GAG-binding variants of tick-borne encephalitis virus.

Previously different authors described various flavivirus mutants with high affinity to cell glycosaminoglycans and low neuroinvasiveness in mice that were obtained consequently passages in cell cultures or in ticks. In present study the analysis of TBEV isolates has shown existence of GAG-binding variants in natural virus population. Affinity to GAG has been evaluated by sorption on heparin-Sepharose. GAG-binding phenotype corresponds to such virus properties, like small plaque phenotype in PEK cells, absence of hemagglutination at pH 6.4, and low neuroinvasiveness in mice. Mutations increasing charge of E protein were necessary but not sufficient for acquisition of GAG-binding phenotype. Molecular modeling and molecular dynamics simulation have shown that the flexibility of E protein molecule could bear influence on the phenotypic manifestation of substitutions increasing charge of the virions.

[Change in the antigenic structure of surface glycoprotein E of tick-borne encephalitis virus during its adaptation to ticks and mammals].

Enzyme immunoassay and immunoprecipitation test of viral proteins, by applying a tick-borne encephalitis (TBE) viral protein E monoclonal antibody kit, have shown that TBE adaptation to ticks and mammals may cause a considerable change in the antigenic structure of surface glycoprotein E, by involving different antigenic domains, including a neutralizing epitope that seems to participate at the early stages of interaction of virions with the cell membrane.

[Residual neurovirulence of the chimera of Langat and Denge-4 flaviviruses in intracerebral infection of monkeys].

The residual neurovirulence of the previously obtained chimeric virus Tp21/DEN4, containing the RNA region encoding the pre M and E structural enzymes, strain Tp21, virus Langat, and the remaining part of the genome from the Denge 4 virus, was studied in experiments with monkeys Cercopithecus aethiops involving the intracerebral administration of the virus. The tick-borne encephalitis virus, strain Absettarov, was used as positive control. A comparative analysis of the experimental and published data showed the chimeric virus to be less virulent by its degree of morphological affection in the CNS zones, its spread into the CNS and by a percentage share of animals with viremia versus the Tp21 parent strain and Elantsev strain of the Langat virus.

[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.

[Genomic changes in strains of the tick-borne encephalitis virus as a result of passages in mice]. / Izmenenie genomov shtammov virusa kleshchevogo éntsefalita v rezul'tate passazhei na myshakh.

Changes in genomes of TBE strains isolated from various sources at early stages of laboratory adaptation to white mice brain were demonstrated by molecular hybridization of nucleic acids with synthetic oligonucleotide probes, complementary RNA sites of reference TBE strains Sofyin and Neudorf. Of the 4 TBE strains passed 6 times through white mice brain in only 1 the level of RNA hybridization with 2 oligonucleotide probes was found changed, whereas in all the 3 tested strains the level of RNA hybridization with 1-2 oligonucleotide probes complementary to strain Sofyin was increased after 7-16 passages in the same system. The most noticeable changes in the genome were detected during readaptation to white mice of a TBE variant selected in passages on H. marginatum marginatum ticks.

[The electrophoretic mobility of the virus-specific proteins of tick-borne encephalitis virus strains isolated in different geographical regions of the CIS Nations]. / Izuchenie élektroforeticheskoi podvizhnosti virusspetsificheskikh belkov shtammov virusa kleshchevogo éntsefalita, vydelennykh v razlichnykh geograficheskikh regionakh SNG.

Tick-borne encephalitis (TBE) virus strains isolated in different geographic areas were characterized by changes in electrophoretic mobility (EPM) of virus-specific proteins. Analysis of EPM of intracellular virus-specific protein E and other high molecular proteins of 42 TBE virus strains revealed similarity among the majority of them, with the exception of several strains including the Sophyin strain (Far-Eastern subtype). The most marked variability in the electrophoretic behavior of virus-specific proteins was observed with low molecular proteins prM, ns4b, ns2a.

[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.

[The properties of the particles formed in the reproduction of an acute in vitro infection with the tick-borne encephalitis virus adapted to H. plumbeum ticks]. / Svoistva chastits, formiruiushchikhsia pri vosproizvedenii ostroi infektsii in vitro virusom kleshchevogo éntsefalita, adaptirovannym k kleshcham H. plumbeum.

The properties of virions produced in pig embryo kidney (PEK) cells inoculated with tick-borne encephalitis (TBE) virus strain EK-328 which had been passaged in white mice and its variant obtained by passages of TBE in H. plumbeum ticks (clone 718/574 H. pl.17) were found to be different. The clone 718/574 H. pl17 virus particles had no hemagglutinating or precipitating activities, greater sedimentation and density heterogeneity in sucrose density gradient centrifugation, and differences in movements to electrodes in electrophoresis and immune electrophoresis. In mixed infection in PEK cultures with EK-328 strain and clone 718/574 H. pl17, the infective dose-dependent interference was observed which affected the infectious virus titre and the size of cathode antigen precipitate.

[Changes in the host-dependent characteristics of the tick-borne encephalitis virus during its adaptation to ticks and its readaptation to white mice]. / Izmenenie zavisimykh ot khoziaina kharakteristik virusa kleshchevogo éntsefalita pri ego adaptatsii k kleshcham i pereadaptatsiii k belym mysham.

Features of multiplication in pig embryo kidney (PEK) cells of a variant of tick-borne encephalitis (TBE) virus previously selected by passages in H. plumbeum ticks and changes in the properties of the variant upon its repassages in white mouse brains were studied. The tick-adapted TBE variant differed from the original strain in the following characteristics: a lower yield of infectious virus and physical virus particles, altered time-course of infectious virus release from the cell, the lack of virions moving towards cathode in rocket immunoelectrophoresis (RIEP), reduced hemagglutinating activity of the virus, small plaque size in PEK culture, reduced level of virus replication in white mouse brain at a high cytopathic activity of the virus, and low synthesis of virus particles moving towards anode in RIEP. The acquired characteristics were stable at the level of 3 passages in white mice. In the course of further readaptation to these animals the virus recovered its capacity for synthesis of "cathode" virions in PEK cell culture, differing from the original strain by a higher level of virus reproduction in mammalian cells, altered electrophoretic mobility of the structural virion protein V1, and plaque size.

[Action of tunicamycin on the infectivity and protein synthesis of the tick-borne encephalitis virus]. / Deistvie tunikamitsina na infektsionnost' i belkovyi sintez virusa kleshchevogo éntsefalita.

Tunicamycin added in concentrations of 0.5-1 microgram/ml into the maintenance medium with tick-borne encephalitis virus (TBE) propagated in PEK cell culture inhibited incorporation of labeled carbohydrates into glycoprotein V3 as well as into virus-specific proteins NV41/2, NV3, p22, p18, p13, and p12 and to a lower degree into proteins NV5 and NV4 irrespective of the time of its addition (immediately or 17 hours after virus adsorption on the cells). In the presence of tunicamycin, incorporation of radioactive amino acids into the intracellular virus-specific proteins was inhibited, and proteins with altered electrophoretic mobility appeared. Tunicamycin in doses of 0.5-1 microgram/ml added to the infected cells immediately after virus adsorption reduced the production of extracellular virus by 2 lg PFU/ml and of intracellular virus by 1 lg PFU/ml, but exerted no inhibitory effect on virus production when added 17 hours after infection. The results indicate that protein V3 and probably all other intracellular virus-specific proteins of TBE virus exist in PEK cells in N-glycosylated form, tunicamycin selectively inhibits the stage of glycosylation of virus-specific proteins, and glycosylation of proteins affects the infectious properties and virus release from the cell.

[Electron microscopic study of a tick-borne encephalitis virus-infected cell culture exposed to tunicamycin]. / Elektronno-mikroskopicheskoe izuchenie infitsirovannoi virusom kleshchevogo éntsefalita kletochnoi kul'tury pri vozdeistvii tunikamitsina.

Ultrastructural features of tick-borne encephalitis virus-infected pig embryo cell culture treated with different concentrations of tunicamycin at various intervals after infection were studied electron microscopically. The inhibition of glycosylation did not prevent virion formation in the infected cells. At the same time, treatment with tunicamycin led to marked accumulation of virus particles in cisterns and vacuoles of the Golgi complex and to a decrease in the number of virions released into the extracellular space. It is assumed that inhibition of glycosylation leads to disorders in the regulation of the final stages of virus particles transportation and release from the infected cell. The results of the study indicate an important role of the Golgi complex in realization of the final stages of flavivirus morphogenesis.

[Immunochemical characteristics of the antigens of a variant tick-borne encephalitis virus adapted to Hyalomma plumbeum ticks]. / Immunokhimicheskie kharakteristiki antigenov varianta virusa kleshchevogo éntsefalita, adaptirovannogo k kleshcham Hyalomma plumbeum.

A comparative study of immunochemical properties of a preparation of the virion antigen of a variant of tick-borne encephalitis (TBE) virus adapted to H. plumbeum ticks (clone 718/574) and the parental TBE strain revealed the following features: (1) immunoelectrodiffusion test revealed no immunoprecipitation rockets directed towards cathode in the clone 718/574 preparation; anodic precipitates differed in shape from those of the parental strain; (2) diffuse precipitation in agar showed a reaction of incomplete identity between the virion antigen of clone 718/574 and the parental strain antigen, the clone 718/574 antigen forming only one precipitation band in contrast to two precipitation bands of the parental strain; (3) immune electron microscopy demonstrated a peculiar nature of the halo-overlay by immunoglobulins of clone 718/574 virions differing from that of the parental strain.

[Differences in the electrophoretic mobility of the low-molecular virus-specific proteins of viruses of the tick-borne encephalitis complex]. / Razlichiia v élektroforeticheskoi podvizhnosti nizkomolekuliarnykh virusspetsificheskikh belkov virusov kompleksa kleshchevogo éntsefalita.

Analysis of low-molecular virus-specific proteins of all the known members of the tick-borne encephalitis (TBE) complex viruses: TBE (2 strains), Langat, louping-ill, Negishi, Kyasanur Forest disease, Omsk hemorrhagic fever (2 strains), and Powassan, was performed. The cells infected with the above viruses were found to contain low-molecular virus-specific NVX (with the exception of Powassan, Omsk hemorrhagic fever, and Kyasanur Forest disease viruses), NV21/2, NV2, NV11/2, and NV1 proteins. These proteins (except NV1) differ in the electrophoretic mobility in the viruses under study and in TBE strains No. 256 and Sophyin. Thus, electrophoresis may demonstrate differences in the characteristics of proteins of different TBE complex viruses and strains of one virus.

[Characteristics of a cell-culture infection with the tick-borne encephalitis virus with periodic replacement of the culture medium]. / Osobennosti infektsii kul'tur kletok virusom kleshchevogo éntsefalita pri periodicheskoi zamene kul'tural'noi serdy.

The following features were revealed in pig embryo kidney cell cultures infected with tick-borne encephalitis (TBE) virus in which the maintenance medium was changed periodically: more rapid accumulation of TBE virus-specific proteins, active proliferation of spherical vesicular structures and membrane elements of the endoplasmic reticulum, transformation of the latter into regularly arrayed complexes, a statistically significant increase in the yield of the infectious virus.

[Comparative study of the oligopeptide maps of virus-specific proteins of the viruses of the tick-borne encephalitis complex]. / Sravnitel'noe izuchenie oligopeptidnykh kart virusspetsificheskikh belkov virusov kompleksa kleshchevogo éntsefalita.

A comparative analysis of tryptic hydrolysates of virus-specific proteins NV5, V3 and NV3 of the tick-borne encephalitis complex viruses showed proteins NV5 to have considerable regions of similar amino acid sequences, V3 proteins to have significantly differing primary structures, and NV3 proteins to have different amino acid sequences.

[Comparative analysis of the electrophoretic mobility of the high-molecular virus-specific proteins of flaviviruses]. / Sravnitel'nyi analiz élektroforeticheskoi podvizhnosti vysoko-molekuliarnykh virusspetsificheskikh belkov flavivirusov.

A comparative study of high molecular virus-specific proteins in cells infected with flaviviruses from 5 serological subgroups was carried out. Proteins NV5, NV4, and V3 were found to have similar electrophoretic mobility in viruses of the tick-borne encephalitis (TBE) complex with the exception of Powassan virus protein. Proteins NV5 and NV4 of mosquito-borne flaviviruses differ in electrophoretic mobility both from the corresponding proteins of the TBE complex viruses and from each other. Protein NV4 1/2 is formed only in cells infected with TBE complex viruses. Varying electrophoretic mobility of protein NV3 in different flaviviruses attests to its virus-specific nature.

[Electron microscopic study of swine embryonic kidney cells infected with the tick-borne encephalitis virus]. / Elektronno-mikroskopicheskoe izuchenie kletok pochek émbriona svin'i, infitsirovannykh virusom kleshchevogo éntsefalita.

Electron microscopic studies of morphological lesions in pig embryo kidney cells (PEK) infected with tick-borne encephalitis (TBE) virus as well as morphology and features of TBE morphogenesis after treatment with actinomycin D, cycloheximide and hypertonic NaC1 concentrations in the medium were carried out. Most marked morphological lesions were observed in the cells after combined effect of high NaC1 concentrations in the medium and inhibitors of protein synthesis. After all kinds of treatment, smooth-contour membrane structures were observed in TBE-infected cells. Their number increased considerably with increasing ionic strength of the medium and subsequent return to normal accompanied by treatment with actinomycin D and cycloheximide. No "budding" particles were found in any case and after any treatment. Features of TBE virus morphogenesis are discussed.

[Characteristics of a low-molecular nonvirion ("soluble") antigen from the tick-borne encephalitis virus]. / Kharakteristiki nizkomolekuliarnogo nevirionnogo ("rastvorimogo") antigena virusa kleshchevogo éntsefalita.

Pig embryo kidney and BHK-21 cells infected with tick-borne encephalitis virus synthesize a nonvirion antigen differing in its immunological properties from the virion antigens. The antigen has a high thermostability. According to the results of ultrafiltration and gel filtration, its molecular weight is approximately 70-100 kilodaltons. Electrophoretic analysis in polyacrylamide gel showed the molecular weight of a polypeptide isolated from the precipitation band formed by this low molecular antigen to differ from molecular weights of virion and high molecular virus-specific nonvirion proteins and to be about 20 kilodaltons. This polypeptide is assumed to be a subunit of the analyzed nonvirion low molecular antigen.

[Heterogeneity of virus-specific flavivirus proteins]. / Geterogennost' virusspetsificheskikh belkov flavivirusov.

The polyacrylamide gel analysis of large intracellular virus-specific proteins NV5, NV4, and the intracellular form of structural protein V3 established differences in the electrophoretic mobility of each of these proteins formed in cells infected with tick-borne encephalitis, Powassan, Langat, and West Nile viruses. It is assumed that these differences in the electrophoretic mobility of NV5, NV4 proteins, and the intracellular form of V3 protein reflect the differences in the primary structure of each of these proteins in the viruses examined.