Understanding spillover potential of European and African bat sarbecoviruses

摘要 2019年底爆发的SARS-CoV-2已导致全球超过600万人丧生。从那时起， 人们一直高度关注疫苗和临床治疗的开发，以增加 这种病毒引起的疾病的存活率。然而，在自然界中， 其可能呈现未来的溢出潜力并且这些治疗可能对其无效。进化枝3 Sarbecoviruses起源于非洲和欧洲，在被认为是Sarbecovirus热点的地区之外， 东南亚地理位置和缺乏天然人类ACE 2利用从最早的 在该进化枝中发现的病毒导致该进化枝被忽略。然而，我们最近发现， 该进化枝的一个成员，来自肯尼亚的BtKY72，具有在一个细胞内获得人ACE2结合的能力。 氨基酸突变和两个突变内的细胞进入。此外，我们首次证明了 该进化枝的另一个成员，来自俄罗斯的科斯塔-2，可以天然结合野生型的人ACE 2 顺序总之，我们最近的观察表明，需要开发工具来研究和抑制潜在的 人类感染这种被忽视的病毒分支。进化枝3可能是未来肉瘤病毒的起源 溢出，但由于进化枝之间的遗传差异，目前的工具可能具有有限的保护能力 3和先前的人肉瘤病毒的刺突蛋白，病毒表面糖蛋白负责 受体结合和病毒包膜与宿主细胞膜的融合。我假设所有成员 该进化枝的受体结合结构域中的几个突变内可以获得人ACE2利用， 但是目前疫苗和抗体治疗将降低抗 分支3肉瘤病毒。在本提案的目标1中，我将揭示进化枝所有当前成员的受体使用情况 3在非洲，欧洲和亚洲分布的菊头蝠物种中鉴定出使人类能够生存的突变 利用安全的非复制假病毒系统，这些病毒的ACE2结合和细胞进入。在 目标2，我将建立疫苗和单克隆抗体治疗方面的临床工具 有效地防止分支3肉瘤病毒进入细胞。了解当前天然受体的使用情况 结合可能能够共同感染特定物种的肉瘤病毒的序列评估， 菊头蝠将深入了解这些病毒的进化可能性。此外，本发明还提供了一种方法， 鉴定能够使人ACE2结合并进入人细胞系的突变将提供 这些病毒距离实现人类传播所需的第一步有多近。最后 评估现有工具对进化枝3型肉瘤病毒的有效性， 进化枝3刺突胞外结构域的表征，将使我们在这些病毒跨越物种时领先一步 未来的屏障。

Abstract The outbreak of SARS-CoV-2 in late 2019 has resulted in the loss of over 6 million lives worldwide. Since then, there has been an intense focus on the development of vaccines and clinical treatments to increase the survivability of the disease caused by this virus. However, in nature other diverse sarbecoviruses circulate which may present future spillover potential and for which these treatments may be ineffective. Clade 3 sarbecoviruses originate in Africa and Europe, outside of the regions considered to be sarbecovirus hotspots in Southeast Asia. The geographical location and the absence of native human ACE2 utilization from the earliest viruses discovered in this clade resulted in this clade being discounted. However, we have recently shown that one member of this clade, BtKY72 from Kenya, has the capacity to gain human ACE2 binding within one amino acid mutation and cellular entry within two mutations. Furthermore, we demonstrated for the first time that another member of this clade, Khosta-2 from Russia, can natively bind human ACE2 as a wildtype sequence. Together, our recent observations indicate the need to develop tools to study and inhibit potential human infection by this overlooked clade of viruses. Clade 3 may be the origin of a future sarbecovirus spillover, but current tools might have limited protective capacity due to the genetic divergence between Clade 3 and the prior human sarbecoviruses in the spike protein, the viral surface glycoprotein responsible for receptor binding and fusion of the viral envelope and the host cell membrane. I hypothesize that all members of this clade can gain human ACE2 utilization within a couple of mutations in the receptor binding domain of the spike glycoprotein but that current vaccines and antibody treatments will have reduced efficacy against clade 3 sarbecoviruses. In Aim 1 of this proposal, I will uncover receptor usage of all current members of clade 3 in Rhinolophus bat species with ranges in Africa, Europe, and Asia identify mutations that enable human ACE2 binding and cellular entry of these viruses making use of safe non-replicating pseudovirus systems. In Aim 2, I will establish what clinical tools in terms of vaccines and monoclonal antibody treatments would be effective at preventing cellular entry of clade 3 sarbecoviruses. Understanding current native receptor usage combined with a sequence assessment of sarbecoviruses that may be able to coinfect a specific species of Rhinolophus will give insight into the evolutionary possibilities available to these viruses. In addition, identification of mutations that enable human ACE2 binding and cellular entry in human cell lines will provide context on how close these viruses are to achieving this first step necessary for human spillover. Finally, the assessment of current tools for their effectiveness against clade 3 sarbecoviruses and the structural characterization of clade 3 spike ectodomains, will give us a head start should these viruses cross the species barrier in the future.