Biology subjects

Loveday, E. K.

Publications and source records attributed to Loveday, E. K..

3 recordsLinked to original sources

bioRxiv2021

Single cell infection with influenza A virus using drop-based microfluidics

Influenza A virus (IAV) is an RNA virus with high genetic diversity which necessitates the development of new vaccines targeting emerging mutations each year. As IAV exists in genetically heterogeneous populations, current studies focus on understanding population dynamics at the single cell level. These studies include novel methodology that can be used for probing populations at the single cell level, such as single cell sequencing and microfluidics. Here, we introduce a drop-based microfluidics method to study IAV infection at a single cell level by isolating infected host cells in microscale drops. Single human alveolar basal epithelial (A549), Madin-Darby Canine Kidney cells (MDCK) and MDCK + human siat7e gene (Siat7e) cells infected with the pandemic A/California/07/2009 (H1N1) strain were encapsulated within 50 m radii drops and incubated at 37{degrees}C. We demonstrate that drops remain stable over 24 hours, that 75% of cells remain viable, and that IAV virus can propagate within the drops. Drop-based microfluidics therefore enables single cell analysis of viral populations produced from individually infected cells.

microbiology↗

bioRxiv2020

Effects of inactivation method on SARS-CoV-2 virion proteins and structure

The risk posed by Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) dictates that live-virus research is conducted in a biosafety level 3 (BSL3) facility. Working with SARS-CoV-2 at lower biosafety levels can expedite research yet requires the virus to be fully inactivated. In this study, we validated and compared two protocols for inactivating SARS-CoV-2: heat treatment and ultraviolet irradiation. The two methods were optimized to render the virus completely incapable of infection while limiting destructive effects of inactivation. We observed that 15 minutes of incubation at 65{degrees}C completely inactivates high titer viral stocks. Complete inactivation was also achieved with minimal amounts of UV power (70,000 J/cm2), which is 100-fold less power than comparable studies. Once validated, the two methods were then compared for viral RNA quantification, virion purification, and antibody recognition. We observed that UV irradiation resulted in a 2-log reduction of detectable genomes compared to heat inactivation. Protein yield following virion enrichment was equivalent for all inactivation conditions, but the resulting viral proteins and virions were negatively impacted by inactivation method and time. We outline the strengths and weaknesses of each method so that investigators might choose the one which best meets their research goals.

microbiology↗

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↗