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Anthrax Genome From Autopsy Samples Offers Insights Into 1970s Soviet Biowarfare Program

NEW YORK (GenomeWeb) – Researchers from the US, Argentina, and Italy have sequenced Bacillus anthracis samples linked to an accidental anthrax outbreak at a military facility in the former USSR, demonstrating that the strain had not undergone any obvious genetic engineering.

"While it is known that Soviet scientists had genetically manipulated Bacillus anthracis with the potential to evade vaccine prophylaxis and antibiotic therapeutics, there was no genomic evidence of this from the Sverdlovsk production strain genome," senior author Paul Keim, a microbial genetics, genomics, and pathogen genomics researcher affiliated with Northern Arizona University and the Translational Genomics Research Institute (TGen), and his co-authors wrote.

A cloud of B. anthracis spores was unwittingly released during air filter maintenance at an anthrax spore-producing facility dubbed "Compound 19" in the Russian city of Sverdlovsk (now called Ekaterinburg) in 1979 — years after a 1975 Biological Weapons Convention prohibited efforts to weaponize pathogens such as anthrax or actively produce spores for offensive purposes. Dozens of people died in the resulting outbreak.

To characterize this B. anthracis strain in a global context, the team did deep sequencing on two autopsy samples from individuals who died in the outbreak. The draft genome sequence, which was subsequently compared with strains from the main B. anthracis clades, was similar to wild-type Russian strains and fell into a clade with strains used in two Asian live vaccines for anthrax. The findings appeared online today in mBio.

"This work provides insights into the world's largest biological weapons program and provides an extensive B. anthracis reference," Keim said in a statement. "We now know what the genome for this strain looks like, so if it ever occurs again, we will be able to pinpoint that this originally came from the Soviet weapons program."

The researchers used Illumina's MiSeq and HiSeq instruments to do deep metagenomic sequencing on formalin fixed, paraffin-embedded (FFPE) autopsy samples from two of the 66 people who died during the Sverdlovsk outbreak.

When they mapped the tens of billions of resulting sequence to the genome of the so-called Ames strain, which was behind several mail attacks in the US in 2001, they found that it covered more than 99 percent of the genome at an average depth of 24-fold.

The team then compared the draft genome with a phylogenetic map of the Ames strain and almost 200 more previously characterized B. anthracis isolates. Based on patterns at nearly 12,000 SNPs, the group defined relationships between strains in the three main clades, which could be further sub-divided into several sub-clades.

The Sverdlovsk genome fell into a B. anthracis A sub-clade known as the Trans-European radiation, neighbouring the Russian Tsiankovskii vaccine strain and another strain used in a live vaccine. The results are consistent with past reports that the strain being produced by the Soviet military in the 1960s and 70s was originally isolated from nature, the team reported.

From the available sequence data, the study's authors did not find evidence of genetic engineering in the strain, though they noted that genome was not complete and contained some errors related to FFPE storage of the samples.

"A higher than normal error rate was observed in the Sverdlovsk samples, likely due to the nature of the specimen preservation, but sufficient depth of coverage was still obtained to accurately genotype known SNP loci and to identify strain-specific polymorphisms," they wrote.

The team argued that the genome sequence patterns were "highly suggestive of a weapons program that identified a suitable strain, maintained master cell stocks to avoid extensive passage, and performed minimal manipulations in order to maintain virulence."