Investigation of enterovirus D68 pathogenesis in the human spinal cord

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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.