Based on phylogenetic analysis of whole-genome sequencing of Yersinia pestis 2.MED1 strains of the medieval biovar, combined with epizootic, epidemiological, and climatic data over a 100-year period, we have reconstructed the most probable directions of distribution of plague in Eastern Europe and Central Asia (EECA) in the 20th and 21st centuries. The data suggest the important role of three great lakes—the Caspian, Aral, and Balkhash—in the circulation and preservation of Y. pestis 2.MED1 in EECA. Three main directions of Y. pestis 2.MED1 expansion have been identified: Caspian (Caspian Sea region foci, 1912–1945; Caucasus, 1953–1986), North Aral (Northern Aral Sea region foci, 1945–1959; Caspian Sea region foci, 1945–2015; Pre-Caucasus, 1999–2003; Karakum, 1949–1965) and Central Asian (Kyzylkum, 1924, 1983–2020; Balkhash foci, 1939–2020; Northern Aral Sea region foci, 1967–2020; Eastern Caspian Sea region foci, 1968–1985). Favorable climatic conditions in the Caspian Sea region, the Northern Aral Sea region, and the Balkhash region in the 20th and 21st centuries contributed to the rapid formation of stable natural plague foci and the long-term persistence of Y. pestis 2.MED1 strains of the medieval biovar, with their further introduction into other foci of EECA. Periodic introductions of the pathogen are one of the reasons for the plague re-emergence and activation of plague foci in the EECA region.

Multilocus variable number tandem repeat analysis (MLVA) is one of the highest-resolution methods for subtyping genetically monomorphic pathogens of especi

The aim of the work is a retrospective molecular examination of plague outbreaks that occurred on the northern coast of the Aral Sea in the middle of the past century, and a phylogenetic analysis of the Yersinia pestis strains that caused them. Materials and methods. Whole-genome sequences of 39 Y. pestis strains from foci of the Northern Aral Sea region, isolated in 1945–1974, were used. Whole-genome sequencing was performed in the Ion S5 XL System (Thermo Fischer Scientific, USA). Data processing and sequence assembly were carried out using the Ion Torrent Suite software package, v5.12, FastQC v0.12.0, unicycler v0.5.0. Phylogenetic reconstruction was carried out using the wholegenome SNP analysis method, the dendrogram was constructed using the Maximum Likelihood algorithm using the PhyML v3.1 program. Molecular typing of strains was performed using the MLVA25 method. Results and discussion. Y. pestis strains isolated in the Northern Aral Sea region between 1945 and 1974 belong to the phylogenetic branch 2.MED1 of the medieval biovar of the main subspecies. Plague outbreaks in the Northern Aral Sea region in 1945, 1955, 1966, and 1969 were caused by strains of the Caspian subbranch 2.MED1, and the outbreak of 1967 – by strains of the Central Asian subbranch, which successively reached this region from the Northern Caspian and Balkhash regions. It is shown that the sources of human infection were epizootics occurring on the northern coast of the Aral Sea. It is established that in the 1960s, Y. pestis strains of the Caspian and Central Asian subbranches 2.MED1 medieval biovar circulated simultaneously in the Northern Aral Sea region. A characteristic feature of the plague outbreak of 1945 was the presence of a large number of SNPs specific to each strain, possibly due to the process of rapid adaptation of the Caspian subbranche 2.MED1 strains to the conditions of the natural biocenosis of the Northern Aral Sea region. Subsequently, the rate of evolution of the emerging Northern Aral population 2.MED1 significantly decreased, which was accompanied by the formation of individual clusters of strains with a small number of individual SNPs.

Multilocus analysis of variable number of tandem repeats, MLVA, is one of the high-resolution methods for typing genetically monomorphic pathogens of particularly dangerous infections. High efficiency in genotyping Yersinia pestis strains was achieved using MLVA25 method of typing by 25 VNTR loci, which is widely used in molecular-epidemiological studies. However, the issue of the effectiveness of different VNTR loci in MLVA25 analysis of Y. pestis strains of medieval biovar of the main subspecies, distributed in the majority of natural plague foci in Eastern Europe and Central Asia, remains open. The aim of this work was to evaluate the efficiency of different VNTR loci and optimize the MLVA25 method for differentiation of Y. pestis strains of the medieval biovar from plague foci of Russia and other CIS countries located in Eastern Europe and Central Asia. Material and Methods. This study analyzed the whole-genome sequences of 220 Y. pestis strains of medieval biovar of the main subspecies and 31 Y. pestis strains of other phylogenetic lineages. The sequences of VNTR loci of those strains were entered into the Bionumerics 7.6.3 program for subsequent phylogenetic analysis by the UPGMA method. Results. MLVA25 genotypes of 251 Y. pestis strains under study have been determined. According to the results of statistical analysis of the loci, 126 individual genotypes are detected in the strains of medieval biovar, which corresponds to the discriminatory power of the typing system (DP=0.98). The effective loci with high discriminatory power, which have the greatest influence on the differentiation of strains of medieval biovar by genotypes, have been identified. Conclusion. The system of MLVA typing of Y. pestis strains of medieval biovar has been optimized. It includes 12 VNTR loci.

Based on phylogenetic analysis of whole-genome sequencing of Yersinia pestis 2.MED1 strains of the medieval biovar, combined with epizootic, epidemiological, and climatic data over a 100-year period, we have reconstructed the spread of plague in Eastern Europe and Central Asia (EECA) in the 20th and 21st centuries. The data suggest the important role of three great lakes—the Caspian, Aral, and Balkhash—in the spread and preservation of Y. pestis 2.MED1 in EECA. Three waves of Y. pestis 2.MED1 spread have been identified: Caspian (Caspian Sea region foci, 1912-1945; Caucasus, 1953-1986), North Aral (Northern Aral Sea region foci, 1945-1959; Caspian Sea region foci, 1945-2015; Pre-Caucasus, 1999-2003; Karakum, 1949-1965) and Central Asian (Kyzylkum, 1924, 1983-2020; Balkhash foci, 1939-2020; Northern Aral Sea region foci, 1967-2020; Eastern Caspian Sea region foci, 1968-1985). Favorable climatic conditions in the Caspian Sea region, the Northern Aral Sea region, and the Balkhash region in the 20th and 21st centuries contributed to the rapid formation of stable natural plague foci and the long-term persistence of Y. pestis 2.MED1 strains of the medieval biovar, with their sporadic spread into other foci of EECA. Periodic introductions of the pathogen are one of the reasons for the plague re-emergence and activation of plague foci in the EECA region.