Nanobody toolkit for human coronavirus classification

摘要 地方性人类冠状病毒(HCoV)NL63、OC43、229E和HKU1通常 与健康人群中类似普通感冒的相对轻微和自限性的呼吸道疾病有关 个人。然而，新出现的人畜共患冠状病毒(SARS-CoV、MERS-CoV和SARS-CoV-2) 会导致更严重的呼吸窘迫，尤其是那些有潜在健康状况的人，可能会 致命的。冠状病毒种类繁多，可从人畜共患病的蓄水池直接或通过 中间宿主。由于人口不断增长，溢出效应可能会更加频繁 生态系统侵蚀，从而确保自然和非自然宿主之间发生更多冲突。我们的探索者 一项研究提案寻求开发一个小型免疫反应剂工具包，它将形成逻辑门的基础，以 对冠状病毒进行鉴定和分类。一种单一的免疫反应剂，可广泛识别所有共享的蛋白质 架构，将允许识别冠状病毒属。分离免疫反应物与绝对 人类冠状病毒和人畜共患冠状病毒之间独特的氨基酸变异的特异性将允许进一步 已知病原体的分类。这样的工具包将很快确定一种新出现的病毒是否是一种 我们熟悉的冠状病毒是一种新的属或谱系，以前从未蔓延到 并可能要求对流行病或大流行的可能性采取紧急后续行动。我们的免疫反应剂 以骆驼单域抗体或纳米抗体为基础，在E. Coli和高度模块化，允许与酶报告酶融合，一步探测受感染的病毒 细胞和夹心检测方法的简易建立。纳米体序列在电子手段中的永久化 在Genbank中存放后，这些数据立即可用于更广泛的研究社区，不同于 分泌免疫球蛋白的多克隆血清或杂交瘤。单锅库技术使快速响应成为可能 针对未来出现的任何新出现的冠状病毒，确保我们更加主动，而不是被动 以帮助维护人类健康。

ABSTRACT The endemic human coronaviruses (HCoV) NL63, OC43, 229E and HKU1 have typically been associated with relatively mild and self-limiting respiratory disease resembling the common cold in healthy individuals. However, the newly emerging zoonotic coronaviruses (SARS-CoV, MERS-CoV and SARS-CoV-2) can cause more severe respiratory distress, especially in those with underlying health conditions and can be fatal. Coronaviruses are diverse and spillover from zoonotic reservoirs either directly to humans or via an intermediate host. Spillover is likely to be more frequent due to the ever-expanding human population and ecosystem erosion, thereby ensuring more collisions between natural and unnatural host. Our exploratory research proposal seeks to develop a small toolkit of immunoreagents that will form the basis of a logic gate to identify and classify coronaviruses. A single immunoreagent, with broad recognition of overall shared protein architecture, will allow the identification of the coronavirus genera. Separate immunoreagents with absolute specificity for unique amino-acid variation between the human and zoonotic coronaviruses will allow the further classification to a known pathogen. Such a toolkit will quickly establish if a newly emerging virus is a coronavirus we are familiar with or is a novel genus or lineage that has never before spilled over into the human population and may demand urgent follow up for epidemic or pandemic potential. Our immunoreagents are based on llama single domain antibodies or nanobodies that are inexpensive to produce at high yields in E. coli and highly modular, allowing fusion to enzymatic reporter enzymes for one-step probing of virus infected cells and facile establishment of sandwich assays. Immortalization of nanobody sequences in silico means these are immediately available to the wider research community following deposition in Genbank unlike polyclonal sera or hybridomas secreting IgG. Single-pot library technology enables a rapid response to be mounted against any newly emerged coronavirus in the future, ensuring we are more pro-active than re-active to help safeguard human health.