[Humoral immune response to structural and nonstructural proteins of tick-borne encephalitis virus in the natural history of the disease].

A hundred and ninety-one patients who had fallen ill with tick-borne encephalitis (TBE) after bites by virus-carrying ticks were examined to reveal the characteristics of antibody production of the major classes of immunoglobulins (IgM and IgG) against structural protein E of the TBE virus and nonstructural protein NS1 of the TBE viral replicative complex in the natural history of an infectious process in different forms and variants of the disease. In the two-wave course of TBE, the infectious process is characterized by a delayed antibody production, lower levels of specific antibodies against virion protein E in the acute phase of disease during the first wave of fever. The development of meningocerebral syndromes at the peak of the second fever wave is observed in patients with the delayed accumulation and low levels of IgG antibodies. Of great diagnostic and prognostic value is the detection of nonstructural protein NS1 antibodies on the first days of TBE, by taking into account the uncertainty of clinical criteria for the disease at its first (febrile) wave and the low titers (or their absence) of specific antibodies against virion protein E, detectable at that time in the sera of patient with the two-wave course of TBE.

Epitope analysis of tick-borne encephalitis (TBE) complex viruses using monoclonal antibodies to envelope glycoprotein of TBE virus (persulcatus subtype).

The arrangement of envelope protein epitopes of tick-borne encephalitis viruses (TBEV) (persulcatus or eastern subtype, Sofjin strain and ricinus or western subtype, Minsk-256 strain) and Kyasanur Forest disease virus (KFDV) was investigated using competitive binding of monoclonal antibodies against the Sofjin E protein. The E protein of TBEV Sofjin strain forms three antigenic domains: E1, E2 and E3, represented by 12, 9 and 2 epitopes respectively; two additional epitopes stand alone. Domains E1 and E2 are heterogeneous. On the epitope map of the Minsk-256 strain domain E3 remains intact, domains E1 and E2 overlap and the relative arrangement of virus-neutralizing epitopes from E1 and E2 domains is changed. The epitope map of KFDV is significantly dissimilar to TBEV. The viruses can be distinguished by epitopes with identical serological reactivity. A satisfactory agreement between our epitope maps and previously published antigenic models of flavivirus envelope protein (Guirakhoo et al., 1989; Mandl et al., 1989a) was observed. The main difference of our map is that domains corresponding to domains B and C (Sofjin strain) and A, B and C (Minsk-256 strain) in Heinz's model are overlapping. The results of competition analysis depend on the nature of the antigen (virion or purified protein) and the immunoassay technique.

Tick-borne encephalitis virus strains of Western Siberia.

Tick-borne encephalitis virus (TBEV) strains were isolated from ticks in Western Siberia for 12 years. Molecular hybridization of the 46 viral RNA with the TBEV cDNA and oligonucleotide probes revealed differences between the Siberian and Far Eastern strains. A comparison of the viral E gene fragment nucleotide sequence showed 89-98% homology between Siberian TBEV strains, whereas their similarity with strains from other populations was less than 83%. However, the viral E and NS1 glycoprotein antigenic structures appeared to be conservative because of the degenerate genetic code. This was shown by enzyme-linked immunosorbent assay with the corresponding monoclonal antibodies (MAb). The single exception was the MAb 17C3 against nonstructural glycoprotein NS1, which could distinguish Siberian from Far Eastern strains. Moreover, the neurovirulence differed between strains from the two natural populations. Lower neuroinvasiveness of the Siberian strains in comparison with Far Eastern Sofyin strain might be caused by both E and NS1 glycoprotein mutations.

Comparative analysis of serological activity of non-structural protein (NS1) from tick-borne encephalitis virus and its analog expressed in bacterial cells.

By means of immunoaffinity chromatography and expression of the gene in Escherichia coli, non-structural glycoprotein NS1 of tick-borne encephalitis virus (TBEV) and its recombinant analog were prepared. Antisera against these proteins were obtained by hyperimmunisation of rabbits. The antisera were tested by means of complement fixation, agar diffusion, hemagglutination inhibition and virus neutralization. Although both antisera are reacted with natural antigen, the recombinant analog of NS1 did not bind antibodies against natural protein in complement fixation and immunoprecipitation. Nevertheless the NS1 analog was rather active in ELISA. Neither the natural nor the recombinant protein protected experimental animals from lethal virus infection. A contamination of natural NS1 antigen with small amounts of structural glycoprotein E may be responsible for both antibody formation and virus neutralization. This can be relevant for the design of a subunit vaccine.

Probability models of the rate of infection with tick-borne encephalitis virus in Ixodes persulcatus ticks.

A total of 3,254 adults of Ixodes persulcatus tick were collected in a taiga forest habitat situated in the Amgun river basin (Khabarovsk region, the Far East, USSR) and examined individually for the presence and amount of tick-borne encephalitis virus. The over-all proportion of infected ticks was 6.6% and it varied between 3.4% and 9.4% in the years 1982 to 1985. The amount of virus per tick was approximated by the gamma distribution determining a probability that the number of plaque-forming units (PFU) per tick is not greater than a selected value. The frequency distribution of infected ticks followed a model of the negative binomial distribution, enabling the estimation of probability of the occurrence of a given number of infected ticks in the area. However, the parameters of both probability models (i.e., the PFU content per tick, and the frequency of infected ticks) varied for particular years.

Detection of Crimean haemorrhagic fever virus antigen by solid phase enzyme immunosorbent assay.

Solid phase enzyme immunosorbent assay (SPEIA) is described for detection of the Crimean haemorrhagic fever (CHF) virus antigen in suspensions of ticks collected in the natural focus.

[Study of the antigenic structure of tick-borne encephalitis virus using synthetic peptides]. / Izuchenie antigennoi struktury virusa kleshchevogo éntsefalita s pomoshch'iu sinteticheskikh peptidov.

A number of peptides, fragments of the envelope protein E of the tick-borne encephalitis virus (Sofjin strain), were synthesized. Their binding to the polyclonal antiserum to protein E was studied. Rats were immunized with both the free peptides and their KLH-conjugates, and the resulting antisera were tested for their reactivity toward protein E and for their neutralizing activity toward the virus in cell culture. The only peptide corresponding to the 98-113 sequence of protein E was shown to be bound by the protein E antiserum in EIA. Two-fold immunization of rats with KLH-conjugates of the peptides corresponding to the 98-113, 130-143, and 394-403 sequences of protein E resulted in antipeptide antibodies capable of binding the native protein E, and the antibodies to the 98-113 and 394-403 peptides were capable of neutralizing the virus.

[Cases of Crimean-Congo hemorrhagic fever in Astrakhan Province]. / O sluchaiakh Krymskoi--Kongo gemoragicheskoi likhoradki v Astrakhanskoi oblasti.

The paper briefly presents clinical, epidemiological, virological and serological data on cases of Crimean-Congo hemorrhagic fever (CCHF) in Astrakhan Province. The results indicate annual infection of the regions population with CCHF virus, the main mode of transmission being vector-borne. The advantages of the fluorescence antibody technique and enzyme immunoassay-for diagnosis of the disease were demonstrated.

[Demonstration of the tick-borne encephalitis virus by a solid-phase immunoenzyme method in materials from natural foci]. / Indikatsiia virusa kleshchevogo éntsefalita tverdofaznym immunofermentnym metodom v materialakh prirodnykh ochagov.

Large-scale examinations of specimens from natural foci of tick-borne encephalitis (TBE) demonstrated that sensitivity of solid-phase enzyme immunoassay (EIA) in detecting TBE virus antigen was as high as that of the fluorescent antibody technique. The EIA takes less time and may be used for examinations of specimens after a preliminary passage in cell cultures. The EIA was also shown to be highly effective in the detection of TBE virus antigen in the cerebral tissue of patients post mortem and in the confirmation of the clinical diagnosis.

[Determination of the degree of infectiousness of adult ixodid ticks with the tick-borne encephalitis virus]. / Opredelenie stepeni infitsirovannosti vzroslykh iksodovykh kleshchei virusom kleshchevogo éntsefalita.

It was shown to be principally possible to determine the content of tick-borne encephalitis virus in ticks in a certain territory by total collection of these arthropods in permanent areas followed by individual virological examination and titration of virus in the infected ticks.

[Monoclonal antibody study of antigens E and NS1 of Western Siberian tick borne encephalitis virus strains]. / Issledovanie s pomoshch'iu monoklonal'nykh antitel antigenov E i NS1 shtammov virusa kleshchevogo éntsefalita Zapadnoi Sibiri.

Antigenic structure of tick-borne encephalitis virus proteins was studied by ELISA with monoclonal antibodies (MAb) to E and NS1 glycoproteins of strain Sofyin. Envelope proteins appeared to be conservative which corresponded to previously determined nucleotide sequences of E gene fragments and deduced primary structures of the corresponding E protein. Five of six studied MAb to NS1 nonstructural glycoprotein of strain Sofyin reacted with this protein of all studied strains. The only exception was MAb 17C3 which discriminates West Siberian strains from Far Eastern strain Sofyin.

[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.

[The immunogenic and hemagglutinating activities of tick-borne encephalitis viral strains isolated from patients in different parts of the geographic range of the disease]. / Immunogennaia i gemaggliutiniruiushchaia aktivnost' shtammov virusa kleshchevogo éntsefalita, vydelennykh ot bol'nykh v raznykh chastiakh nozoareala.

Considerable variability of the immunogenic and antigenic (in HA test) activity of tick-borne encephalitis virus strains was shown. No correlation was observed between the hemagglutinating activity of strains and their capacity to stimulate humoral immunity in the infected animals. The results of the study did not allow one to place strains into groups according to the area of isolation in relation to their immunogenic and antigenic activity.

[Biological properties of tick-borne encephalitis strains isolated in different parts of its geographic range]. / Izuchenie biologicheskikh svoistv shtammov virusa kleshchevogo éntsefalita, vydelennykh v raznykh chastiakh ego areala.

A comparative study of over 200 tick-borne encephalitis (TBE) virus strains isolated from patients and ticks in different regions varying in the proportion of severe clinical forms of TBE was carried out. The strains were tested for their pathogenicity for white mice, cytopathic and plaque-forming properties as well as for the interferon-inducing capacity in SPEV cells. All the strains were found to be highly neurovirulent for mice, to produce plaques with diameters varying form less than 1 to 10 mm, and to induce interferon production in SPEV cell. Among the TBE virus strains isolated in the Baltic republics and Vologda region there occurred strains causing complete destruction of SPEV cells at later intervals than the prototype strains. No association of the examined properties of TBE virus strains with the region of isolation or with the clinical forms of human disease was established.

[Multi-year observations of the humoral immunity status of patients with tick-borne encephalitis in 3 endemic regions]. / Mnogoletnie nabliudeniia za sostoianiem gumoral'nogo immuniteta u bol'nykh kleshchevym éntsefalitom v trekh éndemichnykh regionakh.

Analysis of the results of the laboratory diagnosis of tick-borne encephalitis (TBE) by HI test over many years proved a statistically significant decrease by the 80's in the intensity of humoral immunity in the subjects with a history of tick-borne encephalitis in Kemerovo Province, Udmurtia and Latvia. A decrease in antihemagglutinin levels in the blood of convalescents after TBE was detected in endemic foci located in different geographical regions differing by epidemiological parameters; it did not depend on the portion of infected ticks and ratio of the clinical forms of the disease in a focus. Parallel tests by HI of the diagnostic value of several antigens of TBE virus strains isolated from different sources in different regions with sera from TBE patients revealed no differences among them. The antigenic activity of the strains in HI test did not depend on the source and region of isolation (Kemerovo Province, Udmurtia and Latvia).

[Intrapopulation differences in the infectivity of adult Ixodes persulcatus P. Sch. with the tick-borne encephalitis virus and an assessment of its total content in ticks]. / Vnutripopuliatsionnye razlichiia infitsirovannosti vzroslykh Ixodes persulcatus P. Sch. virusom kleshchevogo éntsefalita i otsenka ego summarnogo soderzhaniia v kleshchakh.

At the present stage of investigations on the role of vectors in foci of tick-borne encephalitis and of other transmissible infections it is necessary not only to operate with extensive indices of arthropod infection rate but also to take into account the degree of their infection. It has been demonstrated that adult empty Ixodes persulcatus ticks at the time of activation differ significantly by the degree of their infection with tick-borne encephalitis virus. In the Khabarovsk Territory, annual changes in the relative and absolute numbers of infected ticks did not lead to sharp variations in the virus population portion associated with ticks. It is suggested that vector individuals containing different virus doses are of different functional importance in providing long-term existence of virus population.

[Identification of individual antigenic epitopes of the envelope protein in the tick-borne encephalitis virus using monoclonal antibodies]. / Identifikatsiia otdel'nykh antigennykh épitopov belka obolochki virusa kleshchevogo éntsefalita s primeneniem monoklonal'nykh antitel.

A variant analysis of virus antigens of tick-borne encephalitis complex was carried out by enzyme immunoassays and topographic mapping of this protein using a panel of monoclonal antibodies to the structural glycoprotein of tick-borne encephalitis (TBE) virus of the Far East subtype. The results of the study confirmed the existence on the structural protein of both identical determinants typical of all the antigens of the complex and of subgroup-specific determinants. The topological analysis of the epitopes binding monoclonal antibodies revealed 3 separate domains possessing different functional properties. The results of topological mapping and immune typing of TBE virus antigen were compared.

[Preparation and properties of monoclonal antibodies to tick-borne encephalitis viral nonstructural proteins]. / Poluchenie i svoistva monoklonal'nykh antitel k nestrukturnym belkam virusa kleshchevogo entsefalita.

Hybridomas secreting monoclonal antibodies (Mab) to tick-borne encephalitis (TBE) virus are obtained. Immunodiffusion showed that 3 Mabs to TBE protein NS3 belong to class IgM and the rest to IgG1. Mabs to TBE protein NS1 were tested in hemagglutination inhibition, complement fixation, neutralization, and protection tests. Only 1 hybridoma produced Mab specific for protein NS1 of TBE strain Sofyin, the rest reacted with the common antigenic determinants of nonstructural TBE complex.

[Monoclonal antibodies against tick-borne encephalitis virus glycoprotein: preliminary characterization]. / Monoklonal'nye antitela k glikoproteidu virusa kleshchevogo éntsefalita: predvaritel'naia kharakteristika.

Characterization of 12 clones of monoclonal antibodies (MAb) generated for the main immunogen of tick-borne encephalitis virus, glycoprotein E, is presented. The following MAb parameters have been determined: constants of binding with antigen, classes and subclasses of immunoglobulins, the activity in two variants of solid-phase enzyme-immunoassay, binding with protein A, and MAb behavior in serologic tests: hemagglutination-inhibition, diffuse precipitation in agar, and virus neutralization. The preliminary studies revealed the presence in these MAb of at least three groups of antibody complementary to various nonoverlapping sites on the structural virus glycoprotein. Further employment of these MAb in practical and research work is discussed.

[The immune status of the population of the Crimea to the tick-borne encephalitis virus]. / Immunnyi status naseleniia Kryma k virusu kleshchevogo éntsefalita.

The immune status of the Crimean population with respect to tick-borne encephalitis (TBE) virus has been studied. The results of the study confirm the existence of natural foci of TBE in the Crimea. The most active and potentially dangerous foci are located in forests of the mountain area of the peninsula. The study has revealed that humans are mainly exposed to the risk of contacting TBE virus infection during their work and rest in the forest.