Characterization of a cerebral organoid model of astrovirus VA1 infection

项目总结/摘要 星状病毒最近被认为是中枢神经系统感染的一个新兴原因 在人类中，星状病毒VA 1（VA 1）是最常见的星状病毒基因型。我们之前 描述了VA 1感染原代人星形胶质细胞并诱导炎性细胞因子表达的能力。 细胞因子如CXCL 10可直接诱导神经元凋亡。VA 1例的组织学检查显示， 脑炎鉴定了神经元损伤和凋亡，但在细胞培养中，VA 1不能在原代人类中复制， 神经元这些结果表明，VA 1可以通过间接机制引起神经元凋亡， 包括感染星形胶质细胞导致CXCL 10表达和诱导细胞凋亡。也有可能 神经元细胞培养不能反映VA 1复制的必要条件，需要更好的模型。 为了进一步研究神经致病性，我们没有在脑组织中检测到明显的病毒复制， 接种VA 1的小鼠。相比之下，用VA 1接种人脑类器官（hCO）， 接种后72小时病毒RNA增加>500倍。这些结果证明了hCO能够 支持VA 1复制，现在我们可以利用这个系统来更好地了解细胞与细胞之间的相互作用， 在VA 1感染期间是重要的。hCO由来自中枢神经系统的多种细胞类型组成，包括 成熟/未成熟神经元、星形胶质细胞和神经祖细胞。我们的协议可以生成具有鲁棒性的hCO 星形胶质细胞和神经元的群体，使细胞类型之间的串扰。在目标1中，我们将研究细胞 VA 1的取向。我们建立了一种新的荧光原位杂交（FISH）和免疫组化（IHC） 用于检测可与不同细胞谱系的宿主标志物共定位的VA 1的测定。使用FISH和IHC， 我们将确定支持VA 1感染的细胞谱系。基于我们之前的研究结果，我们假设 星形胶质细胞而不是神经元支持VA 1复制。尽管如此，hCO将使我们能够确定其他 细胞类型，包括神经元，支持VA 1感染。在目标2中，我们将确定VA 1的结果 感染考虑到VA 1在体内诱导神经元凋亡，我们假设VA 1也会诱导凋亡 hCOs中的神经元。我们将通过对感染后的活化细胞进行染色， caspase-3和TUNEL法。细胞凋亡阳性的细胞将与细胞谱系标志物共染色以鉴定细胞凋亡。 正在经历凋亡的细胞类型。我们还将进行单细胞RNA-seq，以进一步描述细胞内的 对感染的反应。该分析将确定VA 1感染是否诱导炎性细胞因子的表达， 就像CXCL 10一样。此外，RNA-seq将确定其他信号通路，包括细胞死亡， 在VA 1感染和未感染的细胞中激活的途径。通过剖析 hCO中的VA 1感染，这是先前资助的K 08奖项的新扩展，并将建立一个 了解星状病毒引起的神经病理学的基本工具。这一应用也将使进一步的研究 主要研究者的独立性，实现PAR-20-291规定的目标。

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.