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Working in the Hot Zone

‘Better safe than sorry,’ UTMB researchers labor cautiously to neutralize some of nature’s deadliest microbes

By Jim Kelly

Whenever she enters the Robert E. Shope, M.D., Laboratory, researcher Nadya Yun first takes a quick counterclockwise lap around the buffer corridor between the lab and the building that separates it from the outside world. The corridor’s outer walls are slabs of off-white concrete block, but large oval windows grace its ten-inch-thick reinforced concrete inner walls—to Yun’s left as she circles the lab—and she peers through each to make sure that all’s well inside. Her check complete, she grabs a towel, a petite set of blue coveralls, and a pair of socks from a rack by a windowless metal door labeled “Biosafety Level 4.” With a swipe of her security badge, the mechanical door swings open; as soon as she passes through, it closes with a solid “thud.”

researchers at work in the Shope lab When researchers are wearing the Shope lab's "space suits," even routine lab procedures require special care. One researcher compares being in the suit to "working inside a big plastic bubble." The hoses dangling from the lab's ceiling provide air for breathing and to keep the suit pressurized.

Behind that door is the “clean change room,” where Yun will trade her street clothes for the sterilized blue coveralls she brought in with her. “Then she’ll go into the room next door to put on her suit,” Shope lab director Michael Holbrook tells me. “It’s just like one of these, which we’re sending for maintenance.” On the floor outside the door lie two similar white plastic “space suits,” each topped with a transparent flexible helmet.

Around the corner, another oval window provides a view of the lab’s main room. Soon Yun appears, her head looking preternaturally small inside its clear bubble above the now inflated suit. She reaches up to grab one of the many yellow air-supply hoses that dangle in loose coils from the ceiling, plugging it into a valve just above her hip. Her suit plumps up a bit more as she meanders around making routine checks. Then she disconnects the hose and strides perhaps ten feet to a glass-fronted biosafety cabinet, where she plugs her suit into yet another yellow air hose and sits down to work.

The cabinet’s glass front descends to within about six inches of its steel working surface. Along the front edge of this surface, vents constantly draw a thin layer of air down the inside of the glass and across the gap, creating an invisible “laminar flow” barrier that seals around Yun’s gloved hands as she puts them inside the cabinet. Picking up a blue-handled pipette, she begins drawing up precise amounts of a pinkish-red liquid containing the deadly H5N1 avian influenza virus and dispensing them into small vials.

H5N1—the notorious “bird flu” virus—has killed over half of the more than two hundred people it’s known to have infected in Vietnam, Thailand, Cambodia, Indonesia, China, Turkey, Iraq, Azerbaijan, Egypt, and Djibouti; so far, the disease has spread to poultry and wild birds throughout Asia, Europe, and Africa. Still, it’s not H5N1’s lethal past that most concerns infectious disease experts. They focus on the danger that lies in the future, the possibility that the ever-changing virus could suddenly mutate into a form that will give it the ability to easily do some things it now only does with great difficulty: move from bird to human or human to human.

Nadya Yun prepares her biocontainment suit

The last time an avian flu virus did that was in 1918. The result was a global pandemic estimated to have killed as many as one hundred million people. No one knows how many people would die if H5N1 mutated into a pandemic strain today. “We don’t know whether to stock up on boxes of Kleenex or boxes of body bags,” says UTMB professor C.J. Peters. But one advantage we should have over those who suffered previous pandemics are antiviral drugs such as oseltamivir (better known by the trade name Tamiflu) and, perhaps, peramivir, the drug being tested by Yun and the leader of her lab group, Assistant Professor Slobodan Paessler.

Paessler’s team wants to find out whether peramivir can be used as an alternative to oseltamivir if necessary. “We don’t know how clinically effective Tamiflu is, and we worry that resistance might develop fast,” Paessler says. “It’s also possible that in an outbreak we might run out.” If a flu pandemic occurs, public health authorities want as many different tools to work with as possible. So Paessler’s group is testing peramivir in ferrets, whose response to H5N1 is similar to that of humans. In its current form H5N1 is not classified as a biosafety level 4 (BSL4) virus, but rather as an “enhanced” biosafety level 3 (BSL3) virus. (UTMB does not yet have an “enhanced” BSL3 facility, but will will get one when the new Galveston National Laboratory opens in 2008.) But working with ferrets infected with H5N1 is much more dangerous than working with cells infected with the same virus; ferrets can exhale infectious viruses and bite through gloves, and the blood and tissue studies necessary to determine how well an antiviral drug works in animals can more easily expose a researcher to infection by a lethal agent. In this case, it’s better to be safe than sorry.

“Better safe than sorry” could be the motto of the Shope lab. In this unique facility, which began operating in July 2004, UTMB researchers can safely study some of the most dangerous microbes on Earth. Many of these pathogens are responsible for the emerging and reemerging infectious diseases that the lab’s namesake, the late UTMB Professor Robert E. Shope, foresaw almost a decade and a half ago would become a growing threat with rises in human population size and density, rapid environmental change, and increases in the speed and volume of transportation. As Shope, Joshua Lederberg, and Stanley Oaks predicted in their classic 1992 book Emerging Infections: Microbial Threats to Health in the United States, microbes once limited to remote regions of the globe have shown a newfound ability to penetrate to the heart of the developed world, causing death, sickness, and massive economic disruption. Think of viruses like SARS and West Nile, for example, and the H5N1 bird flu itself.

It was this threat that led David Walker, chair of UTMB’s pathology department, to propose the creation here of the United States’ first full-scale maximum-containment laboratory on a university campus. “This was Dave’s dream,” Holbrook says, remembering the first time he heard about the possibility that UTMB would build a BSL4—in 1998, while interviewing for a position as a postdoctoral fellow in pathology professor Alan Barrett’s lab. At the time, UTMB’s infectious disease programs were undergoing an unprecedented expansion, galvanized by Walker’s recruitment of Robert Shope and Robert Tesh. The two eminent virologists had left Yale for Galveston in 1995, bringing with them the World Reference Center for Arboviruses, a priceless collection of thousands of different virus strains collected from all over the globe and freeze-dried for storage. They were followed several years later by Professor Peters, the legendary virus hunter lured to UTMB from the Centers for Disease Control and Prevention. To take full advantage of the talented researchers being drawn to UTMB, Walker wanted a lab where they could work on the diseases in which they were interested: Rift Valley fever, tick-borne encephalitis, hantavirus pulmonary syndrome, Lassa fever, Crimean-Congo hemorrhagic fever—a virtual rogues’ gallery of deadly plagues.

The realization of Walker’s dream, constructed with the help of a $7.5 million grant from The Sealy & Smith Foundation, is often described as “a submarine inside a bank vault.” The lab occupies only about 2,000 square feet on the second floor of a three-story free-standing addition grafted onto the Keiller Building. It’s a building within a building, a sealed capsule sandwiched between two floors occupied by the equipment that makes sure its contents stay isolated from the outside world. In the space above the lab, fans roar day and night, keeping the lab air pressure well below that of the outside atmosphere and even regulating pressure differences between different rooms within the lab, so that air always flows from “safer” areas to those where a potential—however remote—exists that infectious particles might escape into the air.

Exhaust air from the Shope lab is pushed through sets of high-efficiency particulate air (HEPA) filters that scrub it clean of particles down to well below virus size before being released to the outside. In the room below the lab, two five hundred gallon tanks receive all liquid wastes generated in the lab, “cooking” them at 275 degrees Fahrenheit to destroy viral contamination.

“The lab workspace itself looks pretty simple, not that different from a BSL3 or BSL2 lab,” says deputy director of institutional biocontainment resources Miguel Grimaldo. “What makes this lab different is everything upstairs and downstairs.”

Just as in lower level labs, Grimaldo points out, “primary containment” in the BSL4 is provided by biosafety cabinets, where all work with infectious agents is carried out. The full-body “space suits”— at first blush the most obvious difference between the Shope Lab and a BSL3 or BSL2 facility—provide only a final line of defense in the unlikely event that any infectious material escapes a biosafety cabinet and gets into the lab’s atmosphere. “Everything should always be contained in the biosafety cabinet,” Grimaldo says.

Still, ensuring that viruses stay inside the biosafety cabinets and out of the lab proper requires careful, disciplined procedures—especially when animals are involved. Take for example the research that visiting professor John C. Morrill, a veteran of the U.S. Army’s Medical Research Institute for Infectious Diseases (USAMRIID), does in the Shope lab with mice infected by the Rift Valley fever virus. RVF, as it’s known, is a mosquito-borne virus normally found in eastern and southern Africa. Fatal to sheep, cattle, goats, and other animals, it can cause deadly hemorrhagic fever in humans. A 1977 RVF outbreak in Egypt infected tens of thousands of people, killed about six hundred, and wrought havoc on livestock; in 2000, the virus migrated to Saudi Arabia and Yemen, where it did similar damage. If, like West Nile virus, RVF were introduced into the United States—whether accidentally, thanks to a mosquito hitching a ride in an airliner, or deliberately, through bioterrorism—the result could be a human and animal epidemic whose impact dwarfed that of West Nile. Vaccines would be vital to any effort to control the outbreak. But the current RVF vaccine requires multiple injections, takes weeks to reach full strength, and exists only in limited quantities; technical problems make manufacturing enough of it to make a difference impractical. So, in concert with UTMB professors Shinji Makino and Peters, Morrill is laboring to perfect new RVF vaccines that can be used in humans or animals.

That means working with RVF-infected mice, and while RVF is considered a BSL3 virus in cell culture, at UTMB its study in animals warrants BSL4 precautions. With both suit-clad arms inside a biosafety cabinet, Morrill handles contaminated mouse cages with practiced skill, the product of nearly two decades’ experience in maximum- containment labs. He sprays the cages—each the size of a shoebox, made from clear plastic—with a microbicide, then seals them inside plastic bags, the outside of which he sprays with the same germ-killing liquid. Then he pulls the bagged cages out through the gap beneath the biosafety cabinet’s glass door, being careful not to disturb the layer of moving air that seals the gap, and places them onto a metal cart to be rolled into the lab’s autoclave for final sterilization at 250 degrees Fahrenheit. Housekeeping done, he checks on nearby racks of mouse-filled cages, looking like an amiable snowman in his puffy white suit.

“The lab workspace itself looks pretty simple, not that different from a BSL3 or BSL2 lab,” says deputy director of institutional biocontainment resources Miguel Grimaldo. “What makes this lab different is everything upstairs and downstairs.”

Morrill makes working in a biosafety suit look much easier than it actually is. The suits impose their own unique stresses, beginning with the minimum of eight minutes that it takes to get out of one once you’ve entered the lab; that’s how long the chemical shower decontamination lasts. Quick trips to the bathroom are out, although, as Holbrook points out, another feature of the suit environment makes that less of a problem than one might think: “You’re getting more than twenty cubic feet per minute of dry air, so you can get really dehydrated, which means you have to urinate a lot less.” The constant rush of air makes it hard to hear anything else, even a radio inside the suit, so researchers communicate with each other and the outside world mainly via hand signals and scribbled notes on Magic Marker whiteboards.

Other details add to the fatiguing nature of the experience. “You have to learn to work in two pairs of thick gloves—it takes an adjustment to learn to manipulate small objects and not knock things over inside the biosafety cabinet,” says research associate Mary Lou Milazzo, who works on hantaviruses and arenaviruses in the Shope lab with her husband, Associate Professor of Pathology Charles Fulhorst. “It’s a physically cumbersome environment, and you have to move carefully and deliberately if you want to be efficient.”

For Milazzo and Fulhorst, though, research in the BSL4 is well worth the hassle. There they study three different rodent-borne hantaviruses, close relatives of the one responsible for the 1993 outbreak of hantavirus pulmonary syndrome (HPS) that killed twenty-seven young, previously healthy people in the Four Corners region of the American Southwest. HPS, a terrifying and deadly condition in which patients’ lungs rapidly fill with fluid, remains poorly understood and very difficult to treat. (The Four Corners outbreak marked the first recognition of human disease caused by a hantavirus indigenous to the New World, and the first time that human infection with a hantavirus was associated with a disease that predominately affected lung tissue. Hantaviruses capable of causing HPS have since been identified all over North and South America —including in rats captured near Galveston Bay.) “It’s been twelve years since HPS emerged in the Americas, and we still have no specific, effective therapy for this highly fatal disease,” Fulhorst says.

“If you’re a patient and you get so sick you’re hospitalized, there’s a high probability that you will succumb to the disease. The problem is that we don’t know very much about the biological processes that occur in the fourteen- to twenty-five-day incubation period and that ultimately result in fatal disease. If we knew that, physicians might be able to treat HPS patients effectively.”

To figure out how hantaviruses cause HPS, Fulhorst and Milazzo are studying the effects on hamsters of two Venezuelan hantaviruses named Caño Delgadito and Maporal, and an Argentinian hantavirus named Andes virus. Hamsters, unlike most other laboratory animals, develop a disease that’s clinically and pathologically very similar to human HPS. Caño Delgadito and Maporal were discovered in the same melon field in southern Venezuela by Fulhorst and Milazzo; Andes virus, the only hantavirus known to be transmitted person-to-person, was identified in 1996 when it infected twenty people and killed eight of them. (Humans usually become infected with hantaviruses by inhaling respiratory secretions or aerosolized urine from infected rodents.) Andes virus kills virtually all the hamsters it infects, while Maporal kills only some, and Caño Delgadito sickens the animals without killing them.

“This is really the spectrum of disease you see in people,” Fulhorst says. “And those three viruses are serving as the foundation for a program to manipulate the genome of hantaviruses to find out what makes them lethal, so we can improve our knowledge and understanding of how hantaviruses cause disease in humans and then develop specific, effective post-exposure therapies for HPS.”

To Fulhorst, there’s something particularly appropriate about working on such a project in a lab named for Shope, a place he sees as embodying the collaborative spirit and scientific and public health vision of the late virologist. Beginning with basic ecological fieldwork in South America, Fulhorst’s effort now has reached the point where he and his collaborators actually can make a direct contribution to human health. “You know, Bob Shope left a great thing here, a really unique spirit,” Fulhorst says. “We identify these viruses, and then we actually do something with them to help people, which was Shope’s mission—that, and training people to work at the highest level possible. Dave Walker’s vision to create this lab was to fulfill that mission, and it has become an incredibly powerful tool to do just that.”

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