权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Characterization of a cerebral organoid model of astrovirus VA1 infection

星状病毒 VA1 感染脑类器官模型的表征

基本信息

批准号：
10593810
负责人：
Andrew Bok Seng Janowski
金额：
$ 23.33万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10593810
关键词：
Address Affect Apoptosis Astrocytes Astrovirus Award Biological Assay Biological Models Brain CASP3 gene CXCL10 gene Cattle Cell Communication Cell Culture System Cell Culture Techniques Cell Death Cell Lineage Cells Cellular Tropism Central Nervous System Central Nervous System Diseases Central Nervous System Infections Cerebrum Clinical Data Detection Disease Dissection Encephalitis Etiology Exposure to Fluorescent in Situ Hybridization Funding Future Genotype Glial Fibrillary Acidic Protein Goals Histology Hour Human Immunocompromised Host Immunohistochemistry Immunosuppression In Situ Hybridization In Situ Nick-End Labeling Individual Induction of Apoptosis Infection Inflammatory Intervention Leukocytes Life Cycle Stages Lymphopenia Mediating Modeling Morphology Mus Neurologic Neuronal Injury Neurons Organoids Pathogenesis Pathology Pathway interactions Population Prevalence Protocols documentation Publishing RNA Research Research Personnel Seroprevalences Signal Pathway Stains Structure Study models Supporting Cell Survivors System TNF gene Testing Therapeutic Tropism Viral Virus Virus Diseases Virus Replication astrocyte progenitor brain tissue burden of illness cell type central nervous system injury chemokine cytokine emerging pathogen human disease in vivo insight mortality nerve stem cell neuron apoptosis neuropathology novel prevent receptor response single-cell RNA sequencing therapeutic development tool transcriptome transcriptome sequencing viral RNA

项目摘要

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 consequences of VA1 infection in hCOs, this is a novel extension of a previously funded K08 award and will establish an essential tool to understand astrovirus induced neuropathology. This application will also enable further research independence of the primary investigator, achieving the goals set forth under PAR-20-291.

项目总结/文摘