3D fluorescence microscope for virus-host interaction studies

蝙蝠作为传粉者和昆虫控制者具有重要的生态学意义。它们是几种新出现和再出现的病毒的天然宿主，包括SARS-CoV-2等冠状病毒，这些病毒是重要的人类和农业感染的病原体。据推测，这些病毒已经从蝙蝠身上溢出或“跳跃”到人类和其他动物身上，导致严重且往往致命的疾病。尽管有证据表明蝙蝠携带这些病毒，但它们似乎没有表现出明显的感染症状或临床体征。人们对蝙蝠不容易受到这些病毒感染的原因以及使它们成为这些病毒携带者或宿主的生理特征知之甚少。莫斯曼实验室在研究病毒与宿主相互作用方面拥有20多年的经验，并与多伦多的临床同事一起，率先从两名患者身上分离出SARS-CoV-2并对其进行了表征，这导致了20多篇论文的发表。我们的发现基金产生了一个独特而创新的计划，利用我们的专业知识（病毒-宿主相互作用和先天免疫）和福尔博士的专业知识，福尔博士管理着世界上唯一一个包含野生捕获和繁殖动物的蝙蝠群落（麦克马斯特蝙蝠实验室）。我们已经开发了细胞系和工具，用于研究病毒与蝙蝠相互作用的机制，包括原代培养和3D类器官，后者由肠道干细胞的多种细胞类型组成。我们经常使用荧光显微镜来监测病毒如何侵入细胞以及细胞如何对病毒入侵作出反应。由于我们目前的倒置荧光显微镜由于缺乏维修部件而过时（它是19年前购买的），我们正在申请一台新的荧光显微镜。所要求的基础设施是THUNDER 3D成像系统，该系统使用新的计算清除技术，从厚样品（如类器官培养物）中生成高分辨率和高对比度的图像。它允许获取组织深处的图像，如类器官。雷系统是徕卡技术，因为是我们目前的显微镜，允许回收一些组件。这项技术将使我们能够实时监测病毒-宿主相互作用的动力学和复杂性，同时保持敏感样品的完整性。了解蝙蝠如何在明显没有症状的情况下藏匿和传播病毒，对动物和人类的疾病控制具有重要意义。由于大棕色蝙蝠自然存在于加拿大，我们的研究结果将直接影响加拿大的农业和卫生部门。此外，这些发现将为疾病控制和开发新的抗病毒疗法的全球战略提供信息，特别是针对蝙蝠似乎携带的新出现的病毒。最后，该方案将为在多学科环境中培养高素质人才奠定基础，这些人才将在科学前沿工作，并对流行病防范和认识等关键领域产生影响。

Bats are ecologically important as pollinators and insect controllers. They are natural reservoirs for several emerging and re-emerging viruses, including coronaviruses such as SARS-CoV-2, that are causative agents of important human and agricultural infections. These viruses are speculated to have spilled over or "jumped" from bats to humans and other animals, causing severe and often fatal disease. Despite evidence of bats harboring these viruses, they do not appear to show overt symptoms or clinical signs of infection. Little is understood why bats are not susceptible to these viral infections and the physiological characteristics that allow them to be carriers or reservoirs of these viruses. The Mossman lab has over 20 years of experience studying virus-host interactions and along with clinical colleagues in Toronto, were the first to isolate and characterize SARS-CoV-2 from two patients, which has led to over 20 publications. Our Discovery Grant has generated a unique and innovative program that leverages our expertise (virus-host interactions and innate immunity) and that of Dr. Faure who runs the world's only bat colony containing wild caught and bred animals (McMaster Bat Lab). We have developed cell lines and tools with which to study mechanisms of virus-bat interactions, including primary cultures and 3D organoids, the latter composed of multiple cell types from intestinal stem cells. We routinely use fluorescence microscopy to monitor how viruses invade cells and how cells respond to viral invasion. As our current inverted fluorescence microscope is obsolete due to lack of availability of components for repair (it was purchased 19 years ago), we are requesting a new fluorescence microscope. The requested infrastructure, a THUNDER 3D Imaging System, uses new computational clearing technologies to generate high resolution and high contrast images from thick samples, such as organoid cultures. It allows for acquisition of images taken deep within tissues such as organoids. The THUNDER system is a Leica technology, as is our current microscope, allowing for recycling of some components. This technology will allow us to monitor the kinetics and complexity of virus-host interactions in real time, while maintaining the integrity of sensitive samples. Understanding how bats harbor and spread viruses with an apparent lack of symptoms has implications for disease control in animals and humans. As big brown bats naturally occur in Canada, our findings will directly impact Canadian agricultural and health sectors. Moreover, findings will inform global strategies for disease control and development of novel antiviral therapies, particularly for new emerging viruses that bats appear to carry. Finally, this program will lay the foundation for training of highly qualified personnel in a multidisciplinary environment, working at the forefront of science and impacting on critical areas such as pandemic preparedness and understanding.