Genetic Evidence of Yersinia pestis from the First Pandemic

Abstract

Background/Objectives: The Plague of Justinian marked the beginning of the First Pandemic (541-750 CE), yet no genomic evidence of Yersinia pestis has previously been recovered from the Eastern Mediterranean, where the outbreak was first recorded. This study aimed to determine whether Y. pestis was present in a mid-6th to early 7th century mass grave in Jerash, Jordan, and to characterize its genome within the broader context of First Pandemic strains. Methods: We analyzed samples from multiple individuals recovered from the Jerash mass grave. Initial screening for potential pathogen presence was conducted using proteomics. Select samples were subjected to ancient DNA extraction and whole genome sequencing. Comparative genomic and phylogenetic analyses were conducted to assess strain identity and evolutionary placement. Results: Genomic sequencing recovered Y. pestis DNA from five individuals, revealing highly similar genomes. All strains clustered tightly with other First Pandemic lineages but were notably recovered from a region geographically close to the pandemic's historical epicenter for the first time. The near-identical genomes across diverse individuals suggest an outbreak of a single circulating lineage at the time of this outbreak. Conclusions: This study provides the first genomic evidence of Y. pestis in the Eastern Mediterranean during the First Pandemic, linking archaeological findings with pathogen genomics near the origin point of the Plague of Justinian. Summary Sentence: Genomic evidence links Y. pestis to the First Pandemic in an ancient city.

Keywords: First Pandemic; Jerash; Justinianic Plague; Plague; Yersinia pestis; ancient DNA.

Figure 1

Jerash mass grave site in the context of the First Pandemic. (A). Regions of aDNA isolated to date prior or at the First Pandemic are marked in dark brown (around First Pandemic). The Jerash site is marked by a star. The Eastern Roman Empire (indicated in dark red) historically documented the plague spreading from North Egypt and the Near East through Mediterranean regions. Yellow dots in dark brown regions denote the current available genetic evidence of Y. pestis recovered from Western European cemeteries. The single Tian Shan Hun Strain (yellow dot) in Central/Western Asia is hypothesized to be one of the early strains present prior to the onset of the Plague of Justinian. (B). Jerash (Gerasa) hippodrome ground plan. The schematic of the ground plan was redrawn based on the illustration of Anton Ostrasz. The mass graves were uncovered in the abandoned Roman hippodrome chambers W2 and W3. (C). One of the chambers (W2) where the bodies were recovered.

Figure 2

Archaeological, proteomic, and genetic evidence of an ancient epidemic in Jerash. (A). Archaeological excavation records narrow the mass grave timeframe. The abandoned Roman Hippodrome site was used as a mass grave site around the time of the Plague of Justinian. (B). Proteomics analysis identified potential peptides from Y. pestis. At least three unique pathogen peptides passing quality control in experimental and analysis protocols were required. (C). Summary of samples that yielded proteomics, stable isotopes of oxygen, and ancient DNA (aDNA) results. Y. pestis-positive proteomics samples are colored in purple. For three individuals, two independent aDNA samples were generated from two teeth of the same individual.

Figure 3

Genome reconstruction and analysis of Y. pestis in Jerash. (A). Whole genome capture recovered the same Jerash Y. pestis strain from different victims of the mass grave. Average nucleotide identity (ANI) analysis revealed differences of equal to or less than 1 base per million among Jerash isolates, contrasting with over 30 bases per million differences in other First Pandemic-related strains like VAL001 and Tian Shan Hun. Sequencing of two teeth from each of three individuals showed no significant differences, indicated by *. (B). The Jerash strain contains many essential virulence factors for causing an epidemic. A set of virulence factor genes from prehistorical, First Pandemic, and Second Pandemic (Black Death) strains are shown. Many prehistorical strains lack the flea transmission-related gene Ymt, while RT5/RT6 does possess the Ymt gene. Both Tian Shan Hun and Jerash strains possess the Ymt gene.

Figure 4

Phylogenetic confirmation of First Pandemic plague strains. The maximum likelihood phylogenetic reconstruction of Y. pestis strains from the First Pandemic. Tree topology bootstrapping was performed with 1000 replicates to assess the robustness of the inferred relationships. The Jerash strain groups with other First Pandemic strains and their branch lengths are represented. Dots along the branches indicate bootstrap support values greater than 80%, providing strong confidence in those relationships. Collapsed branches are shown with triangles. The Jerash strain is highlighted in red, while the Tian Shan Hun strain is shown in dark red, emphasizing their positions within the phylogeny relative to other strains from the same period.