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

使用基于液滴的微流体识别对 HSV-1 感染的异质神经元反应

• 批准号: 10668003
• 负责人: Matthew P. Taylor
• 金额: $ 21.75万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-20 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10668003
• 关键词: Address; Afferent Neurons; Cells; Classification; Cluster Analysis; Cytopathology; Detection; Development; Devices; Disease; Dose; Drops; Encapsulated; Encephalopathies; Environment; Event; Gel; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Genes; Genetic Transcription; Goals; Growth; Herpesvirus 1; Hydrogels; Image; In Vitro; Individual; Infection; Kinetics; Lesion; Lytic; Measures; Mediating; Methods; Microfluidics; Modeling; Neurons; Outcome; Peripheral Nervous System; Phenotype; Process; Productivity; Proteins; Recurrence; Resolution; Structure; System; Testing; Time; Tissues; Transcript; Viral; Viral Genome; Virus Diseases; Virus Replication; cell type; differential expression; experimental study; human pathogen; in vivo; insight; latent infection; microfluidic technology; novel; orofacial; permissiveness; response; single cell analysis; single-cell RNA sequencing; transcriptome; trend; viral RNA; viral detection

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.

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存在 复制表型趋向于生产或无声感染。但是，管理的变量 这些异源感染表型尚不完全了解。识别影响的因素 神经元复制的结果对于开发新的严重疾病治疗并减轻 HSV-1的全球负担。 对异源感染表型的研究的主要限制是，它们在传统中很快消失了 培养模型，通常被从有效感染的细胞中造成的次级病毒扩散所淹没。我们的 解决此问题的解决方案是合并基于Drop的微流体技术，允许检测到不同 单细胞分辨率的病毒复制结果。我们已经开发了一种生长和感染主要的方法 使用微米尺度的水凝胶珠（微凝胶）的神经元，该神经元允许单细胞实验 孤立的环境。我们的建议旨在调查引起异源的条件和因素 神经元HSV-1感染的结果，其最终目标是更好地了解HSV-1的决定因素 疾病。 该建议将结合滴入神经元感染的发展和表征，以解决 有关HSV-1感染和疾病的关键问题。第一个目的将重点放在分类 不同神经元类型的病毒复制。第二个将使用感染的单细胞转录分析 神经元识别促进生产或无声HSV-1感染的成绩单。特定目标1专注于 量化神经元中HSV-1感染的异源表型。我们将利用基于下降的微流体 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入PCR检测。我们期望病毒的程度 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侧重于神经元中生产性或无声HSV-1复制的单细胞转录分析。我们将 实施来自感染神经元的病毒和细胞转录本的滴入单细胞RNA测序。 单细胞转录组的聚类分析和分类将基于病毒RNA的检测。 单个转录本的存在将反映每个细胞中发生病毒复制的程度。下列的 分类，相关分析将识别每种分类的细胞转录本。 感染状态。这些实验将共同识别和量化HSV-1感染的结果范围 神经元。随后，我们将发现促进或抑制通过 识别某些异源表型的决定因素。探索单细胞的神经元感染 水平将为HSV-1复制和疾病提供前所未有的见解。