 98.8% 72.9% 62.4% 96.7% 100.0% 98.2% 96.7% 95.9% 83.7% 27.3% 98.6% 97.4%
RaTG13 NA 98.5% 97.4% 89.3% 97.8% 100.0% 98.6% 100.0% 97.5% 97.7% 95.0% 99.0% 97.4%
Amino acid ZC45 NA 95.6% 80.2% 63.5% 90.9% 100.0% 98.6% 93.4% 87.6% 93.0% 94.2% 94.3% 97.4%
sequences ZXC21 NA 95.2% 79.6% 62.9% 92.0% 100.0% 98.6% 93.4% 88.4% 93.0% 94.2% 94.3% /
Pangolin/GD/20191 NA 97.1% 90.7% 97.4% 97.4% 100.0% 98.6% 96.6% 97.5% - 94.9% 97.6% -
Pangolin/GX/P5L/2017 NA 92.6% 92.4% 86.8% 89.8% 100.0% 98.2% 95.1% 88.4% 72.1% 87.6% 93.8% 84.2%
SARS-CoV GZ02 NA 86.2% 76.2% 74.6% 73.1% 94.7% 89.6% 68.9% 85.2% 79.5% 29.7% 90.5% /
a
Note: Sequence identities for RmYN02 compared with the SARS-CoV GZ02 (accession number AY390556), the bat SARS-like coronaviruses RaTG13 (EPI_ISL_402131), ZC45 (MG772933)
and ZXC21 (MG772934), and the pangolin SARS-like coronaviruses Pangolin/GD/2019 and Pangolin/GX/P5L/2017 (EPI_ISL_410540).
1
Pangolin/GD/2019 represents a merger of GD/P1L and GD/P2S, and these values were adapted from the reference [6].

“-”: No corresponding values in reference [6].

“/”: This orf is not found.

NA not available.