[The reproductive characteristics of the tick-borne encephalitis virus in interspecific somatic hybrids]. / Osobennosti reproduktsii virusa kleshchevogo éntsefalita v mezhvidovykh somaticheskikh gibridakh.

The study dealt with features of tick-borne encephalitis virus reproduction in two series of interspecies somatic hybrids generated by fusion of transformed cells of Chinese hamster (Ag17) with human diploid fibroblasts (KM/3) and with pseudonormal cells of Indian deer (Muntiacus munjak) (KOM). The viral infection in hybrid Ag17 cells ran an acute course with cell damage, but in KM/3 and KOM cells virus multiplication was not accompanied by the development of cytopathic effect. Two other parameters of tick-borne encephalitis virus infection under study: the extent of infectious particles production and electroimmunochemical properties were found to be under control of genomes of different parental cells.

[A comparison of the protective properties of preparations of the virion and nonvirion ("soluble") antigens of the tick-borne encephalitis virus]. / Sopostavlenie protektivnykh svoistv preparatov virionnogo i nevirionnogo ("rastvorimogo") antigenov virusa kleshchevogo éntsefalita.

A comparative assessment of the protective properties of virion (VA) and nonvirion ("soluble") (NA) antigens of tick-borne encephalitis virus prepared as inactivated samples close in their parameters to vaccine preparations was carried out. The NA in the preparations free from VA or containing only trace, nonprotective amounts of it, was shown to have significantly lower protective properties than VA and exerted no booster effect on the protective activity when added to VA preparations.

[Multiple forms of the NS1 protein as the main component of the nonvirion ("soluble") antigen of the tick-borne encephalitis virus]. / Mnozhestvennye formy belka NS1 kak osnovnogo komponenta nevirionnogo ("rastvorimogo") antigena virusa kleshchevogo éntsefalita.

The nonstructural virus-specific NS1 protein of tick-borne encephalitis (TBE) virus was found to be the main antigenic component of the nonvirion ("soluble") antigen which had been shown previously to present a complex protein structure closely associated with the infected cell membranes. In the presence of sodium dodecylsulphate, beta-mercaptoethanol and 8M urea NS1 protein could be detected in the form of oligomeric molecules which disintegrated to monomers after heating at 100 degrees C for 5 min. The infected cell membrane-associated NS1 protein was shown to differ in electrophoretic mobility from the NS1 protein released into the culture fluid in the course of virus infection. The conditions for detection of NS1 protein and its oligomeric forms by PAG electrophoresis and immune blotting were determined. Dimeric forms of NS1 protein were found to contain products of its proteolysis. NS5 protein was found together with NS1 protein in TBE nonvirion antigen and also possessed antigenic activity.

[Slowly-sedimenting hemagglutinin of the tick-borne encephalitis virus]. / Medlennosedimentiruiushchii gemaggliutinin virusa kleshchevogo éntsefalita.

Ring-shaped particles of 5-10 mm in diameter considered by research workers to be a slowly-sedimenting hemagglutinin (SSHA) of flaviviruses (an antigen immunologically related to virions) were detected in the precipitation band formed in immunoelectrophoresis by the non-virion ("soluble") antigen but not in the precipitation band formed by the virion antigen. The slowly sedimenting (SS) virions of tick-borne encephalitis virus previously found in a SSHA fraction did not differ in the set of structural proteins from virions of the main population (rapidly sedimenting). It is concluded from the foregoing that the hemagglutinating activity of the SS-structures is realized not by a hypothetic SSHA ("natural" ring-shaped fragment of virion envelope, precursor or a by-product of virus morphogenesis, according to other workers) but by SS-virus particles.

[Detection of the virus-specific proteins of the tick-borne encephalitis virus by immunosorption]. / Vyiavlenie virusspetsificheskikh belkov virusa kleshchevogo éntsefalita s pomoshch'iu immunosorbtsii.

The use of immunosorption of tick-borne encephalitis (TBE) virus preparations on polyspecific antibodies covalently bound with sepharose permits good identification of virus-specific protein synthesis in cell cultures in acute and latent infection. Immune affinity separation of virus-specific proteins p93, p79, p69, p53 (V3), p24, p23, p21, p18, and p13 (NVI 1/2) attests to the high polyspecificity of the employed immune preparation, a hyperimmune anti-TBE horse serum gamma-globulin. From a virion antigen preparation, structural V3 (E) protein is isolated but not other structural proteins, V2 (C) or V1 (M). p93 protein (NV5) is one of the proteins recovered from preparations of nonvirion ("soluble") antigen (NA) alongside with heterogeneous p80 protein which may represent a product of p93 protein proteolysis or protein(s) of pig embryo kidney cells separated in immunosorption together with p93 within HA.

[Nonvirion (soluble) antigen of the tick-borne encephalitis virus]. / Nevirionnyi ("rastvorimyi") antigen virusa kleshchevogo éntsefalita.

Immunoelectrophoretic analysis of the cells destroyed as a result of infection with tick-borne encephalitis virus showed a considerable portion of nonvirion ("soluble") antigen to remain associated with cell membranes and to be released after treatment of the cells with detergents. After treatment with nonionic detergents, the nonvirion antigen showed electrophoretic heterogeneity in immunoelectrophoresis, and after treatment with sodium dodecyl-sulphate behaved as a homogeneous population of protein molecules. Using radioactively labeled carbohydrate precursors, lipids, and RNA, the nonvirion antigen was demonstrated to be a complex structure: a membrane-containing ribonucleoprotein. The immunoprecipitation of proteins by means of antibodies ligated to CNBr-activated sepharose confirmed that protein NV 5 (p93) of tick-borne encephalitis virus was an antigenically active component of the nonvirion antigen. This protein was found to undergo proteolytic cleavage in immunochemical reaction. The possible role of the nonvirion antigen in reproduction of flaviviruses is discussed.

[Dependence of the synthesis of virion structures in the tick-borne encephalitis virus on the cell culture type]. / Zavisimost' sinteza virionnykh struktur virusa kleshchevogo éntsefalita ot vida kletochnykh kul'tur.

Variations in synthesis of antigenic structures are observed in tick-borne encephalitis virus replication in cell cultures of different origin. In a number of cell cultures: pig and Syrian hamster embryo kidney cells, as well as in Chinese striped hamster cells and Tasmanian rat kangaroo cells, virion antigens (VA) are synthesized which differ in the direction of movement in the electric field, namely, anode and cathode VA. In other cell cultures: green monkey kidney, barking deer kidney, and human fibroblasts, only cathode VA is synthesized. In chick embryo fibroblast cultures, in addition to the above-mentioned VA, considerable amounts of a VA which does not move in the electric field are synthesized. In all the cultures, a low molecular nonvirion antigen (NA) is actively produced, the virus-containing fluids of human fibroblast cultures and Chinese striped hamster kidney cell cultures contain lower amounts of high molecular NA, while rat kangaroo and green monkey kidney cell cultures have no high-molecular NA of tick-borne encephalitis virus.

[Changes in the synthesis of the virion antigen of the tick-borne encephalitis virus after passage through ixodid ticks and small mammals]. / Izmeneniia sinteza virionnogo antigena virusa kleshchevogo éntsefalita posle passirovaniia cherez iksodovykh kleshchei i melkikh mlekopitaiushchikh.

Large-plaque strains of tick-borne encephalitis (TBE) virus possess a high peripheral activity, and in acute infection in continuous pig embryo kidney cells (PEK) synthesize a virion antigen (VA) whose subpopulations differ in their mobility in agarose gel electrophoresis: the bulk portion of VA moves towards the cathode and a small one towards the anode. After long-term passages of these strains in Ixodid ticks they lose their peripheral activity and produce only small plaques. In reproduction in PEK cells the amount of the infectious virus remains the same but VA synthesis changes significantly. There is almost no synthesis of VA moving towards the cathode and that of VA moving towards the anode remains unchanged or decreases insignificantly. The decrease in the synthesis of the major portion of VA is accompanied by the reduction of the hemagglutinating properties of the strains and retention of the nonvirion antigen production. Reversion to the initial properties of the strains passaged in ticks occurs after their inoculation into natural hosts of tick-borne encephalitis virus: small mammals (bank vole and common vole).

[Glycosylated and nonglycosylated proteins of the tick-borne encephalitis virus synthesized in continuous pig embryonic kidney cells]. / Glikozilirovannye i neglikozilirovannye belki virusa kleshchevogo éntsefalita, sinteziruiushchiesia v perevivaemykh kletkakh pochki émbriona svin'i.

Synthesis of virus-specific proteins p93, p79, p69, p53, p47, p34, p24, p23, p21, p18, p15, p13, and p12 of which p53 and p13 are analogues of virion proteins V3 (E) and V2 (C) occurs in continuous pig embryo kidney (PEK) cells infected with tick-borne encephalitis virus. The third structural protein, p8 (V1, M) is found in virions but not in the cells. Treatment of the cells with cycloheximide and hypertonic NaCl solution, virtually depressing the radioactive label incorporation into PEK cell proteins, also inhibits the synthesis of virus-specific proteins p69, p21, p15, and p12. Protein p53 is present in the cells in glycosylated and nonglycosylated forms differing in electrophoretic mobility. Intensive glycosylation of not only p53 protein but also of proteins p47 and p21 was observed, and poor glycosylation of proteins p93, p79, and p69.

[Characteristics of incorporation of carbohydrate radioactivity into structural proteins of the tick-borne encephalitis virus]. / Osobennosti vkliucheniia radioaktivnosti uglevodov v strukturnye belki virusa kleshchevogo éntsevalita.

The propagation of the tick-borne encephalitis virus in the culture of the porcine embryo kidney cells containing the radioactive mannose and glucosamine results in incorporation of radioactive label into hemagglutinin V3(E) as well as into other structural proteins, the nucleocapsid protein V2(C) and membrane protein V1(M). The possible reasons for carbohydrates borne radioactivity incorporation into the proteins V2 and V1 are discussed.

[Electrophoretic and sedimentation heterogeneity of the virion antigen of the tick-borne encephalitis virus]. / Elektroforeticheskaia i sedimentatsionnaia geterogennost' virionnogo antigena virusa kleshchevogo éntsefalita.

Analysis of tick-borne encephalitis virus virion antigen by cross immunoelectrophoresis showed it to be separated into 2 subpopulations of virions differing by their mobility in the electric field. It was demonstrated that in immunoelectrophoresis the formation of precipitation bands typical of virion antigen occurred due to the movement of soluble immune complexes in the electric field. The immunoelectrophoretic characteristics of TBE virus virions are determined by the membrane E protein which was also found to be heterogeneous in its immunoelectrophoretic characteristics. The nucleocapsid of tick-borne encephalitis virus virions formed no precipitation bands in immunoelectrophoresis. The virion antigen not sedimenting in ultracentrifugation was represented by morphologically intact virions the structural protein composition of which differed from that of the structural proteins of virions sedimentable by ultracentrifugation. The possible causes of electrophoretic and sedimentation heterogeneity of tick-borne encephalitis virus virion antigen are discussed.

[Characteristics of the precipitation of the antigenic structures of the tick-borne encephalitis virus using polyethylene glycol and ammonium sulfate]. / Kharakteristiki osazhdeniia antigennykh struktur virusa kleshchevogo éntsefalita poliétilenglikolem i sul'fatom ammoniia.

When immunoelectrophoretic methods were used for analysis of tick-borne encephalitis (TBE) virus antigens, TBE virus virion antigen (VA) from the virus-containing tissue culture fluid sedimented upon addition of polyethylene glycol (PEG) up to 7% whereas upon addition of ammonium sulphate (AS) the bulk of VA sedimented in the range of 40-70% salt saturation. The non-virion antigens (NA) sedimented at all PEG (up to 22%) and AS (up to 70% saturation) concentrations. In stringent ultracentrifugation procedure (12-13 X 10(6) g X min), the bulk of VA and a small portion of NA are pelleted. Repeated ultracentrifugation results in sedimentation of additional amounts of VA and NA. The sedimentable NA are pelleted by low PEG (up to 5-7%) and AS (up to 20-30%) concentrations and differ in their interaction with antibodies in the course of immunoelectrophoresis from nonprecipitatable ("soluble") NA. Combined sedimentation of antigens with AS (in the range of 40-70% saturation), intensive dialysis and subsequent sedimentation with low and high PEG concentrations produces homogeneous antigenic preparations containing VA or "soluble" NA.

[Synthesis and membrane-dependent processing of a precursor of tick-borne encephalitis virus (flavivirus) structural proteins in cell-free systems]. / Sintez i membran-zavisimyi protsessing predshestvennika strukturnykh belkov virusa kleshchevogo éntsefalita (flavivirusa) v beskletochnykh sistemakh.

Conditions that permitted cell-free synthesis of at least one of the non-structural, in addition to two-structural, polypeptides of tick-borne encephalitis virus have been found. The time course of accumulation of virus-specific polypeptides in extracts of Krebs-2 cells and reticulocyte lysates as well as the peptide maps of the products synthesised were studied. A model of generation of viral structural polypeptides has been proposed, according to which a common precursor of these proteins while in a nascent form, is processed in a membrane-dependent reaction into a C-terminal segment, corresponding to the polypeptide moiety of envelope glycoprotein E, and an N-terminal segment, doublet p36/33. Subsequently, an N-terminal segment, corresponding to the core polypeptide C, is cleaved off from p36/33. The remaining C-terminal segment of p36/33 is possibly a precursor of the membrane polypeptide M. The translational strategy of flaviviruses is compared to that of other positive-stranded RNA viruses.

[Nonstructural protein NV5 of the tick-borne encephalitis virus possesses precipitating activity characteristic for the nonvirion (soluble) antigen]. / Nestrukturnyi belok NV5 virusa kleshchevogo éntsefalita obladaet pretsipitiruiushchei aktivnost'iu, kharakternoi dlia nevirionnogo ("rastvorimogo") antigena.

A preparation of tick-borne encephalitis virus proteins obtained by destruction of the infected cells under strong denaturating conditions possesses the precipitating activity in immunodiffusion characteristic of nonvirion ("soluble") antigen. This precipitating activity was shown to be provided by protein P93 (NV5).

[Immunochemical and electron microscopic analysis of the high-molecular structures in the tick-borne encephalitis virus]. / Immunokhimicheskii i élektronno-mikroskopicheskii analiz vysokomolekuliarnykh struktur virusa kleshchevogo éntsefalita.

High-molecular structures of tick-borne encephalitis virus were sedimented by centrifugation from virus-containing culture fluid and separated by sucrose concentration gradient centrifugation into 3 main fractions: rapidly sedimenting virions, the fraction sedimenting at a moderate rate, represented by "stellate" structures, and the fraction found on the top of the gradient and represented by structures similar in size and shape to virions, designated slowly sedimenting virions. The latter are first described in the paper. In immunodiffusion, the rapidly sedimenting virions form 1 of the 2 precipitation bands closer to the antigen-containing well, and in immunoelectrophoresis give 2 precipitation bands, anodic and cathodic, which fuse, indicating immunological identify of the 2 subpopulations of rapidly sedimenting virions. The slowly sedimenting virions in immunodiffusion form one of the 2 precipitation bands closer to the antigen well, and in immunoelectrophoresis form the cathodic precipitation band. The material sedimenting in the gradient at a moderate rate gives in immunodiffusion one precipitation band, the 3rd from the antigen well, and in immunoelectrophoresis forms a separate anodic precipitation band. This high-molecular non-virion antigen is found in small amounts also in fractions containing the rapidly sedimenting and slowly sedimenting virions. It is at least partially antigenically identical to previously studied low molecular non-virion ("soluble") antigen of tick-borne encephalitis virus.