Neutralizing a bio-terrorist threat
BIOINFORMATICS | Ingenuity Pathway Analysis (IPA)
Neutralizing a bio-terrorist threat

Kylene Kehn-Hall, Ph.D. carefully puts on her personal protective equipment (PPE) – a Tyvek suit, gloves, overshoes and a specialized helmet with a built-in respirator designed to protect the wearer from exposure to airborne pathogens. With an almost meditative vigilance, she periodically checks herself for any potential gaps or ruptures in the material—a necessary precaution when working with the Venezuelan equine encephalitis virus (VEEV).

The arbovirus VEEV is classified as a bioterrorist threat due to the aggressive onset of symptoms caused by its aerosolized form. Researchers in Virginia and Maryland are on a mission to find a therapeutic to stop the severe and often debilitating disease.

Ingenuity Pathway Analysis (IPA) is used to understand pathways that trigger the innate immune response and cell death, giving the team a target for treatment.

First discovered in the 1930s, this alphavirus can cause a serious and potentially fatal form of encephalitis. If inhaled, the virus is known to elicit debilitating brain inflammation in survivors with consequential long term effects on the central nervous system that can result in cognitive, sensory or motor damage. Known for the aggressive onset of symptoms, VEEV was weaponized back in the 1970s by both the United States and the former Soviet Union into a manipulated aerosolized form, which is particularly deadly.

Kylene Kehn-Hall, Ph.D.

worked at the U.S. Army Research Institute on Infectious Disease and is now an associate professor at George Mason University where she studies host-pathogen interactions in viruses. As a key member of the National Center for Biodefense and Infectious Diseases, she has spent the last few years investigating the Venezuelan equine encephalitis virus (VEEV) and how it wreaks havoc on the brain after infection. Her goal is to develop further insights into treating these kinds of diseases in the future.

“We think about our first responders and military personnel,” says Jonathan Dinman, Ph.D., an expert on ribosome structure and function from the University of Maryland who works with Kehn-Hall on the VEEV project. “If we can better understand how this virus does what it does, we can better protect them and also gain insights that could help us develop treatments for other dangerous viruses.”

“If we can better understand how this virus does what it does, we can better protect first responders and develop treatments for other dangerous arboviruses.”
JONATHAN DINMAN, PH.D., MOLECULAR AND CELLULAR BIOLOGY DEPARTMENT AT THE UNIVERSITY OF MARYLAND

After confirming she is safely suited up, Kehn-Hall makes her way through another heavy door into the containment laboratory. She focuses on the loud, rhythmic whooshing from the respirator’s airflow in her headgear to mentally prepare for the work ahead. “It’s hot and it’s loud, and sometimes you can feel quite isolated when you are in here,” she explains as she reaches for a vial filled with a monolayer culture of astrocytes, star-shaped cells from the brain and spinal cord, that will be infected with VEEV. This all occurs inside the restrictive environment of the biosafety cabinet. “Once you are here, you kind of get in the zone. You can focus and really become one with your science.”

Venezuelan equine encephalitis virus (VEEV)

is a member of the genus Alphavirus,  naturally transmitted by mosquitos, and related to such viruses as the Western equine encephalitis virus (WEEV) and the Eastern equine encephalitis virus (EEEV) found in the Americas. In around 1% of human cases, VEEV can be lethal, while the other 99% of people who are infected may have to endure a terrifying disease course. The infection itself only lasts a few weeks but it can drastically impact a person for life. It’s not uncommon for people who have had VEEV to develop photophobia, confusion or even personality changes – due to the virus triggering massive cell death throughout the brain. VEEV is classified as a biodefence pathogen because it can induce severe disease and it is readily aerosolizable.

Tireless work

Kehn-Hall is a key member of the National Center for Biodefense and Infectious Diseases at the Biomedical Research Laboratory at George Mason University. Funded by the Defense Threat Reduction Agency (DTRA), she researches the intricacies of how the virus fully takes over host cells with Dinman, professor and chair in the molecular and cellular biology department at the University of Maryland. The pair were introduced by Jonathan Jacobs, Ph.D., Director of Global Product Management at QIAGEN, and the group has tirelessly worked to better understand VEEV from an “’omics” perspective. In other words, understanding how the virus acts on cells, from the genomic, transcriptomic, proteomic, and metabolomic perspectives. 

The scientists study and compare changes in messenger RNA (mRNA) on a global level due to infection. They hope to identify specific factors that may help the virus trigger the aggressively fast programmed cell death (apoptosis) it is known for after it has infected the brain cells. “We use astrocytes in order to model infection of the brain,” says Kehn-Hall. “After we infect the cells with VEEV in the containment lab, we can manipulate the cells to cross-link the DNA to the protein. This renders the virus non-infectious, so we can take samples and prep them for next-generation sequencing.”

“We have tried other freeware, but ultimately IPA is visually more appealing, manually curated, and we feel much more confident in the pathway ID we get out of it.”
Kylene Kehn-Hall, Ph.D., The National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University

Two new pathways

In a recent study, the group used QIAGEN’s CLC Genomics Workbench, to review raw data, and the Ingenuity Pathway Analysis (IPA) platform, a software that enables analysis, integration and understanding of complex ‘omics data, to analyze NGS results and identify which astrocyte genes were most strongly affected by VEEV infection. The analysis highlighted one key transcription factor—the early growth response 1 (EGR1) factor—which was critically linked to two pathways previously implicated in apoptosis; the innate immune response pathway and the unfolded protein response (UPR) pathway.

“While there is some freeware out there to help analyze this kind of data, ultimately, IPA gives us curated manuals and appealing visuals that made it very easy to investigate the data and decipher what’s going on,” said Kehn-Hall. The IPA platform allows researchers to overlay their data on an enormous knowledge base made up of millions of different findings regarding the human genome. Without that knowledge base, the team may not have discovered EGR1’s vital role in VEEV pathology.

Jonathan Dinman, Ph.D.,

professor and chair at the Department of Molecular and Cell Biology at the University of Maryland, has spent his career studying how viruses alter messenger RNA (mRNA) decoding in order to change and regulate gene expression in host cells. His research focuses on how viruses can direct the ribosome, the cellular organelle that synthesizes proteins, to shift reading frame, and how this can lead to apoptosis or programmed cell death.

Kehn-Hall was intrigued as soon as Jacobs sent her the initial data analyses: “It was my discovery of EGR1.” She delved into literature to try to better understand the gene’s function and the more she read, the more significant the gene appeared to be. “It really hadn’t been looked at in terms of an infectious disease before,” she says. “But once I started looking into the research, it made a lot of sense. It had been implicated in cell death before in other areas of cell biology. I wanted to grab hold of that discovery and push the boundaries of host-pathogen interaction.”

“VEEV blows your brain up...It causes pathology. It causes pain and suffering and I think this humanistic desire to alleviate or prevent pain and suffering is a strong motivator.”
Jonathan Dinman, Ph.D., Molecular and Cellular Biology Department at the University of Maryland

Analyzing very complex data

Kehn-Hall regularly uses QIAGEN’s extraction kits in the laboratory. “They are the industry standard,” she says. “But the IPA software has really made all the difference.  When you get back all the NGS data, especially RNA-related information, it can be really overwhelming. The IPA platform helped us analyze this very complex data set, allowing us to visualize the data and see the pathways that are critical to infection. We would not be able to focus on these key factors, validating and perhaps identifying them as potential therapeutic targets, without it.”

Dinman is convinced that this discovery adds an important piece to understanding the puzzle of VEEV host-cell interactions, and agrees that it makes sense to have EGR1 as a mediator of apoptosis after VEEV infection. “Viruses like VEEV are invaders,” he says, loudly tapping his fingers on the table in front of him. “When the brain cells get stressed by such an invasion, they turn on the innate immune response. Now we see EGR1 is turning on that innate immune response, signaling that the cell has a problem. The cell can’t fix that problem, so it’s programmed to kill itself for the good of its neighbors – sacrificing one for the good of many. So, all of a sudden, your brain cells start to pop like popcorn, resulting in major neurological problems. ”

The Biomedical Research Laboratory

is managed by the George Mason University National Center for Biodefense and Infectious Diseases (NCBID). The $50 million, 52,000-square foot, stand-alone, high-security facility is located adjacent to Mason’s Science and Technology Campus in Manassas, Virginia. The facility features more than 18,500 square feet of lab space comprising BSL-2 open-design laboratories with cell culture suites, preparation areas, and a microscopy room and is also equipped with ABSL-2 spaces, a surgery suite, BSL-3 laboratories, ABSL-3 suites, and a necropsy suite.

Drawn to the mystery

The biosafety level three (BSL3) laboratory at the George Mason University’s Science and Technology Campus is a second home for Kehn-Hall. When asked why she chose virology as her specialty, she quickly says, “love” with a slight laugh.

“Viruses are so small, yet they can do so much damage,” she says earnestly. “They aren’t considered living, and the viruses I work with only encode maybe six to ten different genes, but they can come in and essentially bend cells to their own will, taking those cells over and telling them to produce a million or more copies of the virus in a very short period of time. I suppose I’m drawn to the mystery of how they do what they do – and what we can do to stop them from doing it.”

“We want to first understand how EGR1 is turning on the signaling pathways leading to cell death [after VEEV infection]. Once we understand those pathways we will understand where to intervene in those pathways with small molecule inhibitors or drugs.”
Jonathan Dinman, Ph.D., Molecular and Cellular Biology Department at the University of Maryland

Given how easy it is to weaponize VEEV by creating an aerosolized form of the pathogen, Kehn-Hall argues that it’s vital to understand how VEEV infections result in such severe brain swelling. “If we can dive deep into the mystery of how VEEV wreaks such havoc on the brain, we have the potential to develop a therapeutic drug that can intervene early and stop it from causing too much damage,” she argues.

Unfolded protein response (UPR and EGR1)

After VEEV infection, the cell responds with a reaction called the unfolded protein response (UPR), which is responsible for the induction of the transcription factor EGR1. A cell is not equipped to handle the large amount of proteins created by the virus after infection. Proteins like glycoproteins overwhelm the cell, building up in the endoplasmic reticulum. This triggers the UPR, turning on EGR1 in the process. EGR1 stimulates transcription of various other genes – ultimately inducing signaling pathways leading to programmed cell death.

Identifying genetic targets

The team plans to take a closer look at EGR1’s role in programmed cell death after VEEV infection, hoping to uncover which other genes may be involved. Their goal is the identification of potential targets where drugs could stop the chain of apoptosis before the disease progresses too far, leading to VEEV’s hallmark neurological deficits. “Those findings may also provide new insights on Zika, Dengue or other more prevalent viral diseases that are related to VEEV and could have a real impact on how we treat these diseases in the future…I think that can save lives,” says Dinman.

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