Wearing blue surgical gowns and white respirator hoods, research scientist Pradeep Uchil and post-doctoral fellow Irfan Ullah carry an anesthetized mouse to the lab’s imaging unit. Two days ago, the mouse was infected with a SARS-CoV-2 virus engineered to produce a bioluminescent protein. After an injection of a bioluminescence substrate, a blue glow starts to emanate from within the mouse’s nasal cavity and chest, visible to the imaging unit’s camera and Uchil’s eyes.
“We were never able to see this kind of signal with retrovirus infections.” Uchil is a research scientist at the Yale School of Medicine whose work focuses on the in vivo imaging of retroviral infections. Normally, the mouse would have to be sacrificed and “opened up” for viral bioluminescent signals from internal tissues to be imaged directly.
Before the COVID-19 global pandemic began, Dr. Xuping Xie, Assistant Professor of the University of Texas Medical Branch at Galveston, TX has been studying viruses, such as Dengue and Zika, for more than 10 years. Once the pandemic hit in early 2020, he was prepared to join the fight against the virus. “There was an urgent need to know: Is there a quicker way to develop therapeutics or antibodies to target SARS-CoV-2?” says Dr. Xie. “That’s why we immediately launched our SARS-CoV-2 project.”
His goal was to create an assay that could 1) screen for antiviral drugs and 2) quickly measure neutralizing antibody levels. The assay could be used to determine the immune status of previously infected individuals and to evaluate various vaccines under development. To achieve this, he wanted to create a reporter virus that is genetically stable and replicates similarly to the wild-type virus in cell culture.
Canine distemper virus (CDV) is a highly contagious pathogen that is the etiological agent responsible for canine distemper (CD), a systemic disease that affects a broad spectrum of both domestic dogs and wild carnivores. While there are commercially available vaccines for CDV that can provide immunity in vivo and protect canines from contracting CD, there is a strong demand for effective canine distemper antivirals to combat outbreaks. Such drugs remain unavailable to date, largely due to the laborious, time-consuming nature of methods traditionally used for high-throughput drug screening of anti-CDV drugs in vitro. In a recent study published in Frontiers in Veterinary Science, researchers demonstrated a new tool for rapid, high-throughput screening of anti-CDV drugs: a NanoLuc® luciferase-tagged CDV.
Coronavirus (CoV) researchers are working quickly to understand the entry of SARS-CoV-2 into cells. The Spike or S proteins on the surface of a CoV is trimer. The monomer is composed of an S1 and S2 domain. The division of S1 and S2 happens in the virus producing cell through a furin cleavage site between the two domains. The trimer binds to cell surface proteins. In the case of the SARS-CoV, the receptor is angiotensin converting enzyme 2. (ACE2). The MERS-CoV utilizes the cell-surface dipeptidyl peptidase IV protein. SARS-CoV-2 uses ACE2 as well. Internalized S protein goes though a second cleavage by a host cell protease, near the S1/S2 cleavage site called S2′, which leads to a drastic change in conformation thought to facilitate membrane fusion and entry of the virus into the cell (1).
Rather than work directly with the virus, researchers have chosen to make pseudotyped viral particles. Pseudotyped viral particles contain the envelope proteins of a well-known parent virus (e.g., vesicular stomatitis virus) with the native host cell binding protein (e.g., glycoprotein G) exchanged for the host cell binding protein (S protein) of the virus under investigation. The pseudotyped viral particle typically carries a reporter plasmid, most commonly firefly luciferase (FLuc), with the necessary genetic elements to be packaged in the particle.
To create the pseudotyped viral particle, plasmids or RNA alone are transfected into cells and the pseudotyped viruses work their way through the endoplasmic reticulum and golgi to bud from the cells into the culture medium. The pseudoviruses are used to study the process of viral entry via the exchanged protein from the virus of interest. Entry is monitored through assay of the reporter. The reporter could be a luciferase or a fluorescent protein.
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