Yersinia pestis Lineages in Mongolia

BACKGROUND: Whole genome sequencing allowed the development of a number of high resolution sequence based typing tools for Yersinia (Y.) pestis. The application of these methods on isolates from most known foci worldwide and in particular from China and the Former Soviet Union has dramatically improved our understanding of the population structure of this species. In the current view, Y. pestis including the non or moderate human pathogen Y. pestis subspecies microtus emerged from Yersinia pseudotuberculosis about 2,600 to 28,600 years ago in central Asia. The majority of central Asia natural foci have been investigated. However these investigations included only few strains from Mongolia. METHODOLOGY/PRINCIPAL FINDINGS: Clustered Regularly Interspaced Short Prokaryotic Repeats (CRISPR) analysis and Multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) with 25 loci was performed on 100 Y. pestis strains, isolated from 37 sampling areas in Mongolia. The resulting data were compared with previously published data from more than 500 plague strains, 130 of which had also been previously genotyped by single nucleotide polymorphism (SNP) analysis. The comparison revealed six main clusters including the three microtus biovars Ulegeica, Altaica, and Xilingolensis. The largest cluster comprises 78 isolates, with unique and new genotypes seen so far in Mongolia only. Typing of selected isolates by key SNPs was used to robustly assign the corresponding clusters to previously defined SNP branches. CONCLUSIONS/SIGNIFICANCE: We show that Mongolia hosts the most recent microtus clade (Ulegeica). Interestingly no representatives of the ancestral Y. pestis subspecies pestis nodes previously identified in North-western China were identified in this study. This observation suggests that the subsequent evolution steps within Y. pestis pestis did not occur in Mongolia. Rather, Mongolia was most likely re-colonized by more recent clades coming back from China contemporary of the black death pandemic, or more recently in the past 600 years

A case series of fatal meningoencephalitis in Mongolia: epidemiological and molecular characteristics of tick-borne encephalitis virus

In Mongolia, the incidence and fatality rates of tick-borne encephalitis (TBE) have been increasing. We aimed to identify the epidemiological and molecular characteristics of tick-borne encephalitis virus (TBEV) associated with fatal meningoencephalitis in Mongolia. We conducted a descriptive study of 14 fatal cases of TBE that occurred between 2008 and 2017 in Mongolia. Reverse transcription polymerase chain reaction (RT–PCR) was used to detect viral RNA in brain tissue. RT–PCR products from six patients who died from TBE between 2013 and 2017 were directly sequenced and analysed phylogenetically. Ticks collected from Selenge and Bulgan provinces were also tested for TBEV by RT–PCR. Between 2008 and 2017, there were 14 fatal TBE cases in hospitals in Mongolia. The 14 patients who died reported receiving tick bites in Bulgan or Selenge province; 71.4% of deaths resulted from tick bites in Bulgan province. The TBE case fatality rate was 28.6% for patients in Bulgan province and 2.7% for those in Selenge province. All of the fatalities were men; the median age was 45 ± 12.6 years. Tick bites occurred between April and June in forested areas. In 2013, a 388 base pair fragment of the envelope (E) gene was obtained from a hospitalized patient. The closest relatives of this virus are Far-Eastern TBEV isolates. The case fatality rate differed between two provinces where tick bites occurred. A higher number of TBE cases and the virulent Far-Eastern subtype occurred in patients in Bulgan province. This province should increase vaccination coverage, training, education and investigations

MLVA clustering and SNP branch assignment of 68 previously published <i>Y. pestis pestis</i> branches 1 and 2.

<p>Sixty-eight strains from the 1 and 2 branches previously investigated by both MLVA25 and SNP analysis are displayed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Li1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Morelli1" target="_blank">[6]</a>. For completion, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone-0030624-t001" target="_blank">Table 1</a> gives further information about assignment of biovar, genotype, and origin. Colors reflect MLVA clustering as suggested by Li et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Li1" target="_blank">[5]</a>. The SNP branch assignment of each strain as defined by Morelli et al. is indicated (column <i>Morelli2010</i>) together with the strain ID and biovar designation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Morelli1" target="_blank">[6]</a>. Bootstrap support values are indicated for each node. The results of CRISPR analysis according to Cui et al. are given in column <i>group </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Cui1" target="_blank">[18]</a>. * This strain shows a Medievalis phenotype due to a different mutation in the napA gene compared to the mutation causing the Medievalis phenotype in the Medievalis biovar, as demonstrated by Pourcel et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Pourcel1" target="_blank">[13]</a>.</p

MLVA25 assignment of four clusters of the investigated Mongolian <i>Y. pestis</i> strains.

<p>MLVA25 tree of 16 investigated Mongolian <i>Y. pestis</i> strains (marked with color and boxes) representing four of the 6 clusters, and various <i>Y. pestis</i> strains originating from <i>microtus</i> and <i>pestis</i> biovars. For each strain, the tentative SNP branch or node according to Morelli et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Morelli1" target="_blank">[6]</a> as deduced by the presence of a linking strain in the same MLVA cluster is indicated by a question mark. Strain name, CRISPR profile as investigated in this study, and the sampling site (Focus) are listed.</p

MLVA clustering and SNP branch assignment of 66 previously published <i>Y. pestis microtus</i> and <i>pestis</i> 0, 1 and 3 branches.

<p><i>Microtus</i> and strains from the 0 and 1 branches so far investigated by MLVA25 and by SNP analysis are shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Li1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Morelli1" target="_blank">[6]</a>. Three Ulegeica, two Hissarica and nine Altaica strains not investigated by SNP analysis are also included. For completion, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone-0030624-t001" target="_blank">Table 1</a> gives further information about assignment of biovar, genotype, and origin. Colors reflect MLVA clustering as suggested by Li et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Li1" target="_blank">[5]</a>. The SNP branch assignment of each strain as defined by Morelli et al. is indicated (column <i>Morelli2010</i>) together with the strain ID and biovar designation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Morelli1" target="_blank">[6]</a>. The results of CRISPR analysis according to Cui et al. are shown in column <i>group </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Cui1" target="_blank">[18]</a>. Bootstrap support values are indicated. The figure shows the satisfying terminal branches clustering achieved by MLVA but the sometimes incorrect and usually low bootstrap values of deep branching nodes illustrating the complementarity of the two methods.</p

CRISPR genotypes.

<p>CRISPR genotypes.</p

Overview of <i>Y. pestis</i> subspecies, biovar, genotype, and natural foci as suggested by different authors [4], [5], [6], [7], [18], and as deduced in this study.

<p>*abbreviations as defined by Achtman et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Achtman1" target="_blank">[7]</a> and Morelli et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Morelli1" target="_blank">[6]</a>: PE – pestoides (<i>microtus</i>), ANT – Antiqua, IN – Intermedium, ORI – Orientalis, and MED – Medievalis; Intermedium in Morelli et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Morelli1" target="_blank">[6]</a> has not the same meaning as intermedium defined by Li et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Li1" target="_blank">[5]</a> which refers to Rhamnose positive <i>Y. pestis pestis</i> isolates.</p><p># prefix refers to foci as described by Anisimov et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Anisimov1" target="_blank">[4]</a>. Numbers without # refer to Mongolian foci as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone-0030624-g004" target="_blank">Figure 4</a>.</p

CRISPR spacer signatures.

<p>*<b>bold print:</b> first described in this study.</p

MST tree of the main Mongolian clade.

<p>MST tree of the 78 Mongolian <i>Y. pestis</i> strains, determined as 3.ANT genotype and showing CRISPR spacer b48 (blue). The seven strains previously described by Li et al. and associated to the Mongolian cluster were included (yellow) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Li1" target="_blank">[5]</a>, suggesting distinct clustering. Numbers refer to the sampling sites given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone-0030624-g004" target="_blank">Fig. 4</a>.</p

MLVA25 assignment of two clusters of the Mongolian <i>Y. pestis</i> strains.

<p>MLVA25 tree of two clusters comprising 84 investigated Mongolian <i>Y. pestis</i> strains (marked with color and boxes) compared to various previously typed <i>Y. pestis</i> strains. For each strain, the tentative SNP branch or node according to Morelli et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030624#pone.0030624-Morelli1" target="_blank">[6]</a> as deduced by the presence of a linking strain in the same MLVA cluster is indicated by a question mark. Strain name, CRISPR profile as investigated in this study, and the sampling site (Focus) are listed.</p