Biology subjects

Loveday, E. K.

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

7 recordsLinked to original sources

bioRxiv2025

Primary bovine embryonic fibroblasts support seasonal influenza A virus infection and demonstrate variable fitness of HPAI H5N1

The emergence of highly pathogenic avian influenza (HPAI) H5N1 (clade 2.3.4.4b, genotype B3.13) in dairy cattle presents substantial challenges to the agricultural sector and public health. Mechanistic studies of infection and transmission in cattle have proven difficult due to animal handling restrictions as well as limited availability of established cell culture models. Primary Bovine Embryonic Fibroblasts (BeEFs) were collected from a Montana cow and are investigated here as a model to study influenza A virus (IAV) infection dynamics. We compared sialylation profiles, infectious virus production, viral replication, and plaque morphology in both BeEFs and chicken DF-1 cells following infection with the bovine HPAI H5N1 and an earlier 2.3.4.4b genotype (B1.1) isolated in 2022. The data presented here show increased viral fitness of the bovine origin HPAI H5N1 strains across multiple species and bovine susceptibility to human seasonal IAV. This study highlights the ability of BeEFs to serve as a model for studying IAV infections in bovine hosts.

microbiology↗

bioRxiv2024

Starve a cold or feed a fever? Identifying cellular metabolic changes following infection and exposure to SARS-CoV-2.

Viral infections induce major shifts in cellular metabolism elicited by active viral replication and antiviral responses. For the virus, harnessing cellular metabolism and evading changes that limit replication are essential for productive viral replication. In contrast, the cellular response to infection disrupts metabolic pathways to prevent viral replication and promote an antiviral state in the host cell and neighboring bystander cells. This competition between the virus and cell results in measurable shifts in cellular metabolism that differ depending on the virus, cell type, and extracellular environment. The resulting metabolic shifts can be observed and analyzed using global metabolic profiling techniques to identify pathways that are critical for either viral replication or cellular defense. SARS-CoV-2 is a respiratory virus that can exhibit broad tissue tropism and diverse, yet inconsistent, symptomatology. While the factors that determine the presentation and severity of SARS-CoV-2 infection remain unclear, metabolic syndromes are associated with more severe manifestations of SARS-CoV-2 disease. Despite these observations a critical knowledge gap remains between cellular metabolic responses and SARS-CoV-2 infection. Using a well-established untargeted metabolomics analysis workflow, we compared SARS-CoV-2 infection of human lung carcinoma cells. We identified significant changes in metabolic pathways that correlate with either productive or non-productive viral infection. This information is critical for characterizing the factors that contribute to SARS-CoV-2 replication that could be targeted for therapeutic interventions to limit viral disease.

microbiology↗

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↗

bioRxiv2023

Single-cell Herpes Simplex Virus type-1 infection of neurons using drop-based microfluidics reveals heterogeneous replication kinetics

Single-cell analyses of viral infections often reveal heterogeneity that is not detected by traditional population-level studies. This study applies drop-based microfluidics to investigate the dynamics of HSV-1 infection of neurons at the single-cell level. We used micron-scale Matrigel beads, termed microgels, to culture individual murine Superior Cervical ganglia (SCG) neurons or epithelial cells. Microgel-cultured cells are subsequently enclosed in individual media-in-oil droplets with a dual fluorescent-reporter HSV-1, enabling real-time observation of viral gene expression and replication. Infection within drops revealed that the kinetics of initial viral gene expression and replication were dependent on the inoculating dose. Notably, increasing inoculating doses led to earlier onset of viral gene expression and more frequent productive viral replication. These observations provide crucial insights into the complexity of HSV-1 infection in neurons and emphasize the importance of studying single-cell outcomes of viral infection. The innovative techniques presented here for cell culture and infection in drops provide a foundation for future virology and neurobiology investigations.

bioengineering↗

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↗