Single cell heterogeneity of influenza A virus genetic diversity and host adaptation using drop-based microfluidics

项目摘要 当突变使宿主适应时，可能出现新的大流行性甲型流感病毒（IAV）毒株。的突变 克服宿主范围限制在病毒出现和人畜共患病感染中是重要的。人畜共患病外溢 感染禽流感病毒亚型如H5N1和H7N9的人的死亡率高达60%。而 重大进展已经确定了许多突变，使IAV适应新的宿主物种，我们有一个 对病毒复制过程中产生的病毒突变深度的不完全理解。定义 病毒突变的异质性将揭示使人畜共患病溢出的病毒遗传多样性。 重要的是，人类IAV感染发生在呼吸道中的异源细胞群中， 与病毒传播的可能性不同。这些不同细胞类型的单细胞分析， 人类和禽类IAV毒株将使我们能够探索病毒毒株和细胞类型如何影响病毒多样性。 基于液滴的微流体技术是一种将宿主细胞和病毒在皮升内区室化的方法， 大小的液滴，创造数百万个微环境，允许高通量分析。基于丢弃 因此，微流体技术为从快速进化的RNA中研究病毒遗传多样性提供了理想的平台 实验室里的病毒我们的长期目标是了解IAV的演变， 适应、毒力、传播和最终的人畜共患病传播。开始解决这个长期的 目的我们将通过1.）量化遗传多样性 来自禽类和季节性人IAV感染的个体人原代细胞和2.）执行 通过在单细胞水平上连续传代IAV病毒进行进化研究。这两个独立的，但 互补的目的是为了理解：（目的1）特定的细胞类型如何影响病毒遗传 多样性和人畜共患病风险，以及（目标2）当系统和人口水平 瓶颈被改变。拟议的研究将广泛影响单细胞病毒学领域， 表征病毒多样性在病毒繁殖、传播和进化中所起的作用。这些研究 将产生对病毒宿主细胞动力学的基本机制见解，这可能有助于开发 可以针对快速进化的IAV和其他RNA病毒的有效疫苗和治疗剂。

Project Summary New pandemic influenza A virus (IAV) strains can arise when mutations enable host adaptation. Mutations that overcome host range restrictions are important in viral emergence and zoonotic infections. Zoonotic spillover into humans with avian IAV subtypes, such as H5N1 and H7N9, have mortality rates as high as 60%. While significant progress has identified many mutations that allow IAV to adapt to new host species, we have an incomplete understanding of the depth of viral mutations generated during viral replication. Defining the heterogeneity of viral mutations will shed light on the viral genetic diversity that enables zoonotic spillover. Critically, IAV infection in humans occurs in heterologous cell populations in the respiratory tract that correlate differently with the likelihood of virus transmission. Single cell analysis of these different cell types with both human and avian IAV strains will allow us to explore how virus strain and cell type influences viral diversity. Drop-based microfluidics is a method in which the host cell and virus are compartmentalized within picoliter- sized drops, creating millions of micro-environments, allowing for high-throughput analysis. Drop-based microfluidics therefore provides an ideal platform for the study of viral genetic diversity from fast evolving RNA viruses in the laboratory. Our long-term objective is to understand the evolution of IAV that leads to host adaptation, virulence, transmission, and ultimately zoonotic spread. To begin to address this long-term objective we will evaluate single cell IAV genomic heterogeneity by 1.) quantifying the genetic diversity arising from avian and seasonal human IAV infections of individual human primary cells and 2.) performing evolutionary studies by serial passaging IAV viruses at a single cell level. These two independent, but complementary aims are directed at understanding: (Aim 1) how specific cell types impact viral genetic diversity and zoonotic risk, and (Aim 2) how viral diversity evolves when system and population level bottlenecks are altered. The proposed research will broadly impact the field of single cell virology by characterizing the role that viral diversity plays in virus propagation, transmission, and evolution. These studies will yield fundamental mechanistic insights into virus-host cell dynamics, which may aid in developing efficacious vaccines and therapeutics that can target rapidly evolving IAV and other RNA viruses.