目前一般认为新型冠状病毒（严重急性呼吸综合征冠状病毒2，SARS-CoV-2）起源于蝙蝠。多个蝙蝠冠状病毒跟新型冠状病毒序列高度同源，其中RaTG13与新冠病毒的核苷酸序列同源性达到了96.2%，位列所有已知蝙蝠冠状病毒中的第二位，仅次于老挝发现的BANAL-20-52；此外，RaTG13也可以使用人类血管紧张素转换酶2 （hACE2）作为受体，然而RaTG13在蝙蝠中的宿主范围以及其通过其它动物实现跨物种传播的风险以及机制并不清楚。

针对这两个科学问题，本论文首先对东南亚广泛分布的18个不同蝠种对RaTG13的易感性进行了研究，发现只有表达中菊头蝠的ACE2（Rhinolophus affinis bat ACE2，RaACE2）的细胞对RaTG13 S假病毒的转导高度敏感，表明RaTG13病毒的蝙蝠宿主范围可能比较有限。RaACE2和RaTG13受体结合域（RBD）的亲和力跟hACE2相似，而RaACE2与SARS-CoV-2 RBD的结合要明显弱于hACE2。RaACE2存在至少7个多态突变体（polymorphic variants），RA-01到RA-07，我们发现表达不同RaACE2的细胞对RaTG13 S假病毒转导表现出不同的敏感性，其中表达RA-07ACE2的细胞敏感性最高，而表达RA-03 ACE2的细胞敏感性最差。令我们感到惊讶的是，新型冠状病毒和多个SARS-CoV-2相关的冠状病毒 （SC2r -CoV），包括BANAL-20-52和BANAL-20-236，对RaACE2的多态性并没有表现出显著差异，无论是在结合、膜融合以及假病毒入侵方面。进一步的突变分析RaACE2中的34、38和83位点与RaTG13 S蛋白501和505位点在相互作用中起关键作用。另外，我们还发现RaTG13 S蛋白中T372A突变不仅会显著增加对所有RaACE2突变体的转导，而且还可以显著增强了对多个蝙蝠ACE2的入侵，包括Rhinolophus sinicus YN、Rhinolophus pearsonii和Rhinolophus ferrumeiqum。然而，T372A突变体对BANAL-20-52 S免疫的小鼠血清的中和敏感性提高了约4倍，这表明在蝙蝠冠状病毒中免疫逃逸能力可能使T372优于A372。

随后，我们对RaTG13在其它动物里的宿主范围和跨物种传播风险进行了较详细评估，我们选取了16种不同动物物种的ACE2s，分别从S蛋白的结合、膜融合和假病毒入侵方面评估了它们对RaTG13的敏感性。同时我们也选择了SARS-CoV和SARS-CoV-2作为对比。我们发现，RaTG13 spike （S）蛋白整体的融合能力明显弱于SARS-CoV和SARS-CoV-2。在 16种不同的ACE2s中，有7个ACE2能介导所有三种S蛋白假病毒的入侵。值得注意的是，RaTG13 S假病毒可以利用小鼠ACE2但是不能利用穿山甲的ACE2，而SARS-CoV-2 S假病毒可以利用穿山甲ACE2但是不能利用小鼠的ACE2。随后的分子模拟以及S蛋白突变分析显示，RaTG13和SARS-CoV-2 S蛋白的484，498，501和505位点对识别小鼠和穿山甲ACE2至关重要。

总之，本文通过研究RaTG13的宿主范围，鉴定了对于不同受体入侵的关键氨基酸位点，这些位点对于病毒的跨物种传播非常重要，监测这些位点的突变有助于我们更好地预防下一次蝙蝠冠状病毒的溢出。本研究有助于我们了解蝙蝠冠状病毒的进化路径和变异方式，为开发更有效的广谱抗冠状病毒疫苗提供基础。

SARS-CoV-2 is believed to have been originated from bats, because multiple bat coronaviruses (CoVs) share high homology in genome sequences with that of SARS-CoV-2. RaTG13 was initially discovered in TongGuan, Yunan, China in 2013, and shares about 96.2% similarity with SARS-CoV-2, ranking the second closest to SARS-CoV-2 among all known bat CoVs (only behind BANAL-20-52 from Laos). In addition, RaTG13 can also use human angiotensin-converting enzyme 2 (hACE2) as the receptor. However, the host range and zoonotic potential of RaTG13 among bats and other animals remains elusive.

In this study, we first determine the susceptibility of 18 different bat species commonly found in Southeast Asia to RaTG13, and find that only the RaACE2 's ACE2 (Rhinolophus affinis bat ACE2, RaACE2) is highly sensitive to the transduction of RaTG13 S pseudovirus, suggesting that the potential bat host for  RaTG13 virus might be very limited. RaTG13 S protein binds to RaACE2 at a level similar to hACE2, whereas SARS-CoV-2 S protein interacts with RaACE2 significantly weaker than hACE2. There are at least seven polymorphic variants of RaACE2, RA-01 to RA-07, and they show different levels of susceptiblities to RaTG13 S pseudoviral transduction, with RA-07 the highest and RA-03 the least. Of note, RaACE2 polymorphisms show little impact on receptor binding, membrane fusion, and virus entry of SARS-CoV-2 and its related coronaviruses (SC2r-CoV), such as BANAL-20-52 and BANAL-20-236. Further mutational analysis reveals residues 34, 38, and 83 of RaACE2 and residues 501 and 505 of RaTG13 S protein are critical for the interactions. Interestingly, substitution of T372 with alanine (T372A) of the RaTG13 S protein not only markedly enhances the transduction on all RaACE2 variants but also increases virus entry on several bat ACE2s, including Rhinolophus sinicus YN，Rhinolophus pearsonii and Rhinolophus ferrumeiqum. However, the T372A mutant exhibits approximately a four-fold increase in neutralization sensitivity to the mouse serum, which were immunized with BANAL-20-52 S, indicating that immune evasion might play important role in selection of T372 over A372 during evolution.

We also assess the animal host range and zoonotic transmission risk of RaTG13. We evaluated the receptor susceptibility of ACE2s from 16 different animal species to RaTG13 regarding S protein binding, membrane fusion, and pseudoviral invasion. We found that the fusion potential of RaTG13 S protein was significantly lower than that of SARS-CoV and SARS-CoV-2. Moreover, only 9 out of the 16 ACE2s tested were susceptible to transduction by RaTG13 S pseudovirions, comparing to 13 and 12 for SARS-CoV and SARS-CoV-2, respectively, indicating narrower host range for RaTG13. It is worth noting that RaTG13 S pseudovirus was able to use mouse ACE2 but not pangolin ACE2, while SARS-CoV-2 S pseudovirus could use pangolin ACE2 but not mouse ACE2. Additional mutational analyses showed that residues 484, 498, 501 and 505 of S protein play critical roles in interacting with mouse and pangolin ACE2.

Based on our investigation of the bat host range of RaTG13 and its susceptibility to different ACE2 receptors, as well as the receptor sensitivity of ACE2s from other animal species to RaTG13, SARS-CoV, and SARS-CoV-2, we have identified important amino acid sites that play a key role in receptor recognition and invasion. This information can provide valuable insights into coronavirus invasion, host range, and vaccine design, as well as aid in the study of virus evolution.