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In the Kurgan region, the Siberian subtype of the tick-borne encephalitis virus (TBEV) is dominant. The vaccines prepared from Far-Eastern TBEV subtype are used in this area. Among TBE patients in 2007-2011, 23.79% were vaccinated according to complete or incomplete course. 76.9% of persons were vaccinated with Encevir vaccine, Tomsk. An unusual focal form of TBE with fulminant disease with lethal outcome was developed in a patient who was vaccinated 6 times with heterotype vaccines produced using the strains of the Far-Eastern TBE subtype. Inoculation of immunoglobulin in hospital produced aggravation of clinical symptoms, development of convulsions, brain oedema, and respiratory distress syndrome. The disease continues only 55 hours from first symptoms to fatal outcome. Siberian subtype of TBEV was isolated from patient spinal cord (Kurgan-118-2010 strain). Possible mechanisms of this disease are discussed.

Eighteen polytypic tick-borne encephalitis virus (TBEV) strains containing the fragments of E and NS1 protein genes of Siberian and Far Eastern, occasionally Siberian and European subtypes were isolated in the European and Asian parts of the tick-borne encephalitis (TBE) area. They were identified using real-time polymerase chain reaction, hybridization-fluorescence detection with genotype-specific probes, restriction fragment length polymorphism analysis, and E protein sequencing. The polytypic strains were isolated from individual Ixodes persulcatus ticks, their pools, from the blood of patients and the brain of dead patients. The isolation rates of the polytypic strains in the sympathry area of different TBEV subtypes ranged from 4.4% (the Irkutsk Region) to 15.1% (the Yaroslavl Region). In addition to 2 polytypic strains, a strain similar to the TBEV 886-84 strain was isolated. The TBEV subtypes entering into the composition of the polytypic strains show nongenetic interactions, such as neutral replication or competition. The polytypic strains are stable during passages in the cultured pig embryo kidney epithelial cells and on cloning. Mouse brain passage promotes dissociation of polytypic strains. The conditions for the formation of polytypic strains and their role in the etiology of TBE are discussed.

Long-term monitoring of natural tick-borne encephalitis virus (TBEV) populations could reveal the change of TBEV subtypes, the displacement of the Far Eastern (FE) subtype, and its substitution for the Siberian (Sib) subtype. Acute and inapparent mixed infections were studied in Syrian hamsters to understand this phenomenon. The animals were inoculated with the Sib subtype and then with the FE one of TBEV (JQ845440-YaroslavI-Aver-08 and Fj214132-Kemerovo-Phateev-1954 strains). The inapparent form developed more frequently in mixed infection. Viral progeny was genotyped by reverse transcription polymerase chain reaction and hybridization fluorescence detection using genotype-specific probes. Independent reproduction of strains in the brain gave way to competition. The FE subtype dominated in hamster youngsters with acute infection. The Sib subtype had selective benefits in asymptomatic infection (adult hamsters infected intracerebrally and subcutaneously and youngsters infected subcutaneously). The competition of the subtypes was imperfect.

Polytypic strains containing the fragments of genes of Siberian and Far Eastern tick-borne encephalitis (TBE) virus subtypes were isolated from the brain of fatal TBE patients, the blood of TBE patients, and Ixodes persulcatus ticks in the foci of concomitant circulation of the two subtypes. The interaction of the Siberian and Far Eastern TBE virus subtypes was studied in the neural phase of the infection of albino mice and Syrian hamsters in order to understand conditions for formation of these strains and their role in the etiology of acute TBE. Their viral progeny was genotyped by reverse transcription-polymerase chain reaction and fluorescence hybridization assay with genotype-specific probes. Mixed infection showed an effect of synergism, independent reproduction of the two subtypes in the brain and spleen, competitive exclusion of one subtype from the viral population. The type of the Interaction depended on the species of animals, the properties of partner strains, and the target organ.

Tick-borne encephalitis virus (TBEV) causes human epidemics across Eurasia. Clinical manifestations range from inapparent infections and fevers to fatal encephalitis but the factors that determine disease severity are currently undefined. TBEV is characteristically a hemagglutinating (HA) virus; the ability to agglutinate erythrocytes tentatively reflects virion receptor/fusion activity. However, for the past few years many atypical HA-deficient strains have been isolated from patients and also from the natural European host tick, Ixodes persulcatus. By analysing the sequences of HA-deficient strains we have identified 3 unique amino acid substitutions (D67G, E122G or D277A) in the envelope protein, each of which increases the net charge and hydrophobicity of the virion surface. Therefore, we genetically engineered virus mutants each containing one of these 3 substitutions; they all exhibited HA-deficiency. Unexpectedly, each genetically modified non-HA virus demonstrated increased TBEV reproduction in feeding Ixodes ricinus, not the recognised tick host for these strains. Moreover, virus transmission efficiency between infected and uninfected ticks co-feeding on mice was also intensified by each substitution. Retrospectively, the mutation D67G was identified in viruses isolated from patients with encephalitis. We propose that the emergence of atypical Siberian HA-deficient TBEV strains in Europe is linked to their molecular adaptation to local ticks. This process appears to be driven by the selection of single mutations that change the virion surface thus enhancing receptor/fusion function essential for TBEV entry into the unfamiliar tick species. As the consequence of this adaptive mutagenesis, some of these mutations also appear to enhance the ability of TBEV to cross the human blood-brain barrier, a likely explanation for fatal encephalitis. Future research will reveal if these emerging Siberian TBEV strains continue to disperse westwards across Europe by adaptation to the indigenous tick species and if they are associated with severe forms of TBE.

The evolution of tick-borne encephalitis (TBE) is marked by the expanded nosological area, the transformation of landscapes, the formation of anthropurgic foci, the change of environmental systems, the increase of mortality rate mainly among urban dwellers, as well as pathomorphism. The evolution of natural TBE virus (TBEV) populations was studied in Eastern and Western Siberia, Middle Urals, and the European part of the nosological area. The paper first describes the types of evolutionary transformations of viral populations under the conditions of a varying environmental and epidemiological situation. These include: 1) the change of TBEV subtypes over 50-60 years; substitution of the Far-Eastern subtype for its Siberian subtype (the Sverdlovsk and Kemerovo regions); 2) the steady-state circulation of one Siberian subtype with mutanttypes being accumulated (the Vologda region); 3) co-existence of the Far-Eastern and Siberian subtypes with the common vector Ixodes persulcatus (the Yaroslavl and Irkutsk regions, etc.); 4) original mixed TBEV strains including the gene sites of proteins E and NSI of two subtypes. There is new evidence that the Siberian subtype is able to induce focal TBE forms, leading to death.

The Siberian subtype of the tick-borne encephalitis virus (TEV) is different from the Far-East subtype by a moderate virulence observed in Siberian hamsters and by a low infection development rate (100 strains were compared). No differences were found in neuro-invasiveness. Clinical findings and experiments with monkeys denote the ability of the Siberian subtype to provoke severe forms of tick-borne encephalitis (TBE). The inflammation-and-degenerative changes were localized in the brain cortex, subcortical ganglions, nuclei of medulla oblongata, in the cortex and nuclei of the cerebellum as well as in the anterior horns of the spinal cord. 18 disease cases triggered by the Siberian TEV subtypes in residents of the Western and Eastern Siberia and of Central Russia (Yaroslavl Region), including 7 acute TBE cases (5 lethal outcomes), as well as 11 chronic TBE cases are analyzed. The viral RNA was found in the cortex, medulla oblongata, horn and in the cervical part of the spinal cord of those diseased of acute TBE. Sequences of genotyped strains were presented to Gen Bank, NCBI (AY363846-AY363865).

Agar gel precipitation test with cross-adsorbed immune sera was used for the antigenic differentiation of strains of tick-borne encephalitis virus (TBEV). Fifty strains of the Far East TBEV serotype and 46 strains of the Siberian (Aina) TBEV serotype were isolated from Ixodes persulcatus, which is the main vector of the above TBEV subtypes in the Asian and European parts of Russia. The fragment of the envelope protein gene was sequenced for TBEV strains. Sequences of new-group strains of the Siberian subtypes isolated from 3 patients with chronic TBE and from brain tissues of 4 deceased patients were determined. Lethal TBE outcomes were registered in Siberia (Irkutsk Region and Krasnoyarsk Territory) and in Russia's European part (Yaroslavl Region).

The authors studied the effect of synthetic gestagens on spontaneous contractility of the myometrium in pregnant rats. The direction of the effect of progesterone and its analogs on the contractility is determined by stereochemistry of the linkage of rings A and B of the steroid molecule. Compounds possessing translinkage (5 beta-steroids) increase the amplitude of myometrium contraction, whereas their analogs with cms-coupling of rings A and B (5 alpha-steroids), and 4,5-unsaturated steroids reduce it.

Structural (E) and three nonstructural (NS1, NS3, and NS5) proteins of persistent and antigen-defective strains of tick-borne encephalitis virus are compared by immunoblotting with monoclonal antibodies to the corresponding proteins of strain Sofyin. Appreciable phenotypical differences were revealed between antigen-defective strains, but no immunological modifications as concerns the studied antigenic structures. The size of the reference NS3 protein differed from that of the persistent virus strains. Similar proteins detected in the antigenic preparations of strain Zausaev may have notable modifications.

Typical and atypical forms of Japanese encephalitis (JE) in the Russians visiting Asian countries endemic for JE are described. A patient who contracted the disease in China developed 5 months after returning to Russia acute meningoencephalitis with mental disorders and a lethal outcome on day 5 with bulbar symptoms. JE virus (strain SP-69) was isolated from his brain. By antigenic and genetic properties this strain occupies an intermediate position between Jagar-01 and Nakayama serotypes. A pregnant woman (6 months gestation) who lived in Birma for 3 years suffered from encephalitis running a protracted (more than 6 months) course; mildly manifest pyramidal signs were detected in her one-year-old infant with a normal mental status. Serologic studies showed that the disease was caused by infection with Jagar-01 serotype of JE virus. A patient contracting the disease 1 month after arrival in Japan developed a recurrent pattern of the illness: the diagnosis of JE was confirmed by repeated detection of virus-neutralizing anti-bodies in the blood and liquor. None of the patients was vaccinated against JE. Indications to prophylactic vaccination of subjects leaving for countries endemic for JE are discussed.

The Greek strain Vergina representing an individual third serotype of tick-borne encephalitis (TBE) virus has been compared with 13 TBE strains isolated on the territory of Russia and Central Asia (in Kirghizia). A kit of deoxyoligonucleotide probes complementary to genome sites of Neudorfle strain of the TBE Central European subtype (protein C and prM genes) and of strain Sofyin of the Eastern subtype (protein E, C, M, prM, ns 1, ns 2b, ns 4b genes) was used in molecular hybridization of nucleic acids. Vergina strain was referred to the genetic variant VI prevalent in the western part of the East European plain, in Udmurtia, in the Altai mountains, and in West Siberia. By its antigenic properties Vergina strain is most close to strain Yar-90 isolated in the Yaroslavl district from Ixodes persulcatus ticks.

The Greek Vergina strain of tick-borne encephalitis (TBE) virus was studied in comparison with 7 other strains by molecular hybridization of nucleic acids and by clinicomorphological markers of pathogenicity for monkeys and Syrian hamsters. By the genetical features the Vergina strain differed from the eastern and western TBE subtypes but was found to be similar to the strains of other subtypes of the Urals-Siberian, east-Siberian (Aina-1448) and Central Asian antigenic variant. This group of strains hybridized with cDNA at 65 degrees C only in the absence of 50% formamide, reacted with probe 1115 complementary to protein E gene, with 1-3 probes complementary to the conservative region of the genome but did not react with the probes corresponding to the variable regions of the genome. The Vergina strain is close to TBE genotype III. The Vergina strain was found to be virulent inducing subacute meningoencephalomyelitis which developed slowly and was accompanied by less marked morphological lesions in the cerebral cortex than those induced by the eastern subtype. The Vergina strain was demonstrated to persist in the brain, liver, spleen, and lymph node tissues.

Ten strains of tick-borne encephalitis (TBE) virus isolated from single specimens of I. persulcatus ticks were studied. The strains were divided into antigenically complete (AC) and antigenically defective (AD), depending on the presence or absence of some virus antigens in concentrated virus preparations, characteristics in rocket immune electrophoresis (RIEP), rate and intensity of humoral immune response in monkeys and rabbits, and plaque size in SPEV cell culture. The AC-strain markers include high activities of precipitating, hemagglutinating (HA), and complement-fixing (CF) antigens, formation of precipitates moving in rocket shape towards anode and cathode in RIEP, rapid development of antihemagglutinins and virus-neutralizing antibodies, large plaques (3-5 mm). The AD variants are characterized by the lack of HA and precipitating activity, low titres of CF antigen, slow and poor immune response, the lack of cathode precipitate "rocket", very small plaques. The antigenic defectiveness is transitory and shows in early passages; after 10-11 passages in SPEV cell cultures or in white mice, transformation AD----AC occurs. A transformed strain is neutralized, like standard TBE strains, by blood sera of a typical patient with poliomyelitis-like form of TBE. Examinations of blood sera from the population of an endemic zone (Yaroslavl Province) and 67 TBE patients (Kurgan Province) demonstrated the association of AC and AD variants with the formation of immune portion of the population and TBE etiology. Cases of the disease confirmed by seroconversion in HI with commercial diagnosticum are associated with AC variants, whereas AD variants are associated with those TBE cases which are difficult to diagnose using the commercial diagnosticum.

Myelopeptides (MP), bioregulatory molecules of bone marrow origin, exert a protective effect in persistence of tick-borne encephalitis virus in cynomolgus monkeys (Macaca fascicularis). The experiments involved 32 monkeys. The effect of MP was observed after one or two subcutaneous injections in a dose of 1 mg within 1.5-2 months after virus infection. The effect consists in 25-fold reduction of the frequency of virus persistence, marked limitation of the zone of spread of the persisting virus, including the central nervous system (CNS), decrease in virulence of the persisting virus, and lack of morphological signs of progress of the pathological process in the CNS. The protective effect was also observed when the infected monkeys were treated with MP and inactivated concentrated TBE vaccine. At the same time, the vaccine alone exerted a much less marked effect on the persisting TBE virus producing only a 2-fold reduction in the frequency of persistence without limitation of the zones of virus spread. In acute TBE in BALB/c mice, the effect of MP is observed irregularly. The marked protective effect of MP in TBE virus persistence in monkeys is not associated with stimulation of humoral immunity but is mediated by other immunological mechanisms requiring further study.

Blood sera (1488 specimens) of the human population collected in the zone of isolation of tick-borne encephalitis (TBE) virus strains belonging to the Aina/1448 serotype were tested by the neutralization, hemagglutination-inhibition, agar gel diffusion and precipitation, and complement fixation tests with antigens of the Aina/1448 strain and prototype TBE virus strains representing the eastern and western serotypes. The dominating role of the Aina/1448 serotype in the formation of herd immunity in different areas of the forest-steppe and taiga zones of the Irkutsk region was established. Antibody to the Aina/1448 strain was found in the blood of the normal population and patients with acute and chronic forms of TBE. In TBE patients the percentage of virus-neutralizing antibody to the Aina/1448 strain was significantly higher (36.8%) than in the general population (16.6%). These are differences in the structure of immunity in vaccinees and nonvaccinees with TBE vaccine. Sera from the nonvaccinees reacted with the Aina/1448 strain more frequently, sera of the vaccinees more frequently contained antibody to different serotypes of TBE virus. Diagnostic problems and criteria of evaluation of the local antigenic variants of TBE virus are discussed.

Sixty-seven Macaca rhesus monkeys were inoculated with 2 mutants and 3 virulent strains of tick-borne encephalitis (TBE) virus including strains isolated from patients with a chronic form of TBE. A model of the clinical course of acute, subacute, and chronic encephalitis was produced by intracerebral inoculation and that of asymptomatic infection was produced by subcutaneous inoculation [with or without administration of cyclophosphane (CP)]. Virus persistence developed after inoculation with all the strains, after non-fatal encephalitis and inapparent infection irrespective of CP administration. In monkeys recovering from encephalitis the virus persisted for at least 783 days. After asymptomatic infection, it persisted for 302 days; neither virus nor virus-specific antigen was detected at 356, 367, and 620 days.

Tick-borne encephalitis (TBE) virus persists in experimentally infected rhesus monkeys in the presence of humoral antibodies. Various dynamics of the humoral response (stable, increasing, decreasing, undulatory titres) have been noted, associated with complete or incomplete set of antibodies. Always present were the virus-neutralizing antibodies, often the complement-fixing antibodies, less frequently precipitating and haemagglutination inhibition (HI) antibodies were found. There was a correlation between the set of antibodies present and the virus-specific antigens expressed; the persisting TBE virus was usually deficient in haemagglutinin synthesis. In cases of asymptomatic infection a more expressed and long-lasting immunity as observed with the persistence of TBE virus in organs of the immune system.

Tick-borne encephalitis (TBE) virus was isolated from the brains and spinal cords, blood, livers, lymph nodes and kidneys from Macaca rhesus monkeys showing acute and subacute fatal encephalitis. In subacute encephalitis, virus titres in the CNS were lower than in acute disease (3.0--6.2 against 3.8--8.3 log LD50/ml). TBE virus localization in chronic encephalitis was largely the same as in acute and subacute disease. In monkeys with a chronic course and stable paralysis of the upper extremity, infectious TBE virus was isolated on day 383 from subcortical ganglia and spinal cord. In lymph nodes and spleen, it could be detected only by a combination of methods (co-cultivation in association with fluorescent antibody technique and complement-fixation test, explantation of organ fragments) more sensitive than is the inoculation of mice with organ homogenates. TBE virus was detected by the same methods on day 90 in the CNS and internal organs of a monkey with chronic encephalitis in the stage of remission.

The method of explantation was used to examine 63 organs from M. rhesus monkeys 92-783 days after intracerebral and subcutaneous inoculation with the Vasilchenko, Aina/1448 and 41/65 strains of tick-borne encephalitis virus. The optimal time for examination of the explants by tests of the hemagglutinating, cytopathogenic activity of the virus and its pathogenicity for mice was found to be the 15th day of cultivation. A comparative study of the properties of 3 isolates obtained from explants of the spleen, liver and subcortical cerebral ganglia 202 and 307 days after inoculation of monkeys was carried out. The isolates differed from the parental TBE virus strains by their capacity to form small plaques in PEKV cell cultures (pig embryo kidney cells in versen medium).

Reciprocal stimulation or inhibition are observed in double infection of mice with Rauscher leukemia virus (RLV) and togaviruses Sindbis or West Nile (WN). Stimulation of togavirus infections is manifested by the enhancement of the visceral phase of pathogenesis without subsequent activation of togarivus reproduction in the CNS. This effect is accompanied by enhanced togavirus replication in splenocytes, a decrease in the number of antibody-producing cells in the spleen, a decrease of antihemagglutinins titer in the blood without any significant change in the virus-neutralizing antibody and interferon titers. RLV-induced immunosuppression is termporary and of varying intensity with regard to individual parameters of immune response and to different variants of WN virus (highly virulent, poorly virulent). It is assumed that the differentiated and temporary nature of the immunosupressive effect of RLV is conducive to selective stimulation of the visceral phase of togavirus reproduction followed by inhibition of the neural phase under the influence of restored immune mechanisms and interferon. Because of the defects of humoral and cellular immunity, however, no complete elimination of togavirus occurs and conditions for its long-term persistence are created.

Fifty monkeys (Macaca rhesus) inoculated with tick-borne encephalitis (TBE)virus intracerebrally or subcutaneously were examined. The efficiencies of different virus detection methods at 90 to 783 days after inoculation were: explantation of organs - 41.2%; co-cultivation of trypsinized organ cells and indicator cells in the presence of 5-bromo-2-iododeoxyuridine with detection of the virus-specific antigen by immunofluorescence - 43.7%, detection of infectious virus and complement-fixing antigen - 13.6%; immunofluorescence examination of organ impression smears - 29.3%; and examination of organ homogenates with detection of virus pathogenic for mice - 1.7%. the latter method was the least sensitive for the detection of persisting TBE virus, in spite of that in the first 3 weeks of infection its efficacy was 88.1% in examinations of clinically ill monkeys and 23.8% in the inapparent form of the infection.

In 28 Macaca rhesus monkeys inoculated subcutaneously (s.c.) with different strains and mutants of tick-borne encephalitis (TBE) virus and developing asymptomatic infection, TBE virus and the virus-specific antigen were found at different intervals up to 302 days post inoculation (p.i.) in the CNS and internal organs (liver, spleen, lymph nodes, kidneys). When cyclophosphane (CP) was used as an immunosuppressor, no significant changes in virus distribution were observed with the exception of more frequent isolations at early intervals from kidneys and at late intervals from spleen. With or without CP administration virus was found in the spinal cord at 3--6 days, in the cerebellum and subcortical ganglia at 11--14 days, in the cerebral cortex at 19 days p.i. Early after inoculation the virus was more frequently isolated from the CNS than from the internal organs; later (93--302 days p.i.) the persisting virus or virus-specific antigen were predominantly found in internal organs. The set of sensitive methods used facilitated to detect the persistence of TBE virus in noninfectious form in monkeys with asymptomatic infection.

Syrian hamsters subcutaneously inoculated with tick-borne encephalitis (TBE) virus were given transfer-factor (TF) preparations derived by different methods. The preparation of specific TF was obtained from the blood leukocytes of TBE convalescents. The nonspecific TF preparations were made of the lymphocytes of the tonsils removed from children with chronic tonsillitis outside the TBE focus. The effect of the TF preparations depended on the TBE virus strain and dose, TF preparation dosage schedule and characteristics. The specific TF preparations stimulated the development of acute fatal TBE after 3 injections at intervals of 0, 48 and 96 hours postinoculation with the virus. The preparations of nonspecific TF potentiate the infection after preliminary (24 hours) and simultaneous inoculation of the virus, producing transformation of asymptomatic infection to subacute TBE or exacerbation of the subacute process. A significant inhibition of TBE virus reproduction in spleen and brain tissues is observed after inoculation of a nonspecific TF F150 preparation 72 hours after virus inoculation, that is at the time when the virus has been already localized in the central nervous system. The results of the study indicate that the protective effect of TF is mainly associated with nonspecific immunopharmacological activity of the preparations.

The properties of tick-borne encephalitis (TBE) virus persisting for 90-383 days after intracerebral and subcutaneous inoculation of Macaca rhesus monkeys were studied, namely (1) the type of infection produced directly in the tissues of the experimental monkeys; (2) the activating effect of co-cultivation and explantation procedures; and (3) the phenotype of the isolates by a set of markers. The virus was detected and analysed in 52 instances. Directly in monkey tissues the virus induced a productive infection rarely (5.8%) but more frequently (71.2%) an abortive infection detectable by immunofluorescence (presence of virus-specific antigen). In 23% of instances a nonproductive infection was observed in monkey tissues. Like abortive infection it could be activated by the co-cultivation of cells and explantation procedures. The latter exerted a more marked activating effect than co-cultivation. The strains isolated from monkey tissues in productive infection or activated by explanation or co-cultivation were heterogeneous in their properties. The following virus phenotypes were found: virus highly virulent for mice, cytocidal and antigenically complete; a cytocidal virus of low virulence, possessing haemagglutinin; and a cytocidal virus apathogenic for mice, devoid of haemagglutinin but synthesizing complement-fixing antigen and an antigen detectable by immunofluorescence.