Project Summary/Abstract Astroviruses have been recently recognized as an emerging cause of central nervous system infections in humans, with astrovirus VA1 (VA1) as the most frequently identified astrovirus genotype. We previously described the capacity of VA1 to infect primary human astrocytes and induce expression of inflammatory cytokines like CXCL10, which can directly induce neuronal apoptosis. The histology from the VA1 cases of encephalitis identified neuronal injury and apoptosis, but in cell culture, VA1 cannot replicate in primary human neurons. These findings suggest that VA1 could cause neuronal apoptosis through an indirect mechanism, including infection of astrocytes leading to CXCL10 expression and induction of apoptosis. It is also possible that neuronal cell culture does not reflect necessary conditions for VA1 replication and a better model is needed. To further study the neuropathogenicity, we did not detect significant viral replication in brain tissue from mice inoculated with VA1. In contrast, inoculation of human cerebral organoids (hCOs) with VA1 resulted in a >500-fold increase in viral RNA 72 hours post-inoculation. These results demonstrate the capacity of hCOs to support VA1 replication and now we can leverage this system to better understand cell-to-cell interactions that are important during VA1 infection. hCOs consist of multiple cell types from the central nervous system, including mature/immature neurons, astrocytes, and neural progenitor cells. Our protocol can generate hCOs with robust populations of astrocytes and neurons, enabling cross-talk between cell types. In Aim 1, we will study the cellular tropisms of VA1. We developed a novel fluorescent in situ hybridization (FISH) and immunohistochemical (IHC) assays to detect VA1 that can co-localize with host markers of different cellular lineages. Using FISH and IHC, we will define the cell lineages that support infection by VA1. Based on our previous results, we hypothesize astrocytes but not neurons support VA1 replication. Nonetheless, the hCOs will allow us to determine if other cell types, including neurons, support VA1 infection. In Aim 2, we will determine the consequences of VA1 infection. Given that VA1 induces neuronal apoptosis in vivo, we hypothesize that VA1 will also induce apoptosis of neurons in hCOs. We will identify cells that are undergoing apoptosis after infection by staining for activated caspase-3 and by TUNEL. Cells positive for apoptosis will be co-stained with cell lineage markers to identify the cell types that are undergoing apoptosis. We will also perform single-cell RNA-seq to further describe the cellular response to infection. This analysis will determine if VA1 infection induces expression of inflammatory cytokines, like CXCL10, in hCOs. In addition, RNA-seq will identify if other signaling pathways, including cell death pathways, that are activated in VA1 infected and uninfected cells. By dissecting the tropisms and consequenc...