Bikos, D. A. | bioRxiv Science

bioRxiv2024

Influenza A Viral Burst Size from Thousands of Infected Single Cells Using Droplet Quantitative PCR (dqPCR)

An important aspect of how viruses spread and infect is the viral burst size, or the number of new viruses produced by each infected cell. Surprisingly, this value remains poorly characterized for influenza A virus (IAV), commonly known as the flu. In this study, we screened tens of thousands of cells using a microfluidic method called droplet quantitative PCR (dqPCR). The high-throughput capability of dqPCR enabled the measurement of a large population of infected cells producing progeny virus. By measuring the fully assembled and successfully released viruses from these infected cells, we discover that the viral burst sizes for both the seasonal H3N2 and the 2009 pandemic H1N1 strains vary significantly, with H3N2 ranging from 101 to 104 viruses per cell, and H1N1 ranging from 101 to 103 viruses per cell. Some infected cells produce average numbers of new viruses, while others generate extensive number of viruses. In fact, we find that only 10% of the single-cell infections are responsible for creating a significant portion of all the viruses. This small fraction produced approximately 60% of new viruses for H3N2 and 40% for H1N1. On average, each infected cell of the H3N2 flu strain produced 709 new viruses, whereas for H1N1, each infected cell produced 358 viruses. This novel method reveals insights into the flu virus and can lead to improved strategies for managing and preventing the spread of viruses. Author summaryViruses infect and exploit host cells to reproduce and spread. The viral burst size, or the number of viral particles released from an infected cell, plays a critical role in understanding infection dynamics and overall viral fitness. However, accurately determining burst size for many single cells using conventional laboratory methods can be challenging. Here, we introduce dqPCR, a droplet microfluidic method for the rapid measurement of influenza virus numbers produced by thousands of individual cells. Our findings revealed that only a small proportion of infected cells are responsible for producing a significant portion of the total viral population. By utilizing this method in future studies, we can gain a deeper understanding of the role of diversity in rapidly evolving viruses.

bioengineering↗

bioRxiv2020

Screening of additives for droplet qRT-PCR thermocycling enables single influenza A virus genome quantification

The miniaturization of real time quantitative polymerase chain reaction (qPCR) using drop-based microfluidics, or droplet qPCR, allows for quantification of single nucleic acids. The nucleic acids are compartmentalized into aqueous microdroplets, picoliters in volume, separated by an immiscible oil, and stabilized by a surfactant. In droplet qPCR, accurate data can only be obtained if the drops remain stable to coalescence upon thermocycling and drop contents do not diffuse to neighboring drops. In this work, we present a droplet qRT-PCR assay for quantifying influenza A virus (IAV) following systematic testing of different PCR additives, resulting in the optimal combination of Tween-20 / BSA / betaine to maintain drop stability and limit dye diffusion. We use a standard qPCR machine to generate real time amplification curves of hundreds of thousands of drops and correlate this data with constructed amplification curves obtained from hundreds of drops sampled at various cycle numbers and imaged using epifluorescence microscopy. To demonstrate the utility of our method, we tested a range of in vitro transcribed M gene and IAV viral supernatant from infected cells. We directly amplified IAV genomes from infected supernatant without an RNA extraction step. Our droplet qPCR assay enables detection of IAV down to 0.274 cpd, or a single viral genome per drop, establishing the high sensitivity and precision of our method.

bioengineering↗