coronavirus

RNA Extraction for Clinical Testing—Do Not Try this with Home-brew

Posted on by Michele Arduengo

This blog was written with much guidance from Jennifer Romanin, Senior Director IVD Operations and Global Service and Support, and Ron Wheeler, Senior Director, Quality Assurance and Regulatory Affairs at Promega.

A Trip Down Memory Lane

Back in the day when we all walked two miles uphill in the snow to get to our laboratories, RNA and DNA extraction were home-brew experiences. You made your own buffers, prepped your own columns and spent hours lysing cells, centrifuging samples, and collecting that fluorescing, ethidium bromide-stained band of RNA in the dark room from a tube suspended over a UV box. Just like master beer brewers tweak their protocols to produce better brews, you could tweak your methodology and become a “master isolater” of RNA. You might get mostly consistent results, but there was no guarantee that your protocol would work as well in the hands of a novice.

Enter the biotechnology companies with RNA and DNA isolation kits—kits and columns manufactured under highly controlled conditions delivering higher quality and reproducibility than your home-brew method. These systems have enabled us to design ever more sensitive downstream assays–assays that rely on high-quality input DNA and RNA, like RT-qPCR assays that can detect the presence of a specific RNA molecule on a swab containing only a few hundred cells. With these assays, contaminants from a home-brew isolation could result in false positives or false negatives or simply confused results. Reagents manufactured with pre-approved standard protocols in a highly controlled environment are critical for ultra sensitive tests and assays like the ones used to detect SARS-CoV-2 (the virus that causes COVID-19).

The Science of Manufacturing Tools for Scientists

There are several criteria that must be met if you are producing systems that will be sent to different laboratories, used by different people with variable skill sets, yet yield results that can be compared from lab to lab.

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Which Came First: The Virus or the Host?

Posted on by Ken Doyle

They existed 3.5 billion years before humans evolved on Earth. They’re neither dead nor alive. Their genetic material is embedded in our own DNA, constituting close to 10% of the human genome. They can attack most forms of life on our planet, from bacteria to plants to animals. And yet, if it wasn’t for them, humans might never have existed.

3D structure of a coronavirus, viral evolution
A depiction of the shape of coronavirus as well as the cross-sectional view. The image shows the major elements including glycoproteins, viral envelope and helical RNA. This file is licensed under the Creative Commons Attribution-Share Alike 4.0 International license.

No, that’s not the blurb for a new Hollywood blockbuster, although recent developments have proven, once again, that truth is decidedly more bizarre than fiction. Now that “coronavirus” has become a household word, the level of interest in all things virus-related is growing at an unprecedented rate. At the time of writing, coronavirus and COVID-19 topics dominated search traffic on Google, as well as trends on social media. A recent FAQ on this blog addresses many of the questions we hear on these topics.

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Investigation of Remdesivir as a Possible Treatment for SARS-CoV-2 (2019-nCoV)

Posted on by Kyle Hooper

Remdesivir (RDV or GS-5734) was used in the treatment of the first case of the SARS-CoV-2 (formerly 2019-nCoV ) in the United States (1). RDV is not an approved drug in any country but has been requested by a number of agencies worldwide to help combat the SARS-CoV-2 virus (2). RDV is an adenine nucleotide monophosphate analog demonstrated to inhibit Ebola virus replication (3). RDV is bioactivated to the triphosphate form within cells and acts as an alternative substrate for the replication-necessary RNA dependent RNA polymerase (RdRp). Incorporation of the analog results in early termination of the primer extension product resulting in the inhibition.

Note the spikes that adorn the outer surface of the virus, which impart the look of a corona surrounding the virion, when viewed electron microscopically. In this view, the protein particles E, S, M, and HE, also located on the outer surface of the particle, have all been labeled as well. A novel coronavirus virus was identified as the cause of an outbreak of respiratory illness first detected in Wuhan, China in 2019.
This illustration, created at the Centers for Disease Control and Prevention (CDC), reveals ultrastructural morphology exhibited by coronaviruses. Photo Credit: Alissa Eckert, MS; Dan Higgins, MAM CDC

Why all the interest in RDV as a treatment for SARS-CoV-2 ? Much of the interest in RDV is due to a series of studies performed by collaborating groups at the University of North Carolina Chapel Hill (Ralph S. Baric’s lab) and Vanderbilit University Medical Center (Mark R. Denison’s lab) in collaboration with Gilead Sciences. 

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The Race to Develop New Therapeutics Against Coronaviruses

Posted on by Michele Arduengo

Once the purview of virology researchers, the word “coronavirus” is now part of the vernacular in the mainstream media as reports of quarantined cruise ships (1) and makeshift hospitals (2) fill our online news feeds. While there is currently no approved anti-viral treatment for coronavirus infection (3), a team led by researchers from Vanderbilt University recently published work characterizing the anti-CoV activity of a compound, which they now plan to test against 2019-nCoV (4).

Developing New Therapeutics Against Coronaviruses

Coronaviruses (CoVs) are enveloped, single-stranded RNA viruses that exhibit cross-species transmission—the ability to spread quickly from one host (e.g., civet) to another (e.g., human). Scientists classify CoVs into four groups based on the nature of the spikes on their surface: alpha (α), beta (ß), gamma (γ) and delta (δ, 1). Only the alpha- and beta-CoVs can infect humans. Four coronaviruses commonly circulate within human populations: Human CoV 229E (HCoV229E), HCoVNL63, HCoVOC43, and HCoVHKU1. Three other CoVs have emerged as infectious agents, jumping from their normal animal host species to humans: SARS-CoV, MERS-CoV and most recently, 2019-nCoV (5).

TE micrograph of a single MERS-CoV
Digitally colorized transmission electron micrograph reveals ultrastructural details of a single Middle East respiratory syndrome coronavirus (MERS-CoV) virion. Image credit: National Institute of Allergy and Infectious Diseases

The need for an effective, broad spectrum treatment against HCoVs, has been brought into sharp focus by the recent outbreak of the 2019 Novel Coronavirus (2019-nCoV; 6).

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