Genetic diversity and geographical distribution of the Siberian subtype of the tick-borne encephalitis virus.

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.

DEFINITION AND COMPARATIVE ANALYSIS OF THE GENOMIC STRUCTURE OF SIBERIAN STRAINS OF TICK-BORNE ENCEPHALITIS VIRUS OF THE EUROPEAN SUBTYPE.

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).

Comparative analysis of complete genome sequences of European subtype tick-borne encephalitis virus strains isolated from Ixodes persulcatus ticks, long-tailed ground squirrel (Spermophilus undulatus), and human blood in the Asian part of Russia.

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.

[Detection of potential sites of recombination in the Tick-borne encephalitis virus by the methods of comparative genomics].

The results of the bioinformatic search for the potential sites of the recombination in the genome-wide structures of the tick-borne encephalitis virus (TBEV) through a series of software techniques were presented in this work. The genomes of the 55 TBEV strains were assayed, 21 of them showed the presence of the recombination sites. Recombinant strains belonged to the Far Eastern (19 strains) and European (2 strains) genotypes. 22 sites of the recombination attributed were identified to five types based on position, strain, and regional characteristics. The parental strains were identified based on the genotypic and geographical parameters, which do not contradict the possibility of the formation of the recombinants. Nearly two-thirds of the sites are located in the regions of NS4a and NS4b genes, which are the "hot spots" of the recombination, most of them being concentrated in the gene NS4. It was shown that the recombination processes did not occur at the level of the genotypes (European genotype) or certain groups within the genotype (Far East) and were typical of the peripheral populations.

[Genotypes 4 and 5 of the tick-borne encephalitis virus: features of the genome structure and possible scenario for its formation].

On the basis of the comparison of complete genome structures of 32 strains and gene E fragments (160 ndt) of 643 strains and RNA isolates of tick-borne encephalitis (TBE) virus, we confirmed our previously expressed assumption (Zlobin V.I. et al, 2001) of existence, along with the three major genotypes, of genotypes 4 (strain 178-79) and 5 (strain 886-84). "Mosaic" structure of the polyprotein in the two strains was established. It manifests itself in particular in the sequences of 14 positions (C-3, E-206, NS1-54, NS-285, NS2A-100, NS2A-127, NS2A-174, NS2A-175, NS2A-225, NS3-376, NS4B-28, NS4B-96, NS5-18, NS5-671) containing the amino acids strictly conserved for each of the three major genotypes and is consistent with a uniform pattern of distribution of nucleotide substitutions that are specific for genotypes 1, 2 and 3. Possible scenario of the origin of TBE genotypes 4 and 5 was suggested.

[Detection of recombinations in tick-borne encephalitis virus by using computer analysis of viral genomes].

Computer programs were used to search for tick-borne encephalitis (TBE) virus recombinants among the isolates whose complete nucleotide sequences are deposited in the GenBank database. The application of RDP, Chimaera, Maximum chi2, and TOPAL programs has revealed recombinant sites in a number of sequences, which indicates that TBE virus has recombinations and that the programs are suitable for their detection.

Genotyping and characterization of the geographical distribution of tick-borne encephalitis virus variants with a set of molecular probes.

A panel of deoxyoligonucleotide probes for studying the genetic variability and genotyping of Tick-borne encephalitis virus (TBEV) strains by molecular hybridization of nucleic acids (MHNA) was created. This panel allows to estimate the genetic structure of individual TBEV strains, as the targets for probes are both variable and genotype (subtype)-specific sequences of all TBEV genes. With the help of this panel using the method of molecular hybridization of nucleic acids 268 archived TBEV isolates were investigated and the distribution of its genotypes and subgenotypes of genotype 3 was made more precise in the territory of Eurasia. The conclusion made earlier has confirmed that five genotypes of TBEV co-circulate in Eastern Siberia. It is generally recognized that the Far Eastern (TBE-FE), European (TBE-Eu), and Siberian (TBE-Sib) genotypes are widespread and epidemiologically important. The fourth genotype is presented by only one isolate, TBE178-79, originated from Irkutsk region, Russia. The fifth genotype includes 10 isolates, 1 of them, TBE886-84, was found earlier and recognized as unique [Zlobin et al. (2001b): Vopr Virusol 1:12-16 (Russian)].

[The genetic variability and genotyping of tick-borne encephalitis virus with deoxyoligonucleotide probes].

A panel of genotype-specific molecular probes has been designed, which is used to indicate and differentiate tickborne encephalitis (TBE) virus. It assesses the individual genetic structure of each strain since the targets for the probes are the variable sequences of all 10 virus genes, which are specific for each of three genotypes. The molecular nucleic acid hybridization by means of the panel was used to study 273 TBE virus strains. Isolated from a Eurasian area; along with the representatives of three genotypes, the virus strains, the genomic structures of which do not fit in the established concept on three genotypes, circulate in Eastern Siberia.

[Molecular epidemiology of tick-borne encephalitis].

The review presents information on the development of studies into the molecular epidemiology of tick-borne encephalitis (TBE) in Russia and foreign countries. The existence of three major virus genotypes has been established by various techniques, such as genomic fragment sequencing, molecular hybridization using genotype-specific probes, and restriction fragment length polymorphism test. Each of the genotypes prevails in different parts of a natural habitat; the Ural-Siberian genotype (a Siberian subtype) is most commonly encountered. The genetic differences between the strains belonging to different genotypes are great and comparable with differences between some mammalian flaviviruses transmitted by ticks (viruses of a TBE complex). Further studies of the molecular epidemiology of TBE are of importance in understanding the evolution of the causative agent, improving the taxonomy and the classification of flavivuruses, and designing highly effective methods for the specific diagnosis, prevention, and treatment of the disease.

[Current approaches to emergency specific prevention of tick-borne encephalitis].

Emergency specific prevention of tick-borne encephalitis (TBE) by using homologous immunoglobulin is an important element in the package of controlling measures against this viral natural and focal infection. There are annually a few hundred thousand referrals for health care facilities for tick bites. Their maximum coverage tactics via immunoglobulin prevention is medically unreal and unjustifiable. The paper presents the results of a long-term application of another approach based on preliminary rapid studies of the ticks taken from victims or the blood of patients in the period of possible development of virusemia and preventive immunoglobulin use only in the persons bitten with TBE virus-infected ticks. Examination of the material available from more than 56 thousand referrals indicated the high epidemiological (more than 99%) and economic effectiveness of the target administration of an immunological drug. By taking into account the accumulated data on a wide spread of combined foci of TBE and other tick-borne infections and the authors' own experience, it is suggested that it is necessary to organize a comprehensive differential laboratory diagnosis and emergency prevention against the whole complex of Ixodes tick-borne infections.

[Specific determination of flaviviruses by molecular hybridization with synthetic deoxyoligonucleotide probes]. / Spetsificheskoe opredelenie flavivirusov metodom molekuliarnoi gibridizatsii s sinteticheskimi dezoksioligonukleotidnymi zondami.

Molecular probes were designed for the purpose of specific determination of flavioviruses transmitted by the ticks of tick-borne encephalitis (TBE) and Omsk hemorrhagic fever (OHV) as well as by mosquitoes of Japanese encephalitis (JE), North Nile (NN), Murrey Valley encephalitis (MVE), Saint-Lois encephalitis (SLE), dengue 1-4 and of yellow fever (YF). The probes are synthetic deoxyoligonucleotides with the 18-20 long basis and complementary for the RNA fragments defined by computer analysis. The thus obtained probes, which specifically hybridize themselves with the sets of the TBE virus or of the OHV virus and do not hybridize themselves with other TBE viruses' sets. Group-specific probes for YE and dengue viruses as well as virus-specific probes, which are able to detect each of the above viruses without any cross effects, were suggested for indexing and identifying the flaviviruses transmitted by mosquitoes.

[Analysis of genetic variability of strains of tick-borne encephalitis virus by primary structure of a fragment of the membrane protein E gene]. / Analiz geneticheskoi éntsefalita po pervichnoi strukture fragmenta gena belka obolochki E.

Primary structures of gene fragments of E protein (160 n.b.) have been determined for 29 tick-borne encephalitis (TBE) strains isolated from different parts of a territory. Analysis of homology of nucleotide sequences of these strains and data on 6 TBE strains published by other authors showed that they can be divided into 6 groups (genotypes) by the following gene typing criteria: strain structure within the genotypes differing by no more than 9%, differences between strains of different genotypes are at least 12%. Based on these criteria, the prototype strains of the Far Eastern antigenic variant (Sofyin), Central European antigenic variant (Neudoerfle), and Vergina strain form different genotypes 1, 2, and 6, respectively. East Siberian strain Aina and Ural Siberian strain Lesopark-II belong to the same TBE virus genotype 3; two-thirds of analyzed strains belong to this genotype. Genotype 4 is represented by one strain 178-79, and genotype 5 by strain 886-84, both isolated in East Siberia.

[Genetic typing of tick-borne encephalitis virus based on an analysis of the levels of homology of a membrane protein gene fragment]. / Genticheskoe tipirovanie shtammov virusa kleshchevogo éntsefalita na osnove analiza urovnei gomologii fragmenta gena belka obolochki.

All heretofore known genomic structures of tick-borne encephalitis (TBE) virus are analyzed. The authors prove the adequacy of using short fragment of E protein gene for characterization of philogenetic relationships between TBE strains. Three main genotypes of the virus are distinguished, one corresponding to Far Eastern variant, one to West, and the third includes strains belonging to Ural Siberian and Central Siberian and Transbaikal variants. Results of genetic typing by nucleotide sequences are confirmed by analysis of amino acid sequences of E protein fragments, specific marker amino acids in definite positions being determined for each genotype.