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Home , Biocontainment, Biodefense, Biosafety, Biosafety level

technology feature The basics of Studying potentially dangerous microbes in animal models of the disease they cause takes some extra considerations, as researchers and staf must perform their work under the careful restrictions of diferent level laboratories. Jim Kling

n early 2020, when the first concerns arose about a SARS-CoV-2 pandemic, Iresearchers at biosafety facilities realized they would soon be pivoting to work with the novel coronavirus. Fortunately, many had previous experience to draw on, such as working with or other airborne , or with SARS-CoV-1. That 2002 outbreak resulted in a more severe, though less transmissible, disease than the current virus. Preprints from Chinese and Asian researchers guided early efforts, said Kenneth Palmer, director of the University of Louisville (UoL) Regional Biocontainment Laboratory and the UofL Center for Predictive for and Emerging Infectious Diseases. The team in Kentucky had also established a ferret model during their Keeping microbes, such as SARS-CoV-2, contained is a critical consideration for those studying work with SARS-CoV-1. “We found that dangerous agents in biosafety facilities. Credit: E. Dewalt, Springer Nature our experience with the ferret model of SARS-CoV-1 was very similar to the published experience [with SARS-CoV-2], and the procedures we are using for research are tiered from (BSL) 1 to are reserved for agents with a high fatality are very similar between SARS-CoV1 4. BSL-1 can be used for agents that don’t rate, such as . They require full body and SARS-CoV2, and consistently cause disease. Work can be positive pressure suits, decontamination virus,” said Palmer. done on open benches, and little personal of all materials, and a shower upon But whether a virus is entirely novel protective equipment (PPE) is required, exiting the lab. Work is performed in a or has some semblance to pathogens of though the lab is separated from the rest of , and the lab is housed in a prior outbreaks, there are two essentials: the facility. BSL-2 labs work with moderately completely separate building (Table 1). “How is it transmitted, and how do we kill hazardous agents such as Staphylococcus SARS-CoV-2 falls into the BSL-3 it?” said Brian O’Shea, biological safety aureus, a common cause of skin and other category. The Centers for Disease Control program manager at Battelle’s Biomedical . PPE use is still minimal, but has not classified it as a – that Research Center. the work occurs behind self-closing doors is, a microbe with the potential to be used Answering those questions takes and animals are often handled in biosafety in biowarfare or as a terrorist weapon. some extra effort and preparations. cabinets, which are enclosed, ventilated The pandemic has made the designation ‘Biocontainment’ is often the stuff of science workspaces with a sash that can also be moot, according to O’Shea: “It’s not hard fiction and zombie apocalypses, but in real used to house the cages; this ensures those for someone to get hold of.” Still, Battelle life it plays a central role in biomedical animals share the same exposure status and carefully tracks its viral samples to select research. “Under normal circumstances, for environmental conditions. agent standards, just in case that designation potential biowarfare agents, we’re hoping Things start to get interesting at level should change. we’re testing products that will sit in the 3. BSL-3 facilities are designed to house BSL facilities come in a range of sizes. strategic national stockpile and collect potentially lethal, airborne microbes. The Battelle BSL-3 facility is fairly large, with dust,” said Dan Sanford, a senior research The that causes tuberculosis is 16 holding rooms and 3 rooms reserved leader at Battelle. one example, as is the virus behind avian for challenges and other procedures, along Active pandemics can up the ante, but the influenza. Two sets of doors isolate a BSL-3 with associated wet labs. It basics of biocontainment remain the same. lab. If a vaccine exists, staff may be required is all connected within one building today, to be immunized against the microbe being though it started out as individual units. To the biosafety lab studied within and they should wear PPE as “It was built up like a Lego™ set over time as The first critical decision to be made well as . All work is done within research demanded,” said O’Shea. The BSL-3 with any new is what level of biosafety cabinets, and the air must be facility is connected to supporting BSL-2 containment it will require. Biosafety labs filtered before it’s exhausted. BSL-4 facilities labs via airlocks, where animals may be

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Table 1 | Biosafety breakdown | Some of the differences between the four levels of biosafety laboratories BSL-1 BSL-2 BSL-3 BSL-4 Use For studying microbes that don’t For studying moderately For studying potentially lethal, For studying highly lethal microbes consistently cause disease hazardous microbes airborne microbes Example Non-pathogenic , Staphylococcus aureus; Mycobacterium tuberculosis; Ebola virus; Lassa virus microbes Saccharomyces cerevisiae Hepatitis SARS-CoV-2 Containment Basic safety measures, but Controlled access; Double-door ; biosafety Housed in entirely separate buildings, measures open benches and no special PPE worn and biosafety cabinets & air filtration required; with air filtration; biosafety cabinets equipment required cabinets used for PPE and respirators worn; required; staff wear full body, positive containment decontamination measures pressure suits; decontamination taken measures taken kept before they are exposed to an agent or The cages are all wire, similar to a pet store PPE to keep that risk level low so that our vaccine. The BSL-2 lab reduces the burden or home cage, with a perforated plastic floor staff can still work safely with the animals,” on the more sensitive BSL-3 area. “If you and a pan underneath to catch feces and said O’Shea. For example, in some cases don’t need to work inside the BSL-3, then we urine. A sealed plexiglass wall and door the team decided it was safe to substitute don’t want you to be back there. There are surround the entire system, which rests boot covers for shoe covers. In other cases, support facilities all over the place, but the inside the BSL-3 facility. a brand of safety equipment that the lab BSL-3 is centrally housed,” he added. To test infectivity, researchers insert had long used might be sold out, forcing a Within the labs themselves, the animal dividers to partition each cage, placing switch. “We need to analyze that alternative cages are a vital component of the unit. They infected animals on the outside, where the to make sure it meets our standards as well,” must meet regulatory standards for housing fresh air flows in. The naïve animal is on the O’Shea added. the animal, but also minimize risk to the inside of the system, closer to the plenum “We haven’t gotten to a point where we’ve handlers. Some cages are closed systems, and outflow. It’s critical to avoid physical said: ‘We can’t do this procedure,’ because with air that is filtered before entry and then contact between the animals, to ensure that there is some variability in the PPE we can exhausted. That prevents infected animals any occurs through respiration. use,’ said O’Shea. “It’s just one of the things from prematurely exposing vaccinated or Despite their apparent simplicity, these we’ve had to deal with.” control animals. structures pose engineering challenges, Engineering caging systems can be tricky. according to Bilecki. Any object that Reward in risky environments Brian Bilecki, director of Vivarium Design reduces airflow, like a divider, can cause Insufficient PPE or other failures Services at Allentown, Inc, described one suspended droplets to fall out of the air and could lead to exposure. Equipment can system that he helped develop, which used on to surfaces. “I have to manage all of that, fail. The University of Louisville staff latches and compression gaskets to maintain keeping the physical separation, and still undergoes training to ensure workers react seals. Users must dock and undock the cage have enough bioload to get that naïve animal appropriately. “For training purposes, we from racks in order to clean it or work with to test the transmission that the researcher is would take out somebody’s [] the animals. While developing the system, looking for,” said Bilecki. battery and see how they respond to that the team used tracer gas to test whether Aside from the rooms and animal situation, to make sure they don’t get scared the cage was completely evacuated of air cages, respirators are key to working with and take it off. So the staff are familiar before a user opened it. “We found a small airborne pathogens in BSL-3 and BSL-4 with how to respond if something goes burst of contaminated air inside the cage at facilities. Two types of respirators are wrong,” said Jennifer Kraenzle, manager of one of the ports in the back,” said Bilecki. commonly used in BSL-3 facilities for the Biocontainment Animal Facility at the That prompted a change. “We redesigned studying SARS-CoV-2: N95 masks and University of Louisville. the ports and interface such that the last powered air-purifying respirators (PAPRs). The highest risk procedures involve thing to close was the exhaust on the rack, The latter has a breathing tube, and a direct handling of a in so that was evacuated before the cage was battery-operated blower that draws in air high concentration, such as challenging an disengaged. That’s the level of concern with and moves it through a high-efficiency animal with agent in a syringe or nebulizer. these types of products,” he said. particulate air (HEPA) filter to a full or “Any direct interaction with a challenged, Design is also critical for experimental half face-piece. Due to the pandemic, the awake, alert animal – we want to limit the set ups. To investigate how transmissible shortage of N95 masks has led Battelle to direct interaction between an animal care SARS-CoV-2 is in physical spaces, such as move to PAPRs for some procedures, since technician and a challenged animal because indoor restaurants, researchers are using the devices can be readily decontaminated they’re unpredictable. They could bite, they ferrets to work out how long it takes for multiple times. could scratch, they could shed virus or other one animal to infect another, and even how As PPE became increasingly short in organisms, and we really want a barrier much virus needs to be expelled to raise the spring of 2020 due to the pandemic, between the animal and the technician,” the infection risk. A typical setup for a there were delays in some shipments that said O’Shea. transmission experiment would be six large had the potential to delay research. “When Generally speaking, animals are cages that are enclosed together around a we have a standard operating procedure in anesthetized when possible during such central enclosed space, or plenum. Airflow place that employs a certain type of PPE, procedures. “When we’re using high comes from the sides, through the cages, and we can no longer get that PPE, we need concentrated material, we make sure the and then out the center plenum where to reevaluate our risk assessment for that animal is not moving and there’s less risk to it is exhausted into the building’s outflow. procedure, or find some kind of augmented the staff,” said Sanford.

286 Lab Animal | VOL 49 | October 2020 | 285–287 | www.nature.com/laban technology feature

“The biggest risk is when you’re handling a high containment lab. That makes the it’s very black and white with no grey area. the cages. Always,” said Bilecki. When it’s standard operating procedures we have I like that, it’s definitive. It has a different time for cleaning, for example, handlers very important. If one thing is forgotten or feel [than other animal work]: very slow, move the animal to a clean cage, then mistaken, it can cause a cascade effect in the very methodical. I like that in a work remove contaminated bedding before research,” said Sanford. environment,” she said. sterilizing the cage. But does the exterior Indeed, the work environment in The intense attention to detail can be of the cage need to be wiped down? What a BSL-3 or BSL-4 requires a special difficult to keep up for long periods, so about contamination in the drinking water? kind of individual. “There is no tolerance workers at Battelle are given regimented All of those factors and more for going off protocol. It’s best suited to breaks. Workflow management among staff underline the human element of working people who are very rigorous and is also an important consideration. “It’s a biocontainment facility. In 2014, the meticulous,” said Palmer. Researchers not something you want to do for nine or National Institutes of put in and staff start out working in BSL-2 and 10 consecutive hours. Staff go in, do their place a moratorium on gain-of-function must first demonstrate proficiency in morning activities, then come out and get experiments on influenza, SARS-CoV-1 that lower risk environment. “It’s sort a break, some water or food, and then go (known simply as SARS-CoV at the time), of a tiered experience, so it selects for back,” said Sanford. and Middle East respiratory syndrome people who are well suited to working But the work is rewarding, said coronavirus (MERS). Such experiments in a very regulated environment,” Sanford, because of the high probability aim to introduce changes to the virus in he said. of the research having a direct impact order to better understand its mode of The extra layers of security and on . He recalled his days in transmission or infectivity. But several protection mean that everything moves graduate school, working to understand incidents involving mishandling of H5N1 at a slower pace. “It takes a lot longer the function of a gene involved in tumor influenza and samples unnerved to do routine maintenance. A cage change cells. If it ever became the target of a drug, some, leading to the pause. The moratorium at animal BSL-2 might take 2 or 3 minutes, he figured he would never know about it was lifted in 2017, and research proposals where in the animal BSL-3 it might take because pharmaceutical companies often are now evaluated by an NIH committee 6-10 minutes,” said Karen Powell, supervisor keep that information proprietary. “But that determines if the experiments are really of the Large Animal Clinical Services and especially on the animal side, we’re testing necessary, or if the same information could Regional Biocontainment Laboratory at the counter-measures. Knowing that you had be gained through less risky means. University of Louisville. a hand in evaluating products that in an Bilecki echoes those concerns about safe It’s also vital that researchers and staff emergency could be deployed and you’ve handling. He makes his living designing come prepared. “If you have to move shown to be efficacious, it’s very rewarding. cages with the lowest possible risk to staff backwards into the hall, maybe because The technicians who understand that and researchers, but he knows his work you need biohazard bags or some kind of generally do better. They’re very focused could be undermined by sloppy practice. supplies, it might take five or six minutes on their specific task, but at the end of “The honest truth is that all the mechanical just to get through the doors because you the study they love to see the big picture,” systems in the world that we can design, have to wait for interlocks. Everything just said Sanford. ❐ without really solid training and standard takes a tremendous amount of additional operating procedures for everyone to follow, time,” she said. Jim Kling ✉ you can throw that out the window. It has to “You learn very quickly to be very Freelance science writer, Bellingham, WA, USA. be a very regimented day when you’re in a organized before you go into the facility,” ✉e-mail: [email protected] space like that,” said Bilecki. added Kraenzle, who said she enjoys “It takes a lot of organization, a lot working in biocontainment despite the Published online: 9 September 2020 of planning, just to do a simple task in challenges. “Everything is in your control, https://doi.org/10.1038/s41684-020-0644-8

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