New Genotype of Yersinia pestis Found in Live Rodents in Yunnan Province, China

Abstract

Yunnan Province, China is thought to be the original source of biovar Orientalis of Yersinia pestis, the causative agent of the third plague pandemic that has spread globally since the end of the 19th century. Although encompassing a large area of natural plague foci, Y. pestis strains have rarely been found in live rodents during surveillance in Yunnan, and most isolates are from rodent corpses and their fleas. In 2017, 10 Y. pestis strains were isolated from seven live rodents and three fleas in Heqing County of Yunnan. These strains were supposed to have low virulence to local rodents Eothenomys miletus and Apodemus chevrieri because the rodents were healthy and no dead animals were found in surrounding areas, as had occurred in previous epizootic disease. We performed microscopic and biochemical examinations of the isolates, and compared their whole-genome sequences and transcriptome with those of 10 high virulence Y. pestis strains that were isolated from nine rodents and one parasitic flea in adjacent city (Lijiang). We analyzed the phenotypic, genomic, and transcriptomic characteristics of live rodent isolates. The isolates formed a previously undefined monophyletic branch of Y. pestis that was named 1.IN5. Six SNPs, two indels, and one copy number variation were detected between live rodent isolates and the high virulence neighbors. No obvious functional consequence of these variations was found according to the known annotation information. Among genes which expression differential in the live rodent isolates compared to their high virulent neighbors, we found five iron transfer related ones that were significant up-regulated (| log₂ (FC) | > 1, p.adjust < 0.05), indicating these genes may be related to the low-virulence phenotype. The novel genotype of Y. pestis reported here provides further insights into the evolution and spread of plague as well as clues that may help to decipher the virulence mechanism of this notorious pathogen.

Keywords: Yersinia pestis; iron content; live rodent; low virulence; pathogenicity.

FIGURE 1

Geographic locations of the new isolates in Heqing County and neighboring Lijiang City. Red circles indicate the locations of the newly sequenced isolates described in this study. The size of the yellow circle indicates the scope of the epidemiological investigation. The map was created using ggmap (Kahle and Wickham, 2013) based on the public geographical data downloaded from OpenStreetMap (http://openstreetmap.org).

FIGURE 2

Liver section from Heqing County live rodents positive for bacterial culture. (A), Lymphocyte infiltration in the portal area (a) and hepatocyte cords (b). (B) Hepatic sinusoids (c) were significantly dilated and congested and there was patchy necrosis of hepatocytes (d).

FIGURE 3

Phylogenetic tree of 388 Y. pestis genomes and phylogeny of the new 1.IN5 phylogroup. (A) Maximum likelihood phylogenetic tree for Y. pestis consisting of 368 public genomes and 20 newly sequenced isolates. The tree was built using PhyML. A total of 5,458 polymorphic sites were used to construct the tree, which was visualized and edited using FigTree. Main branches/sub-branches were collapsed for clarity. (B) Minimum spanning phylogeny of 20 Y. pestis strains in Heqing County (HQ) and Lijiang City (LJ), based on 52 SNPs. The labels are the strain IDs. Red circles indicate the LJ isolates; blue circles indicate the HQ isolates; white circle indicates the outgroup strain.

FIGURE 4

Distribution of SNPs, indels, and CNVs in the genomes of Heqing County (HQ) and Lijiang City (LJ) strains. The LJ935 genome was used as the reference. Dotted frames indicate different variations; red letters indicate the base sequences of the variation; gray numbers indicate the variation sites.

FIGURE 5

Differentially expressed genes (DEGs) between live rodent isolates (HQ) and the high virulence isolates from wild rodents (LJ). (A) Volcano plots of the DEGs between the HQ and LJ isolates. Red and blue spots indicate the DEGs; black spots indicate genes that were not differentially expressed. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation of the DEGs. The top 14 enriched pathways are shown; pathways with less than five genes are not shown.