Understanding spillover potential of European and African bat sarbecoviruses

摘要 2019年末爆发的SARS-CoV-2疫情已导致全球600多万人丧生。从那以后， 疫苗和临床治疗的发展一直是人们关注的焦点，以增加 这种病毒引起的疾病的存活率。然而，在自然界中，其他不同的肉瘤病毒正在传播 这可能会带来未来的溢出潜力，而这些治疗方法可能对其无效。分支3 肉瘤病毒起源于非洲和欧洲，不在被认为是肉瘤病毒热点的地区。 东南亚。人类最早利用ACE2的地理位置和缺失 在该分支中发现的病毒导致该分支被打折。然而，我们最近已经表明， 这个分支中的一个成员，来自肯尼亚的BtKY72，有能力在一个月内获得人类ACE2结合 氨基酸突变和两个突变内的细胞进入。此外，我们还首次进行了演示 这个分支的另一个成员，来自俄罗斯的Khosta-2，可以天然地将人类ACE2结合为野生型 序列。总而言之，我们最近的观察表明，需要开发工具来研究和抑制潜力 人类感染了这种被忽视的病毒分支。分支3可能是未来肉瘤病毒的起源 溢出，但由于Clade之间的遗传差异，目前的工具可能具有有限的保护能力 而先前人类肉瘤病毒中的刺突蛋白、病毒表面糖蛋白负责 病毒被膜和宿主细胞膜的受体结合和融合。我假设所有成员 在受体结合域的几个突变中，可以获得人类ACE2的利用 尖峰糖蛋白，但目前的疫苗和抗体治疗将降低对 3分支肉瘤病毒。在这项提案的目标1中，我将揭示所有Clade当前成员的受体使用情况 在分布在非洲、欧洲和亚洲的蝙蝠物种中，发现了能够使人类 利用安全的非复制型伪病毒系统，ACE2结合和细胞进入这些病毒。在……里面 目标2，我将确定疫苗和单抗治疗方面的临床工具是什么 有效防止分支3肉瘤病毒进入细胞。了解当前本地受体的使用情况 结合对肉瘤病毒的序列评估，这些病毒可能能够同时感染特定物种的猪 犀牛将对这些病毒可用的进化可能性提供洞察。此外, 鉴定能够与人ACE2结合并进入人类细胞系的突变将提供 关于这些病毒距离实现人类溢出所需的第一步有多近的背景。最后， 评估现有工具对抗分支3肉瘤病毒的有效性和结构 分支3棘突外区的特征，将给我们一个领先的开始，如果这些病毒跨越物种 未来的障碍。

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.