Investigation of enterovirus D68 pathogenesis in the human spinal cord

梅根·弗里曼，医学博士，匹兹堡大学儿科传染病(ID)研究员 医学中心(UPMC)匹兹堡儿童医院(CHP)。她获得了医学博士学位和博士学位 在范德比尔特大学，她研究了冠状病毒感染的细胞生物学和发病机制(Mark 医学博士丹尼森)。在CHP接受了儿科学和儿科ID的临床培训后，她加入了卡罗琳·科因的实验室， 他是肠道病毒和病毒感染的组织屏障方面的专家。她的研究重点是理解 肠道病毒D68(EV-D68)致病机制是一种类似脊髓灰质炎的急性弛缓性脊髓炎(AFM)的假想原因 既往健康儿童的瘫痪综合征。弗里曼博士使用了肠道有机化合物和初级 与AFM相关的一些当代EV-D68分离株能够 除呼吸道外，还可使用肠道进行复制，建议采用另一种感染途径。 这项工作构成了本提案中所描述的脊髓器官模型的基础。 弗里曼博士将在研究病毒-宿主相互作用的10年经验的基础上，同时获得新的 精通免疫学、神经科学和成像技术，这将有助于她过渡到 独立资助的儿科医生兼科学家。弗里曼博士已经召集了一个多学科的导师和 顾问小组，由主要导师John Williams博士(呼吸道病毒免疫/发病机制)担任主席，并与 导师特伦斯·德莫迪博士(病毒致病/脑炎)。其他顾问成员包括Dr。 Carolyn Coyne(肠道病毒/类有机物)、Philana Lin博士(免疫学)和Clayton Wiley博士 (神经科学/脑炎)。该委员会将促进技术获取、职业发展和 合作。培训计划包括课程内容、技术专长、书面和口头技能。 沟通，负责任的研究行为，在会议上的陈述，以及寻求 额外的资金。匹兹堡大学医学院是一个出色的培训环境 关于科学成就、资源、对医生科学家的承诺和专业知识的记录。 AFM在2012-2018两年一次达到顶峰，与EV-D68的发行量一致，然而，原因是 当代EV-D68的神经系统靶点尚不清楚。这项提议的中心假设是 当代EV-D68毒株直接感染脊髓运动神经元，导致神经元损伤 和瘫痪。利用创新的人类脊髓器官模型，AIMS将1)定义脊髓 复制EV-D68，鉴定感染细胞类型(S)，并发现对发病机制的贡献，2)定义 脊髓类器官对EV-D68感染的宿主细胞反应；3)评估谷氨酸的变化 在EV-D68感染脊髓器官的过程中。这项提案中的研究使用了先进的组织 建模和成像技术、病毒复制分析以及宿主细胞因子、细胞死亡和RNA 转录分析以了解EV-D68感染中枢神经系统如何导致AFM。

Megan Freeman, MD, PhD, is a Pediatric Infectious Diseases (ID) Fellow at the University of Pittsburgh Medical Center (UPMC) Children’s Hospital of Pittsburgh (CHP). She earned MD and PhD degrees at Vanderbilt University where she investigated the cell biology and pathogenesis of coronavirus infection (Mark Denison, MD). After clinical training in Pediatrics and Pediatric ID at CHP, she joined the lab of Carolyn Coyne, PhD, an expert in enteroviruses and tissue barriers to viral infections. Her research focused on understanding enterovirus D68 (EV-D68) pathogenesis, the hypothesized cause of acute flaccid myelitis (AFM), a polio-like syndrome of paralysis in previously healthy children. Dr. Freeman used intestinal organoids and primary bronchial epithelial cells to show that some contemporary EV-D68 isolates associated with AFM were able to use the intestinal tract for replication in addition to the respiratory tract, suggesting an alternate infection route. This work formed the basis for the spinal cord organoid model featured in this proposal. Dr. Freeman will build on >10 years of experience studying viral-host interactions while gaining new proficiencies in immunology, neuroscience, and imaging techniques which will facilitate her transition into an independently-funded pediatrician-scientist. Dr. Freeman has assembled a multidisciplinary mentorship and advisory team, chaired by primary mentor Dr. John Williams (respiratory virus immunity/pathogenesis) and co- mentor Dr. Terence Dermody (viral pathogenesis/encephalitis). Additional advisory members include Dr. Carolyn Coyne (enteroviruses/organoids), Dr. Philana Lin (immunology) and Dr. Clayton Wiley (neuroscience/encephalitis). The committee will facilitate technique acquisition, career development, and collaborations. The training plan incorporates coursework, technical expertise, skills in written and oral communication, responsible conduct of research, presentation at conferences, and a timeline for seeking additional funding. The University of Pittsburgh School of Medicine is an outstanding training environment due to the record of scientific achievement, resources, commitment to physician scientists, and expertise. AFM peaked biennially from 2012-2018 coincident with EV-D68 circulation, however, reasons why contemporary EV-D68 targets the nervous system are unknown. The central hypothesis of this proposal is that contemporary strains of EV-D68 directly infect motor neurons of the spinal cord, resulting in neuronal damage and paralysis. Using an innovative human spinal cord organoid model, the Aims will 1) define spinal cord replication of EV-D68, identify infected cell type(s), and discover contributions to pathogenesis, 2) define the host cellular response of spinal cord organoids to infection with EV-D68, and 3) evaluate glutamate alterations during infection of spinal cord organoids with EV-D68. The studies in this proposal utilize advanced tissue modeling and imaging techniques, viral replication assays, as well as host cytokine, cellular death, and RNA transcript analysis to understand how CNS infection with EV-D68 leads to AFM.