Go fISH! Using in situ Hybridization to Search for Expression of a SARS-CoV-2 Viral Entry Protein

Loss of smell (olfaction) is a commonly reported symptom of COVID-19 infection. Recently, Bilinska, et al. set out to better understand the underlying mechanisms for loss of smell resulting from SARS-CoV-2 infection. In their research, they used in situ hybridization to investigate the expression of TMPRSS2, a SARS-CoV-2 viral entry protein in olfactory epithelium tissues of mice.

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NanoBRET™ Assays to Analyze Virus:Host Protein:Protein Interactions in Detail

Recently, Gordon et al. published an atlas of protein:protein interactions of all proposed SARS-CoV-2 proteins expressed individually in HEK 293 cells (Table 1). The study tagged each of the viral proteins with an epitope tag and performed a pull-down of the expressed protein followed by trypsin digestion and mass spec analysis, a process referred to as affinity purification–mass spec analysis. The group identified 332 human proteins interacting with 27 SARS-CoV-2 proteins.

The interactions identified in the HEK 293 cells helped Appelberg et al. analyze interactions over time in SARS-CoV-2-infected Huh7 cells. Gordon et al. used the PPI data to identify FDA-approved drugs, drugs in clinical trials, and pre-clinical compounds that bound to the identified human proteins and labs in New York and Paris tested some of these drugs for antiviral effects.   

Table 1. The general functional area of human proteins identified to interact with individually expressed SARS-CoV-2 proteins as reported by Gordon, et al. (1). The SARS-CoV-2 proteins are classified as non-structural proteins (nsp#), structural proteins (E, M, and N) and accessory proteins (orf#).  
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Using Wastewater Surveillance to Track COVID-19 Outbreaks

Today’s blog is written by guest blogger, Sameer Moorji, Director, Applied Markets.

Even as countries are now gradually starting to reopen after lockdown, the COVID-19 pandemic is far from over. Researchers around the world continue to find new ways to monitor, prevent and treat the disease. One new way of monitoring COVID-19 outbreaks relies on a somewhat unexpected source: sewage water.

In March 2020, researchers at the KWR Water Research Institute found the presence of SARS CoV-2 RNA in wastewater samples collected near Schiphol airport in Amsterdam and several other sites in Netherlands. The result came within a week after the first case of COVID-19 in the country was confirmed. This study opened the door to the possibility of using wastewater-based epidemiology to determine population-wide infections of COVID-19.

What is Wastewater-based epidemiology?

Wastewater based epidemiology (WBE), or sewershed surveillance, is an approach using analysis of wastewater to identify presence of biologicals or chemicals relevant for public health monitoring. WBE is not new, as wastewater has previously been used to detect the presence of pharmaceutical or industrial waste, drug entities (including opioid abuse), viruses and potential emergence of super bugs. In fact, several countries have been successful in containing Polio and Hepatitis A outbreaks within their geographic locations.

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Choices for Measuring Luciferase-Tagged Reporter Pseudotyped Viral Particles in Coronavirus Research

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).  

CDC / Alissa Eckert, MS; Dan Higgins, MAMS

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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Understanding the Structure of SARS-CoV-2 Spike Protein

Glycosylation is the process by which a carbohydrate is covalently attached totarget macromolecules, typically proteins. This modification serves various functions including guiding protein folding (1,2), promoting protein stability (2), and participating signaling functions (3).

ribbon structure of SARS-CoV-2 protein
Ribbon Structure of SARS-CoV-2 Spike Protein

SARS-CoV-2 utilizes an extensively glycosylated spike (S) protein that protrudes from the viral surface to bind to angiotensin-converting enzyme 2 (ACE2) to mediate host-cell entry. Vaccine development has been focused on this protein, which is the focus of the humoral immune response. Understanding the glycan structure of the SARS-CoV-2 virus spike (S) protein will be critical in the development of glycoprotine-based vaccine candidates.

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Antibody From Humanized Mice Blocks SARS-CoV-2 Infection in Cells

As the SARS-CoV-2 coronavirus continues to spread throughout the world, the race is on to produce antivirals and vaccines to treat and prevent COVID-19. One potential treatment is the use of human monoclonal antibodies, which are antibodies engineered to target and block specific antigens. A recent study by Wang, C. and colleagues published in Nature Communications showed that human monoclonal antibodies can be used to block SARS-CoV-2 from infecting cells.

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RiboMAX and the Effort to Find Antiviral Drugs to Fight Coronaviruses and Enteroviruses

Prior to 2020, there were two major outbreaks of coronaviruses. In 2003, an outbreak of SARS-CoV sickened 8098 people and killed 774. In 2012, an outbreak of MERS-CoV began which so far has sickened 2553 and killed 876. Although the overall number of MERS cases is low, the disease has a high fatality rate, and new cases are still being reported. Even though fatality rates are high for these two outbreaks, containment was quickly achieved. This makes development of a treatment not commercially viable so no one had undertaken a large effort to develop an approved treatment for either coronavirus infection.

Fast forward to late 2019/2020… well, you know what has happened. There is currently no reliable antiviral treatment for SARS-CoV-2, the coronavirus that causes COVID-19 infections.

Zhang, et al. thought of a way to make an antiviral treatment commercially viable. If the treatment is actually a broad-spectrum antiviral, it could be used to treat more than one infection, meaning, it can be used to treat more people and thus be seen as more valuable and worth the financial risk to pharmaceutical companies. So, they decided to look at the similarities between coronaviruses and enteroviruses.

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Just Keep Swimming: How the Wisdom of a Blue Cartoon Fish Can Inspire Us Amid COVID-19

Today’s blog is written by guest blogger Karen Stakun, Global Brand Manager at Promega.

Wise words from a forgetful blue fish are uniting Promega employees during these trying days. Initiated by our VP of Operations as a rallying call to employees and reinforced through a kind gesture from the Hollywood writer and director who dreamed up the fish, I invite you to join Promega as we “Just Keep Swimming.”

Those words were uttered by Dory, a blue tang with short-term memory loss, in the 2003 animated movie Finding Nemo. Now a classic, it tells the story of Marlin, an overprotective clownfish, who searches the ocean for his missing son Nemo. Dory is his sometimes-unwelcome companion. Desperate to find his son, Marlin grows exhausted and begins to feel defeated, but Dory will not let him give up. Her motivation is simple yet potent. “Just Keep Swimming.”

Setting the Scene

As COVID-19 was emerging in China, Promega began scaling up manufacturing in January to meet the growing global need for testing products. As epidemic became pandemic, and demand quickly became unprecedented, we moved swiftly to increase capacity and add more shifts at our Madison manufacturing facilities, all while ensuring the safety of our employees.

All of this takes dedicated people, especially those on our operations team, working long hours in an atmosphere of global uncertainty. Dedication is in abundance at Promega, as every employee feels a deep commitment to humanity’s struggle against this disease. However, Chuck York, our VP of Operations, says he began seeing the team struggle with the never-ending increases in demand. Despite record product totals, it could be demoralizing for a group that prides itself on always being able to deliver what customers need.

That’s when Chuck recalled one of his family’s favorite movies. “I love the never-give-up aspect of Finding Nemo and in particular the net scene.” Toward the end of the movie, Dory and several other fish find themselves caught in a fishing net. With Nemo’s help, the fish realize they can turn Dory’s mantra into action. They keep swimming together in the same direction and break free of the net.   

“I wanted the team to focus on what we could control, doing all we can each day to keep product flowing. And we were and are doing an outstanding job of that. I also hoped to lighten the mood and bring a smile to peoples’ faces. Our ‘net’ is the ever surging COVID-19 demand, but eventually we will overcome if we just keep swimming.”

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The Cytokine Storm: Why Some COVID-19 Cases Are More Severe

coronavirus

Blog Updated on June 16, 2020

One of the biggest outstanding questions of the COVID-19 pandemic is why symptoms vary so much among patients. Some patients have no symptoms at all; some symptoms are mild, while others are extremely severe. Among the more severe cases, a common pattern of disease progression happens like this: A patient gets through the first week with some signs of recovery—then suddenly they rapidly deteriorate. In some cases, they go from needing just a tiny bit of oxygen to requiring a ventilator within 24 hours.

This pattern, often seen in young and otherwise healthy patients, has baffled doctors. What causes these patients to suddenly crash? Research now suggests that the patient’s own immune system may be to blame. It’s called cytokine release syndrome—also known as the “cytokine storm”.

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Wisconsin’s Public-Private Partnership to Increase COVID-19 Testing Capacity

This blog is written by Sara Mann, General Manger, Promega North America Branch

Promega is part of a new public-private partnership among Wisconsin industry leaders to increase the state’s laboratory testing capacity for COVID-19. I am pleased to represent Promega in this effort. The valuable insight we at Promega are gaining every day through our participation in this innovative partnership not only benefits Wisconsin labs, it also provides unique understandings about how we can best meet the testing needs of our customers around the world.

Promega Maxwell Instrument shown in a laboratory.

The new partnership includes laboratory support from Exact Sciences, Marshfield Clinic Health System, UW Health, as well as Promega. These organizations, along with the Wisconsin Clinical Lab Network, are sharing knowledge, resources, and technology to bolster Wisconsin’s testing capacity. Our goal is to help labs find the quickest approach to the most tests with their validated methods.

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