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Three Years after the Anthrax Letters, Are We Safer?

By Edward R. Winstead

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One of the gravest fears about terrorist threats against the United States is that enemies will use biological weapons. We know anthrax is in the terrorist arsenal. Quite likely, plague is too. But three years after the as yet unsolved case of the anthrax letters, whose senders remain unknown, there is not enough reliable intelligence about how to detect or disarm biological weapons of war.

On this point everyone agrees. Therefore, the United States government is spending billions to generate information about a long list of lethal pathogens. In the research community, business is booming for those scientists who may be able to develop tools for identifying microbial pathogens and the drugs or vaccines to render them harmless.

This dish was used to identify anthrax in fluid collected from the site of a bioterrorism incident in Kameido, Toyko, in 1993.
At the National Institutes of Health alone, the budget for biodefense research topped $1.6 billion for the fiscal year 2004, and spending on all types of anthrax research will be $144 million.

Anthrax is perhaps the most interesting case to date, partly because there was a real episode and because scientists have now developed a genomic model for investigating the pathogen that can be applied to other agents of biological war—and possibly help in the unsolved letters case.

So are we more prepared for a biological attack today than we were three years ago, when someone sent letters laced with anthrax to government offices and the media, killing five people and injuring about two dozen others?

In some respects the answer is “perhaps,” but in a broader sense, the fact is there’s a long way to go.

Still, while nothing would prevent someone from dropping an anthrax letter in a mailbox today, the technology exists to detect anthrax in at least some buildings and post offices. And if anthrax from a research laboratory were used in an attack today, there’s a good chance investigators would identify the source laboratory in a matter of days or weeks.

Thatís because anthrax researchers cooperating with the FBI on the letters case have developed an experimental system for rapidly identifying anthrax strains. It can be used to match a sample of anthrax DNA to any known strain or to its nearest genetic relatives, according to a new study that appears in Proceedings of National Academy of Sciences.

“The goal was to develop DNA signatures of the anthrax bacterium that could be used in forensic studies,” says Paul Keim, a researcher at Northern Arizona University in Flagstaff and also at the Translational Genomics Research Institute (TGEN) in Phoenix.

Keim was one of the few researchers who had money to study anthrax prior to 9/11, and his laboratory may have the world’s largest collection of anthrax. By studying five genetically diverse strains in great detail, he and his colleagues laid the foundation for the new identification system.

“We were able to identify very rare genetic differences among the strains, and this will enable us to come up with really effective diagnostic tools,” Keim says. “Certainly the only time you would use this information is if you were investigating an anthrax case or using it in a clinic to see what kind of anthrax a person has.”

With funding from the Department of Homeland Security, Keim’s laboratory is developing new detection technology that he says could be used in buildings to distinguish between anthrax and one of its harmless cousins, thereby preventing potentially expensive false alarms.

“Let’s imagine you have a detector in a post office today that’s monitoring DNA in spores of anthrax,” says Keim. “It could be that a relative of Bacillus anthracis is floating around in there, and it isn’t pathogenic—but it may still set off a false positive in the post office. The ramification is that the nation’s entire network of post offices could be shut down—so you need the detector to be sensitive.”

Prototypes of the detectors are in his laboratory today, he says, and if all goes as planned the detectors could be in post offices next year. “Sooner,” he adds, “if there’s a crisis.”

Asked whether he feels safer today, Keim is a bit evasive, saying that the science today is light years ahead of where it was two and a half years ago and that the new knowledge and forensic tools will make it easier to deal with a crisis. And it may serve as a deterrent.

“Whoever perpetrated the first crime must realize that we have the capability to identify material and to track the material back to its source,” he says. “Whoever did this is presumably aware of what’s going on, and if the person is a scientist, they can read the study.”

“Hopefully,” Keim adds, “the person is out there thinking: When am I going to get caught?”

Anthrax from Around the World

Keim’s laboratory has more than 1,200 anthrax “isolates” from around the world. The main architect of the collection is Martin Hugh-Jones, a veterinary epidemiologist at Louisiana State University in Baton Rouge who since the mid-nineties has obtained anthrax from North and South America, Europe, Australia, and Asia.

The collection started after Hugh-Jones traveled to the former Soviet Union with the team investigating an anthrax outbreak that occurred in 1979 because a secret factory producing biological weapons accidentally released anthrax spores into the air. Sixty-six people in the city of Svredlovsk died.

Today Hugh-Jones is helping map anthrax outbreaks in Kazakhstan and expects to receive 20 cultures soon. Over the years he’s persuaded colleagues around the world to share their cultures. Samples have arrived in “dribs and drabs,” with 200 strains coming from China at one point.

The easiest place to get anthrax is in the refrigerator of a laboratory. --Martin Hugh-Jones
The major holes in the collection are India, West Africa, and Russia, whose scientists are forbidden by the government to share strains. India, he says, will not share strains with “anyone, anywhere, at any time and this has held them back when they’ve had an outbreak of disease” among animals.

“Anthrax is basically an animal disease and if it gets into humans it’s basically due to veterinary stupidity or because people are slaughtering infected animals,” he says. “Those of us who deal with anthrax know how to handle it.”

As long as humans have been trading animal hides and products made of animal bones, Bacillus anthracis has moved around the globe, as genetic analyses can show. Anthrax can exist in spore form for long periods of time before reproducing and causing an outbreak.

“Lots of people think you can go out and pick up anthrax anywhere, and I tell them, ‘Hey, it’s not like that,’” says Hugh-Jones. “The easiest place to get anthrax is in the refrigerator of a laboratory.” The “Ames” strain linked to the letter attacks was widely used to develop vaccines, he notes.

The Ames strain was initially recovered from a dead cow in Texas in 1981 and sent to College Station, Texas, for analysis. At about the same time, the US Army Medical Research Institute in Fort Detrick, Maryland, was looking for anthrax for its program on “defensive” biological weapons.

The strain came to be associated with a town in Iowa because the Texas researchers sent it to Fort Detrick in a prepaid envelope that said “Ames” on it. The strain was subsequently sent to other laboratories in the U.S. and Europe. Tests have shown that the anthrax used in the letter attacks is related to Ames.

Asked whether we are safer today than we were three years ago, Hugh-Jones responds that the cumulative effect of all the activity in recent years “has been to make the situation more dangerous than it was at the beginning.”

Apart from some clever diagnostic tests like the ones Keim is developing, he’s not impressed by the new research he’s seen and thinks that the vaccine they’ve been using for years is still probably the best.

When Hugh-Jones started working on anthrax eight or nine years ago, everyone in the field knew each other by their first names. “There were no more than ten labs in the nation working with the organism, and now it’s about 310—and they all want virulent strains,” he says.

“In the old days virtually everyone was paid by Department of Defense to do their research because that’s the only place where money came from because the organism wasn’t thought to be of economic importance,” he says. “Now that it’s a bioterrorist threat and money’s available for research, experts have come out of the walls.”

“The whole damn thing is bizarre.”

Sequencing Anthrax Genomes

Back in the fall of 2001, Keim’s collaborators on the anthrax work, scientists at The Institute for Genomic Research (TIGR) in Rockville, Maryland, were sequencing the anthrax genome. Soon after the attacks they received federal money to sequence the strain that killed a photo editor in Florida.

The project was an experiment to see if they could get useful information from a more comprehensive analysis that looked at the entire genome rather than just regions. When the answer turned out to be yes, they embarked on the project with Keim to develop a picture of the genetic diversity of the species.

Anthrax is one of the most genetically similar species known, and surveying the entire genome—all five million letters of DNA—was the only practical way to pick up rare genetic differences.

“We could only have done this through genome sequencing because we never would have found the differences any other way,” says Jacques Ravel, who led the sequencing at TIGR. The project was funded by the National Institute of Allergy and Infectious Diseases (NIAID), which is the main distributor of biodefense dollars within the National Institutes of Health.

Anthrax spore, magnified 92,000 times.
After sequencing the five strains, Ravel and his colleagues identified about 1,000 genetic differences among the strains. The differences are sites in the genome where a single genetic letter differs from the norm—what’s called a single nucleotide polymorphism or SNP (pronounced “snip”).

These were sent to Keim’s laboratory, where his team boiled them down to the 24 most informative markers. As a demonstration, the researchers used the markers to accurately place 26 diverse strains on the anthrax family tree.

“Before the study we had a pretty good idea of how different strains were related to each other, but this work provided much greater detail with much more confidence,” says Talima Pearson, a colleague of Keim’s at the University of Northern Arizona who worked on the study.

The study shows that a small panel of markers can provide a great deal of diagnostic information, and this will make it a model for research on other organisms, says Pearson.

Making an Example Out of Anthrax

In the coming months TIGR will use the same strategy in projects on plague and the influenza virus.

“We’re taking the anthrax paradigm and repeating it for other pathogens,” says Claire M. Fraser, president of TIGR. “The more we do this the more convinced we are that you can’t begin to answer questions about these organisms with just one genome sequence.”

And others are likely to follow their example, according to Maria Y. Giovanni, NIAID’s assistant director for microbial genomics and advanced technologies. “What’s important about the anthrax study is that we can now use it as a model for other organisms, whether they are bioterrorism threats or not,” she says.

Keim has already been in touch with scientists who are drafting proposals for genome projects on pathogens, and he tells them that the key is to select diverse strains for sequencing at the outset, because this determines the utility of genetic markers in forensics.

The genetic differences need to be representative of all the branches of the family tree, he says. If you select closely related strains at the beginning, then you’ll miss signature differences in more distantly related strains.

“In the field of genomics we think this insight will be widely appreciated,” Keim says.

The insight comes as NIAID-funded microbial sequencing centers at TIGR and the Broad Institute at the Massachusetts Institute of Technology are churning out data on pathogens to be used by the larger scientific community.

“Our big push is to make tools available for the scientific community so they can develop the drugs, vaccines, and diagnostics that we desperately need,” says Giovanni.

Not everyone thinks it’s a good idea to make the genome sequences of anthrax and plague public. But last week a panel of scientists that included Keim and Fraser issued a report that urged the U.S. government to continue its policy of open access to genomic information.

The panel argued that more good than harm would come from keeping genomic information in the hands of scientists everywhere who are working on drugs and vaccines despite the risk that the information could be misappropriated for other ends.

A National Center for Biodefense

Although the researchers are under a court order not to talk about the anthrax case, they are cooperating with the FBI so it’s safe to assume that their new tools and information have been in the hands of investigators.

Similarly, the researchers are almost certainly working with the new National Bioforensics Analysis Center at Fort Detrick in Frederick, Maryland, which is supposed to be a hub of resources for dealing with biological attacks. The center will eventually house genomic data and materials for conducting forensic tests, among other things.

Just the sort of thing that Keim’s team has developed, with help from TIGR.

If their system had been in place three years ago, would the case of the letters be solved by now?

Again the answer is “perhaps.”

Investigators certainly would have had better leads, but sequencing a few anthrax genomes isn’t going to reveal a killer. In the end, it seems, police work still matters.

Pearson, T. et al. Phylogenetic discovery bias in Bacillus anthracis using single-nucleotide polymorphisms from whole-genome sequencing. Proceedings of the National Academy of Sciences 101, 13536-41 (September 14, 2004).
Read, T. D. et al. Comparative genome sequencing for discovery of novel polymorphisms in Bacillus anthracis. Science Express. Published online May 9, 2002.

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