[A full-size DNA copy of the tick-borne encephalitis virus genome. I. Analysis of the 5'- and 3'-terminal noncoding regions of the genome]. / Polnorazmernaia DNK-kopiia genoma virusa kleshchevogo éntsefalita. I. Analiz 5'- i 3'-kontsevykh nekodiruiushchikh oblastei genoma.

Using reverse transcription and the polymerase chain reaction, cDNA fragments of noncoding regions of the tick-borne encephalitis virus (TBEV) genome were obtained. These fragments were cloned into a pGEM3 vector, and their nucleotide sequences were determined. The heterogeneity of the 3'-terminal untranslated region of the TBEV RNA was revealed. To create a stable full-size DNA copy of the TBEV genome, four cDNA variants differing in length and structure of the 3'-terminal fragment of the viral RNA were cloned into a pBR322-derived vector.

[Multiplicity of affinity modification of RNAse during its alkylation with a reactive analog of 5-deoxyribonucleotide]. / Mnozhestvennost' affinnoi modifikatsii RNKazy pri alkilirovanii ee reaktsionnosposobnym analogom 5'-dezoksiribonukleotida.

The interaction of pancreatic RNase with 5'-deoxyribodinucleotide alkylating derivative, 4-(N-2-chloroethyl-N-methylamino)benzylamide of d(pTpA) d[(ClRCH2NH)pTpA], was studied. The unreactive oxyanalogue d[(HORCH2NH)pTpA] was shown to act as competitive inhibitor of cCMP hydrolysis by RNase. d[(ClRCH2NH)pTpA] irreversibly inactivated RNase. A protective effect was exerted by d(pTpA) and d[(HORCH2NH)pTpA]. The modification, although having an affinity character, was not accompanied by total inactivation of the enzyme. It was supposed that covalent bonding between the reagent and enzyme induced the dinucleotide displacement from the recognition site. The formation of four RNase monolabeled forms retaining the activity in the hydrolysis of cCMP and poly(U) was demonstrated.

[Highly-sensitive nonradioactive detection of the tick-borne encephalitis virus]. / Vysokochuvstvitel'naia neradioaktivnaia detektsiia virusa kleshchevogo éntsefalita.

The non-radioactive reverse dot-blot method was used for the detection of tick-borne encephalitis virus (TBEV) in clinical specimens. The method involves reverse transcription (RT) and polymerase chain reaction (PCR) using a pair of biotin-labelled oligonucleotide primers. These primers flank a region in the gene of the envelope protein E, which is more conserved than other regions, and initiate the polymerisation with RNAs of all the investigated strains. The amplified cDNA was captured from solution on a solid support using complementary oligonucleotides covalently bound to a polyamide membrane. The biotin labels of the resulting hybrids were visualized by means of the streptavidin-horseradish peroxidase conjugate. The detection limit of the test was about 10(3)-10(4) molecules of target RNA. The sensitivity was comparable to that obtained by dot-hybridization of PCR-product with 32P-labelled DNA probe. The method was used for the detection of RNA in specimens of tick and blood.

[Genetic analysis of tick-borne encephalitis virus strains from West Siberia]. / Geneticheski'i analiz shtammov virusa kleshchevogo éntsefalita Zapadno'i Sibiri.

Tick-borne encephalitis (TBE) virus strains were isolated in West Siberia in the forest-steppe region near the Ob river in 1981-1992. Hybridization of genome RNA of 46 TBE strains with [32P]cDNA of TBE Sofyin strain revealed essential differences in the genomes of West-Siberian and Far-Eastern Sofyin strains of TBE virus. Nucleotide sequences of 6 TBE strains (1348-1503 n.) have been determined. A 89-98% homology of Siberian TBE strains has been shown, while the similarity of the respective fragment of E gene for West Siberian and Sofyin strains was no more than 81%. No significant changes in E gene of TBE strains have been detected over a 12-year period.

[Interaction of tick-borne encephalitis virus RNA with proteins]. / Vzaimodeîstvie RNK virusa kleshchevogo éntsefalita s belkami.

Binding of tick-borne encephalitis (TBE) virus RNA to proteins was studied by 3 methods: gel retardation, RNA-protein cross-linking under UV light, and RNA-protein blotting. Two cellular proteins with molecular weights about 30 and 22 kDa were detected in the nuclear fraction of two cell strains (PS and RH) and in blood mononuclear cells of patients with TBE. Weak interaction between the studied RNA and viral proteins can result from trace amounts of TBE virus proteins in infected cells and by lack of additional binding factors. Bacterial super-products and affinity chromatography for isolation of native glycoproteins were used to increase the amount of viral proteins. Purified E and NS1 proteins did not react with viral RNA, while incubation with recombinant nonstructural TBE virus NS3 protein led to a shift in the mobility of TBE virus RNA in gel in the absence of cross-linking under conditions of UV exposure after addition of heparin. Poor binding of nonstructural TBE virus NS3 protein is based on electrostatic binding. Oligoribonucleotide 14 nucleotide residues long corresponding to 5'-terminal of TBE virus genome did not react with NS3 protein, probably because of its small size or absence of certain sequences.

[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.

[Comparison of 3 express-methods of detection of tick-borne encephalitis virus]. / Sravnenie trekh ékspress-metodov indikatsii virusa kleshchevogo éntsefalita.

Comparative studies of the diagnostic value of three express methods for detection of tick-borne encephalitis virus in ticks (fluorescent antibody technique (FAT) enzyme immunoassay (EIA), and molecular hybridization of nucleic acids) and the traditional method (bioassay in white mice) showed all the three express methods to be rapid, specific, sensitive, and useful for large-scale epidemiological surveys. Notable was the high effectiveness of the method of nucleic acids hybridization which was not inferior to bioassays in suckling mice and exceeded FAT and EIA. The results of the latter seem to be affected by antigenic variations among tick-borne encephalitis virus strains.