BRC-BIO: Discovery and characterization of novel viral pathogens of Caenorhabditis elegans

秀丽隐杆线虫和其他相关的自由生活线虫是一种流行和强大的模型系统，用于研究病原体如何感染宿主细胞以及宿主免疫系统如何在细胞水平上对感染作出反应。秀丽隐杆线虫为这些类型的研究提供了许多优势：动物体积小，易于大量生长，生命周期快，易于基因操作，并且是透明的，因此可以观察活体动物的感染情况。然而，迄今为止仅鉴定出一种能够自然感染秀丽隐杆线虫的病毒，这严重限制了秀丽隐杆线虫作为专门研究病毒感染的模型系统。该项目旨在通过在野生捕获的线虫中识别可以传播给秀丽隐杆线虫的新病毒，并研究新发现的病毒感染的细胞生物学来解决这一空白。野生线虫的收集将通过“线虫猎人计划”进行，这是一项将真实的生物学研究带入小学课堂的外展计划。“线虫猎人”项目的参与者将从当地环境中收集野生的自由生活的线虫，并将它们提交给感染筛查，这将由参加基于课程的研究经历的本科生进行。将对新发现的病毒进行研究，以确定它们感染的组织和感染过程。最终，这些新病毒将提供给研究界，从而使秀丽隐杆线虫模型的力量能够应用于更广泛的关于病毒如何与宿主细胞相互作用的问题。本项目旨在鉴定野生捕获的自由生活线虫中可传播给秀丽隐杆线虫的新型病毒，并表征其基因组结构、感染动力学和组织趋向性。将通过“线虫猎人”外展计划收集地理上不同的样本，并通过与表达荧光感染报告的秀丽隐杆线虫共培养来筛选病毒对秀丽隐杆线虫的传播能力。潜在的病毒感染将通过对显示感染传播证据的共培养物进行匀浆，过滤匀浆以排除比病毒大的病原体，并测试这些滤液传播感染的能力来确定。这种高通量方法将允许快速筛选许多野生线虫分离株，以鉴定新的病毒感染。对于潜在的病毒感染，将从感染滤液和被感染的蠕虫中提取RNA和DNA，并测序以识别与病毒RNA/DNA相对应的reads。鉴定的病毒序列将用于设计引物和荧光原位杂交探针，用于确定新发现的病毒感染的组织趋向性和感染动力学。通过建立病毒发现和表征的管道，该项目将发现能够自然感染秀丽隐杆线虫的新病毒，并将它们提供给研究界进行比较研究。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Caenorhabditis elegans and other related free-living nematodes are a popular and powerful model system used to study how pathogens infect host cells and how the host immune system responds to infection on a cellular level. C. elegans offers many advantages for these types of studies: the animals are small and easy to grow in large numbers, have a fast life cycle, are easy to genetically manipulate, and are transparent, thus allowing observation of infections over time in live animals. However, only one virus capable of naturally infecting C. elegans has been identified to date, which severely limits C. elegans as a model system for studying viral infections specifically. This project aims to address this gap by identifying new viruses in wild-caught nematodes that can be passed to C. elegans, and studying the cell biology of the newly identified viral infections. Wild nematode collection will be conducted through the Nematode Hunters program, an outreach program that brings authentic biology research into elementary school classrooms. Nematode Hunters participants will collect wild free-living nematodes from their local environment and submit them for infection screening, which will be conducted by undergraduate students enrolled in a course-based research experience. Newly identified viruses will be studied to determine what tissues they infect and the course of the infection. Ultimately, the new viruses will be made available to the research community, thereby enabling the power of the C. elegans model to be applied to a wider range of questions about how viruses interact with host cells. This project aims to identify novel viruses in wild-caught free-living nematodes that are transmissible to C. elegans and to characterize their genomic structure, infection dynamics, and tissue tropism. A geographically diverse sampling will be collected via the Nematode Hunters outreach program, and screening for viral transmissibility to C. elegans will be conducted by co-culturing with C. elegans expressing fluorescent infection reporters. Potential viral infections will be identified by homogenizing co-cultures showing evidence of infection transmission, filtering the homogenate to size-exclude pathogens larger than a virus, and testing the ability of these filtrates to transmit the infection. This high-throughput method will allow rapid screening of many wild nematode isolates to identify novel viral infections. For potential viral infections, RNA and DNA will be extracted from infectious filtrates and infected worms, and sequenced to identify reads corresponding to viral RNA/DNA. Viral sequences identified will be used to design primers and fluorescence in situ hybridization probes, which will be used to determine the tissue tropism and infection dynamics of newly discovered viral infections. By establishing a pipeline for viral discovery and characterization, the project will uncover new viruses capable of naturally infecting C. elegans and make them available to the research community for comparative studies.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.