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是否 复制表型倾向于生产性或沉默感染。然而，决定性的变量 这些异源感染表型还不完全清楚。确定影响 神经元复制的结果对于开发严重疾病的新治疗方法和减轻神经元的损伤是至关重要的。 HSV-1的全球负担。 异源感染表型研究的一个主要限制是它们在传统的免疫学中很快丢失。 培养模型，通常被来自生产性感染细胞的二次病毒传播所淹没。我们 该问题解决方案是结合基于液滴的微流体技术， 单细胞分辨率的病毒复制结果。我们已经开发出一种方法， 神经元使用微米级，水凝胶珠（微凝胶），允许单细胞实验内， 孤立的环境。我们的建议旨在研究引起异源的条件和因素， 神经元HSV-1感染的结局，最终目标是更好地了解HSV-1的决定因素 疾病 该建议将联合收割机的发展和表征的滴神经元感染，以解决 关于HSV-1感染和疾病的关键问题。第一个目标将侧重于对 病毒在不同类型神经元中复制。第二个将使用单细胞转录谱的感染 神经元识别促进生产性或沉默HSV-1感染的转录本。具体目标1侧重于 定量神经元中HSV-1感染的异源表型。我们将利用基于液滴的微流体 HSV-1感染神经元的分离和分析。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内PCR检测。我们预计， 复制反映了不同细胞环境支持HSV-1感染的能力。具体目标 2集中于神经元中生产性或沉默HSV-1复制的单细胞转录谱。我们将 实施液滴内单细胞RNA测序以分析来自受感染神经元的病毒和细胞转录物。 单细胞转录组的聚类分析和分类将基于病毒RNA的检测。 单个转录物的存在将反映每个细胞中发生的病毒复制的程度。以下 分类，相关性分析将确定细胞转录本的增加或减少与每个 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 的神经元。随后，我们将发现细胞基因，促进或抑制生产性病毒复制， 鉴定某些异源表型的决定因素。探索单细胞的神经元感染 水平将提供前所未有的了解HSV-1复制和疾病。

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.