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

BRC-BIO：秀丽隐杆线虫新型病毒病原体的发现和表征

• 批准号: 2218079
• 负责人: Jessica Sowa
• 金额: $ 50.22万
• 依托单位: West Chester University of Pennsylvania
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2218079&HistoricalAwards=false
• 关键词: BRC; BIO; Discovery; characterization; novel

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.

秀丽隐杆线虫和其他相关的自由生活线虫是一种流行且强大的模型系统，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做出反应。线虫为此类研究提供了许多优势：动物体型小，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随时间的感染情况。然而，迄今为止，仅发现了一种能够自然感染线虫的病毒，这严重限制了线虫作为专门研究病毒感染的模型系统。该项目旨在通过识别野生捕获的线虫中可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，来弥补这一空白。野生线虫收集将通过线虫猎人计划进行，该计划是将真实的生物学研究带​​入小学课堂的外展计划。线虫猎人的参与者将从当地环境中收集野生自由生活的线虫，并将其提交进行感染筛查，该筛查将由参加基于课程的研究经历的本科生进行。将研究新发现的病毒，以确定它们感染哪些组织以及感染过程。 最终，新病毒将提供给研究界，从而使线虫模型的力量能够应用于更广泛的有关病毒如何与宿主细胞相互作用的问题。 该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、感染动态和组织向性。将通过线虫猎人外展计划收集不同地区的样本，并通过与表达荧光感染报告基因的线虫共培养来筛查病毒对线虫的传播性。潜在的病毒感染将通过均质化显示感染传播证据的共培养物、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识别。这种高通量方法将能够快速筛选许多野生线虫分离株，以识别新型病毒感染。对于潜在的病毒感染，将从感染性滤液和感染的蠕虫中提取 RNA 和 DNA，并进行测序以识别与病毒 RNA/DNA 相对应的读数。鉴定出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线虫的新病毒，并将其提供给研究界进行比较研究。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。