Understanding virus jumping and cross-species infection

When the COVID-19 pandemic struck, scientists worldwide mobilized their efforts to understand the SARS-CoV-2 virus and prevent its spread. Sarah Roelle, a research assistant at Case Western Reserve University School of Medicine, investigated how the virus infects humans and identifying potential cross-species transmission. Leveraging a cell engineering platform developed by her PI Dr. Kenneth Matreyek, Roelle and her team studied the interactions between the virus’s spike protein and human cells’ receptors.

Roelle and her team employed a cell engineering platform to genetically modify human embryonic kidney cells (HEK293T) and create stable cell lines. By expressing various human or bat ACE2 proteins in these cells, they tested their susceptibility to infection by coronaviruses isolated from different bat species. This approach enabled them to investigate the potential for cross-species transmission between bats and humans.

The team’s experimental workflow involved several key steps. They used Gibson Assembly to create plasmids containing ACE2 genes from humans and various bat species, as well as spike proteins from different coronaviral clades. The Thermo Scientific™ MaxQ™ Benchtop Orbital Shaker facilitated bacterial culture for plasmid production, while plasmid sequencing was confirmed using an Applied Biosystems™ 3730 DNA Analyzer. Recombining these plasmids into transgenic cell lines with selection markers ensured high expression rates. Pseudoviruses, which express GFP upon infecting a host cell, were engineered and produced for studying spike protein infectivity. Flow cytometry was then employed to quantify infectivity rates.

To design efficient screens, the team incorporated internal controls into their experiments. They grew two types of transgenic cells within one well: one set expressed full-length ACE2, while the other set expressed a modified version lacking its ectodomain, making it less susceptible to coronavirus infection. Fluorescent reporters were used to detect both ACE2 proteins simultaneously, serving as an internal control and saving time. The Invitrogen™ Attune™ NXT Flow Cytometer was utilized for optimized sample analysis, allowing pre-set acquisition volumes to avoid air bubbles and ensure sufficient sample volume for analysis.

The laboratory’s work in understanding virus-host switching and cross-species infection has contributed to our knowledge of SARS-CoV-2 and other coronaviruses. By leveraging innovative cell engineering platforms and optimizing experimental workflows, they have shed light on the mechanisms underlying viral transmission.

To read the full article on Roelle’s research download the article Predicting Viral Pandemic Potential with Cell Engineering read it online at TheScientist Magazine. To learn more about products and solutions for academic research, visit thermofisher.com/academiclabs.

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