Droplet-Assisted RNA targeting by single-cell sequencing to dissect the single-cell heterogeneity of RNA virus infection

快速进化的RNA病毒，如轮状病毒、流感病毒、人类免疫缺陷病毒和寨卡病毒，是全球主要的死亡原因，对全球疾病控制构成重大挑战。尽管RNA病毒的基因组很小，通常只由几千个核苷酸和少数几个基因组成，但通过现代基因测序技术研究RNA病毒的感染生物学仍然非常困难。RNA病毒进化得很快。速度如此之快，以至于即使在单个感染细胞的水平上，RNA病毒也不是作为一个单一的病毒基因型出现，而是作为一群密切相关的基因型出现。这种遗传多样性是RNA病毒逃避细胞免疫、进化抗药性和导致紧急疾病能力的关键决定因素。受感染细胞的自然免疫反应又是高度异质性的，并取决于细胞类型和状态。RNA病毒感染的极端异质性很难用目前的分子技术进行调查，这些技术在很大程度上仅限于分析受感染细胞的群体。为了克服这一限制，我们最近创造了一种单细胞RNA测序技术，它结合了多路扩增子测序和单细胞转录图谱：液滴辅助单细胞测序靶向RNA(DART-SEQ)。DART-SEQ实现了基于液滴-微流体的单细胞与条形码引物珠的共封装和超深DNA测序的组合。使用DART-SEQ可以对单个感染细胞内病毒基因组序列的多样性进行分类，同时记录细胞对病毒感染的反应。DART-SEQ的每个细胞成本不到一美元，一次DART-SEQ测试可以在生物样本中产生数千个细胞的测量结果。在一项原则验证研究中，我们使用DART-SEQ分析了感染哺乳动物正病毒(REOV)3型Dering株(T3D)的单个细胞中病毒与宿主的相互作用和病毒基因组动力学。在这里，我们建议扩展这些最初的技术开发实验，以询问REOV和其他节段RNA病毒的感染生物学，包括轮状病毒，一种常见的胃肠炎原因，以及甲型流感病毒。我们还将进一步研究DART-SEQ作为一种工具的实用性，以高通量筛选病毒基因型和单细胞感染性之间的关系，以及宿主细胞类型或状态与感染传播率之间的关系。这一创新项目引入了一种高通量单细胞分析技术，具有研究RNA病毒感染生物学的转化潜力。这些研究具有很高的翻译性：DART-SEQ可以带来新的诊断方法，并可以作为使用病毒载体的基因治疗的读出工具。

Fast evolving RNA viruses, such as rotavirus, influenza virus, human immunodeficiency virus, and zika virus, are a leading cause of death worldwide and represent a major challenge for global disease control. Despite their miniscule genomes, often comprised of only a few thousand nucleotides and a handful of genes, it remains exceedingly difficult to study the infection biology of RNA viruses via modern gene sequencing technologies. RNA viruses evolve quickly. So quickly that even at the level of the individual infected cell, RNA viruses do not occur as a single viral genotype but rather as a swarm of closely related genotypes. This genetic diversity is a key determinant of the capacity of RNA viruses to escape cellular immunity, evolve drug resistance and cause emergent disease. The natural immune response of the infected cell in turn is highly heterogeneous, and dependent on cell type and state. The extreme heterogeneity of RNA virus infections is difficult to survey with current molecular technologies which are largely limited to analyzing populations of infected cells. To overcome this limitation, we have recently created a single cell RNA sequencing technology that combines multiplexed amplicon sequencing with single cell transcriptional profiling: Droplet Assisted RNA Targeting by single-cell Sequencing (DART-seq). DART-seq implements a combination of droplet-microfluidics based co- encapsulation of single cells with barcoded primer beads and ultra-deep DNA sequencing. With DART-seq it is possible to catalog the diversity of viral genome sequences within single infected cells, and at the same time record the cellular response to viral infection. The cost per-cell of DART-seq is less than one dollar, and a single DART-seq assay can yield measurements across thousands of cells in a biological sample. In a proof-of-principle study, we have used DART-seq to profile viral-host interactions and viral genome dynamics in single cells infected with mammalian orthoreovirus (REOV) strain Type 3 Dearing (T3D). Here we propose to expand upon these initial technology development experiments to interrogate the infection biology of REOVs and other segmented RNA viruses including rotavirus, a common cause of gastroenteritis, and influenza A virus. We will furthermore investigate the utility of DART-seq as a tool to perform high-throughput screens of the relationship between viral genotypes and single-cell infectivity and the relationship between host cell type or state and infection permissivity. This innovative project introduces a high-throughput single-cell analysis technology with transformative potential for investigations of the infection biology of RNA viruses. These studies are highly translational: DART-seq can lead to novel diagnostic approaches and can find application as a read-out tool for gene therapies that employ viral vectors.