(88g) High-Throughput Quantification of Influenza A Virus RNA Using Novel Drop-Based qRT-PCR Analysis

Influenza A virus (IAV) causes seasonal epidemics and sporadic worldwide pandemics that have resulted in over 100 million deaths during the past century. IAV populations are dynamic with high mutation rates and short replication times. A better understanding of IAV diversity at the single cell level would provide greater insight into how heterogeneity of infection affects population-level dynamics. Investigating IAV and host cell dynamics is typically performed using bulk culture and may not fully capture cell-to-cell differences in virus production. Thus, there is a need for high-throughput quantification of the IAV output, or burst size, from single cells to elucidate individual host-cell dynamics at the population scale. Here, we have developed a novel drop-based quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) method to measure the amount of IAV RNA contained in individual drops. Concentrations of RNA are encapsulated in tens of thousands of picoliter-sized drops. These drops are processed on a microfluidic device which splits a small portion of the drop and merges it with qRT-PCR solution. The merged drops are thermocycled using a standard qPCR machine. Drops are randomly sampled at different cycles to measure drop fluorescence. Using a novel qPCR analysis method, we quantify the amount of RNA contained in the drops. Our method is the first to measure IAV RNA using a high-throughput drop-based assay, which will allow for future work in investigating the heterogeneity of IAV infection.