[Polytypic strains in the genofund of tick-borne encephalitis virus].

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.

[Evolution of tick-borne encephalitis and a problem of evolution of its causative agent].

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.

[Comparative analysis of virulence of the Siberian and Far-East subtypes of the tick-born encephalitis virus]. / Sravnitel'nyi analiz virulentnosti sibirskogo i dal'nevostochnogo podtipov virusa kleshchevogo entsefalita.

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).

[The Siberian and Far-Eastern subtypes of tick-borne encephalitis virus registered in Russia's Asian regions: genetic and antigen characteristics of the strains]. / Sibirskii i dal'nevostochnyi podtipy virusa kleshevogo éntsefalita v evropeiskikh i aziatskikh regionakh Rossii: geneticheskaia i antigennaia kharakteristika shtammov.

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).

[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.

[A comparative analysis of proteins from persistent and antigen-defective strains of tick-borne encephalitis virus]. / Sravnitel'nyi analiz belkov persistiruiushchikh i antigenno-defektnykhshtammov virusa kleshchevogo éntsefalita.

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.

[Japanese encephalitis in citizens of Russia who travel abroad]. / Iaponskii éntsefalit u grazhdan Rossii, vyezzhaiushchikh za rubezh.

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.

[Vergina-like strains of tick-borne encephalitis in Russia]. / Verginapodobnye shtammy virusa kleshchevogo éntsefalita v Rossii.

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 genetic characteristics of the Vergina serotype of the tick-borne encephalitis virus and its pathogenetic traits]. / Genetichskaia kharakteristika serotipa Vergina virusa kleshchevogo éntsefalita i osobennosti ego patogennosti.

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.

[The phenomenon of antigenic defectiveness in naturally circulating strains of the tick-borne encephalitis virus and its possible connection to seronegative forms of the disease]. / Iavlenie antigennoi defektnosti u tsirkuliruiushchikh v prirode shtammov virusa kleshchevogo éntsefalita i ego vozmozhnaia sviaz' s seronegativnymi formami zabolevaniia.

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.

[Deoxyoligonucleotide probes that differentiate antigenic and pathogenetic variants of the tick-borne encephalitis virus]. / Dezoksioligonukleotidnye zondy, differentsiruiushchie antigennye i patogeneticheskie varianty virusa kleshchego éntsefalita.

Experiments on molecular hybridization were carried out using a panel of 11 deoxyoligonucleotide probes complementary to different parts of tick-borne encephalitis (TBE) virus, strain Sophyin, genome. Under study were the TBE virus strains differing by 3 criteria: (1) source of isolation (patients with acute and chronic TBE, Ixodes persulcatus and D. nuttalli ticks, small mammals); (2) serotype (eastern and Siberian Aina/1448), (3) virulence for Syrian hamsters. RNA of all the strains was hybridized with kDNA, 90% of strains with probe Sh5 complementary to protein E gene, nucleotide positions 1285-1311. The highest differentiating capacity was observed with probes P131 and Sh3 complementary to genes of proteins ns2b and M. These probes reacted with RNA of 100% of highly virulent strains of the eastern serotype and only with 20-30% of strains of the Aina/1448 serotype of lower virulence. A certain differentiating capacity was demonstrated by probes Sh2 and P10 complementary to genes of prm and C proteins: they hybridized with RNA of 80% of eastern serotype strains highly virulent for hamsters and with only 20% of Aina/1448 serotype strains of low virulence. The panel of probes used revealed no significant differences among strains in relation to their isolation source, with the exception of a strain isolated from D. nuttalli ticks which reacted only with kDNA and probe P2 complementary to nsI protein gene, but not with other probes. The TBE virus strains isolated from patients with chronic TBE were shown to represent a genetically heterogeneous group.

[Immunogenicity and stability of a Soviet inactivated cultured vaccine against Japanese encephalitis]. / Immunogennost' i stabil'nost' otechestvennoi kul'tural'noi inaktivirovannoi vaktsiny protiv iaponskogo éntsefalita.

Six immunologically active vaccine batches inducing a specific antibody to Japanese encephalitis (JE) virus were obtained in serial manufacture of the preparation. In HI tests, the minimal antibody titre was 1:80, the maximal 1:320, neutralization index 1g was 3.7 to 5.2. The data on the stability of the antigenic potency of the vaccines in relation to the duration of storage at 4 degrees-6 degrees C are presented (the follow-up period 3 years). A certain relationship was found between the antigenic potency of the preparation and the titre of the initial infectious tissue culture virus. Also, a definite correlation was found between the initial immunogenic potency of the vaccines and their stability in storage. After 3 years of storage, three vaccine lots remained antigenically active, namely those which after manufacture had induced antihemagglutinins in titres 1:160 to 1:320. The antigenic activity of 6 vaccine batches prepared from the production strain Peking-1 (Nakayama serotype) was studied against the predominant strain of Jagar-10 serotype. All the freshly prepared vaccine batches were found to induce production of antihemagglutinins to both serotypes of JE virus, whereas virus-neutralizing antibodies were found only to the test strain Nakajama-NIH homologous to the vaccine Peking-1 strain. After 1 year of storage, four vaccine batches lost their capacity to induce production of antihemagglutinins to Jagar-01 strain, two batches induced antibody in low titres. This fact should be considered in evaluation of postvaccination immunity status in humans.

[The Aina/1448 serotype of the tick-borne encephalitis virus]. / Izuchenie serotipa Aina/1448 virusa kleshchevogo éntsefalita.

The antigenic structure of 48 tick-borne encephalitis virus (TBE) strains isolated in the Irkutsk region and the Buryat ASSR in 1960-1981 was studied. Antigenic analysis was performed by the agar gel diffusion-precipitation test using adsorption of the immune sera. A new group of 31 TBE virus strains was found to belong to the Aina/1448 antigenic type. Close ecological relationship between this serotype and Muridae was found. Ix. persulcatus ticks are vectors of 2 TBE virus serotypes, Aina/1448 and eastern serotype. Data on combined circulation of these 2 serotypes of TBE virus in the Irkutsk region and the Buryat ASSR were obtained, the Aina/1448 serotype definitely dominating in 3 areas of the Irkutsk region. The areas of circulation of the Aina/1448 serotype strains are associated with forest and forest-steppe landscapes disturbed by human economic activity.

[Characteristics of tick-borne encephalitis virus strains isolated from patients with chronic diseases of the central nervous system]. / Kharakteristika shtammov virusa kleshchevogo éntsefalita, izolirovannykh ot bol'nykh s khronicheskimi zabolevaniiami tsentral'noi nervnoi sistemy.

Two groups of tick-borne encephalitis (TBE) virus strains were studied: Group 1, 5 strains isolated from patients with chronic TBE with progressive course, Group 2, 13 strains isolated from residents of an endemic locality, with chronic diseases of the CNS (amiotrophic lateral sclerosis, epidemic encephalitis, polyoencephalomyelitis, syringomyelia, etc.). Strains of both groups belong to two serotypes of TBE virus: mid-Siberian and Transbaikal (synonym Aina/1448) and eastern. Group 1 strains were heterogeneous in their virulence, immunogenic and surface properties of the virions. The latter characteristic was demonstrated in studies of elution from macropore glass and sensitivity of hemagglutinin to the effect of detergents (Bridge-96, Tween-80). Eight of 13 Group 2 patients had concurrent diseases (tuberculosis, toxoplasmosis, tumors, etc.). Streptomycin was demonstrated to activate asymptomatic infection with TBE virus in hamsters. It is assumed that isolation of TBE virus from Group 2 patients could be due to activation of persistent infection under the effect of concurrent diseases and drugs.

[Variants of the host immune response to the tick-borne encephalitis virus]. / Varianty immunnogo otveta khoziaina na virus kleshchevogo éntsefalita.

Syrian hamsters intracerebrally infected with tick-borne encephalitis (TBE) virus were used to study the correlation between virulence of the strains, their serotype, form and course of the disease, average survival time (AST) of hamsters, features of CNS pathomorphology, and the changes of values of cell-mediated and humoral immunity. Thirty one strains of TBE virus isolated in Siberia and Far East were studied. Virulence of the strains ranged from 100% to 5%, AST from 5 to 237 days. Hamsters developed acute lethal encephalitis, subacute encephalitis, or asymptomatic infection. Most virulent strains produced early and extensive lesions in the brain stem. In encephalitis with subacute course pathomorphological changes in the CNS developed slower and the brain stem was less involved. Each variant of TBE course was associated with a certain pattern of host immune response formed as early as the first day of infection. Highly virulent strains inducing acute encephalitis and 100% lethality within 5-9 days produced immune response characterized by high antigen reactivity of lymphocytes by the level of rosette-forming cells, marked specific sensitization in splenocyte migration-inhibition test, moderate antibody-producing cell reaction, increased thymus weight index, low values of nonspecific resistance with low levels of antihemagglutinins and virus-neutralizing antibody by the end of the first week. Different immunologic condition was associated with asymptomatic infection and increase of AST to 100-237 days.

Persistence of tick-borne encephalitis virus in monkeys. VII. Some features of the immune response.

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.

Study on West Nile virus persistence in monkeys.

Experiments in M. rhesus showed persistence to be a typical property of West Nile virus. This property was exhibited by strains belonging to different antigenic types, and varying in virulence and in the isolation area (U.S.S.R., Uganda, India). The duration of persistence was at least 5 1/2 months in asymptomatic infection and in convalescence after encephalitis or a febrile disease. The virus isolated within the first 2 weeks after inoculation of monkeys has the standard properties. The virus persisting for 2 months retains its cytopathic and antigenic activity, however, is non-pathogenic for white mice. After 5 1/2 months of persistence the virus has no neurovirulence or cytopathic properties but is capable of infecting the susceptible cells and induces in them the synthesis of virus-specific antigen detectable by immunofluorescence. The persisting virus has been isolated by cocultivation of trypsinized monkey organ cells and cells of the indicator culture. This virus was located mostly in the cerebellum, cerebral subcortical ganglia, lymph nodes, and kidneys. The monkeys experiencing encephalitis, febrile, or asymptomatic infection showed in morphological examinations a subacute inflammatory-degenerative process in the central nervous system. The results suggest that West Nile virus, one of the most widely spread arboviruses in Africa, Asia, and Europe, may be implicated in the etiology of subacute diseases of the CNS.

[Characteristics of the persistence of the tick-borne encephalitis virus against a background of experimentally induced allergic encephalomyelitis in M. rhesus monkeys]. / Osobennosti persistentsiii virusa kleshchevogo éntsefalita na fone éksperimental'no indutsirovannogo allergicheskogo éntsefalomielita u obez'ian M. rhesus.

Asymptomatic persistence of tick-borne encephalitis (TBE) virus was modeled by subcutaneous inoculation; the virus and the antigen were detected up to 307 days by the cell co-cultivation, tissue explants, and immunofluorescence methods. At early and late periods of the persistent infection (1 1/2, 8 1/2, or 10 1/2 months) for the purpose of its stimulation, an autoimmune disease, experimental allergic encephalomyelitis (EAE), running a course of the type of delayed hyperergic reaction was induced in the animals by inoculation of an encephalitogenic mixture (EM). After EM inoculation, a chronic demyelinating process developed in the CNS without the involvement of neurons; proliferation of immunocompetent cell elements was observed in the spleen and lymph nodes; a short-time or stable stimulation of TBE-specific humoral immunity was observed. Against the background of the development of chronic EAE and a short-time stimulation of humoral immunity, persistence of TBE virus was prolonged for over 2 years, its location being unchanged (different parts of the brain, spleen). Despite long-term persistence of TBE virus in the CNS, no clinical and morphological symptoms of chronic TBE were found. Possible mechanisms of the effect of this stimulating factor favouring the activation of the persisting TBE virus on the one hand and limiting this process on the other are discussed.