Peek Inside a Level 3 Biosafety Facility at Tufts

The exterior of the Regional Biosafety Laboratory at Cummings School of Veterinary Medicine at Tufts University. Photo: Joanie Tobin / Tufts University

The Regional Biosafety Laboratory at Cummings School of Veterinary Medicine is on the forefront of researching infectious diseases—and keeping them contained

Tucked away on a quiet road near Cummings School of Veterinary Medicine at Tufts University sits a stoic brick building, surrounded by a wrought iron fence. The unassuming exterior belies the critical research taking place inside the New England Regional Biosafety Laboratory (RBL), a level-three biosafety facility in Grafton that safely allows for research of highly contagious microorganisms.

The National Institutes of Health (NIH) selected Tufts University as one of 12 sites across the United States to house one of these special laboratories, which were mandated by Congress in the aftermath of the terrorist attacks on September 11, 2001. The original intent was for the laboratories to focus on responding to bioterrorism, such as the anthrax attacks that occurred that same year. However, many of the labs have since evolved into researching emerging and reemerging infectious diseases.

"The NIH had considerable foresight in putting these laboratories out there, because they’re now at the front lines of our country's response to the COVID-19 pandemic," said Sam Telford III, a professor in the Department of Infectious Disease and Global Health (IDGH) at Cummings School of Veterinary Medicine and director of the RBL.

Telford was one of many Tufts faculty members who contributed to the 200-page proposal to the NIH requesting that Tufts be granted a facility. The project was spearheaded by Saul Tzipori, distinguished professor and Agnes Varis University Chair in Science and Society at Cummings School of Veterinary Medicine. The initial effort began in 2003, when Tzipori received a $25 million grant from the National Institute of Allergy and Infectious Diseases to develop a biodefense countermeasure portfolio—work that would require a level three biosafety (BSL-3) facility, which Tufts did not have at the time. This funding opportunity offered an incentive for the university to permit Tzipori to apply for the facility.

Some of the people in this archive photo, from left: Congressman Richard Neal, Michael Kurilla from the NIH, Saul Tzipori, CEO of the Massachusetts Life Sciences Center Susan Windham-Bannister, Representative George Peterson, State Senator Michael Moore, Cummings School Dean Emerita Deborah Kochevar, and Tufts President Emeritus Lawrence Bacow. Photo: Courtesy of Saul Tzipori

Some of the people in this archive photo, from left: Congressman Richard Neal, Michael Kurilla from the NIH, Saul Tzipori, CEO of the Massachusetts Life Sciences Center Susan Windham-Bannister, Representative George Peterson, State Senator Michael Moore, Cummings School Dean Emerita Deborah Kochevar, and Tufts President Emeritus Lawrence Bacow. Photo: Courtesy of Saul Tzipori

The NIH ultimately provided approximately 75% of the total construction cost of $32 million, with the required matching funds provided by the Commonwealth of Massachusetts through a Massachusetts Life Science Center grant of $9.5 million. Tufts University set aside $7 million of university funds in an endowment to partially offset annual operating costs. Over the last two years, Cummings School received more than $7 million in grants from the NIH for infrastructure improvements to upgrade the facility's technologic and scientific capabilities. This influx of funding ensured that critical building systems are safely operational and sustainable for the next 10 years.

Tzipori, who has spent his career researching infectious disease, was the first director of the RBL and stepped down in 2016. He keeps a shovel from the RBL's groundbreaking ceremony in his office.

"It took a pandemic, but we are finally seeing the RBL live up to its full potential as a bustling hub of cutting-edge infectious disease research focused on the health of humans and animals alike," said Tzipori. "It's a career dream come to fruition. For years to come, the RBL will enable Cummings School and Tufts University to be a safe place for investigators to advance research efforts on diseases that affect millions of people worldwide."

Biosafety levels (BSL) refer to lab practices and procedures, while risk groups refer to the microbes used in research. While most risk groups match the biosafety level, this is not always the case.

BSL-1: Microbes used at this level are of minimal risk to lab staff and generally do not cause disease in healthy adults. There are often treatments or vaccines available for these microbes, which include some strains of E. coli. Researchers can work with these on an open lab bench or table while wearing standard laboratory PPE such as gloves and lab coats.

BSL-2: Microorganisms used at this level pose a moderate risk of infection for researchers but are often difficult to contract through the air, such as Salmonella or HIV. Researchers must wear personal protective equipment (PPE) such as gloves, facial and/or eye protection, and a gown. Work is typically conducted in a biosafety cabinet which collects and filters the air to prevent escape of the organisms to the environment.

BSL-3: Microbes used at this level can cause serious or possibly fatal disease through respiratory transmission, but vaccines and treatments are generally available for many risk group three organisms. Examples include SARS-CoV-2, Mycobacterium tuberculosis, and Eastern equine encephalitis (EEE). Work must be conducted in a biosafety cabinet. Researchers often wear a full Tyvek coverall suit plus disposable booties, multiple pairs of gloves, and an N95 respirator—or sometimes, a Powered Air Purifying Respirator (PAPR), which filters air to prevent infectious organisms from coming through.

BSL-4 is reserved for the most dangerous and exotic microbes that have a high risk of airborne transmission and do not have vaccines or treatment available, such as the Ebola virus. This work is not conducted in the RBL or in any other labs at Tufts.

It wasn't always a hub of activity. When the RBL was first proposed, some people in the community questioned whether such a facility was safe. In discussions with local residents, Cummings School faculty explained that the RBL would be used to investigate bacteria or viruses that they might already have in their backyards, such as West Nile virus or Eastern equine encephalitis (EEE). That helped mitigate their concerns, the facility was completed in 2010, and the initial research with West Nile virus and EEE helped demonstrate to the community that the lab is safe.

Today the RBL is full of scientists from Cummings School and third-party companies that rent lab space in the facility. The companies are major research organizations involved in a range of activities, including those related to SARS-CoV-2.

The building has limited access, and federal security rules limit photography inside, too. But the researchers are happy to talk about their work. From cutting-edge COVID-19 research to investigating a vaccine for tuberculosis, here's a look what goes on behind closed doors at the RBL.

All work with live infectious agents is conducted in a biosafety cabinet designed to contain the virus or bacteria in that space. BSL-3 lab spaces are under negative air pressure relative to the hallways, and pressure sensors feed data continuously to a building automation system. Alarms go off if there are any issues with negative air pressure, at which point work is stopped and the problem is promptly identified and resolved.

"Working under BSL-3 conditions is restrictive and labor intensive," said Wendy Puryear, senior scientist and manager of the Runstadler Lab at the RBL. "Ideally, we take care of non-infectious prep or setup, if possible, in a lower biosafety level area, which is a more physically comfortable space."

In the RBL's level-three spaces, researchers change into scrubs and wear a full Tyvek coverall suit over them, plus disposable booties, two pairs of gloves, and an N95 respirator—or sometimes, a Powered Air Purifying Respirator (PAPR), which filters air through a HEPA filter to prevent infectious organisms from coming through.

"That's where things get more uncomfortable. PAPRs have battery packs that can be a little noisy, and it gets quite hot inside the [Tyvek] suit. The air coming down from the PAPR blows you up like the Stay-Puft Marshmallow Man," Puryear laughed. "But we try to tolerate it, because to take a break, you’d have to fully disinfect yourself before leaving the space and then re-dress in all the gear to go back in, both of which are very time consuming."

"Such efforts are worth the trouble, because Tufts scientists and the outside companies that work in the RBL are striving to develop treatment and prevention strategies for diseases that continue to cause deaths across the globe, and that work can be done only in BSL-3 spaces," said Jonathan Runstadler, professor and chair of the Department of IDGH at Cummings School.

All the PPE hangs from a wall in the anteroom, a pass-through support area with a sink and bins for personal belongings, which cannot enter the BSL-3 lab. Photo: Courtesy of Wendy Puryear

Level-three spaces at Tufts have a pass-through support area referred to as an anteroom, much like surgical rooms in hospitals, with a sink and bins for personal belongings which cannot enter the BSL-3 lab. All the PPE hangs from a wall in the anteroom; everyone has their own PAPR, as those are not shared. A glass window in the anteroom allows for visibility into the lab space. Each lab also has a pass-through autoclave through which laboratory waste exits so that the waste is decontaminated before it leaves the area.

"This level of research requires extensive training and is conducted with exquisite care by experienced faculty members and other scientists," said Bernard Arulanandam, vice provost for research at Tufts. "The RBL positions Tufts as a leading academic research institution making significant contributions to public health in general and the future of infectious disease research."

About 30 million people in the U.S. get the flu every flu season. But influenza doesn't just affect humans. A highly pathogenic strain of avian influenza is currently circulating among birds in North America. Since it arrived in late 2021, it has been attributed to the reported death of over 3,000 wild birds and more than 50 million poultry in the U.S. It's also been attributed to equivalent numbers of both in Europe. Research on high pathogenicity influenza, a risk group three virus, is one of the biggest projects in the Runstadler Lab.

In the Runstadler Lab, the influenza virus is grown in embryonated chicken eggs or cell cultures. Photo: Courtesy of Wendy Puryear

In the Runstadler Lab, the influenza virus is grown in embryonated chicken eggs or cell cultures. Photo: Courtesy of Wendy Puryear

"The RBL is an invaluable resource that allows Tufts and Cummings School to do unique types of research on infectious disease," said Runstadler. "Our lab collects a lot of samples from wildlife, not only birds but also seals in the Northeast, which transmit and carry influenza viruses. We bring those samples into the RBL because it allows us to conduct our work at an enhanced biosafety level, and if we isolate a virus that is a biosafety concern, we’re already in containment and we can deal with it safely."

Runstadler and his colleagues, including senior scientist and lab manager Wendy Puryear, analyze samples of tissue or swabs to detect any viruses present, sequence the viruses they find, and look for differences between virus strains to determine the ability of those viruses to infect different species or change over time in their wild animal hosts. They grow the influenza virus in embryonated chicken eggs or cell cultures, which is the primary way to amplify and propagate viral samples for research on influenza, inside an incubator in their lab.

Before the COVID-19 pandemic, much of the work in the Runstadler Lab was related to influenza, but in 2020, his team expanded their work into parallel research with SARS-CoV-2, which is also a risk group three virus. However, with SARS-CoV-2, they are studying the potential for the virus to move into wildlife hosts which could become a reservoir for human infection.

In the spring of 2020, Tzipori was eager for Cummings School to set up animal models for SARS-CoV-2 research in order to test vaccines and therapeutics. Pathologist Amanda Martinot, assistant professor in the Department of IDGH and Department of Comparative Pathobiology at Cummings School, was one of the first to dive in when she received a Fast Grant with colleagues from Harvard Medical School to analyze tissues from COVID-19 vaccine studies in animal models.

During the height of the pandemic, Puryear says she and other lab members frequently clocked 12- to 16-hour days, often working inside the BSL-3 lab space for 4–6 hours at a time. Nearly every day, they processed samples coming into the lab from a variety of sources, including Tufts Wildlife Clinic, the Henry and Lois Foster Hospital for Small Animals, the Hospital for Large Animals, home kits that were sent to people to test their pets, third-party wildlife clinics and rehabilitators, as well as from humans via clinical studies in which the lab was participating. The results contributed to a variety of efforts, including understanding transmission between people and animals, both domestic and wildlife, as well as developing a better understanding of how features of the host can impact susceptibility to infection and/or disease.

More recently, Martinot and virologist Sally Robinson in Tzipori's lab received COVID seed funding from Tufts to start doing their own COVID challenges in hamster models at the RBL. Earlier this year, Martinot and other authors published a paper on their work.

A researcher prepared to work with SARS-CoV-2 diagnostic samples inside the RBL. Photo: Courtesy of Wendy Puryear

A researcher prepared to work with SARS-CoV-2 diagnostic samples inside the RBL. Photo: Courtesy of Wendy Puryear

"It was a really easy transition to start analyzing our own tissues instead of tissues that were being sent to us from elsewhere," Martinot said. "And it was a huge step forward because now we are getting the virus, propagating the virus, and making viral stocks from the different variants out there."

Robinson does most of the work monitoring the animal subjects. After they are infected with the virus, she weighs them daily to track body weight and documents their journey from being sick to recovery. All animal research is approved in advance by the Institutional Animal Care and Use Committee, which requires investigators to carefully monitor infected animals. She has collaborated on this research not only with fellow Tufts scientists, but also with companies that work on campus and companies outside of Tufts. Robinson also supports the Martinot lab with a study going with Tufts Medical Center developing an animal model to study long-term effects of COVID-19.

"We maintain stocks of the different SARS-CoV-2 virus variants," Robinson said. "We give those to the animals and characterize the type of infection and the corresponding clinical signs and pathology that we see. When we’re testing vaccines and therapeutics, we’re looking for whether the animals are protected from those clinical signs or whether they’re protected from chronic pathology."

Martinot splits her time between SARS-CoV-2 research and tuberculosis (TB) research, for which she uses mouse models. Because of this, she was intent on setting up a mouse model for SARS-CoV-2 in addition to the hamster model in order to perform infection studies and test vaccines and therapeutics. They now have the MA10 "mouse-adapted SARS-CoV-2" model up and running in the RBL.

"This opens up opportunities for us to ask interesting questions about co-infections," said Martinot. "We know people all over the world have other diseases, like diabetes, TB, or parasitic infections, but we don't understand how those other diseases could be impacting their susceptibility to COVID-19, or how well COVID vaccines may work on someone with that background. With the hamster and mouse models, we’ve been getting fantastic data, and we’ve started testing vaccines and monoclonal antibodies."

Martinot admits she has somewhat of an ulterior motive with the new SARS-CoV-2 mouse models set up at the RBL: She hopes to employ them in her work to develop a new tuberculosis vaccine with funding from the Bill and Melinda Gates Foundation. The current TB vaccine is called Bacillus Calmette–Guérin, or BCG, which is a live attenuated vaccine, meaning it uses a weakened form of the bacteria Mycobacterium bovis, which causes TB. Historically, attenuated vaccines have been extremely effective, she pointed out, citing the polio vaccine as an example. But these days, most vaccines are killed vaccines or mRNA-based, and do not actually give people a live organism. Martinot is hoping to develop a new live attenuated TB vaccine that may offer a side benefit in offering some additional protection against COVID-19.