Article Extended Data Fig. 7 | Analysis of 2019-nCoV receptor usage. Determination of virus infectivity in HeLa cells with or without the expression of human APN and DPP4. The expression of ACE2, APN and DPP4 plasmids with S tag were detected using mouse anti-S tag monoclonal antibody. ACE2, APN and DPP4 proteins (green), viral protein (red) and nuclei (blue) are shown. Scale bars, 10 μm.

The 2002–3 pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV) was one of the most significant public health events in recent history1. An ongoing outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV)2 suggests that this group of viruses remains a major threat and that their distribution is wider than previously recognized. Although bats have been suggested as the natural reservoirs of both viruses3–5, attempts to isolate the progenitor virus of SARS-CoV from bats have been unsuccessful. Diverse SARS-like coronaviruses (SL-CoVs) have now been reported from bats in China, Europe and Africa5–8, but none are considered a direct progenitor of SARS-CoV because of their phylogenetic disparity from this virus and the inability of their spike proteins (S) to use the SARS-CoV cellular receptor molecule, the human angiotensin converting enzyme II (ACE2)9,10. Here, we report whole genome sequences of two novel bat CoVs from Chinese horseshoe bats (Family: Rhinolophidae) in Yunnan, China; RsSHC014 and Rs3367. These viruses are far more closely related to SARS-CoV than any previously identified bat CoVs, particularly in the receptor binding domain (RDB) of the S protein. Most importantly, we report the first recorded isolation of a live SL-CoV (bat SL-CoV-WIV1) from bat fecal samples in Vero E6 cells, which has typical coronavirus morphology, 99.9% sequence identity to Rs3367 and uses the ACE2s from human, civet and Chinese horseshoe bat for cell entry. Preliminary in vitro testing indicates that WIV1 also has a broad species tropism. Our results provide the strongest evidence to date that Chinese horseshoe bats are natural reservoirs of SARS-CoV, and that intermediate hosts may not be necessary for direct human infection by some bat SL-CoVs. They also highlight the importance of pathogen discovery programs targeting high-risk wildlife groups in emerging disease hotspots as a strategy for pandemic preparedness.

A large number of SARS-related coronaviruses (SARSr-CoV) have been detected in horseshoe bats since 2005 in different areas of China. However, these bat SARSr-CoVs show sequence differences from SARS coronavirus (SARS-CoV) in different genes (S, ORF8, ORF3, etc) and are considered unlikely to represent the direct progenitor of SARS-CoV. Herein, we report the findings of our 5-year surveillance of SARSr-CoVs in a cave inhabited by multiple species of horseshoe bats in Yunnan Province, China. The full-length genomes of 11 newly discovered SARSr-CoV strains, together with our previous findings, reveals that the SARSr-CoVs circulating in this single location are highly diverse in the S gene, ORF3 and ORF8. Importantly, strains with high genetic similarity to SARS-CoV in the hypervariable N-terminal domain (NTD) and receptor-binding domain (RBD) of the S1 gene, the ORF3 and ORF8 region, respectively, were all discovered in this cave. In addition, we report the first discovery of bat SARSr-CoVs highly similar to human SARS-CoV in ORF3b and in the split ORF8a and 8b. Moreover, SARSr-CoV strains from this cave were more closely related to SARS-CoV in the non-structural protein genes ORF1a and 1b compared with those detected elsewhere. Recombination analysis shows evidence of frequent recombination events within the S gene and around the ORF8 between these SARSr-CoVs. We hypothesize that the direct progenitor of SARS-CoV may have originated after sequential recombination events between the precursors of these SARSr-CoVs. Cell entry studies demonstrated that three newly identified SARSr-CoVs with different S protein sequences are all able to use human ACE2 as the receptor, further exhibiting the close relationship between strains in this cave and SARS-CoV. This work provides new insights into the origin and evolution of SARS-CoV and highlights the necessity of preparedness for future emergence of SARS-like diseases.