ISSN 10227954, Russian Journal of Genetics, 2015, Vol. 51, No. 3, pp. 238–244. © Pleiades Publishing, Inc., 2015. Original Russian Text © L.M. Kukleva, N.Yu. Shavina, G.N. Odinokov, E.G. Oglodin, N.Yu. Nosov, N.A. Vinogradova, N.P. Guseva, G.A. Eroshenko, V.V. Kutyrev, 2015, published in Genetika, 2015, Vol. 51, No. 3, pp. 298–305.
GENETICS OF MICROORGANISMS
Analysis of Diversity and Identification of the Genovariants of Plague Agent Strains from Mongolian Foci L. M. Kukleva, N. Yu. Shavina, G. N. Odinokov, E. G. Oglodin, N. Yu. Nosov, N. A. Vinogradova, N. P. Guseva, G. A. Eroshenko, and V. V. Kutyrev Russian Research Antiplague Institute “Microbe,” Saratov, 410005 Russia email:
[email protected] Received April 30, 2014; in final form, July 7, 2014
Abstract—The genetic diversity of Yersinia pestis strains from the Mongolian natural plague foci has been investigated. A total of 32 strains isolated from western, eastern, and central aimaks, as well as from the terri tory of the Gobi region, have been studied. Twentyfour strains belong to the main Y. pestis subspecies, while eight belong to nonmain subspecies. There is only one strain of biovar medievalis (genovariant 2.MED1) among the strains of the main subspecies, while the rest of the subspecies belong to the biovar antiqua. Biovar antiqua strains are split into three groups. Strains from the eastern part of the country were classified as gen ovariant 2.ANT3, and those from the western and central regions were classified as genovariant 3.ANT2, which was endemic for Mongolia. One strain from the BayanUlegeiskii aimak had the rare genovariant 4.ANT. None of the strains of the biovar antiqua belonged to its ancient 0.ANT branch, which is inconsistent with the commonly accepted idea that ancient marmot’s plague agent race originates from Mongolia. Six out of eight strains of the nonmain subspecies belonged to the ulegeica subspecies, which are endemic to Mon golia, one strain belonged to the microtus group, and the last belonged to a previously uncharacterized variant of the minor subspecies. DOI: 10.1134/S1022795415010068
INTRODUCTION Natural plague foci are located in many countries, including the Russian Federation and other CIS coun tries—Kazakhstan, Tajikistan, Kyrgyzstan, Uzbeki stan, Turkmenistan, and Armenia—as well as in countries neighboring Russia—Georgia, Mongolia, and China [1]. While the phenotypic and genetic fea tures of Yersinia pestis strains circulating in Russia, other CIS countries, and China are quite well studied, the Y. pestis strains from Mongolia are less investi gated. However, in Mongolia many plague foci are active, which, together with the intensive economic and cultural relations between Russia and Mongolia, creates a real threat of the introduction of this espe cially dangerous infection into our country. There is a precedent for such an introduction; it occurred in 2012 when a Y. pestis virulent strain was introduced in the Altai Mountain foci [2]. In light of such danger, it is necessary to have a clear idea about the properties of the plague agent strains from Mongolia in order to identify them correctly in time and to determine the source and possible paths of infection introduction. The research on Y. pestis strains from Mongolia also has an obvious theoretical interest, because the most virulent marmot Y. pestis originated in Mongolia according to classical ideas about the origin of the plague agent.
According to the domestic classification, plague agent strains include the main (highly virulent and epidemically dangerous) strains and minor (selectively virulent strains with low epidemic significance)— caucasica, altaica, hissarica, and ulegeica subspecies [3, 4]. According to the foreign classification, the strains of the main subspecies are divided into three biovars—antiqua, medievalis, and eastern, and the strains of the nonmain subspecies are called the Pes toides. The biochemical activity of the Y. pestis strains of all subspecies and biovars of the main subspecies is different. The strains of the main subspecies in con trast to all minor subspecies do not ferment rhamnose and melibiose. The biovar medievalis strains do not reduce nitrates, the orientalis do not ferment glycerin, and antiqua are active for both features. The strains of altaica, hissarica, and ulegeica subspecies also do not reduce nitrates, and strains of the first two also do not ferment arabinose. Sequencing of several genomes of Y. pestis strains recently detected more significant intraspecific diver sity of plague agent than had been previously consid ered. Phylogenetic lines and corresponding genovari ants are distinguished within the subspecies and bio vars; they are separate populations of an agent existing in different landscapes and geographical areas. Thus, biovar antiqua strains belong to three phylogenetic lines: 0.ANT, 1.ANT, and 2.ANT. Strains of the ancient line 0.ANT exist in high mountain foci of Tien
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Shan and China, 1.ANT are in Africa, and 2.ANT are in Central Asia. In Eurasia strains of 2.MED line of biovar medievalis, which are also found in Africa, are widespread [5–7]. At the same time, the area of distri bution of the strains of different subspecies, biovars, and genovariants in the natural foci of Mongolia remains little explored. This knowledge may shed light on possible means of forming the modern boundaries of the plague agent area during the Y. pestis species ori gin and expand the view on the global genetic diversity of Y. pestis. The aim of the present work is to genetically char acterize Y. pestis strains from different Mongolian regions and to determine the genovariants of these strains for the detection of their place in the common scheme of intraspecies evolution of the plague antigen. MATERIALS AND METHODS Strains and culturing conditions. The investigated Y. pestis strains isolated from the territory of Mongolia in different periods of time are shown in the table. All of the strains were obtained from the state collection of pathogenic bacteria of the Russian Research Anti plague Institute, Microbe. They were cultured in broth and LB agar (pH 7.1–7.2) at 28°C for 24–38 h. The research on the culturalmorphological and biochemical properties of the strains was performed
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according to the practical guide, The Laboratory Diag nostics of Particularly Dangerous Infections [8]. Ability to ferment carbohydrates and alcohols. The enzymatic activity against rhamnose, melibiose, ara binose, and glycerin was studied in Hiss’s media con taining 1% of these substrates and 1% of Andrade indi cator. The strain was cultured for 24 h at 28°C on the LB agar plates (pH 7.2). The bacterial suspension was prepared with a concentration of 1 × 109 CFU/mL, and 0.1 mL of it was inoculated in tubes with 5 mL of Hiss’s medium. The tubes with the inoculations were incubated at 28°С. A change of the indicator color was evidence of fermentation. Detection of the denitrifying activity. The ability to reduce nitrates was studied by culturing bacteria in LB broth (pH 7.2) with 0.1% KNO3. A suspension of cells of the tested strain (in concentration 5 × 108 CFU) cultured for 18 h at 28°С was prepared. Two hundred microliters of bacterial suspension were inoculated into 4 mL of the broth with KNO3 and incubated for 72 h at 28°С following by the adding of 0.5 mL of Griess reagent. Staining of the medium (from pink to darkred) indicated the denitrifying activity of the analyzed culture. PCR analysis and detection of nucleotide sequence. To determine the Y. pestis strains of the main and non main subspecies via multilocus PCR, the following primers were used:
89S AATCAAATCTCGCCCAGC/89As GCTGCGTATCATTTCACC, 45S AGTGGTCTGCTTCTCTGG/45As СGGCATACACAGAATACC, inv839 TACCTGCACTCCCACAAC/inv1007 СCCATACGCTGATCTACC; in the following temperature regime: 1 cycle 94°С for 5 min, 35 cycles at 94°С for 45 s, 55°С for 1 min, and 72°С for 45 s, and the final cycle 3 min at 72°С. The obtained PCR products were analyzed by electro phoresis in 2% agarose gel at 10–15 V/cm. Detection of the nucleotide sequences was per formed using a Genetic Analyzer CEQ 8000 (Beck man Coulter). Comprehensive analysis of the bacterial genomes was performed using the nucleotide sequences of Y. pestis and Y. pseudotuberculosis strains from NCBI GenBank database with BLAST algo rithm and DNA STAR v. 11.2.1.25 and Vector NTI Advance v. 11.5 software. VNTR analysis. To detect the phylogenetic rela tionship of Y. pestis strains, an analysis was performed for seven loci of variable tandem repeats— yp0120ms01, yp1290ms04, yp2769ms06, yp2916ms07, yp1335ms46, yp4280ms62, and yp1580ms70. Primers published elsewhere were used to obtain amplificates [9]. The samples obtained by traditional PCR were sequenced by a CEQ 8000 genetic analyzer (Beckman Coulter). Data were analyzed using the resources of the computer database “The Microorganism Tandem RUSSIAN JOURNAL OF GENETICS
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Repeats Database,” http://minisatellites.upsud.fr. Construction of the MLVA7 dendrogram was per formed with the Ward algorithm of BioNumerics 7.1 software (Applied Maths, Belgium). RESULTS AND DISCUSSION Thirtytwo Y. pestis strains isolated in different regions of Mongolia were studied (table). According to the used administrative division, the territory of the country was divided into 21 aimak. The natural plague foci area occupies the territories of many aimaks of Mongolia, and the strains of many of them were included in the present study (Fig. 1). In the studied collection, there were isolates from eight aimaks local ized in the western (BayanUlegeiskii—two strains, Ubsunurskii—five strains, and Dzabkhanskii—three strains), central (Central—two strains and Uver Khangaiskii—three strains), and eastern (Khenteiskii— seven strains) parts of the country and in the Gobi region (SouthernGobiiskii—three strains and BayanKhongorskii—three strains), as well as four strains without a definite region of isolation in their 2015
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Seleginskii
Khubsugulskii
Ubsunurskii
Orkhonskii
Bayan Ulegeiskii
Bula nski i
GobiAltaiskii
Ba ya n Kh on go rsk ii
Khenteiskii Central
2.ANT3
0.PE4 0.PE5
3.ANT2 4.ANT
GobiSumberskii SukheBatorskii
Srednegobiiskii
SouthernGobiiskii
0.PE4m
Eastern
Ea ste rn G ob iis ki i
Kobdonskii
UlanBator
AraKhangaiskii
Uv er Kh ang aisk ii
Dzabkhanskii
Darkhaskii
2.MED1
Fig. 1. Map of Mongolia aimaks with genovariants of Y. pestis strains.
passports. The presence of the strains from different parts of Mongolia made it possible to obtain a clear picture of the diversity of the Y. pestis strains circulat ing there. Characterization of the differential biochemical signs of all investigated Y. pestis strains from Mongolia allowed a determination of their systematic affiliation according to the applied phenotypic schemes of clas sification. According to the differential signs—rham nose and melibiose fermentation—it was shown that 24 out of the 32 investigated strains belonged to the main subspecies and 8 belonged to the nonmain sub species (table). For the following systematization of the strains of the main subspecies, the capacity of these strains for glycerin fermentation and nitrate reduction was analyzed. All of the strains had the ability to ferment glycerin, and therefore there were no strains of biovar ori entalis of the main subspecies among them. Three Y. pes tis strains—231(14), 235(21), and 239(25)—could be attributed to the biovar medievalis by the absence of the ability among the strains of the main subspecies to reduce nitrates. However, we had shown before that only the last of them indeed belonged to the biovar medievalis, and strains 231(14) and 235(21) belong to the biovar antiqua, in spite of the absence of the capac ity for nitrate reduction [10]. All of the remaining strains of the main subspecies fermented glycerin and reduced nitrates and therefore belonged to the Y. pestis biovar antiqua (table). Among the eight strains of the nonmain subspe cies (rhamnose and melibiosedependent strains), six were attributed by their biochemical properties to the ulegeica subspecies because they did not reduce
nitrates but fermented arabinose, which corresponded to the differential biochemical signs of ulegeica sub species and passport data of these strains (table). The strains of the ulegeica subspecies are isolated only on the territory of Mongolia. Two Y. pestis strains, I3085 and I3086, by passport data belonged to the altaica subspecies, because they did not reduce nitrates and did not ferment arabinose. It should be mentioned that microtus strains from two China foci have the same biochemical properties. They also do not reduce nitrates and do not ferment arabinose. Recently Chi nese researchers suggested distinguishing them into separate biovar [11]. To confirm the intraspecific belonging of the stud ied Y. pestis strains from Mongolia, their genetic char acteristics were obtained based on the developed stan dard algorithm of molecular typing of the plague agent [12]. Using the multilocus PCR developed earlier, which provided the division of Y. pestis strains of the main and nonmain subspecies and the closely related Yersinia pseudotuberculosis, we confirmed a relation, determined based on phenotypic characteristics, between 24 strains from Mongolia and the main sub species and between eight strains and the minor sub species. The terC and ilvN genes of the strains of the main subspecies contained deletions typical for this subspecies. They were 89 and 45 bp in size and were absent in strains of the minor subspecies. All of the Y. pestis strains contained the inserted sequence IS1541 were 708 bp in size and were absent from the strains of the pseudotuberculosis microbe. A relation of the only selected strain of the Y. pestis biovar medievalis 239(25) to this biovar was confirmed
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231(14)*, Mongolia, 1931
235(21)*, Dzabkhanskii aimak (a/m), 1936
236(22), Dzabkhanskii aimak a/m, 1936
239(25), Dzabkhanskii aimak a/m,1936
I839, I840, I878, I880, I899, Ubsunurskii a/m, 1960, 1962, 1963
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1562, 1864, BayanKhongorskii a/m, 1956
I3227, I3230, Mongolia, 1988
I3244, BayanUlegeiskii a/m, 1988
I3085, UverKhangaiskii a/m, 1982
I3086, BayanKhongorskii a/m, 1983
I3068, I3069, UverKhangaiskii a/m, 1982
I3130, I3131, I3071, SouthernGobiiskii a/m, 1982, 1984
I2422, BayanUlegeiskii a/m, 1974
* The strains of biovar antiqua which do not reduce nitrates.
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I3101, I3102, I3033, I3030, I3036, I3197, I3217, Khenteiskii a/m, 1983, 1984, 1988
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I846, I847, Central a/m, 1960
Genovariant, locus, and the presence of the marker nucleotide
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Nitrate 4.ANT, 3.ANT2, 0.PE4a, 0.PE4m, 0.PE5, reduction 2.MED1, 2.ANT3, rhamnose, arabinose YPO2744, YPO3506, YPO1418, YPO3183, YPO1548, YPO2223, YPO1120, melibiose Т А A A A T Т
137(19), Mongolia, 1931
Strain, place, and year of isolation
Sugars’ fermentation
Studied Y. pestis strains form Mongolia and their complex description
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genetically based on marker mutation in the napA gene of periplasmic nitrate reductase—a single nucle otide substitution G → T at position 613 from the beginning of the gene. This mutation causes an inabil ity in the biovar medievalis strains to reduce nitrates. Two strains incapable of nitrate reduction, 231(14) and 235(21), did not have this mutation, which con firms that these strains belong to the biovar antiqua. All of the rest strains of the main subspecies did not have this mutation, which, together with the ability to reduce nitrates and ferment glycerin, indicates that they are strains of biovar antiqua. We used our scheme, based on the detection of sin gle nucleotide substitutions—markers for separate Y. pestis genovariants—to determine the genovariants of strains of the main and minor subspecies from Mongolia [7]. Single nucleotide substitutions, mark ers for the genovariants of biovars antiqua and medi evalis of the main subspecies, for altaica subspecies and microtus strains were found. The genovariants of Y. pestis strains from Mongolia were determined (table). A single medieval Y. pestis strain 239(25) was shown to belong to genovariant 2.MED1. In the locus YPO2744 of its genome, there is a marker mutation for this genovariant—a single nucleotide substitution C → T. The strains of this genovariant are widespread in Russia, neighboring countries, and throughout all of the territory of central Asian natural plague foci [5–7]. All of the strains of biovar antiqua from Mongolia were divided into three groups. The strains of 2.ANT line were isolated in Khenteiskii aimak in eastern Mongolia (Fig. 1). All seven strains from Khenteiskii aimak belonged to genovariant 2.ANT3, because they have the marker for this genovariant single nucleotide substitution G → T in the YPO3506 locus. Two more strains belonged to this genovariant, I3227 and I3230; their place of isolation is not accurately known. The strains of genovariant 2.ANT3 also circulate in the TransBaikal steppe focus adjacent to the eastern Mongolia [13] as well as in eastern China. According to the published data, strains of genovariant 2.ANT3 are not found in other Eurasian regions and on other continents. This means that 2.ANT3 strains are endemic for the region including eastern Mongolia and adjacent regions of Russia and China. Among the antique strains, one Y. pestis strain I3244 was isolated in BayanUlegeiskii aimak. Based on the presence of a single nucleotide substitution G → A in the YPO1418 locus, this strain was classified as a 4.ANT line. According to our data, all of the strains from the Tuvinskii Mountain focus in Russia belong to the same phylogenetic line, 4.ANT. This mountain focus is situated close to BayanUlegeiskii aimak, where the strain I3244 was found [7]. The affinity of Y. pestis strain I3244 from Mongolia and strains from Tuvinskii Mountain focus is confirmed by the presence in them of an additional cryptic plasmid about 34 kb in size, which is unique for the strains of
Tuvinskii Mountain focus of plague. Earlier in the scheme of world genetic diversity, there was the only a strain of the genovariant 4.ANT, which was found in Mongolia [6]. The strains of 4.ANT can be found only in this region on the western border between Mongolia and Russia; therefore, they are also endemic for this territory. Eleven strains isolated from western (Dzabkhanskii and Ubsunurskii) and Central aimaks of Mongolia, as well as in the Gobi region (BayanKhongorskii aimak), were classified to genovariant 3.ANT.2, because they have the marker nucleotide substitution T → A in the locus YPO3183. Two more strains were classified as the same genovariant, 137(19) and 231(14), the isolation location of which in Mongolia is not accurately known (table). Strains of genovariant 3.ANT2 were described only for Mongolia and are also endemic for this territory (Fig. 1). Thus, strains of the main subspecies from Mongo lia belonged to genovariant 2.MED1 (1 strain), 2.ANT3 (7 strains), 4.ANT (1 strain), 3.ANT2 (11 strain), and the last three genovariants are endemic for this region and circulate only in Mongolia (genovariant 3.ANT2) or in the territory of eastern regions of Mongolia and border areas of Russia (4.ANT and 2.ANT3) and China (2.ANT3). To confirm Y. pestis I3085 and I3086 classifica tion within the altaica subspecies, the strains of which also circulate in the Altaiskii Mountain plague focus in Russia bordering Mongolia, we determined the geno variants of these strains based on the marker nucle otides in two loci, YPO1548 and YPO2223, that are typical for the line 0.PE4: the genovariants 0.PE4a (altaica subspecies) and 0.PE4m (microtus). As it turned out, the nucleotide substitution C → A, a marker for the microtus strains, was found in locus YPO2223 in the strain I3085. This means that strain I3085 belongs not to the altaica subspecies but to the microtus strains. It did not have the marker for the altaica subspecies nucleotide substitution G → A in locus YPO1548. This substitution also was absent from the genome of another strain, I3086, which was clas sified by passport data to the altaica subspecies. Since this strain of the nonmain subspecies did not have the marker mutations of altaica and ulegeica subspecies and microtus strains, it apparently belonged to another variant of the minor subspecies and its phylo genetic position is to be determined further. To confirm the conclusions about the phylogenetic relationship of Y. pestis strains from different Mongo lian regions, we performed multilocus analysis of a variable number of tandem repeats of seven VNTR loci—yp0120ms01, yp1290ms04, yp2769ms06, yp2916ms07, yp1335ms46, yp4280ms62, and yp1580ms70 [9]. As follows from the MLVA7 dendro gram in Fig. 2, there was a clear clustering of strains according to their genovariant classification and place of isolation. A single strain of Y. pestis biovar medieva
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ANALYSIS OF DIVERSITY AND IDENTIFICATION OF THE GENOVARIANTS 137(19) 1562 l846 l847 l839 l840 l899 l880 l878 1864 231(14) l3244 l2422 l3131 l3068 l3069 l3071 l3130 l3085 l3086 l3033 l3036 l3102 l3227 l3217 l3230 235(21) 236(22) 239(25)
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Mongolian People’s Republic BayanUlegeiskii Central Central Ubsunurskii Ubsunurskii Ubsunurskii Ubsunurskii Ubsunurskii BayanUlegeiskii Mongolian People’s Republic BayanUlegeiskii BayanKhongorskii SouthernGobiiskii UverKhangaiskii UverKhangaiskii SouthernGobiiskii SouthernGobiiskii BayanKhongorskii UverKhangaiskii Khenteiskii Khenteiskii Khenteiskii Mongolian People’s Republic Khenteiskii Mongolian People’s Republic Dzabkhanskii Dzabkhanskii Dzabkhanskii
Fig. 2. MLVA7 dendrogram of Y. pestis strains isolated in different Mongolia regions (Ward algorithm, BioNumerics 7.1 software, Applied Maths, Belgium).
lis, 239(25) from Dzabkhanskii aimak, belonged to a separate branch of the MLVA7 dendrogram, as well as the only strain of genovariant 4.ANT—I3244 from BayanUlegeiskii aimak. Strains of the antique geno variant 2.ANT3 from eastern Mongolia formed a sep arate cluster, including strains from Khenteiskii aimak and two strains, I3227 and I3230, of the genovariant 2.ANT3 from Mongolia, which apparently were also isolated in this region. The biggest cluster included the strains of antique genovariant 3.ANT2, which was identified in the western (Ubsunurskii) and central (Central) aimaks and in the Gobi region (Bayan Khongorskii aimak). A separate cluster was formed by the strains of the same genovariant but from western aimak—Dzabkhanskii aimak. Strains of the nonmain subspecies formed a clus ter separate from all other strains; it included two sub clusters. One of them included all six strains of the ulegeica subspecies, and the second one included strains I3085 (genovariant 0.PE4m) and I3086, the genovariant of which is to be further determined. Therefore, MLVA7 analysis confirmed the structure of the population of Y. pestis strains from Mongolia detected by PCR and multilocus sequencing. It is worth mentioning that, of the 24 studied Y. pestis strains of the main subspecies from Mongolia, not one belonged to the most ancient line, 0.ANT. Thus, the strains circulating in Mongolia are not the oldest of the antique (marmot’s) Y. pestis strains, which in general contradicts the common hypothesis RUSSIAN JOURNAL OF GENETICS
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about the origin of the plague agent in this country. It can be suggested that the formation of genovariants of Y. pestis endemic in Mongolia was the result of the penetration into this and neighboring territories of strains of the ancient phylogenetic line, 0.ANT (possi bly, from the Tien Shan Mountain foci of Kyrgyzstan) and their adaptation to the landscapegeographical conditions of the region, which led to the formation of the lines 2.ANT3, 4.ANT1, and 3.ANT2. REFERENCES 1. Prirodnye ochagi chumy Kavkaza, Prikaspiya, Srednei Azii i Sibiri (Natural Plague Foci in the Territory of the Caucasus, PreCaspian Region, Central Asia and Sibe ria), Onishchenko, G.G. and Kutyrev, V.V., Eds., Mos cow: Meditsina, 2004. 2. Balakhonov, S.V., Afanas’ev, M.V., Shestopalov, M.Yu., et al., The first case of the Yersinia pestis subsp. pestis isolation in the Altai mountain natural foci of plague: 1. Microbiological characterization, molecular genetic and mass spectrometric identification of the isolate, Probl. Osobo Opasnykh Infekts., 2013, no. 1, pp. 12–16. 3. Timofeeva, L.A., On the taxonomy of plague microbe, Probl. Osobo Opasnykh Infekts., 1972, no. 1(23), pp. 15–20. 4. Kutyrev, V.V. and Protsenko, O.A., Classification and molecular genetic studies of Yersinia pestis, Probl. Osobo Opasnykh Infekts., 1998, no. 1, pp. 11–12. 5. Morelli, G., Eppinger, M., Roumagnac, P., et al., Yers inia pestis genome sequencing identifies patterns of glo 2015
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Translated by A. Kashevarova
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