(100f) High-Throughput Assaying of Individual Host-Pathogen Dynamics in Influenza A Virus Infection Using Drop-Based Microfluidics

Influenza A virus (IAV) is notorious for causing seasonal epidemics and sporadic worldwide pandemics that have resulted in over 100 million deaths during the past century. Understanding how IAV interacts with its host cells is typically performed using tissue culture systems or with animal models. These systems provide bulk measurements of virus production and/or cellular responses, such as interferon activation and production. However, individual cells exist as a heterogeneous population, resulting in cell-to-cell differences in virus production and antiviral responses. Conventional methods for measuring the antiviral response in individual cells during IAV infection are labor intensive, incur high reagent costs, and require extensive serial dilutions to isolate singularly infected cells. Thus, there is a need for high-throughput analysis of single cells to elucidate individual host-cell dynamics. Here, we have developed a drop-based microfluidic method to characterize IAV infection and quantify the cellular response to viral infection at the single cell level. This method utilizes drop-based microfluidics to encapsulate infected cells within picoliter sized drops followed by single cell, drop-based qRT-PCR analysis. We successfully formed >10⁶ drops that contained individual human alveolar epithelial (A549) cells infected with the pandemic 2009 H1N1 IAV. Our results show that IAV replicates within the drops and that drops remain stable overnight with high cell viability. We quantified viral RNA and cellular mRNA from these infected drops at kilohertz rates using our novel, multiplexed drop-based qRT-PCR assay. This approach allowed us to measure interferon β production in response to viral RNA output from >10⁵ individually infected cells in a single experiment. Our method is the first to measure individual host-pathogen dynamics in single cells following infection with IAV using a high-throughput, multiplexed drop-based assay.