Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus

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.

自2005年以来，中国不同地区的马蹄蝠体内已检测到大量SARS相关冠状病毒（SARS-related coronaviruses, SARSr-CoV）。然而，这些蝙蝠SARSr-CoV在多个基因（如S、ORF8、ORF3等）上与重症急性呼吸综合征冠状病毒（SARS coronavirus, SARS-CoV）存在序列差异，被认为不太可能是SARS-CoV的直接祖先毒株。本研究报道了我们对中国云南省一处栖息有多种马蹄蝠的洞穴开展的5年SARSr-CoV监测结果。结合此前的研究结果，我们对11株新发现的SARSr-CoV毒株进行全基因组分析后发现，该单一栖息位点内传播的SARSr-CoV在S基因、ORF3与ORF8区域具有高度多样性。尤为重要的是，在S1基因的高变N端结构域（N-terminal domain, NTD）与受体结合结构域（receptor-binding domain, RBD）、ORF3区域及ORF8区域，分别与SARS-CoV具有高度遗传相似性的毒株，均在该洞穴中被发现。此外，本研究首次发现，部分蝙蝠SARSr-CoV的ORF3b基因以及裂解形成的ORF8a与ORF8b区域，与人类SARS-CoV具有高度相似性。相较于其他地区检测到的SARSr-CoV毒株，该洞穴中的SARSr-CoV毒株在非结构蛋白基因ORF1a与ORF1b区域与SARS-CoV的亲缘关系更为密切。重组分析结果显示，这些SARSr-CoV在S基因内部及ORF8区域周边频繁发生重组事件。我们推测，SARS-CoV的直接祖先毒株可能起源于这些SARSr-CoV前体之间的序贯重组事件。细胞进入实验结果表明，3株具有不同S蛋白序列的新发现SARSr-CoV，均能够以人类血管紧张素转换酶2（human angiotensin-converting enzyme 2, ACE2）作为受体，进一步证实了该洞穴内毒株与SARS-CoV之间的密切亲缘关系。本研究为SARS-CoV的起源与演化提供了新的科学视角，同时也强调了为未来可能出现的类SARS疾病做好防控准备的必要性。