To address a critical need for improved human tissue models to study infectious diseases, we propose to form the MIT Center for Human Tissues and Infectious Diseases (MIT.HTMID). The MIT.HTMID Center will study viral infections of the human brain and central nervous system by using biologically relevant two dimensional (2D) human neural cells and three dimensional (3D) human cerebral organoids. The cells are homogeneous preparations of human neuronal progenitors, neurons, oligodendrocytes, astrocytes, and microglia, derived from embryonic stem (ES) cells and induced pluripotent stem cells (iPS cells), and produced in the Human Cell and Tissue Core. Prior work has demonstrated that these cells and the organoids formed are nearphysiological in their biological functions, thereby representing an experimental system that is superior to rodent models. The goal of Project 1, Aim 1A is to define the viral infection phenotypes for five cell types. Two types of viruses, produced in the MIT.HTMID Virology Core, will be used. First, we will study flaviviruses, including Dengue Fever Virus, West Nile Virus, and Zika Virus. Indeed, an important motivation for studying human brain tissue and viruses is to understand the pathogenesis of Zika virus infections, which are causing microcephaly and Guillain Barre syndrome worldwide. Second, we will use pseudotyped vesicular stomatitis viruses (VSV) to study entry of several encephalitic viruses, including Eastern Equine Encephalitis Virus (EEEV), Western Equine Encephalitis Virus (WEEV) and Venezuelan Equine Encephalitis Virus (VEEV). By replacing the VSV viral envelope with that of EEEV, WEEV, or VEEV, we are able to study virus entry in the 2D cells and 3D organoids. These experiments are highly significant because EEEV, VEEV, and WEEV are select agents. Using pseudotyped select viruses and multiple relevant cell types will allow us to study virus tropism, pathology, and potential therapeutic targets. Project 1 Aim 1A will examine virus infection phenotypes; that is, we will analyze virus replication, cell viability, spontaneous electrical activity, transcriptome and secretome changes, and potential to differentiate to other cell types. This approach is significant because it will demonstrate which cell types are most susceptible to virus infections, also revealing similarities and differences among viruses that cause very different clinical diseases. In Project 1, Aim 1B, we propose to study virus infection in the context of the three dimensional human cerebral organoid tissue model. Our group may be unique in having the technical capacity to generate organoids that include microglia, which are the immune cells of the brain. Our goal will be to determine if activated microglia have protective roles by secreting factors that establish an antiviral environment. Alternatively, we will also test a hypothesis that microglia, upon infection with Zika virus, are ab...