Equine piroplasmosis (EP) is a global worldwide infection, which can lead to the death of animals. Despite the causative agents of EP being well studied, there are no data on the distribution and genetic characteristics of EP agents in any region of Russia. In this study, blood samples from 750 horses from Novosibirsk province, Irkutsk province, and Altai region of Russian Siberia were examined for the presence of EP agents. Theileria equi and Babesia caballi were detected in all examined regions, with mean prevalence rates of 60.4

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.

Tick-borne encephalitis virus (TBEV) is a flavivirus which causes an acute or sometimes chronic infection that frequently has severe neurological consequences, and is a major public health threat in Eurasia. TBEV is genetically classified into three distinct subtypes; however, at least one group of isolates, the Baikal subtype, also referred to as “886-84-like”, challenges this classification. Baikal TBEV is a persistent group which has been repeatedly isolated from ticks and small mammals in the Buryat Republic, Irkutsk and Trans-Baikal regions of Russia for several decades. One case of meningoencephalitis with a lethal outcome caused by this subtype has been described in Mongolia in 2010. While recombination is frequent in Flaviviridae , its role in the evolution of TBEV has not been established. Here, we isolate and sequence four novel Baikal TBEV samples obtained in Eastern Siberia. Using a set of methods for inference of recombination events, including a newly developed phylogenetic method allowing for formal statistical testing for such events in the past, we find robust support for a difference in phylogenetic histories between genomic regions, indicating recombination at origin of the Baikal TBEV. This finding extends our understanding of the role of recombination in the evolution of this human pathogen.

Background. Cattle anaplasmosis and babesiosis and theileriosis of horses are a group of natural focal vector-borne diseases that cause significant economic damage to livestock and horse breeding around the world. On the territory of the Baikal region these diseases and their pathogens remain little explored. Purpose – to study the epizootic situation of blood-parasitic diseases of farm animals in the Irkutsk region, to establish the species affiliation of the identified pathogens and their genetic diversity. Materials and methods. For the presence of Babesia spp./Theileria spp. and Anaplasma spp. has been researched659 horse blood samples, 579 sheep blood samples, 25 goat blood samples and 647 cattle blood samples. For the detection of babesia and theileria, nested two-round PCR with primers from the 18S rRNA gene region was used, and for the detection of anaplasma DNA with primers from the 16S rRNA gene region. For study the genetic diversity of the identified pathogens, selective sequencing of samples was carried out with subsequent phylogenetic analysis. Results. The DNA of Anaplasma spp. was detected in blood samples of small ruminants from 12 districts of the region. The infection rate of sheep and goats was 68.7±1.9% and 68%±9.3%, respectively. Nucleotide sequences of the 16S rRNA gene fragment were determined in 68 A. ovis samples from the blood of sheep and goats. The researched sequences were conservative for the studied gene and differed from each other in two heterozygous sites of the gene(T/C and G/A nucleotide substitutions).They corresponded to the sequences of the prototype strain Haibei (CP015994), as well as to the A. ovis sequences previously found in the blood of sheep from Altai (Russia) and Mongolia, deer and D. niveus and D. nuttalli ticks from China. DNA of Babesia spp./Theileria spp. found in blood samples of horses from 13 districts of the region. The average infection of horses was 64.2%±1.9%. Two etiological agents of equine piroplasmosis were found in the blood of the examined horses: T. equi (genetic groups A and E) and B. caballi. Definite sequences of the B. caballi 18S rRNA gene (700–1146 bp) were identical to each other and differed by at least ten substitutions and one deletion from the sequences available in the GenBank database. However, the greatest similarity was observed with B. caballi sequences found in the blood of a horse from Brazil (KY952238), ticks from Kazakhstan (MN907451) and China (MN173021). The results indicate that DNA of Anaplasma and Babesia in the blood of cattle have not been identified. Conclusion: The data received evidence a wide distribution of foci of small cattle anaplasmosis and piroplasmosis of horses in the Irkutsk region and the need to continue monitoring their epizootic condition.

Orthoflavivirus encephalitidis, formerly tick-borne encephalitis virus (TBEV), belongs to the Orthoflavivirus genus. TBEV is transmitted by tick bites and infection with TBEV can lead to serious disorders of the central nervous system. In this study, a new protective monoclonal mouse antibody (mAb) FVN-32, with high binding activity to glycoprotein E of TBEV, was selected and examined in post exposure prophylaxis in a mouse model of TBEV infection. BALB/c mice were injected mAb FVN-32 at doses of 200 μg, 50 μg, and 12.5 μg per mouse one day after a TBEV challenge. mAb FVN-32 showed 37.5% protective efficacy when administered at doses of 200 μg and 50 μg per mouse. The epitope for protective mAb FVN-32 was localized in TBEV glycoprotein E domain I+II, using a set of truncated fragments of glycoprotein E. Additionally, the target site recognized by mAb FVN-32 was defined using combinatorial libraries of peptides. Three-dimensional modeling revealed that the site is dspatially close to the fusion loop, but does not come into contact with it, and is localized in a region between 247 and 254 amino acid residues on the envelope protein. This region is conserved among TBEV-like orthoflaviviruses.

Spiroplasma are vertically-transmitted endosymbionts of ticks and other arthropods. Field-collected Ixodes persulcatus have been reported to harbour Spiroplasma , but nothing is known about their persistence during laboratory colonisation of this tick species. We successfully isolated Spiroplasma from internal organs of 6/10 unfed adult ticks, belonging to the third generation of an I. persulcatus laboratory colony, into tick cell culture. We screened a further 51 adult male and female ticks from the same colony for presence of Spiroplasma by genus-specific PCR amplification of fragments of the 16S rRNA and rpoB genes; 100% of these ticks were infected and the 16S rRNA sequence showed 99.8% similarity to that of a previously-published Spiroplasma isolated from field-collected I. persulcatus . Our study shows that Spiroplasma endosymbionts persist at high prevalence in colonised I. persulcatus through at least three generations, and confirms the usefulness of tick cell lines for isolation and cultivation of this bacterium.

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.

Tick-borne encephalitis (TBE) is caused by a neurotropic RNA virus from the Flavivirus genus. TBE is characterized by a significant variability of clinical manifestations from nonparalytic forms (fever, meningitis) to severe paralytic (focal) forms (meningoencephalitis, poliomyelitis, polioencephalomyelitis). The result of interaction between a virus and a host (and, consequently, the viral disease course and outcome) largely depends on genetically determined ability of the host (particularly, human) organism immune system to suppress the development of viral infection. However, hereditary predisposition to TBE has been rather poorly studied in human populations. In this study, the results of whole exome sequencing of DNA samples from 22 Russian non-immunized TBE patients with severe TBE forms and 17 control individuals from the same populations are presented. Sixteen single nucleotide polymorphisms (SNPs) associated with predisposition to severe forms of TBE were identified. The genotype and allele frequencies for three of these SNPs localized in the ABCB9 (rs4148866, G/A, intron), COL22A1 (rs4909444, G/T, Ala938Asp), and ITGAL (rs1557672, G/A, intron) genes were then studied in larger samples of patients with different forms of TBE (n= 177) and in the control population (n = 215). As a result, the association of the ABCB9 and COL22A1 gene SNPs with the development of severe forms of TBE was for the first time demonstrated in the Russian population. The hypothesis regarding a possible mechanism of the effect of the ABCB9 gene intronic SNP on the process of human infection with TBE virus is considered.

Tick-borne encephalitis virus (TBEV) is the most important tick-transmitted pathogen. It belongs to the Flaviviridae family and causes severe human neuroinfections. In this study, protective efficacy of the chimeric antibody chFVN145 was examined in mice infected with strains belonging to the Far-Eastern, European, and Siberian subtypes of TBEV, and the antibody showed clear therapeutic efficacy when it was administered once one, two, or three days after infection. The efficacy was independent of the TBEV strain used to infect the mice; however, the survival rate of the mice was dependent on the dose of TBEV and of the antibody. No enhancement of TBEV infection was observed when the mice were treated with non-protective doses of chFVN145. Using a panel of recombinant fragments of the TBEV glycoprotein E, the neutralizing epitope for chFVN145 was localized in domain III of the TBEV glycoprotein E, in a region between amino acid residues 301 and 359. In addition, three potential sites responsible for binding with chFVN145 were determined using peptide phage display libraries, and 3D modeling demonstrated that the sites do not contact the fusion loop and, hence, their binding with chFVN145 does not result in increased attachment of TBEV to target cells.

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.

The causative agent of tick-borne encephalitis (a neurotropic RNA virus from the Flavivirus genus) can cause both severe paralytic forms of the disease (meningoencephalitis, etc.) and milder nonparalytic forms (fever and meningitis). The organism response to viral infection (and, as a consequence, the nature and outcome of the disease) significantly depends on individual peculiarities of the human organism protective systems predetermined by genome structure. Human genetic predisposition to tick-borne encephalitis has been poorly studied. In the present work, the results of the search for new genes that predetermine the peculiarities and outcome of tick-borne encephalitis in humans are presented. The aim of the work was to verify the association between three previously detected (using the exome sequencing on a limited sample of tick-borne encephalitis patients with severe forms) SNPs: intronic rs3109675 (C/T) in the COL5A1 gene, intronic rs41554313 (A/G) in the POLRMT gene, and intergenic rs10006630 (C/A), and the predisposition to tick-borne encephalitis in a Russian population (using an extended sample of patients with different forms of tick-borne encephalitis). The association of the rs10006630 SNP located in chromosome 4 between the FABP2 and LINC01061 genes with a predisposition to tick-borne encephalitis was confirmed. This SNP can be considered as a new genetic marker of a human predisposition to severe forms of tick-borne encephalitis. The possible regulatory role of this SNP in the functioning of neighboring genes and a mechanism of its effect on the development of predisposition to severe forms of tick-borne encephalitis require further study.

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.

Tick-borne encephalitis virus (TBEV), belonging to the Flaviviridae family, is the most significant pathogen transmitted by Ixodes ticks and causing one of the most severe human neuroinfections. In Russia, serum immunoglobulin produced from the donor blood is currently used for post-exposure prophylactic and therapy of tick-borne encephalitis virus. However, it is known that preparations obtained from donated blood have certain disadvantages, and therefore development of novel preparations for post exposure prophylaxis and therapy of tick-borne encephalitis is required. To develop an alternative preparation, which does not include donor blood, a chimeric antibody ch14D5 against glycoprotein E of TBEV was constructed.This study was aimed to investigate protective efficacy of the chimeric antibody ch14D5 against the Far-Eastern, Siberian, and European subtypes of TBEV in in vivo experiments.A peripheral mouse model of tick-borne encephalitis was used in this study: the chimeric antibody ch14D5 was administrated intravenously in mice one day after their intraperitoneal infection with TBEV strains Sofjin, Vasilchenko, and Absettarov. Anti-TBEV serum immunoglobulin was used as a control preparation, which was administered in the same way. Protective efficacy of the chimeric antibodies 14D5 was assessed using the log-rank test. In the study, the presence or absence of antibody-dependent enhancement of infection (ADE) was examined when mice, infected with different subtypes of the TBEV, got the antibody ch14d5.Obtained results demonstrated high efficacy of the ch14D5 antibody in post-exposure prophylaxis of the disease in mice infected with any of the used TBEV strains, as well as the absence of ADE.It was shown that protective efficacy of antibody ch14D5 is higher than that of the anti-TBEV serum immunoglobulin, and antibody ch14D5 could be used for development of a therapeutic preparation for post-exposure prophylaxis.