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Genomic Analysis Finds Different Plague Strains Were Behind Plague of Justinian, Black Death

NEW YORK (GenomeWeb Daily News) – The Yersinia pestis strains that caused the Plague of Justinian and the later Black Death stemmed from separate introductions of the bacteria from rodents into humans, according to a genomic analysis of Y. pestis.

As researchers led by McMaster University's Hendrik Poinar reported in the Lancet Infectious Diseases yesterday, they extracted DNA from two individuals who died during the Plague of Justinian, which raged during the sixth and eighth centuries, to recreate that strain's genome.

By comparing that strain to others, the researchers noted that the Justinian strain does not appear to have any contemporary relatives.

"The research is both fascinating and perplexing — it generates new questions which need to be explored. For example, why did this pandemic, which killed somewhere between 50 and 100 million people, die out?" Poinar, an associate professor and director of the McMaster Ancient DNA Centre, said in a statement.

At least three waves of plague — the Plague of Justinian, the Black Death, and another outbreak dating to the 19th and 20th centuries — have caused human pandemics, but Poinar and his colleagues noted that it was unknown whether it was the same strain or different strains of Y. pestis that caused the outbreaks.

Poinar and his colleagues extracted DNA from the teeth of two Justinian plague victims — radiocarbon dating placed the pair as living around 533 AD and 504 AD. Using a one-million Agilent SureSelect DNA capture array, the researchers targeted the core Y. pestis chromosome, three primary plasmids, and 155 Y. pestis genes identified in other strains that are not housed on the core chromosome. They then sequenced the library on the Illumina HiSeq 1500 to produce some 684,000 reads of an average 51.8 base pairs in length that they then mapped to the reference genome.

Based on these Justinian strains and some 130 other plague strains along with Y. pseudotuberculosis as an outgroup, the researchers constructed a maximum likelihood phylogenetic tree. The Justinian branch, they noted, is placed far from the strains that caused the Black Death and the pandemic in the 19th and 20th centuries. Closer by are two strains from China, which supports the theory that the outbreak originated in China, rather than in Africa, and spread to Europe along trade routes like the Silk Route.

The researchers also generated a second phylogeny with the added constraint that the Justinian strain was direct ancestor of the strains that caused the second and third pandemics. The first maximum likelihood tree, however, was a better fit than the second, indicating that the Justinian plague was a separate emergence of Y. pestis than the one causing the Black Death and the 19th and 20th century pandemic.

By contrast, the Black Death strain is placed at the base of a branch that includes that later third pandemic strain, suggesting the Black Death strain gave rise to the other pandemic strain. The strains on that branch include ones that are present in modern-day rodents in Africa and China.

In addition, the researchers found that there is a large variation in the length of the branches making up the phylogenetic tree, but that variation does not correlate with sampling time. For instance, they noted that the Justinian strain lineages are about the same distance from the root of the tree as the Black Death strain, despite the Black Death coming along some 800 years after the Justinian Plague.

Using a Bayesian Markov chain Monte Carlo analysis, Poinar and his colleagues endeavored to estimate the rate of nucleotide substitution or time to common ancestry. However, they found that Y. pestis evolution is marked by large rate variations that they suspect might be due to changes in bacterial generation times during epidemic and endemic cycles, changes in selective pressures, or a combination of the two.

Without a timescale for Y. pestis evolution, the investigators said that they could not determine whether the Plague of Athens in 430 BC or the Antonine Plague of between 165 AD and 180 AD were due to novel Y. pestis strains or strains linked to other plagues.

Before the Plague of Justinian died out, it appeared to have a high mortality rate — some estimates suggest a 40 percent mortality rate. By examining the genome of the bug, the researchers found that it contained a number of SNPs that may partially account for that virulence.

For instance, within the two samples, Poinar and his colleagues identified 23 non-synonymous unique chromosomal or plasmid SNPs, and two of those were within the known virulence factors Ail and YopJ genes. Ail, the researchers noted, is an outer membrane protein linked to promoting bacterial resistance to complement-mediated killing and adhesion to host cells, while YopJ is an effector protein with acetyltransferase activity that is secreted into host cells.

In addition, they reported that the Justinian strain, like the Black Death strain, appears to have included an approximately 15 kb genomic island, dubbed DFR4, that is home to a number of virulence-related genes like ccm2a, which encodes a multidrug ABC transporter ATPase. Other strains of plague have lost this island, the researchers added.

To fully work out the role of those SNPs and genomic island in virulence, more ancient plague genomes will need to be sequenced, Poinar and his colleagues said.

Still, their findings indicate that multiple Y. pestis strains have caused widespread epidemics.

"We know the bacterium Y. pestis has jumped from rodents into humans throughout history and rodent reservoirs of plague still exist today in many parts of the world. If the Justinian plague could erupt in the human population, cause a massive pandemic, and then die out, it suggest it could happen again," said first author Dave Wagner, an associate professor in the Center for Microbial Genetics and Genomics at Northern Arizona University, in a statement. "Fortunately we now have antibiotics that could be used to effectively treat plague, which lessens the chances of another large scale human pandemic."