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26 Aug 2008

Source: USA Today, August 24, 2008.

FBI explains the science behind the anthrax investigation

It wasn't a Redskins game or a Capitol Hill soiree that was the place to be in Washington D.C. last week. It was "an informal, on-the-record roundtable discussion" held by the FBI to discuss the science behind the 2001 mailing of anthrax-containing envelopes that killed five people. The meeting was held to spill scientific, but not investigative, clues from the still-open case.

In late July, anthrax vaccine researcher Bruce Ivins, 62, of the United States Army Medical Research Institute for Infectious Diseases (USAMRIID) in Frederick, Md., committed suicide. FBI investigators revealed that Ivins was a suspect in the anthrax mailings that sent bioterrorism fears nationwide after the toxin was sent to news organizations and the offices of Sen. Pat Leahy (D-Vt.) and Sen. Tom Daschle (D-S.D.)

Earlier this month, the Justice Department released 2007 search warrants that had been issued for Ivins' home and workplace. The warrants linked four mutations in the attack anthrax a sub-type of the "Ames" strain used in anthrax vaccine experiments to a collection held in a flask controlled by Ivins from 1997 to 2004.

U.S. Attorney Jeffrey Taylor has called Ivins' flask of anthrax "the murder weapon." Ivins' attorney Paul Kemp says his client was innocent, suggesting that many researchers had access to the flask. "I have nothing but questions," Kemp said via e-mail Monday. "The science, according to many, can only identify a strain of anthrax. If they can identify it, why didn't they act within the last three years to arrest Ivins?"

To address such questions, the FBI invited news organizations to the science roundtable that was held at 2 p.m. on August 18. Several organizations, including USA TODAY, sent a Justice Department and science reporter.

The J. Edgar Hoover FBI Building, the bureau's headquarters, is a concrete colossus squatting along Pennsylvania Avenue between the U.S. Capitol and the White House. Participants in the roundtable discussion passed through a metal detector booth and walked down an interior driveway before turning into an outdoor hallway that led to a large briefing room, dimly-lit, just like in crime movies. A rectangle of tables waited, with place names for the scientists at the head table.

The scientists included Paul Keim of the University of Northern Arizona in Flagstaff, one of the world's leading anthrax sub-type experts; Claire Fraser-Liggett of the University of Maryland in Baltimore, who led efforts to genetically analyze the attack anthrax; and Jacques Ravel of the University of Maryland School of Medicine, who led the efforts to complete the genomes (the complete mapping of genes) of bacteria colony samples derived from the attack anthrax.

Vahid Majidi, the assistant director responsible for the FBI's Weapons of Mass Destruction Directorate, and Chris Hassell, FBI Laboratory Director, spoke for their agency. And behind the scientists, a gallery of Justice Department investigators, researchers and prosecutors sat in chairs, providing a gallery of reactions, ranging from grimaces to grins, in response to reporter's questions over the next three hours. At certain points, Taylor also answered questions about the investigation, some of it news to the scientists, as well as the reporters.

"After nearly seven years of investigation we have developed a body of powerful evidence that allows us to conclude that we have identified the origin and the perpetrator of the 2001 bacillus anthracis mailings," Majidi said in a opening statement. He then took questions.

FBI STATEMENT: Science Briefing on the Anthrax Investigation

Reporters first asked the scientists to lead them through the studies that produced the four genetic mutations that served as markers to the attack anthrax, starting with a 2002 study in Science led by Fraser-Liggett. This research established that it might be possible to determine an exact genetic match to the attack anthrax, something never attempted before. By 2004, Majidi said that researchers had uncovered two genetic markers that tied the anthrax from the letters to anthrax originating in a collection called RMR-1029 held at USAMRIID. They collected Ivins' flask and took samples of the RMR-1029 it contained, finding a match to the attack anthrax.

RMR-1029, it turns out, was an unusual collection of spores, created in batches of growths from the original "wild type" Ames anthrax collected from a Texas calf that ate contaminated grass (anthrax is fond in the wild) and died in 1981. Researchers at the Army's Dugway Proving Grounds started the collection with 13 "production runs" of anthrax growth, followed by Ivins adding another 22 runs, to create 164 liters of spores in 1997. That collection, dubbed RMR-1029, was concentrated by Ivins down to two flasks by 2001, and to a one-liter flask by 2004.

Keim suggested that such a large number of growths, in which one colony might hold a trillion generations of anthrax spores, offered a larger-than-normal opportunity for mutations to crop up.

From 2002 onward, the FBI had requested samples of Ames anthrax held by 16 U.S. labs, and others in Canada, Sweden and the United Kingdom, to create a collection of 1,070 Ames "isolates." At the same time, the bureau sent Ravel's lab blind copies of anthrax samples to search for distinct genetic mutations that might set the attack anthrax apart from the wild type.

Most bacteria colonies just look like splotches of gunk grown on test-tube material. Some, less than 1% of attack anthrax colonies, had unusual rough edges, Majidi says. Sequencing the genomes of these aberrant colonies revealed a number of mutations, says Ravel, that were distinct to the attack anthrax.

"That's what took so long," Ravel says in a post-discussion interview. Sequencing the genomes of at least seven aberrant colonies took three months at least, longer for high-quality genomes. And they had to be done one at a time in the lab, to prevent cross-contamination that would hinder their use as evidence in court someday. "This was science at the very highest level, the most precision. We learned a great deal from the investigators," Ravel says. "We always knew that someone was looking over our shoulders and would want to see our lab notebooks someday."

A separate "red team" of scientist-consultants selected four "insertion and deletion" mutations, places where genes had been permanently removed from a genome, that were distinct to colonies from the attack anthrax. Those were used in a test that by late 2006 had assessed all 1,070 samples in the FBI repository. Deleted genes, thousands in some cases, wouldn't just reappear in a genome, Ravel says, giving scientists a great deal of confidence in their use as markers. Only eight isolates, either from USAMRIID or one other unnamed lab, had the four markers.

At the FBI discussion, many questions surrounded the presence of silicon, the chief ingredient in sand, in the attack anthrax spores. In the past, some news stories had taken reports of sand or other additives as a sign of complex weaponization of the anthrax spores. Instead, the silicon appears "natural," said Joseph Michael of Sandia National Laboratories in Albuquerque, New Mexico, who led an electron microscope analysis of the spores. One of at least two batches of the attack spores apparently collected silica on their inner skins as they grew, which happens to bacteria sometimes.

Majidi said that Post Office sorting machines crushed the dried anthrax in mailing envelopes, making them very powdery. Another panel scientist, James Burans of the National BioForensic Analysis Center, added that the curious dispersion of the anthrax spores into the air partly resulted from them picking up an electric charge, static electricity, in their travels through the postal system, which made the dried spores repel one another as soon as their envelopes were opened.

The discussion wrapped up with a description of the original samples of RMR-1029 given to investigators by Ivins in 2002. After discussing with investigators the proper procedure for preparing samples they had to be presented in slants, test tubes tilted to the side to grow evenly-spaced bacterial colonies and stopped appropriately Ivins gave RMR-1029 samples that didn't meet the protocol, Majidi said. One of those samples was sent to Keim's lab and the FBI destroyed the other. Ivins later gave another sample under subpoena in April 2002, one investigators realized was bogus two years later. Majidi called this behavior "questionable." The FBI search warrants had labeled it deceptive.

In the end, Majidi and others acknowledged that the science results could only tell part of the story. The researchers hope to publish a collection of related studies in a journal in coming months, says Ravel. But that will still leave questions for prosecutors to answer. "I don't think we're ever going to put all the suspicions to rest," Majidi says. "There's always going to be a spore on a grassy knoll."

Contributing: Donna Leinwand