Genotyping and phylogenetic analysis of Yersinia pestis by MLVA: insights into the worldwide expansion of Central Asia plague foci

Abstract

Background: The species Yersinia pestis is commonly divided into three classical biovars, Antiqua, Medievalis, and Orientalis, belonging to subspecies pestis pathogenic for human and the (atypical) non-human pathogenic biovar Microtus (alias Pestoides) including several non-pestis subspecies. Recent progress in molecular typing methods enables large-scale investigations in the population structure of this species. It is now possible to test hypotheses about its evolution which were proposed decades ago. For instance the three classical biovars of different geographical distributions were suggested to originate from Central Asia. Most investigations so far have focused on the typical pestis subspecies representatives found outside of China, whereas the understanding of the emergence of this human pathogen requires the investigation of strains belonging to subspecies pestis from China and to the Microtus biovar.

Methodology/principal findings: Multi-locus VNTR analysis (MLVA) with 25 loci was performed on a collection of Y. pestis isolates originating from the majority of the known foci worldwide and including typical rhamnose-negative subspecies pestis as well as rhamnose-positive subspecies pestis and biovar Microtus. More than 500 isolates from China, the Former Soviet Union (FSU), Mongolia and a number of other foci around the world were characterized and resolved into 350 different genotypes. The data revealed very close relationships existing between some isolates from widely separated foci as well as very high diversity which can conversely be observed between nearby foci.

Conclusions/significance: The results obtained are in full agreement with the view that the Y. pestis subsp. pestis pathogenic for humans emerged in the Central Asia region between China, Kazakhstan, Russia and Mongolia, only three clones of which spread out of Central Asia. The relationships among the strains in China, Central Asia and the rest of the world based on the MLVA25 assay provide an unprecedented view on the expansion and microevolution of Y. pestis.

Figure 1. Dendrogram based on the 25 VNTR loci.

Clustering analysis was done using the categorical distance coefficient and the Neighbor-Joining clustering method. The Y. pseudotuberculosis representative (blue dot) was chosen as outgroup to root the tree. Red dot, Angola isolate. Red arrows, Y. pestis subsp. pestis isolates with exceptional genotypes. The branches color code is as indicated in legends from Figure 2 and Figures S1, S2, S3.

Figure 2. The Y. pestis bv. Microtus isolates, dendrogram based on the 25 VNTR loci.

From left to right, the columns designate the strain Id, focus of origin, biovar (bv.), subspecies (subsp.), geographic origin (location), host or vector, genomovar based on DFR analysis . The biovar or subspecies designation follows current usage, with inconsistencies in terms of a future nomenclature since “biovar Microtus” contains a number of “subspecies”. The ‘genomovar+DFRX’ and ‘genomovar-DFRX’ respectively indicates that the strain is similar to this genomovar except for DFRX which was present or absent. The branches color code reflects the focus of origin. Five atypical isolates corresponding to essentially two strains fall into two very long and loosely connected branches (red rectangles). At least three were derived from patients.

Figure 3. The close relationship among the foci in China and Central Asia.

The color code reflects some significant genetic relationships as indicated by MLVA clustering. Orange, the bv. Microtus investigated here, including subspecies caucasica (4), ulegeica (BP), and the most closely related hissarica (34) altaica (36), xilingolensis (L) and qinghaiensis (M). Red foci, Y. pestis subsp. pestis biovar Medievalis. Purple foci, bv. Orientalis foci. Other colors, different varieties of Y. pestis subsp. pestis bv. Intermedium and Antiqua strains. The more detailed composition of each focus is presented in Figures 2 and Figures S1, S2, S3.