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

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.