Student Updates

Interdisciplinary BUMC Team Investigates Dialysis Access Malfunction

Patients with chronic kidney disease frequently develop stenosis of their intrathoracic large veins (i.e., central venous stenosis), which jeopardizes their prospects of having a functional dialysis access, while the current modality to diagnose this complication is fraught with inaccuracies.

An interdisciplinary team of clinicians, biomedical engineers and scientists, including members of the Evans Center IBR Thrombosis and Hemostasis ARC, has taken on this challenge. They were awarded a grant by Philips to lead a clinical study focused on assessing the potential of intravenous ultrasound, an emerging technique, to investigate and diagnose the mechanical properties of central venous stenosis. Results from this study will address one of the critical bottlenecks in the management of dialysis access malfunction, particularly in the underserved patient population, and is likely to seed larger clinical trials in the future to guide endovascular therapies.

NEIDL Researchers Create Tool to Study New Virus

Could help determine if Ebola-like LLOV can cause disease in humans

In recent years, traces of new zoonotic viruses—pathogens that live in animals and could potentially cause infectious diseases in humans—have been discovered in bats, fish, and other species around the world. But scientists have been stumped in their search for the complete genome for these viruses, the critical data that is needed to study their biology, and their potential dangers for humans.

But now, researchers at BU’s National Emerging Infectious Diseases Laboratories (NEIDL) have created a tool that they say will unlock many of the mysteries of one of these new pathogens—a filovirus closely related to the Ebola and Marburg viruses—and help them determine whether it could cause disease in humans. Ebola and Marburg are among the most virulent and lethal viruses known to infect humans.

Called Lloviu virus (LLOV), the new filovirus was first discovered in 2002 in dead bats in a cave in Spain; it was found again in 2014, in dead bats in Hungary. It is not known whether LLOV sickened and killed the European bats (known as Schreiber’s long-fingered bats, they are not found in North or South America) or if it causes disease in any animal. As with so many other new viruses, the complete genome has not been found.

The NEIDL team was led by Elke Mühlberger, a School of Medicine associate professor of microbiology, who is one of the world’s leading filovirus researchers. Filoviruses are made up of single-stranded RNA instead of DNA.

Here is how Mühlberger’s team created the new tool: They established a novel minigenome system for LLOV, which allows for the virus to be studied safely in a Biosafety Level-2 lab. Minigenomes are shortened versions of viral genomes, in which all the viral genes—those that cause infection—are removed and replaced by a single nonviral reporter gene. Minigenomes contain all the necessary signals to replicate the virus in cells, but they lack the proteins necessary to produce infectious virus.

“However, in order to make a minigenome, you need the viral start and stop signals that allow for replication,” says Whitney Manhart (MED’20), a PhD student in Mühlberger’s lab, who coauthored with lab manager Jennifer Pacheco a September 4 Cell Reports study on the new tool. “These are exactly what we were missing from LLOV,” she says.

“We currently have no therapeutics or vaccines which would target LLOV, and so answering basic questions about LLOV biology or the disease it might cause could help us be prepared for—or prevent—a major outbreak.” —Whitney Manhart (MED’20)

To make up for the missing elements of the LLOV genome, the NEIDL researchers copied and pasted similar sequences from the related Ebola, Marburg, and Reston filoviruses (Reston causes disease in nonhuman primates, but is not known to cause disease in humans). This technique is called sequence complementation.

“While this work is most interesting to people who study filoviruses, the idea could be applied to any kind of virus,” Pacheco says, noting that researchers studying a bat-derived influenza with an incomplete genome used a similar method to create an infectious viral particle.

Pacheco, Manhart, and their colleagues found that LLOV replication and transcription mechanisms are more similar to Ebola viruses than Marburg viruses. “We found that when the start signal is the same as Ebola viruses, then LLOV can replicate well,” says Pacheco. “When the start signal is the same as Marburg viruses, LLOV cannot replicate.”

“The minigenome helps scientists answer some questions about LLOV,” Manhart says, “but we need the authentic virus to answer all questions.”

Using the LLOV minigenome system, Manhart, who has been trained to work in BSL-4 conditions, will produce the real virus at Rocky Mountain Laboratories, a National Institutes of Health (NIH) BSL-4 lab in Montana, in collaboration with scientists there. Then, she says, Heinz Feldmann, the chief of Rocky Mountain’s Virology Laboratory and an expert in animal models of filovirus infection, will infect nonhuman primates at that lab with the virus as a step in determining if it could cause disease in people. “If it doesn’t cause disease, then we can learn about why LLOV doesn’t make primates sick even though it’s so similar to Ebola and Marburg viruses,” says Manhart. “If they do get sick, then we can start to learn more about the disease.”

LLOV was found in bats that live in northern Africa, and across Europe, Manhart says. “So if a spillover did occur, it could infect a lot of people very quickly. We currently have no therapeutics or vaccines which would target LLOV, and so answering basic questions about LLOV biology or the disease it might cause could help us be prepared for—or prevent—a major outbreak. This minigenome system, and importantly, the infectious virus, will be critical in those studies.”

The other NEIDL researchers who helped develop the tool are PhD candidate Tessa Cressey (MED’19), research assistant Adam Hume, and former research scientist Laure Deflubé.

The work on the new tool was begun with support from the NEIDL director’s fund and is now funded by the NIH.

This BU Today story was written by Sara Rimer

Ebola Research Begins at NEIDL

After years of review, lab receives first BSL-4 pathogen

Eight months after receiving final approval from the Boston Public Health Commission to conduct research at Biosafety Level 4 (BSL-4), Boston University’s National Emerging Infectious Diseases Laboratories (NEIDL) has begun work with its first Level-4 pathogen, the Ebola virus.

“This is clearly an important step for the NEIDL,” says Ronald B. Corley, NEIDL director and a School of Medicine professor of microbiology. “This will permit us to fulfill our mission of studying emerging pathogens and developing diagnostics, therapeutics, and vaccines for these pathogens, even those that require BSL-4 containment. It has taken a very long time to get to this point, but the time that has passed has not dampened our enthusiasm—and excitement—to be able to start BSL-4 work.”

NEIDL microbiologist Elke Mühlberger says the lab’s first Level-4 projects will examine how the Ebola virus damages cells in the liver, and why it triggers such a powerful inflammatory response. Answers to those questions, she says, could speed the development of a therapy for Ebola virus disease, which sickened tens of thousands of people and led to more than 11,000 deaths in West Africa in a 2014–2016 outbreak, and sickened 59 people and killed 29 in an outbreak in the Democratic Republic of Congo this past May.

Ebola virus causes a rare but life-threatening disease that has become a global public health threat, traveling to the United States and Europe during the 2014 outbreak. There is no available FDA-approved vaccine or therapy for Ebola virus, which along with the related Marburg virus, arrived at NEIDL earlier this week.

Mühlberger, a MED associate professor of microbiology, says the researchers’ plans include at least three projects involving the Ebola and Marburg viruses, all funded by the National Institute of Allergy and Infectious Diseases, which also provides more than $10 million a year to help underwrite the cost of operating the BSL-4 in the NEIDL. The first project, which will look at Ebola’s damage to the liver, will use human liver cells generated from induced pluripotent stem cells at BU’s Center for Regenerative Medicine by stem cell biologist and tissue engineer Gustavo Mostoslavsky, a MED associate professor of medicine and microbiology.

“We are excited to use these cells,” says Mühlberger, “because the liver is one of the main target organs of Ebola virus infection. We will use Gustavo’s cells to find out why Ebola virus is so devastating for the liver.”

A second project, which follows up on research conducted by NEIDL researcher John Connor, a MED associate professor of microbiology, will examine why the Ebola virus triggers an inflammatory response in specific immune cells. Mühlberger says researchers will also work with the Marburg virus, hoping to learn why it kills humans but not Egyptian fruit bats, a mystery that has been studied by Thomas Kepler, a MED professor of microbiology.

Safety and Security Are Paramount

“The need for this facility is even more acute today than when it was first built,” says Corley. “And we remain committed to being completely transparent in our operations, and maintain the NEIDL with the greatest attention to safety and security.”

Kevin Tuohey, executive director of research compliance at Boston University and Boston Medical Center, says the arrival of the Ebola and Marburg viruses has been eagerly awaited and was meticulously monitored. Tuohey says transportation of Level-4 agents is regulated by several authorities, including the US Department of Transportation, the Centers for Disease Control and Prevention (CDC), the International Air Transportation Authority, and the Boston Public Health Commission. Deliveries of the agents to the NEIDL, he says, are coordinated with Boston Emergency Management, Emergency Medical Services, and Boston Fire and Police Departments, as well as the Massachusetts State Police.

During transport, Tuohey says, Level-4 agents are packaged in accordance with the Department of Transportation’s “triple packaging” regulations, and BU requires an additional level of packaging in the form of a secured rigid material over the pack case. Shipments are carefully tracked with GPS. BU requires each delivery to have two drivers, both of whom are reviewed in an annual clearance process.

Tuohey says the NEIDL has bolstered its preparation for Level-4 research with the hiring of two new Environmental Health & Safety officers. Shannon Benjamin is the new associate director for high containment/BSL-3 BSO, and Nadya Yun is associate director for maximum containment/BSL-4 BSO. Benjamin comes to BU with 10 years of biosafety experience, most recently at Brown University, and Yun has two years of biosafety and compliance experience following 10 years as a researcher and research manager. She comes to BU from Environmental Health & Engineering, Inc., and Harvard Medical School, after working in BSL-4 at the Galveston National Lab and University of Texas Medical Branch.

In December 2017, after more than three years of review, BSL-4 research at the NEIDL was approved by the CDC, and last December the lab received final approval from the Boston Public Health Commission. All BSL-4 research conducted at the NEIDL must also be approved on a case-by-case basis by BU’s Institutional Biosafety Committee, which was created under National Institutes of Health guidelines to review research involving biohazardous materials.

Mühlberger says NEIDL researchers are excited to finally begin work with Ebola and Marburg viruses. She says the lab’s first step will be to propagate these viruses to produce enough material for experiments. The next step will involve inactivation procedures, so that material can be safely moved from BSL-4 to BSL-2, where most of the research will take place. She says the work will be conducted by several researchers who have been trained to work at BSL-4. They include senior research scientist Judith Olejnik, research scientist Adam Hume, research technician Jennifer Pacheco, and PhD student Whitney Manhart (MED’23), the first student trained in BSL-4 work at the NEIDL.

Those researchers will be joined later this summer by Robert Davey, a highly regarded virologist whose research bridges the fields of virology and cell biology, and by Anthony Griffiths, a virologist who studies how highly pathogenic viruses cause disease. Both come to the NEIDL from Texas Biomedical Research Institute, a private research center in San Antonio, Tex.

This BU Today story was written by Art Jahnke.