key: cord-0783723-kch863gi authors: Guo, Hua; Hu, Ben; Si, Hao-rui; Zhu, Yan; Zhang, Wei; Li, Bei; Li, Ang; Geng, Rong; Lin, Hao-Feng; Yang, Xing-Lou; Zhou, Peng; Shi, Zheng-Li title: Identification of a novel lineage bat SARS-related coronaviruses that use bat ACE2 receptor date: 2021-05-21 journal: bioRxiv DOI: 10.1101/2021.05.21.445091 sha: 8e92182a01f321ef1e31a89bcfc82a4d18a9384c doc_id: 783723 cord_uid: kch863gi Severe respiratory disease coronavirus-2 (SARS-CoV-2) causes the most devastating disease, COVID-19, of the recent century. One of the unsolved scientific questions around SARS-CoV-2 is the animal origin of this virus. Bats and pangolins are recognized as the most probable reservoir hosts that harbor the highly similar SARS-CoV-2 related viruses (SARSr-CoV-2). Here, we report the identification of a novel lineage of SARSr-CoVs, including RaTG15 and seven other viruses, from bats at the same location where we found RaTG13 in 2015. Although RaTG15 and the related viruses share 97.2% amino acid sequence identities to SARS-CoV-2 in the conserved ORF1b region, but only show less than 77.6% to all known SARSr-CoVs in genome level, thus forms a distinct lineage in the Sarbecovirus phylogenetic tree. We then found that RaTG15 receptor binding domain (RBD) can bind to and use Rhinolophus affinis bat ACE2 (RaACE2) but not human ACE2 as entry receptor, although which contains a short deletion and has different key residues responsible for ACE2 binding. In addition, we show that none of the known viruses in bat SARSr-CoV-2 lineage or the novel lineage discovered so far use human ACE2 efficiently compared to SARSr-CoV-2 from pangolin or some of the SARSr-CoV-1 lineage viruses. Collectively, we suggest more systematic and longitudinal work in bats to prevent future spillover events caused by SARSr-CoVs or to better understand the origin of SARS-CoV-2. Southeast Asia, which includes bat CoVs RaTG13 and RmYN02 from Yunnan, Rc-2 1 2 o319 from Japan, RshSTT182 from Cambodia, RacCS203 from Thailand, as well as 2 1 3 two different strains of pangolin-CoVs (Table S2) . It is also conceivable that RaTG15 2 1 4 clusteres with SARSr-CoV-2 in the phylogeny using full-length RdRp gene ( Figure 2 1 5 S1A). In contrast, similarity plot analysis reveals that beyond ORF1b, RaTG15 is 2 1 8 remarkably distinct from both SARSr-CoV-2 and SARSr-CoV-1 in majority of the 2 1 9 genome ( Figure 1A ). It exhibits less than 80% nucleotide identities in ORF1a, M and 2 2 0 N genes and lower than 70% identities in S, ORF3, 6 and 7a/7b to all other SARSr-2 2 1 CoVs (Table S2) . Overall, the full genome of the SARSr-CoV RaTG15 show 74.4% 2 2 2 sequence identity to SARS-CoV-1 and 77.6% to sequence identity to SARS-CoV-2. Notably, RaTG15 show higher sequence identity to SARS-CoV-1 than to SARS-2 2 4 CoV-2 in the spike, E, M, N and ORF6 proteins. It also has almost equivalent 2 2 5 homology to any other known SARSr-CoVs from bat or pangolin CoVs (Table S2 ). This mosaic profile suggests that this novel lineage viruses may be a results of 2 2 7 recombination of different SARSr-CoVs. The result of phylogenetic analysis is in accordance with similarity plot. SARSr-CoVs 2 3 0 mainly consists of two sub-lineages, the SARSr-CoV-1 and SARSr-CoV-2 ( Figure 2 3 1 1B). The latter one includes SARS-CoV-2 from pangolins and different Rhinolophus 2 3 2 bats species recently reported in a wide range of areas in Asia. In the full-length 2 3 3 genome tree and S gene tree, RaTG15 and the related viruses are distant from both of 2 3 4 the two existing sub-lineages, and forms a well-supported novel lineage with the 2 3 5 sarbecoviruses ( Figure 1B and Figure S1B ). to HuACE2, only slightly weaker than SARS-CoV-2 RBD but still higher than pangolin-CoV-GX is slightly weaker than pangolin-CoV-GD. Next, we wanted to These results suggested the SARSr-CoVs we discovered from bat now may be just the The closest bat CoV to SARS-CoV-2 at this stage, RaTG13 only showed very weak 3 1 9 binding affinity to HuACE2. Albeit there is a speculation claiming the possible 3 2 0 leaking of RaTG13 from lab that caused SARS-CoV-2, the experiment evidence 3 2 1 cannot support it. In contrast, the pangolin-CoV shows strong binding capacity to 3 2 2 human or bat ACE2, posing high cross-species potential to human or other species. In The authors declare no competing interests. e n t s . S c i e n c e . 2 0 2 1 J a n 3 4 5 8 ; 3 7 1 ( 6 5 2 5 ) : r i g i n a n d e v o l u t i o n o f p a t h o g e n i c c o r o n a v i r u s e s . 3 9 0 N a t u r e r e v i e w s M i c r o b i o l o g y . 2 0 1 9 M a r ; 1 7 ( 3 ) : 1 8 1 -1 9 2 . 3 9 1 2 0 . D r o s t e n C , G u n t h e r S , P r e i s e r W , e t a l . I d e n t i f i c a t i o n o f a n o v e l 3 9 2 c o r o n a v i r u s i n p a t i e n t s w i t h s e v e r e a c u t e r e s p i r a t o r y s y n d r o m e . N E n g l J 3 9 3 M e d . 2 0 0 3 M a y 1 5 ; 3 4 8 ( 2 0 ) : Bat RacCS203 Bat RshSTT182 Bat RacCS203 Bat CoV RaTG15 SARS-CoV Bat CoV RaTG13 Bat CoV RmYN02 Bat CoV RacCS203 Bat CoV RshSTT182 Pangolin-CoV-GD Pangolin-CoV-GX Bat CoV Roc Bat SARSr-CoV WIV16 Bat SARSr-CoV WIV1 Bat SARSr-CoV SHC014 Bat SARSr-CoV YNLF 31C Bat SARSr-CoV Rp3 Bat SARSr-CoV LYRa11 Bat SARSr-CoV Rf1 Bat SARSr-CoV HKU3-1 Bat CoV RaTG13_China Bat CoV RmYN02_China Bat CoV RacCS203_Thailand Bat CoV RshSTT182_Cambodia Pangolin-CoV-GD Bat CoV RsZC45_China Pangolin-CoV-GX Bat CoV Rc-o319_Japan Bat CoV RaTG15 Bat CoV Rst7931 Bat CoV Rst7905 Bat CoV Rst7907 Bat CoV Rst7924 Bat CoV Rst7952 Bat CoV Rst7896 Bat CoV Rst7921 Bat Hp BetaCoV Zhejiang2013 Rousettus bat CoV HKU9 Bat CoV GCCDC1