Identifying heterogenous neuronal responses to HSV-1 infection with drop-based microfluidics

项目摘要 单纯疱疹病毒1型(HSV-1)疾病的结局在很大程度上取决于神经元 复制表型倾向于多产或静默感染。然而，主宰一切的变量 这些异源感染表型尚不完全清楚。找出影响公司业绩的因素 神经元复制的结果对于开发治疗严重疾病的新方法和缓解 单纯疱疹病毒1型的全球负担。 异源感染表型研究的一个主要局限是它们很快就在传统的 培养模式，通常被来自生产性感染细胞的二次病毒传播所淹没。我们的 这个问题的解决方案是结合基于液滴的微流控技术，该技术允许检测不同的 病毒复制结果与单细胞分辨率。我们已经开发出一种方法来培养和感染初级 使用微米级水凝胶微珠(微凝胶)的神经元，允许在 孤立的环境。我们的建议旨在调查产生异源基因的条件和因素。 神经性HSV-1感染的结局，最终目的是更好地了解HSV-1的决定因素 疾病。 这项建议将结合滴状神经元感染的发展和特征来解决 关于HSV-1感染和疾病的关键问题。第一个目标将侧重于对以下方面的结果进行分类 病毒在不同类型神经元中的复制。第二个将使用感染病毒的单细胞转录图谱 神经元识别促进HSV-1有效或沉默感染的转录本。具体目标1侧重于 量化神经元中HSV-1感染的异源表型。我们将利用基于液滴的微流体 单纯疱疹病毒1型感染神经元的分离与分析。病毒感染的结局将通过HSV-1- 表达的荧光蛋白和病毒基因组的滴入聚合酶链式反应检测。我们预计病毒传播的程度 复制反映了不同细胞环境支持HSV-1感染的能力。特定目标 2侧重于神经元中HSV-1复制的有效或沉默的单细胞转录图谱。我们会 实施滴内单细胞RNA测序，以分析受感染神经元的病毒和细胞转录本。 单细胞转录本的聚类分析和分类将基于病毒RNA的检测。 单个转录本的存在将反映每个细胞中发生的病毒复制的程度。跟随 分类，相关分析将识别细胞转录随每一项的增加或减少 感染状态。总之，这些实验将确定和量化HSV-1感染的结果范围 神经细胞的。随后，我们将通过以下方式发现促进或抑制病毒复制的细胞基因 确定某些异源表型的决定因素。探索单细胞的神经元感染 Level将为HSV-1复制和疾病提供前所未有的洞察力。

Project Summary The outcome of Herpes Simplex Virus type 1 (HSV-1) disease depends heavily on whether neuronal replication phenotypes trend towards a productive or silent infection. However, the variables which govern these heterologous infection phenotypes are not fully understood. Identification of factors which influence the outcome of neuronal replication is essential to develop new treatments for severe disease and alleviate the global burden of HSV-1. A major limitation to the study of heterologous infection phenotypes is that they are quickly lost in traditional culture models, typically being overwhelmed by secondary viral spread from productively infected cells. Our solution to this problem is to incorporate drop-based microfluidic technology that allows detection of diverse viral replication outcomes with single-cell resolution. We have developed a method to grow and infect primary neurons using a micron-scale, hydrogel bead (microgel), that permits single-cell experimentation within an isolated environment. Our proposal aims to investigate the conditions and factors that give rise to heterologous outcomes of neuronal HSV-1 infection with the ultimate goal of better understanding the determinants of HSV-1 disease. This proposal will combine the development and characterization of in-drop neuronal infection to address critical questions about HSV-1 infection and disease. The first aim will focus on classifying the outcomes of viral replication in different neuron types. The second will use single-cell transcriptional profiling of infected neurons to identify transcripts that promote productive or silent HSV-1 infection. Specific Aim 1 focuses on quantifying heterologous phenotypes of HSV-1 infection in neurons. We will utilize drop-based microfluidic separation and analysis of HSV-1 infected neurons. Outcomes of viral infection will be measured by HSV-1- expressed fluorescent proteins and in-drop PCR detection of viral genomes. We expect that the extent of viral replication reflects the capacity for different cellular environments to support HSV-1 infection. Specific Aim 2 focuses on single cell transcriptional profiling of productive or silent HSV-1 replication in neurons. We will implement in-drop single-cell RNA-sequencing to profile viral and cellular transcripts from infected neurons. Clustering analysis and classification of single cell transcriptomes will be based on the detection of viral RNAs. The presence of individual transcripts will reflect the extent of viral replication occurring in each cell. Following classification, correlation analysis will identify cellular transcripts that are increased or decreased with each infection state. Together, these experiments will identify and quantify the range of outcomes for HSV-1 infection of neurons. Subsequently, we will discover cellular genes that promote or inhibit productive viral replication by identifying determinants of certain heterologous phenotypes. Exploring neuronal infections at the single-cell level will provide unprecedented insight into HSV-1 replication and disease.