[The problem of the so-called "change" of the tick-borne encephalitis virus genotype in the middle urals for the last 60 years].

Although the history of the tick-borne encephalitis in Urals dates back to 75 years ago, the problem of the spread and evolution of the tick-borne encephalitis virus (TBEV) largely remains terra incognita. Any research in this area is of great basic and practical importance. A few years ago, a hypothesis about the change of the TBEV genotype in the Sverdlovsk region in the 50-60-year period was put forward. In other words, replacement of the Far-Eastern genotype by Siberian genotype was suggested. The goal of this work was to discuss this hypothesis and the fallacy of this view. Recent large-scale data show the long history of the formation of Siberian TBEV virus population in the Middle Urals, as well as a foreign nature of Far-Eastern genotype through their introduction in the past. The concept of the decisive role of anthropogenic factor in the spread of TBEV in the Urals is discussed as an alternative to the hypothesis of the genotype change.

Tick-borne encephalitis virus: reference strain Sofjin and problem of its authenticity.

The Sofjin strain is one of the first isolates of tick-borne encephalitis virus and, due to its wide distribution in virus collections, it has become the reference strain. Until now, GenBank has recorded several sequences associated with the Sofjin strain that have significant differences between each other. We have sequenced the complete genome of the Sofjin strain from a virus collection and a genome fragment of the two vaccine Sofjin strains. According to phylogenetic analysis, we concluded that the GenBank sequences belong to three independent groups of Sofjin strains of the Far Eastern subtype. Their genetic differences are not a result of microevolution associated with numerous passages. Retrospective analysis of the peculiarities of origin and distribution for each of these groups showed the authenticity of one of them. For the first time, we have determined a complete genome sequence of the authentic reference TBEV strain Sofjin. Two other groups of strains named Sofjin were probably the result of cross-contamination or laboratory error. The high probability of contamination requires the introduction of a new standard for virological laboratories, the key point of which is the obligatory genetic identification of all collection strains.

Distribution of Far-Eastern tick-borne encephalitis virus subtype strains in the former Soviet Union.

European and Asian viruses within the tick-borne encephalitis flavivirus complex are known to show temporal, spatial and phylogenetic relationships that imply a clinal pattern of evolution. However, the isolation of recognized Far-Eastern tick-borne encephalitis virus (TBEV) strains in the European region of the former Soviet Union (SU), i.e. thousands of kilometres west of the region in which they are considered endemic, appears to contradict this concept. Here, we present a parsimonious explanation for this apparent anomaly based on analysis of the dates and regions in which these non-endemic strains were isolated, together with their phylogenetic relationships and the records of redistribution of animals under the All-Union programme for acclimatization of game animals within the former SU. Our evidence supports the concept that the anomalous distribution of Far-Eastern TBEV strains in Europe and Siberia arose primarily as the result of the large-scale westward redistribution of game animals for economic purposes.

Origin and distribution of tick-borne encephalitis virus strains of the Siberian subtype in the Middle Urals, the north-west of Russia and the Baltic countries.

Tick-borne encephalitis virus (TBEV) plays an important role in infectious human morbidity, particularly in Russia and the Middle Urals. The Siberian subtype of TBEV (S-TBEV) is dominant in the Middle Urals. Determining the origin of S-TBEV strains in this territory and also in the European part of Russia and the Baltic countries is very important for understanding the cause of its distribution. The surface glycoprotein E gene was partially sequenced in 165 S-TBEV isolates collected in the Middle Urals between 1966 and 2008. Nucleotide and amino acid sequence identity of the studied isolates is 94 and 97.4 %, respectively. Eighty per cent of them are represented by six clusters with identical amino acid sequences in the glycoprotein E fragment analysed. We have determined four types of isolate distribution in the explored territory: local, split, corridor and diffuse. The average rate of nucleotide substitutions per site year(-1) is estimated to be 1.56 x 10(-4). The age of the S-TBEV population was evaluated to be slightly less than 400 years. Phylogenetic analysis of the data and comparison with historical events indicate that the distribution of S-TBEV strains in the Middle Urals and the European part of Russia originated twice from different foci in western Siberia. This is related to the first land road into Siberia and the Trans-Siberian Way, which functioned at different times. The main reason for such rapid distribution of S-TBEV strains is the anthropogenic factor, i.e. human economic activity during the colonization of new territories in Siberia in the recent past.

[Molecular and epidemiological characteristics of tick-borne encephalitis virus in the Sverdlovsk Region on the basis of genotype-specific RT-PCR].

Studies and complete awareness of the regional and epidemiological properties of tick-borne encephalitis virus (TBEV) allow one to improve methods for preventing, diagnosing, and treating its severe neurological infection. The authors have developed reverse-transcriptase polymerase chain reaction (RT-PCR) systems for the detection of RNA of TBEV and for the determination of its genotype in the ticks and clinical materials. RT-PTC was shown to have a higher sensitivity and specificity than the practically used enzyme immunoassay system. Despite significant variations in the spread of infected ticks in some districts of the Sverdlovsk Region (5-12%), the average regional value was 8% over the study period. The authors have studied more than a thousand of ticks collected from the nature and humans in the epidemic season of 2005-2006. There was a virtually complete predominance (more than 95%) of the Ural-Siberian genotype, with rare cases of the European genotype (slightly more than 4%) being detected. The Far-Eastern genotype was not detected.

[Stimulation of arbovirus reproduction in cell cultures by hormones]. / Stimuliatsiia reproduktsii arbovirusov v kletochnykh kul'turakh gormonami.

The possibility of stimulation of reproduction and antigen production of arboviruses (tick-borne encephalitis, Japanese encephalitis, western equine encephalomyelitis) in cell cultures (SPEV, chick embryo fibroblasts) by using various hormones (insulin, ACTH, hydrocortisone) and optimal temperatures was demonstrated. The stimulating effect depended upon the type of cell culture, virus, method of cell treatment, and manifested by a significant increase of the hemagglutinating and infectious activity of the replicating viruses. Differences in the structure of populations, biophysical and antigenic properties of the viruses associated with the conditions of their reproduction in vitro were observed.

[Radial hemolysis reaction in the diagnosis of tick-borne encephalitis]. / Reaktsiia radial'nogo gemoliza v diagnostike kleshchevogo éntsefalita.

A test of radial hemolysis in gel (RHG) has been developed and first used for serodiagnosis of tick-borne encephalitis (TBE). In examinations of 464 blood serum specimens from 258 patients with TBE and subjects suspected of this disease in RHG and HI tests the diagnosis of tick-borne encephalitis was confirmed in 77 subjects, the results of both tests being in complete agreement. A comparative analysis of antibody levels by RHG and HI tests revealed a complete correlation. An advantage of RHG over HI test is its insensitivity to serum inhibitors and the possibility of quantitation of antibodies in whole sera which omits a complicated procedure of serum treatment to remove inhibitors and serum titrations in serial dilutions mandatory for HI tests. The specificity and sensitivity of RHG, simplicity in running and the possibility of analysing many sera within a short time recommend the RHG test for public health practice for TBE serodiagnosis.

[Antihemagglutinin exhaustion reaction in studying the activity of arbovirus antigenic preparations]. / Reaktsiia istoshcheniia antigemaggliutininov v izuchenii aktivnosti antigennykh preparatov arbovirusov.

A modification of serological tests, the antihemagglutinin exhaustion test, was developed for the study of capacity of different fractions of the antigenic preparations of tick-borne encephalitis and western equine encephalomyelitis viruses to react with antibody. The test is simple to perform, highly sensitive and specific which permits within a very short time to examine the antigenic activity of practically all, including non-hemagglutinating, virus fractions obtained by gel chromatography and ultracentrifugation. Significant functional heterogeneity of virus particles in interaction with antibody was established. Non-hemagglutinating virions were capable of binding antihemagglutinins. The intensity of interaction with antibody of the most active virus fractions in the HI and CF tests was considerably higher than that of nonfractionated preparations.

[Use of suspension adsorption methods for studying arboviruses]. / Ispol'zovanie suspenzionnykh adsorbtsionnykh metodov dlia izucheniia arbovirusov.

Hemagglutinating antigens of Western equine encephalomyelitis (WEE) virus and 5 strains of tick-borne encephalitis (TBE) virus were charcaterized by their capacity to adsorb on inorganic (kaolin, bentonite, barium sulphate) and organic (chick embryo fibroblasts) adsorbents. The adsorption capacity of TBE virus was more marked than that of WEE. The TBE virus strains 4072 and 1524 avid in serological tests adsorbed better on inorganic adsorbents than nonavid strains No. 80 and 1509. In contrast, nonavid strains interacted better with biologic adsorbents. The adsorption capacity of tissue culture antigens was superior to that of brain hemagglutinating antigens. By means of adsorption on kaolin and chick embryo fibroblasts hemagglutinating and nonhemagglutinating infections particles of WEE and TBE viruses which also differed in some biological properties were separated.

Buoyant density of some togaviruses in sucrose density gradient and capacity of their haemagglutinin fractions to interact with antibody.

The buoyant densities of Western equine encephalomyelitis virus (an Alphavirus) and tick-borne encephalitis (TBE) virus (a Flavivirus) antigens prepared by different methods were studied. Sucrose density centrifugation revealed a heterogeneity in the density of the virions. The sedimentation pattern and height of peaks of the haemagglutinating activities and infectivity, other conditions being equal, depended both on the virus species and properties of its strains and on the mode of preparation and treatment of the virus-containing material. Different haemagglutinating antigen fractions differed in their capacity to interact with specific antibody. It was suggested that the kinetics of a serological reaction and its result depend on the functional activity of the antigen preparation and, in particular, on the proportion in the reaction mixture of virus particles with a dissimilar antigenic structure.

[Avidity criteria in assessing the functional activity of antigens, antibodies and non-specific serum inhibitors on a model of the kinetic reaction of hemagglutination suppression with arboviruses]. / Kriterii aviditeta v otsenke funktsional'noi aktivnosti antigenov, antitel i nespetsificheskikh syvorotochnyk ingibitorov na modeli kineticheskoi reaktsii podavleniia gemaggliutinatsii s arbovirusami

The functional activity of some arboviruses of groups A and B, of the antibodies and serum inhibitors was studied on a model of the kinetic hemagglutination inhibition test (HAI) by different avidity criteria (velocity, completeness and stability of formation of a neutral complex). The avidity indices of the antigens, antibodies and the inhibitors proved to depend on the group, species and strain peculiarities of the arboviruses, the method of preparation of the antigen, the biological species of the donor of the immune and normal blood sera, the method of treatment of the sera and a number of other factors. There proved to be no constan-correlation between the avidity of the strain and the avidity of the serum immune to it. Inhibitors of the normal rabbit and human sera were not less effective in comparison with the specific antibodies to a number of viral strains of tick-borne encephalitis and Japanese encephaliti or even exceeded them by the avidity indices to the antigens in the HAI test. The most active (functionally) strains can be recommended for obtaining high-quality viral (antigenic and serum) preparations.

[Factors determining the antigenic activity of tissue cultures from arbovirus-infected diagnostic specimens]. / Faktory, opredeliaiushchie antigennyiu aktivnost' kul'tural'nykh arbovirusnykh diagnostikumov

Formation of arbovirus group A and B antigens is influenced significantly by the number of cells, time of monolayer growth before inoculation, the time of maximum accumulation of hemagglutinins, the infectious dose, the growth and maintenance medium composition, the autointerfering activity of the viruses and their sensitivity to tissue inhibitors. The stability of the hemagglutinating and complement-fixing properties of arbovirus diagnostic preparations does not depend upon the degree of concentration (7-12-fold) and the amount of the stabilizer (2-4%), and storage of group A diagnostic preparations in ampoules under vacuum or in ampoules sealed with air for 2-3 1/2 years (the observation period) does not influence their antigenic activity.