Neutralizing a bio-terrorist threat

Unraveling the mysterious pathways of a biodefense pathogen using IPA in the search for a therapeutic against dangerous arboviruses.

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).

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.

“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.”

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.”

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.”

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.

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.”

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. ”

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.”

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.