Antigenic analysis of tick-borne encephalitis complex viruses by time-resolved fluoroimmunoassay with monoclonal antibodies.

The antigenic structure of 5 strains of tick-borne encephalitis (TBE) virus and 7 other viruses of the TBE complex was examined by the highly sensitive and specific technique of time-resolved fluoroimmunoassay (TR-FIA). A collection of 8 monoclonal antibodies to the Austrian strain. Neudörfl, was used in this study. The findings demonstrate the uniformity of the antigenic structure of TBE viruses from different geographic regions of the USSR. In addition, an epitope was detected which is characteristic of western variants of TBE virus, and another epitope was detected which permits the differentiation of the east-Siberian strain, Aina, from other TBE virus strains. The unique nature of Skalica virus was confirmed, and its similarity, but not identity, to Langat TP-21 virus was shown. Substantial variability in the antigenic structure of some TBE complex viruses was also demonstrated.

[Monoclonal antibodies cross-reacting with the tick-borne encephalitis virus and with the Venezuelan equine encephalomyelitis virus]. / Monoklonal'nye antitela, perekrestno reagiruiushchie s virusom kleshchevogo éntsefalita i virusom venesuél'skogo éntsfalomielita loshadei.

Employment of radioimmunoassay led to the demonstration of serological crossing between tick-borne encephalitis (TBE) virus and Venezuelan equine encephalomyelitis (VEE) virus. Using hybridoma technology, three hybridomas were produced secreting monoclonal antibodies (MAb) cross-reacting with these two viruses. With MAb, the epitope of binding of these antibodies was shown to be located on protein E of TBE virus and protein E1 of VEE virus. Despite the low percentage (14%) of homology of amino acid sequences of these proteins, 12 areas with homology from 24% to 63% were demonstrated. Considering conservative replacements, homology of these areas was 53%-75%. The assumed existence of some of these areas in alpha-helical conformation may explain the observed immunological crossing.

[Antigenic analysis of tick-borne encephalitis virus group using monoclonal antibodies and immunofluorescence]. / Antigennyi analiz virusov gruppy kleshchevogo éntsefalita s ispol'zovaniem monoklonal'nykh antitel i immunofliuorestsentsii.

The study included 18 monoclonal antibodies (MAb) to E- or NS1-antigens tested by immunofluorescence with tick-borne encephalitis (TBE) complex viruses. MAb were induced to 3 strains of TBE virus: the pathogenic 4072 strain isolated from a patient; the Skalica strain of low pathogenicity; and the Neidorf strain isolated from ticks. According to their reactivity to complex viruses, MAb comprised 3 groups: monospecific for TBE virus (T6, T15) which detected tick-borne encephalitis virus alone; widely cross-reactive with 4-6 viruses of the complex (NEK, KEN, T7, T9); and partially complex-reactive (T11, T12, T13, T33/3) and bound to 2-3 viruses of the complex. T13 and T33/3 MAb reacted with the Omsk hemorrhagic fever virus to the same degree or stronger than with TBE virus. The cross-reactivity was more marked in anti-E-than in anti-NS1 MAb. The similarity of the Langat viruses and the Skalica strain was confirmed. Using anti-NS1 MAb in tests with non-fixed cells, the release of NS1-antigen was found to begin at hour 18 (time of observation). The results of the study may be useful for improvement of laboratory diagnosis of TBE and evaluation of the capacity of a vaccine to induce cross immunity to viruses of the TBE complex.

[Monoclonal antibodies to flavivirus antigens induced by a vaccinal strain of yellow fever]. / Monoklonal'nye antitela k flavivirusnym antigenam, indutsirovannye vaktsinnym shtammom zheltoi likhoradki.

Monoclonal antibodies (MABs) YEL-2 induced by the vaccine FNS Dakar yellow fever (YF) virus were characterized for their capacity to enter into serological reactions and to react with heterologous flaviviruses. YEL-2 MABs belong to the IgG2a class of immunoglobulins, possess the antihemagglutination properties, are active in indirect IF test but do not activate complement and have no neutralizing properties. The inability to enter into CFT in the presence of antihemagglutinating properties suggests that YEL-2 MABs are directed for the structural E glycoprotein. YEL-2 MABs reacted similarly with the vaccine 17D strain and the FNS Dakar strain by which they had been induced. In addition to YF virus, YEL-2 MABs reacted with Tyuleniy, Rosio, Ilhéus, Uganda S, Karshi, and Sokuluk viruses, the reaction with Tyuleniy virus reaching the same titer as with the homologous virus but was of one-way nature. No reaction of YEL-2 MABs was observed with the viruses of the tick-borne encephalitis complex, Japanese encephalitis, West Nile, Dengue 2 and 4 viruses. These results specify the antigenic classification of flaviviruses.

[Conditions for forming ascitic tumors in hybridoma cultivation in vivo]. / Izuchenie uslovii obrazovaniia astsitnykh opukholei pri kul'tivirovanii gibridom in vivo.

The conditions of the formation of ascitic cells in BALB/c mice injected with hybridoma cells were studied. All the hybridomas under study, producing monoclonal antibodies to viral antigens, induced the formation of ascitic tumors when introduced into the abdominal cavity of BALB/c mice pretreated with sensitizing agents. In the mice pretreated with pristane hybridoma cells took at a rate of 43-80% and in the mice pretreated with Freund's complete adjuvant, 31-70%. Angara oil and perfume oil, as well as Bayol F, were less effective. The time of the formation of ascites was inversely proportional to the dose of the injected cells, while the volume of ascitic fluid depended rather on the type of hybridoma and not on the dose of the injected cells. The study showed that the use of physiological saline or culture medium without serum for washing the abdominal cavity of mice after withdrawing ascites permitted the additional collection of 2.6-13.7 million hybrid cells, as well as a considerable amount of immunoglobulins.

Preparation and characterization of hybridomas secreting monoclonal antibodies to tick-borne encephalitis virus.

Monoclonal antibodies (MA) were prepared to two strains of tick-borne encephalitis (TBE) virus: strain 4072 isolated from a patient in the U.S.S.R. and low-pathogenic for mice strain Skalica, isolated from a bank vole (Clethrionomys glareolus) in Slovakia. MA specific to the 4072 and Skalica strains were produced by hybridomas of the KEN (60 clones) and NEK (65 clones) series, respectively. Chromosomal analysis of MA producing 114 hybridoma clones of both series revealed a great variability in the number of chromosomes either in the range of given clones or between individual clones. The hybridoma cells under study possessed a high degree of transformation manifested by good growth in the mouse peritoneal cavity and marked accumulation in ascitic fluid (AF).

Two kinds of monoclonal antibodies to tick-borne encephalitis virus.

Two types of monoclonal antibodies (MA) of the KEN and NEK series prepared to tick-borne encephalitis (TBE) virus differed in the spectrum of their reactivity in serological tests and in their ability to react with individual representatives of the TBE virus complex. The KEN series MA were induced to the 4072 strain isolated from the blood of a patient in the U.S.S.R. The NEK series MA were prepared to the Skalica strain isolated from a bank vole in Czechoslovakia. Both groups of MA belonged to IgG class, reacted in immunofluorescence (IF) test, but possessed no haemagglutination inhibiting (HI) activity. MA of the NEK series reacted in the IF and complement fixation (CF) tests with all members of the TBE virus complex, except of the Powassan virus. MA of the KEN series had no CF activity and in the IF test, they did not react with Powassan and Langat TP-21 viruses and with the Skalica strain of TBE virus.

[Serological characteristics of KAMA-51 monoclonal antibodies to the tick-borne encephalitis virus]. / Serologicheskaia kharakteristika monoklonal'nykh antitel KAMA-51 k virusu kleshchevogo éntsefalita.

KAMA-51 monoclonal antibodies to tick-borne encephalitis virus are produced by hybridoma obtained by fusion of splenocytes of mice immunized with this virus with myeloma X-653 cells. The antibody belongs to the IgG class, is active in the immunofluorescence test (IFT), does not react in CFT, and has no antihemagglutinating or neutralizing properties. In the IFT, it reacts with all viruses of the tick-borne encephalitis complex indicating their directivity to the groupspecific determinant of E protein. In the indirect IFT, antibody titres in the culture fluid are within the range of 1: 16-1: 62, in the ascitic fluids 1: 320-1: 640. Because of the wide range of interspecies reactions, KAMA-51 monoclonal antibody may be used for group detection of the tick-borne encephalitis complex viruses.

[Karyological characteristics of L cells and the identification with them, of the antigen of persisting Japanese encephalitis virus]. / Kariologicheskaia kharakteristika kletok L i identifikatsiia v nikh antigena persistiruiushchego virusa iaponskogo éntsefalita.

The immunofluorescence procedure revealed the virus-specific Japanese encephalitis virus antigen in latently infected L cells as well as differences in the percentage of antigen-positive cells and in the localization of the virus-specific antigen under conditions of acute, chromic, and latent infection and in the period of activation of the latent state of the virus at 28 degrees C. Karyological studied demonstrated no marked differences between persistently infected and original lines.

[Use of the immunoperoxidase method for the rapid detection of togaviruses, orthopoxviruses and their antibodies]. / Primenenie immunoperoksidaznogo metoda dlia bystroi indikatsii nekotorykh togavirusov, ortopoksvirusov i antitel k nim.

The successful application of the immunoperoxidase method of (IP) with light microscopy for rapid detection of certain togaviruses and orthopoxviruses and antibodies to them is described. A comparative evaluation of the IP and immunofluorescent (IF) method for these purposes was performed. The results indicate practically similar sensitivity and specificity of both methods. The detection of viral antigen directly depended upon the infective dose and time of examinations (within 4--12 hours postinoculation). In evaluating the IP method, however, the account should be taken of the fact that it does not require expensive equipment and therefore may be used in any practical laboratory or even under field conditions. Rapid detection of both viral antigens and antiviral antibody by this method opens approaches for its wide application in the future.

[Accelerated method of detecting antibodies to tick-borne encephalitis and Isfahan viruses on fixed smears of antigen-containing cells]. / Uskornnyi metod vyiavleniia antitel k virusam kleshchevogo éntsefalita i Isfagan na fiksirovannykh mazkakh antigensoderzhashchikh kletok.

A modification is suggested of the indirect method of fluorescent antibodies (IMFA) for rapid diagnosis of the TBE and Isfagan viruses in fixed smears of antigen-containing cells. Preparation of such smears with equal numbers of antigen-containing cells in a monolayer covering the whole area of the depression inoculated with the infected cellular suspension ensures standard conditions for the test and comparable results. The titer of the sera in the smears determined with the IMFA is higher or similar to that obtained in the infected monolayer cultures grown on slides. One preparation can be used not only for the serum antibody identification but also for its titration. It takes no more than 4-5 hours to analyse the test sera. The fixed preparation with the antigen-containing cells can be stored during 6 months at -20 degrees C with no specificity lost. Availability of such preparations in stock will permit the IMFA to be applied in rapid diagnosis.

[Natural foci of viruses borne by Phlebotomus papatasi in the USSR according to a serologic study of the population]. / Prirodnye ochagi virusov, perenosimykh Phlebotomus papatasi, v SSSR po dannym serologicheskogo obsledovaniia naseleniia.

Seven hundred sixteen blood serum specimens from residents of presumable foci of phlebotomus fevers in Turkmenia, Tajikistan, Uzbekistan, Azerbaijan, and Moldavia were examined by the neutralization, complement fixation, hemagglutionation-inhibition and indirect immunofluorescence tests for the presence of antibody to viruses of the group of phlebotomus fevers (Sicilyan, Neapolitan, and Karimabad) and to rhabdovirus isfahan transmitted by phlebotomus papatasi. For the first time, antibody to Karimabad and Isfahan viruses were found in residents of the Central Asian republics. Antibody to Sicilyan and Neapolitan fevers were found in residents of all the republics examined. Data have been obtained indicating probable pathogenicity of Isfahan virus for man.