After a biological attack, how do recovery teams know for sure that a site is sufficiently decontaminated and safe once again for people? Turns out, it’s not an easy or fast process, but one that biologist Cathy Branda (8621) is hoping to revolutionize with a rather amazing nematode, Caenorhabditis elegans.
Cathy Branda watches for fluorescent expression in a strain of C. elegans that, with a little work, may someday be used for on site clearance sampling. (Photo by Randy Wong)
Cathy is working with Mark Tucker (6327), who for many years has led Sandia’s efforts in developing methods, procedures, and technologies to remediate facilities contaminated in a terrorist release of a biological agent. Mark’s team found that testing for residual live virus or bacteria following initial decontamination efforts, a process called clearance sampling, can be the longest step in the remediation process. Detection of residual live virus is particularly problematic and time-consuming, as it requires evaluation of replication potential within a eukaryotic host such as chicken embryos.
Currently, clearance sampling involves collecting swab samples from across a site and transporting them to a suitable laboratory for measurement of biological activity. Depending on the agent, the samples may need to be processed at a biosafety level 3 or 4 laboratory. In the aftermath of the Senate anthrax attack of 2001, the US Army Medical Research Institute of Infectious Diseases (USAMRIID) performed more than 30,000 individual assays on more than 10,000 samples, processing more than 700 per day at the peak.
The Hart Senate Office Building was closed for three months, more than half of which was for clearance sampling. Some of the postal facilities affected in the attack were closed for years. While closing office buildings and postal facilities is of relatively low consequence — the work can be moved elsewhere — the closure of a major transit center such as an airport could have devastating economic impact.
“As all of our prior work on remediation has shown, the time required for clearance sampling really limits our ability to return facilities or even entire cities to operation,” says Duane Lindner (8120). “Having a rapid method of clearance sampling would relieve a fundamental roadblock to rapid restoration.”
Cathy’s answer is to turn C. elegans loose on the problem and perform clearance sampling on site using the nematode. This Laboratory Directed Research and Development (LDRD) project is attempting to use modified C. elegans that would fluoresce in the presence of live virus. Clearance sampling could be as simple as spreading a C. elegans-laden gel on the affected area and shining a UV light to see if the virus had initiated replication within the animal. Remediators could have their answer in as little as eight hours.
“To revolutionize this process, you need to bring the host to the site of contamination and have an on-site, rapid assay process so you don’t have to take swab samples, transport them to a laboratory, or do culturing,” she says. “That’s the utility we are trying to bring to C. elegans.”
Naturally, this process is easier said than done. But Cathy and her team — chemical engineer Mark Tucker (6327); virologist Oscar Negrete (8621); data analyst Joe Schoeniger (8621); and laboratory technologists Julie Kaiser (8621, year one) and Carrie Kozina (8625, years two and three) — can leverage what is already known about C. elegans and viral replication. They’ll also draw on Mark’s work with decontamination foam in developing the gel.
A worm by any other name
Let’s get one thing straight: C. elegans are roundworms, but they aren’t the parasitic sort that can make people sick. In fact, C. elegans, which are present in large numbers in the environment all around us, have already made significant contributions to medical research.
C. elegans is one of Cathy’s (along with many other scientists’) favorite lab animals for a number of reasons. Despite being just a millimeter long, they are multicellular organisms with complex biology and a well-developed nervous system. They are genetically well-characterized, optically transparent, and replicate in just a few days, producing large brood sizes.
Another advantage of C. elegans is that they are eukaryotes, sharing cellular and molecular structures and control pathways with higher organisms. That’s not all it shares — about 35 percent of the little worm’s genes have human homologs, or, in other words, common ancestry.
They are extremely adept at living in harsh environments and even have an alternative life form called the dauer larval stage, in which they can survive without food or water for months. C. elegans survived the space shuttle Columbia disaster in 2003. This heartiness poses a problem for Cathy’s grand plan — while they are susceptible to infection, they also have a strong antiviral response and natural defenses, such as a strong outer cuticle.
“To make this application sensitive enough for clearance sampling, we need to render these animals as susceptible to viral infection as possible,” says Cathy. “This poses a significant challenge, but I believe it’s possible. There is no doubt we need to use a lot of creativity in our approach.”
The team is testing a large number of C. elegans mutants and experimenting with different chemicals and pressure gradients to see if they can take those natural defenses down a notch. They are also working with Creg Darby, an assistant professor at the University of California, San Francisco, who is using C. elegans to research biofilms of Yersinia pestis, the causative agent of bubonic plague. Darby has shared his library of identified mutations that render the cuticle fragile.
Shine a light
The other half of the C. elegans solution is to modify the animal’s metabolic signaling pathways with a “molecular tag” so that when a specific virus replicates within it, the animal acts as a sensor and fluoresces. Again, the team has the advantage of previous research, but still needs to break new ground.
The team is working with mKate2, a fluorophore that fluoresces red when exposed to green light. Since most background autofluorescence glows green when exposed to blue light, similar to the popularly used green fluorescent protein (GFP), the team’s use of mKate2 should help to eliminate false positives.
As a proof of concept, Oscar is developing molecular tags for two viruses: Rift Valley fever virus (RVFV) and vesicular stomatitis virus (VSV). Here at Sandia, he’s using attenuated, nonvirulent strains that fully conform to Sandia’s rigorous biosafety requirements.
Getting the molecular tag into the animal is not the difficult part — reading it is. “Because C. elegans is only a millimeter long and we are talking about a subset of their cells, the ability to amplify that signal is critical if we are going to be able to detect it,” says Cathy.
Fortunately, another of C. elegans’ talents is communication via a pheromone signaling pathway. The response of the animal depends on the pheromone concentration it detects — enough will send it into the dauer state. This trigger involves an insulin receptor pathway similar to that found in humans, so it has been studied extensively.
The idea is to manipulate the C. elegans so that it produces the dauer signal in cells that have been infected. Instead of sending the recipient into the dauer state, the modified signal would trigger an amplification of the fluorescent response. “While much is known about this trigger, no one has tried to take advantage of its properties for an application,” Cathy says. “To trigger the amplification of the signal is novel, and determining the right way to do that is a challenge.”
By the end of the LDRD project, Cathy aims to have modified strains of C. elegans that can be used as sensors for RVFV and VSV. Her other objective is to develop sufficiently immune-compromised C. elegans. Between these goals and where the researchers are now, there are several significant milestones.
“Even if we can’t render the C. elegans susceptible enough for clearance sampling, the strains we will generate will still be useful tools for both homeland defense and research,” says Cathy. For example, she envisions a pool of C. elegans sensor strains that could be used to quickly identify the viral agent employed in an attack.
The immune-compromised C. elegans could also serve as a model to study questions such as how a virus spreads through tissues, what tissues are targeted, the receptors for viral infections, and mechanisms for limiting infections.
She’s also excited about the potential to share the research within Sandia. C. elegans are an incredibly useful tool that could be applied to many research areas relevant to Sandia’s missions, including cell-cell signaling, genomics, proteomics, environmental and particle toxicity, identification of antimicrobial and antifungal agents, etc.
“There is a lot of excitement about this project because it is such a fresh approach,” says Duane. “If it works — and there is every indication that it will work — this clearance sampling method will revolutionize restoration.”