Background. Isolation of strains from tick-borne encephalitis (TBE) patients proved that the European subtype tick-borne encephalitis virus (TBEV-Eur) in Siberia is involved in regional human infectious pathology and causes a clinical picture similar to Western TBE. However, a comparative analysis of the genomes of TBEV-Eur strains isolated from TBE patients in Eastern Siberia and Europe has not been carried out.The aim. Genome comparative analysis and search for virulence determinants in TBEV-Eur strains isolated from patients in Eastern Siberia and Northern and Eastern Europe.Materials and methods. In current work, TBEV-Eur strain 1G-98 from the collection of Scientific Сentre for Family Health and Human Reproduction Problems (GenBank Acc. No. KY069119) was used. The analysis also included all complete genome sequences of TBEV-Eur strains from patients submitted in GenBank database at the time the study began. Complete genome sequencing of strain 1G-98 was performed using the Sanger method. The virulence of the strain was assessed by intracerebral and subcutaneous infection of laboratory mice.Results. It has been shown that, according to the coding region of the genomes, the level of differences between TBEV-Eur strains from Siberia does not exceed the previously established maximum for this subtype of 3.1 %. The strain 1G-98, isolated from the blood of TBE patient from the Irkutsk region demonstrated high cerebral and peripheral activity. In this strain, D67G mutation in E protein DII domain was revealed, which could potentially be associated with virulence, and a long deletion in the variable part of the 3’-noncoding genome region, comparable in length to the highly virulent strain Hypr from Europe was found.Conclusions. For the first time, a comparative analysis of the genomes of TBEV-Eur strains from TBE patients from the Asian part of Russia and Europe was carried out showing their genetic similarity, and potential virulence determinants were identified. Key words: tick-borne encephalitis virus, European subtype, genome, amino acid sequence, virulence determinants

Background. Piroplasmosis (babesiosis and theileriosis) is a group of natural focal infectious diseases of humans and animals caused by intraerythrocytic parasites transmitted predominantly by ixodid ticks. In the Baikal region, despite the widespread foci of babesiosis and theileriosis, the data about these pathogen vectors is fragmentary.The aim. To identify human and animal babesiosis vectors, study various Babesia spp. tropism to ixodid ticks common in the Baikal region.Materials and methods. A total of 3,239 specimens of I. persulcatus, 1,795 specimens of D. nuttalli and 729 specimens of H. сoncinna ticks collected in natural habitats of the Baikal region from 19 districts of the Irkutsk region and six districts of the Republic of Buryatia, as well as 5 specimens of D. nuttalli ticks removed from horses were examined for the presence of Babesia spp./Theileria spp. DNA. For detection and typing of Babesia and Theileria, a nested PCR with genus- and species-specific primers corresponding to 18S rRNA gene region was used. Selective sequencing of samples was performed to study the genetic diversity of the identified pathogens.Results. Babesia spp. DNA was detected in three species of ixodid ticks in Irkutsk region and in one species in the Republic of Buryatia. In I. persulcatus ticks B. microti US-type, B. venatorum, B. crassa-like and a unique Babesia sp. Irk-Ip655 were found. DNAs of B. crassa-like, B. motasi-like and Babesia spp. were detected in H. concinna ticks. DNAs of Babesia spp. And T. equi were found in D. nuttalli ticks.Conclusion. Vectors of both human and animal babesiosis pathogens were detected in the Baikal region. Tropism of B. microti US-type and B. venatorum in relation to I. persulcatus ticks was established. The establishment of specific relations between other species of Babesia and Theileria and vector ticks in the Baikal region will be continued.

The ixodid tick Haemaphysalis concinna (Koch, 1844) is a carrier of pathogens of vector-borne diseases of viral, bacterial and protozoal etiology. This tick was previously considered an adventive relict species in the Baikal region, but has recently shown a tendency to increase its numbers and expand its habitat. The aim of the study. To generalize the available and newly received information on the distribution of the H. concinna tick in the Irkutsk region and the Republic of Buryatia; to carry out the comparative analysis of the results in order to identify the dynamics of the development of populations of this species of ixodid ticks in the Baikal region. Materials and methods. The coordinates of H. concinna detection points in our studies were obtained using a GPS navigator directly in the field. Ticks were caught during the period of their maximum activity from plants using a flannel flag. Approximate geographic coordinates of H. concinna tick detection points were established when analyzing the maps published by other researchers. Results. As a result of the generalization of our own data and data from literary sources, 52 georeferenced detection points of H. concinna were obtained on the territory of the Baikal region. A map showing the distribution of H. concinna in the territory of the Irkutsk region and the Republic of Buryatia is presented. It is shown that in a number of surveyed areas there are stable populations of this species of tick, which tend to increase in their numbers and expand their range. Conclusions. Considering these data, as well as the fact that H. concinna ticks are actively involved in the circulation of pathogens of natural focal diseases in humans and animals, we can conclude that it is necessary to systematically monitor the populations of this vector species in the Baikal region.

Anaplasmosis of ruminants is a group of natural focal infections caused by bacteria from the genus Anaplasma of the Anaplasmataceae family. The main etiological agent of anaplasmosis in sheep, goats, and wild ruminants is Anaplasma ovis, which parasitizes in the erythrocytes of these animals. The purpose of this study was the finding and identification of Anaplasma spp. in the blood of small ruminants using genetic methods and obtaining data on the distribution of anaplasmosis in the Irkutsk region. 20 goat blood samples, 611 sheep blood samples and 209 Dermacentor nuttalli ticks from 12 districts of the Irkutsk region were examined for the presence of Anaplasma spp. Only one type of anaplasma, A. ovis, was found among the genotyped samples. A. ovis was found in the blood of sheep and goats in all of the studied districts of the Irkutsk region. The proportion of sheep blood samples containing anaplasma DNA varied from 30 % to 85 %, in goats – from 10 % to 100 % in different districts, and averaged 57.8 % in sheep and 55,0 % in goats. Frequency of infection of D. nuttalli ticks with A. ovis was 5.7 %. The nucleotide sequences of the samples detected in the blood of small ruminants on the territory of the Irkutsk region differed from each other by a single nucleotide substitution and were identical to the sequences of the type strain Haibei, as well as the sequences of A. ovis previously found in the blood of sheep from Mongolia, deer from China, and Dermacentor niveus and Dermacentor nuttalli ticks from China. These sequences were also identical to the sequences previously found in the blood of sheep from Altai and in Dermacentor nuttalli ticks from Tuva, which indicates the wide distribution of these A. ovis genovariants in Siberia and the probable role of D. nuttalli as a carrier of the agent of anaplasmosis of small ruminants in the Irkutsk region.

The tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, is currently subdivided into three main subtypes-the European (TBEV-Eu), the Far-Eastern (TBEV-FE), and the Siberian (TBEV-Sib). The TBEV-Sib is the most common subtype and found in all regions where TBEV was detected, except for Central and Western Europe. Currently, four genetic lineages have been described within TBEV-Sib. In this study, detailed analysis of TBEV-Sib genetic diversity, geographic distribution, phylogeography and divergence time of different TBEV-Sib genetic lineages based on E gene fragments, complete genome sequences, and all currently available data in the GenBank database was performed. As a result, a novel Bosnia lineage within the TBEV-Sib was identified. It was demonstrated that the Zausaev lineage is the most widely distributed among the TBEV-Sib lineages, and was detected in all studied regions except the Far East. The Vasilchenko lineage was found from Western Siberia to the Far East. The Baltic lineage is presented from Europe to Western Siberia. The Obskaya lineage was found only in Western Siberia. TBEV strains from a newly described Bosnia lineage were detected in Bosnia, the Crimean peninsula, Kyrgyzstan and Kazakhstan. The greatest divergence of the TBEV-Sib genetic variants was observed in Western Siberia. Within the TBEV-Sib, the Obskaya lineage diverged from the common ancestor the earliest, after that the Bosnia lineage was separated, then the Baltic lineage, and the Zausaev and Vasilchenko lineages diverged most recently.

We have studied 13 enzyme systems encoded by 15 loci of the I. persulcatus tick populations from two suburban areas of the city of Irkutsk. We have identified a high intrapopulation heterogeneity and insignificant differences between the two populations. This indicates a high rate of gene migration.

Introduction.On the territory of the Ekhirit-Bulagatsky district of the Irkutsk region zones of sympatry of four Ixodes ticks species are found, where the species and genetic diversity of infectious agents transmitted through tick bites may be more pronounced than in foci with a mono-dominant type of ticks’ population. In this connection, the study of the species and genetic diversity of representatives of the Anaplasmataceae family in the sympatry zone of the Ixodes ticks of closely related species was of scientific interest.Objective: To study the species and genetic diversity of members of the Anaplasmataceae family in the zones of sympatry of Ixodes ticks Ixodes persulcatus, Dermacentor silvarum, D. nuttalli and Haemaphysalis concinna, to identify the main carriers and potential reservoir hosts of ehrlichia and anaplasma.Methods. In the course of the study, 1106 specimens of adult ticks and 49 samples of small mammalian livers from the Ekhirit-Bulagatsky area were analyzed. Anaplasma and ehrlichia DNA were detected by two-round PCR in the presence of genus- and species-specific primers from the 16S rRNA gene region. The nucleotide sequences of the 16S rRNA gene and the fragment of the groESL operon were identified in some samples. Sequencing was carried out according to the Sanger method. Comparative analysis was performed using the BLASTN program and ClustalW method. Epidemiological data analysis was performed using parametric methods of statistical processing of the material.Results. The DNA of Ehrlichia muris and Anaplasma phagocytophilum were detected in all studied species of ticks in their sympatry area. However, the rate of infection of taiga ticks was significantly higher than that of H. concinna and Dermacentor spp. Potential reservoir hosts of the Anaplasmataceae family members can be classified as Microtus oeconomus, M. gregalis, Myodes rutilus and Sorex spp. When analyzing the nucleotide sequences of the 16S rRNA gene, three genetic variants of anaplasma were detected. The nucleotide sequences of the A. phagocytophilum groESL operon belonged to two genetic groups.

Introduction .On the territory of the Ekhirit-Bulagatsky district of the Irkutsk region zones of sympatry of four Ixodes ticks species are found, where the species and genetic diversity of infectious agents transmitted through tick bites may be more pronounced than in foci with a mono-dominant type of ticks’ population. In this connection, the study of the species and genetic diversity of representatives of the Anaplasmataceae family in the sympatry zone of the Ixodes ticks of closely related species was of scientific interest. Objective: To study the species and genetic diversity of members of the Anaplasmataceae family in the zones of sympatry of Ixodes ticks Ixodes persulcatus, Dermacentor silvarum, D. nuttalli and Haemaphysalis concinna, to identify the main carriers and potential reservoir hosts of ehrlichia and anaplasma. Methods. In the course of the study, 1106 specimens of adult ticks and 49 samples of small mammalian livers from the Ekhirit-Bulagatsky area were analyzed. Anaplasma and ehrlichia DNA were detected by two-round PCR in the presence of genus- and species-specific primers from the 16S rRNA gene region. The nucleotide sequences of the 16S rRNA gene and the fragment of the groESL operon were identified in some samples. Sequencing was carried out according to the Sanger method. Comparative analysis was performed using the BLASTN program and ClustalW method. Epidemiological data analysis was performed using parametric methods of statistical processing of the material. Results. The DNA of Ehrlichia muris and Anaplasma phagocytophilum were detected in all studied species of ticks in their sympatry area. However, the rate of infection of taiga ticks was significantly higher than that of H. concinna and Dermacentor spp. Potential reservoir hosts of the Anaplasmataceae family members can be classified as Microtus oeconomus, M. gregalis, Myodes rutilus and Sorex spp. When analyzing the nucleotide sequences of the 16S rRNA gene, three genetic variants of anaplasma were detected. The nucleotide sequences of the A. phagocytophilum groESL operon belonged to two genetic groups.

Background. During the study of the genetic variability of the tick-borne encephalitis virus (TBEV) in Eastern Siberia, a group of 22 strains with a unique genetic structure significantly different from all known TBEV subtypes was identified. This TBEV variant was tentatively called “group 886”. Therefore, for this original TBEV variant it was necessary to study the genetic, biological properties of the “group 886” strains, clarify its TBEV taxonomic status, its range, evolutionary history, etc.Aim. The generalization of the currently available data on genetic and biological properties of TBEV “886” group.Materials and methods. The genetic structure of “group 886” strains was studied by the complex of molecular-genetic methods (MHNA, sequencing of fragments or the complete genome).Results. It was shown that “group 886” strains form a separate cluster on phylogenetic tree, and the level of genetic differences from other genotypes is more than 12 %. It was defined that this TBEV variant has its own area (Irkutsk region, Republic of Buryatia, Trans-Baikal region, Northern Mongolia). Its ecological connection with all links of the transmissive chain (ixodid ticks, small mammals, human), participation in human pathology, stability and duration of circulation in the Baikal region, individual evolutionary history were proved. Some phenotypic characteristics of the “group 886” strains were considered.Conclusion. The presented data testify to the validity of the “886 group” isolation as an independent genetic type. Taking into account the geographical distribution of this TBEV genotype, we propose to assign it the name “Baikal genotype/subtype”.

Equine piroplasmosis is a natural tick-borne infection caused by hemoprotozoan parasites of the order Piroplasmida, Babesia caballi and Theileria equi. Animals that recover from piroplasmosis remain persistently infected carriers and can transmit pathogens to vector ticks. Cases of equine piroplasmosis are periodically observed in Siberia, however, no agent of equine piroplasmosis has yet been genetically characterized in Russia. The aim of this work was studying the prevalence of the infectious agents of piroplasmosis in horses from Siberia and genotyping the detected agents. Blood samples from 155 horses were examined for the presence of Babesia and Theileria DNA by nested PCR with the subsequent sequencing of positive samples. DNA of T. equi was found in blood samples from 57.9 %, 38.5 % and 65.0 % of horses from Novosibirsk province, Irkutsk province, and the Republic of Altai, respectively. T. equi DNA was found in the samples from almost all sampling sites included in this study, indicating that most of the studied sites are endemic for equine theileriosis. Surprisingly, DNA of B. caballi was not found in any of the samples examined, even though this agent had previously been detected in many regions in Russia, including Altai. The analysis of the determined 18S rRNA gene sequences demonstrated that T. equi samples belonged to two genetic groups, which differed significantly by the sequences of the variable (V4) region of the gene. All T. equi sequences from group B were identical and corresponded to T. equi sequences found in the blood of horses from China and Korea, while T. equi sequences from group A differed by 1–5 nucleotide substitutions and were identical to the sequences from the blood of horses from India and Brazil or differed from them by single mismatches. Notably, in this study the presence of etiological agent of piroplasmosis in blood samples from horses in Russia was genetically confirmed for the first time.

Tick-borne encephalitis virus (TBEV) is classified into three subtypes: Far Eastern (TBEV-FE), European (TBEV-EU) and Siberian (TBEV-SIB). In Russia, these are also called genotypes 1, 2 and 3, respectively. Geographically, TBEV-EU dominates in Central and Northern Europe, but its representatives are also found to the east - along the southern part of the forest zone of extratropical Eurasia - up to Eastern Siberia and South Korea. However, the strains isolated outside Europe remain poorly investigated. In the proposed study, eight full genomes of the Siberian isolates of TBEV-EU were determined and 13 complete genomes were compared. The analysis of 152 full-genome TBEV sequences showed that the TBEV-EU has a higher degree of stability of the genome-coding region in the entire Eurasian area (3.1% of differences) compared to TBEV-FE (6.6%) and TBEV-SIB (7.8%). At the same time, the maximum differences are observed not between European and Siberian strains, as one could expect, but between the representatives from Europe - TBEV strains Mandl-2009 from Norway and Hypr from the Czech Republic. The studied strains from Siberia form the compact genetic cluster of 42 TBEV-EU strains and are divided into two subclusters - West Siberian and East Siberian variants. These variants differ in the combinations of amino acid substitutions in all proteins except NS2B. The West Siberian variant mostly circulates in the territory of Altai, and the closest relative of its representatives is Absettarov strain from the European part of Russia. The strains similar to the East Siberian variant of the European subtype were recorded in the Altai (strain 84.2, 2007) and in Belarus (N256, about 1940).

To find out what is the role of some environmental factors in the formation of a heterogeneous viral population, the analysis of the distribution of tick-borne encephalitis virus (TBEV) genotypes in different types of landscapes of Eastern Siberia has been carried out. In our study, it was shown that TBEV area in all investigated landscapes of Eastern Siberia is represented not only by TBEV of genotype 3, absolute dominance of which is noted in the studied regions, regardless the landscapes where the foci are located, but also by strains of other genotypes. The relationship between genotypes 1,3 and "polytypic" strains with a certain type of landscape and the degree of their development by man has not been established. The prevailing number of strains of TBEV genotype 5 is isolated from the material collected in the Transbaikalia, in the landscapes of the mountain taiga. They, as well as the strain of genotype 4, are found in the foci, where joint circulation of several TBEV genotypes is noted. The strains of TBEV genotype 2 were revealed in the landscapes of plains and plateaus taiga and insular steppes and forest steppes of the southern part of the Baikal region. Moreover, the detection of RNA of TBEV genotype 2 in the "polytipic" 763-87 and 765-87 strains isolated in the floodplain and forest-steppe landscapes of the Barguzin hollow does not exclude the possibility of circulation of TBEV genotype 2 in the Transbaikalian foci. The greatest genetic heterogeneity of TBEV is observed in natural foci located along Lake Baikal and in the southeastern part of Transbaikalia.

The aim of the study was to determine the ecology features of tick-borne encephalitis virus (TBEV) of European subtype circulating in Siberia. The strains of European subtype TBEV from Siberia were isolated in areas with different terrain types - from the plains to the midlands. The strains isolation areas differed in variety of landscapes, flora and fauna and were characterized by the presence of sharp-continental, or in some places - quite harsh climate. The composition of the main vectors and reservoir hosts of TBEV of European subtype in Siberia has its own features and is significantly different from that one in Europe. However, in spite of this, the homology of TBEV strains of European subtype isolated in different parts of habitat range of the virus from the Scandinavian countries in the West to its Eastern borders is much higher than the homology level degree of TBEV strains of FarEastern and Siberian subtypes.

Since ixodid ticks and their feeders (vertebrates) form the basis of the tick-borne encephalitis virus (TBEV) population, it was interesting to obtain a genetic characteristic of strains isolated from various sources in the territory of Eastern Siberia. In our study, it was found that the circulation of TBEV of genotypes 1, 2, 3, 5 and "polytypic" strains in the territory of Eastern Siberia is maintained both by the main vectors (I. persulcatus ticks) and by their feeders (small and large mammals, birds). The distribution of genotypes in strains groups from ixodid ticks and vertebrates varied. TBEV strains of genotypes 1 and 2 were significantly more often isolated from rodents, and genotype 3 from ixodid ticks (p < 0.05). Isolates of genotype 3 (85.5 %) prevailed in the group of strains isolated from ixodid ticks, of which 29.6 % belonged to "Vasilchenko", and 25.6 % to "Zausaev"subgenotypes. Genotypes 1 and 2 were represented by single isolates (3.3 % and 1.3 %, respectively). Among strains isolated from warm-blooded hosts, the proportion of genotype 1 was 35.3 %, genotype 2 -11.8 %, genotype 3 - 35.3 %. More than half of genotype 3 strains (58.3 %) were related to "Vasilchenko" subgenotype, and the strains of "Zausaev" subgenotype were not detected. The obtained data suggest that ixodid ticks and warm-blooded animals are amplifiers of TBEV of genotypes 3 and 1, respectively. The certain differences in the phenotypic characteristics of strains isolated from ticks and warm-blooded animals have been revealed. It has been found that strains isolated from ticks are more homogeneous in their antigenic characteristics, less heterogeneous in S-feature than strains isolated from warm-blooded sources. The strains isolated from warm-blooded animals demonstrate somewhat greater resistance to warming but reducing the reproduction of the virus at 42 °C, and have greater virulence compared to strains isolated from ticks.

The aim of the study was to determine the ecology features of tick-borne encephalitis virus (TBEV) of European subtype circulating in Siberia. The strains of European subtype TBEV from Siberia were isolated in areas with different terrain types - from the plains to the midlands. The strains isolation areas differed in variety of landscapes, flora and fauna and were characterized by the presence of sharp-continental, or in some places - quite harsh climate. The composition of the main vectors and reservoir hosts of TBEV of European subtype in Siberia has its own features and is significantly different from that one in Europe. However, in spite of this, the homology of TBEV strains of European subtype isolated in different parts of habitat range of the virus from the Scandinavian countries in the West to its Eastern borders is much higher than the homology level degree of TBEV strains of FarEastern and Siberian subtypes.

Tick-borne encephalitis virus (TBEV) is divided into three subtypes: European (TBEV-Eu), Siberian (TBEV-Sib), and Far Eastern (TBEV-FE) subtypes. The geographical range of TBEV-Eu dominates in Europe, but this subtype is present focally across the whole non-tropical forested Eurasian belt, through Russia to South Korea. However, the TBEV-Eu strains isolated outside Europe remain poorly characterized. In this study, full-genome sequences of eight TBEV-Eu isolates were determined. These strains were isolated from Ixodes persulcatus ticks, long-tailed ground squirrel (Spermophilus undulatus), and human blood in the natural foci of Western and Eastern Siberia, Russia. A phylogenetic analysis of all available TBEV-Eu genomic sequences revealed that strains from Siberia were closely related to other strains from Europe and South Korea. The closest relation was identified between the Siberian strains and strains from Zmeinogorsk (Western Siberia, Russia) and strain Absettarov (Karelia, Russia), and were most divergent from strains from the Czech Republic and Norway. TBEV-Eu strains isolated in Eastern Siberia were more closely related phylogenetically to strains from South Korea, but strains from Western Siberia grouped together with the strains from Europe, suggesting two genetic TBEV-Eu lineages present in Siberia.

Tissue samples from 228 small mammals captured from 2013 to 2015 in four districts of Irkutsk region and in one district of Novosibirsk region were examined for the presence of Anaplasmataceae bacteria and Babesia parasites by nested PCR method with subsequent sequencing of positive samples. In Ekhirit-Bulagatskiy District of Irkutsk region, Babesia microti DNA was found in 10.9 % of small mammals, Anaplasma phagocytophilum DNA – in 7.3 %, and Ehrlichia muris DNA – in 1.8 %. Infected mammals were not found in the other three examined districts of Irkutsk region. In Novosibirsk region, B. microti DNA was found in 8.9 % of small rodents, A. phagocytophilum DNA – in 8.9 % of small mammals, and Candidatus Neoehrlichia mikurensis DNA – in 8.9 % of small mammals. The identified infection agents corresponded to microorganism species and genetic variants previously found in Ixodes persulcatus ticks but not in ticks of other species.

The aim of the study was to obtain the complex characteristics of tick-borne encephalitis virus (TBEV) of European subtype circulating in Western and Eastern Siberia. Using full-genome sequencing approach it was demonstrated that TBEV strains of European subtype isolated in Siberia are genetically similar to the strains from European part of its habitat range, and with the representatives from South Korea. It was confirmed that the homology of TBEV strains of European subtype isolated in different parts of the virus habitat area from Scandinavian countries in the west to the eastern borders of the area (South Korea) is much higher than the homology level of TBEV strains of Far Eastern and Siberian subtypes. The Siberian population of TBEV of European subtype is presented with two groups of strains called as Eastern Siberian and Western Siberian variants, which differ in the combinations of amino acid substitutions in all proteins except NS2B protein. It is found that TBEV strains of European subtype from Siberia possess high neurovirulence, but some of them, like strains from Europe, demonstrate low invasiveness. It is shown that TBEV strains of European subtype have good adaptive capacity, and therefore, can easily adapt to the circulation in various biocenoses in the territory of different landscape-geographical zones. It was found that the circulation of TBEV of European subtype is fixed in Siberia territory for over 40 years. It was emphasized that in spite of circulation of TBEV of European subtype in the significantly different areas by climatic conditions, topography, landscape, habitat characteristics it possesses a high degree of genome stability.