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

使用基于液滴的微流体技术研究甲型流感病毒遗传多样性和宿主适应的单细胞异质性

基本信息

批准号：
10728192
负责人：
Emma Kate Loveday
金额：
$ 23.4万
依托单位：
MONTANA STATE UNIVERSITY - BOZEMAN
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-05 至 2025-06-30
项目状态：
未结题

项目摘要

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.

项目概要当突变使宿主适应时，可能会出现新的大流行甲型流感病毒（IAV）毒株。突变克服宿主范围限制对于病毒出现和人畜共患感染非常重要。人畜共患病的溢出效应携带禽流感亚型（例如 H5N1 和 H7N9）传染给人类的死亡率高达 60%。尽管重大进展已经确定了许多突变，使 IAV 能够适应新的宿主物种，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了解。定义病毒突变的异质性将揭示导致人畜共患病溢出的病毒遗传多样性。至关重要的是，人类 IAV 感染发生在呼吸道中的异源细胞群中，这些细胞群与与病毒传播的可能性不同。使用两种方法对这些不同细胞类型进行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。基于液滴的微流体是一种将宿主细胞和病毒在皮升内划分的方法大小的液滴，创建数百万个微环境，从而实现高通量分析。基于滴的因此，微流体为研究快速进化的 RNA 的病毒遗传多样性提供了一个理想的平台实验室里的病毒。我们的长期目标是了解导致主机的 IAV 的演变适应、毒力、传播以及最终的人畜共患传播。为了开始解决这个长期问题我们将通过以下方式评估单细胞 IAV 基因组异质性： 1.) 量化产生的遗传多样性来自个体人类原代细胞的禽类和季节性人类 IAV 感染，以及 2.) 执行通过在单细胞水平连续传代 IAV 病毒进行进化研究。这两个独立，但是互补的目标旨在理解：（目标 1）特定细胞类型如何影响病毒遗传多样性和人畜共患风险，以及（目标 2）病毒多样性在系统和群体水平上如何演变瓶颈被改变。拟议的研究将广泛影响单细胞病毒学领域描述病毒多样性在病毒传播、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方法。