Defining mechanisms of Zika-virus associated microcephaly: cell population dynamics and gene expression in infected human cerebral organoids and neural progenitor cells

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PROJECT SUMMARY/ABSTRACT Zika virus swept across the Americas in 2015-16, and it was during this epidemic that the teratogenic consequences of congenital Zika infection were first described. Despite extensive research, it remains unknown how Zika infection causes microcephaly, or whether this pathology is unique to recent strains, Two new tools will facilitate discovery in this area. First, we and others have shown that the human stem cell derived cerebral organoid is a tractable neurodevelopmental model in which to study Zika infection. Second, we have demonstrated that single cell sequencing techniques that enrich for poly-adenylated mRNAs can identify flavivirus-infected cells – an unexpected result given flavivirus genomes do not have poly(A) tails. The goal of this study is to determine the mechanisms by which Zika virus disrupts fetal brain development, building on these technical advances. We will employ single cell RNA-sequencing to identify and compare cellular populations in organoids over time, in the presence and absence of Zika virus. This will allow us to distinguish pathogenic disruptions in a) stem cell abundance, b) cell division, and c) differentiation. We will use single cell RNA-sequencing and ribosome profiling to define gene expression responses to Zika infection in neural progenitor cells, thought to be the target of Zika virus in the fetal brain. By comparing viruses of varying pathogenicity in these studies, we will identify differences in host gene expression and viral replication associated with disease severity. The proposed study will address a major unresolved problem in the field of Zika virus pathogenesis, contribute broadly to our understanding of cerebral development, and mature cutting edge technologies for the investigation of questions at the interface of infection and development. The project will be developed under the mentorship of Dr. Lee Gehrke, a leader and expert in the field of RNA virology with a background in developmental biology. Additional scientific and career guidance will be provided by a scientific advisory committee composed of experts in virology, single-cell sequencing, stem-cell derived tissue models, and pediatric infectious disease pathogenesis. The training program will include coursework in computational biology, training in molecular and tissue culture techniques, workshops in leadership, and didactic learning from local seminars as well as national and international conferences. Research and training will occur at the MIT Institute for Medical Engineering and Science, and at Boston Children’s Hospital at the Harvard Medical School. Together, MIT and Harvard Medical School afford extensive resources and expertise in all aspects of the proposed research. Boston Children’s Hospital is a supportive environment committed to providing 85% protected time for this research, and offers workshops in career development and leadership to prepare early-stage investigators for independence. The project will build on the candidate’s background in virology and high-throughput sequencing, allow her to mature as a physician-scientist, and position her to achieve her goal of an independent research career in pediatric infectious disease pathogenesis.