Forensic Genomics and the 2001 Anthrax Attacks

Seven anthrax-laced letters were sentbetween Sept 18 - Oct 9, 2001.

Targets included news outlets in NY andFL and two US senators.

22 people were infected and 5 died.

Decontamination costs ≥ $250 million.

The FBI-led investigation lasted 7 yearsand involved over 10,000 interviews and5,750 subpoenas.

No one was ever charged, as the main suspect, Bruce Ivins, a USAMRIIDmicrobiologist, committed suicide in July 2008.

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Anthrax is caused by a rod-shaped bacterium

Bacillus anthracis forms highly resistantspores that cause infection when inhaled oringested.

Anthrax spores are found globally andinfect humans and cattle.

Human infections can be pulmonary,gastrointestinal or cutaneous.

Even with treatment, the mortality rate forpulmonary anthrax ≥ 50%.

B. anthracis has a single circular genome containing over 5 million bp.However, there is very little genetic variation: the most distantly-relatedstrains share > 99.99% nucleotide identity.

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The Ames strain was used in the 2001 attacks

B. anthracis Ames was isolated in Texas from a dead heifer.

This isolate was acquired by USAMRIID in 1981 and selected asa challenge strain for vaccine studies.

All Ames strain anthrax cultures used in laboratory research weresourced from the USAMRIID material (Ames ancestor).

Multi-locus VNTR analysis showed that the anthrax used in theattacks was indistinguishable from the Ames strain.

Additional genetic analyses of this material found no evidence thatthe anthrax had been genetically engineered, e.g., no antibioticresistance genes were present.

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Multiple colony morphotypes were present in the attack letters

Analysis of the anthrax samples from the NYP, Daschle and Leahyletters revealed four variants differing in colony morphology and color.

Sporulation was reduced in all four morphotypes.

These morphotypes were stable under cultivation.

Morphotypes A, B, and E were isolated from multiple letters.

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Morphotype A is associated with a tandem duplication

Initial comparison of the assembled genome sequences of morphotypeA and the Ames Ancestor identified no genetic differences.

Careful examination of the morphotype A reads revealed amisassembled tandem repeat region.

Re-assembly of the morphotype A genome identified a 2023-bprepeat.

Analysis of the NYP draft genome identified a 2607-bp repeat in thesame region.

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Morphs B-E are associated with variants in the spo0F pathway

Morphotype B samples all have a unique non-synonymoussubstitution in the spo0F gene.

Morphotypes C/D are associated with a nonsense mutation ordeletion in a sensor histidine kinase gene.

Morphotype E is associated with a 9- or 21-bp deletion in aplasmid-encoded phosphatase gene.

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Colony morphotypes in other Ames strain stocks

The FBI created a repository of anthrax samples obtained from 20laboratories known to work with the Ames strain.

1059 samples were screened for the presence of genotypes associatedwith morphotypes A1/A3, D, and E.

8 samples were positive for all four genotypes: 7 of these werefrom a flask (RMR-1029) at USAMRIID and one was from anotherlaboratory which had recently obtained its stock from USAMRIID.

Bruce Ivins was one of several scientists and technicians at Ft.Detrick with access to RMR-1029.

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Conclusions of the NAS review committee (Feb 2011):

“It is not possible to reach a definitive conclusion about theorigins of the B. anthracis in the mailings based on theavailable scientific evidence alone.”

“The results of the genetic analyses of the repository sampleswere consistent with the finding that the spores in the attackletters were derived from RMR-1029, but the analyses did notdefinitively demonstrate such a relationship.”

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References

Rasko, D. A. et al. (2011) Bacillus anthracis comparative genomeanalysis in support of the Amerithrax investigation. PNAS 109:5027-5032.

National Research Council (2011) Review of the Scientific ApproachesUsed During the FBI’s Investigation of the 2001 Anthrax Letters. TheNational Academies Press.

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