[Genetic diversity of the tick-borne encephalitis virus in Ixodes persulcatus ticks in northeastern European Russia].

The genetic diversity of the tick-borne encephalitis virus (TBEV) in the PCR-positive Taiga ticks collected in the Republic of Komi in 2010 was evaluated. The analyses of nucleotide sequences of the 5'-NCR fragments of viral genome from ticks had shown that 13 isolates of TBEV from 16 sequencing variants were represented by the highly pathogenic Far Eastern genotype of the TBEV and only 3 isolates were identified as the Siberian genotype of TBEV. The nucleotide sequences of 5'-NCR of viral genome strongly varied variable in individual ticks. Variability for the A1 element has been observed in all the tested samples, and for elements C1, B2, CS B--in more than 50%. A2 element and ATG codon of the 5'-NCR remained completely conservative. Computer simulation of conformations of the 5'-NCR of TBEV genome demonstrated the possibility of significant changes of the spatial structure of the 5'-NCR of viral genome in individual taiga ticks. The obtained data confirm the hypothesis that the variability in the 5'-NCR of TBEV genome can be crucial for efficient replication of TBEV in different hosts.

[The role of birds in the maintenance of tick-borne infections in the Tomsk anthropurgic foci].

The role of birds in the focus of tick-borne infections was studied from 2006 to 2011. The frequency index of ticks carried by ground dwelling birds is about 49.7%. The index of their abundance is 3.8. The larvae of ticks have been found on birds in 43.8% of cases. Nymphs and adult ticks have been found in 39.9 and 16.3%, respectively. It was revealed that Ixodex pavlovskyi was transferred and dominated in the urban microfoci because of its ornithophily. The markers of infectious agents have been recorded in 42 of 60 bird species under study.

[Genetic diversity of ixodid tick-borne pathogens in Tomsk City and suburbs].

We studied two urban and two suburban biotypes of Tomsk City for tick-transmitted diseases prevalence in naturally collected ticks. Tick-borne encephalitis virus (TBEV) was found in 6.5% of tick samples, West Nile virus (WNV) in 2.2%, Borrelia spp. in 8%, Rickettsia spp. in 2.5%, and Ehrlichia spp. in 1.7% of samples. Genetic markers of Powassan virus, Bartonella spp., and Balbesia spp. were not found. Analysis of the genetic diversity of revealed pathogens resulted in the following conclusions: 1. TBEV strains belong to Siberian and Far-Eastern subtypes, and Far-Eastern subtype of TBEV is most frequent in urban biotypes (up to 43 % of urban strains of TBEV); 2. WNV strains belong to genotype la; 3. Borrelia spp. were classified as B. garinii; 4. Rickettsia spp. were classified as R. tarasevichiae and probably as a new Rickettsia raoultii subspecies; 5. Ehrlichia spp. were classified as E. muris. The coexistence of several pathogens was found in 5.7% of tick samples, and the most frequent combination was TBEV + Borrelia spp.

[Detection of the West Nile Virus and its genetic typing in ixodid ticks (Parasitiformes: Ixodidae) in Tomsk City and its suburbs].

Four tick species, Ixodes persulcatus, I. pavlovskyi, I. trianguliceps, and Dermacentor reticulatus, were found in Tomsk and its suburbs in 2006. The species I. pavlovskyi was found to be dominant in the localities situated in Tomsk City, and I. persulcatus was dominant in its suburbs. Viral RNA and viral antigen of the West Nile virus (WNV) were detected in the ticks I. pavlovskyi and I. persulcatus collected in the city and its suburbs by the RT PCR method and enzyme immunoassay with monoclonal antibodies against protein E of the WNV. Average rate of the WNV infected ticks varied from 5.2 up to 11.7% in different localities. Identification of the nucleotide sequence of the protein E gene fragment allowed classifying the cDNA obtained as genotype Ia of the WNV. The sequences are proved similar to the strain LEIV-Vlg99-27889-human of the WNV isolated in Volgograd. The obtained data showed that natural foci of the WNV virus can appear in the city and its suburbs probably involving two dominant tick species. The WNV infected imagoes, larvae, and nymphs of I. persulcatus and I. pavlovskyi were collected from small mammals, lizards, and birds. Therefore we presume that these hosts can be involved in the circulation and distribution of WNV on the territory of Tomsk Region.

[Activation of the RIG-I gene, coding for DEXH/D-protein in infection of RH cells by tick-borne encephalitis virus]. / Aktivatsiia gena RIG-I, kodiruiushchego DEXH/D-belok pri infektsii kletok RH virusom kleshchevogo éntsefalita.

It was demonstrated by subtractive hybridization that the infection of a human embryonic kidney cell line with tick-borne encephalitis virus causes an approximately tenfold transcription activation of the RIG-1 gene, which encodes a protein of the DExH/D-box-containing RNA helicase family. A possible involvement of the protein in antiviral cell systems is discussed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.

[Laminin-binding protein as a cellular receptor for the equine Venezuelan encephalomyelitis virus: Report 2. Inhibition of replication of equine Venezuelan encephalomyelitis virus by blocking laminin-binding protein on the surface of Vero cells]. / Lamininsviazyvaiushchii belok kak kletochnyi retseptor dlia virusa venesuél'skogo éntsefalomielita loshadei: Soobshchenie. 2. Ingibiriovanie replikatsii virusa venesuél'skogo éntsefalomielita loshchadei putem blokirovaniia lamininsviazyvaiushchego belka na poverkhnosti kletok Vero.

A possible inhibition of the Venezuelan equine encephalomyelitis (VEE) virus replication in Vero cells through the laminin-binding protein (LBP) blocking in the surface of such cells was investigated in order to verify the LBP value. It was demonstrated, on the basis of the flow scanning cytometry and FITC-labeled antibodies to LBP, that there are at least 263 thousand LBP molecules in the Vero cells' surface. Blocking of the molecules by rabbit polyclonal antibodies to 43 kD of the recombinant LBP (rLBP) was shown to inhibit the VEE virus replication in Vero cells by more than 300,000 times, which made them virtually resistant to the possibility of VEE virus infection. This also confirmed that LBP is a target-molecule for VEE virus in Vero cells with the interplay between VEE virus and LBP in the cells' surface being the initial stage of virions' penetration into the cell and of their replication inside the cell.

[Laminin-binding protein (LBP) as a cellular receptor for the virus of Venezuelan equine encephalomyelitis (VEE): Part 1. A study of the interaction between VEE virus virions and the human recombinant LBP]. / Laminosviazyvaiushchii belok (LSB) kak kletochnyi retseptor dlia virusa venesuél'skogo éntsefalomielita loshadei (VEL): Soobshchenie 1. Issledovanie vzaimodeistviia virionov virusa VEL s rekombinantnym LSB cheloveka.

The interaction of the VEE virus virions with human LBP was investigated. The affinely purified 43 kDa recombinant LBP (rLBP) of man was found to interact effectively with the VEE virus virions purified in immune enzyme assay. The affinity constant of 43 kDa rLBP with virions was equal to 4.3-4.8 x 10(7) M-1. The rabbit antiviral polyclonal antibodies blocked the interaction of rLBP with the VEE virus virions. According to Western blot, rLBP is capable of interacting with the E1 glycoprotein of the VEE virus, which suggests the presence of a specific epitope of binding with LBP in the surface of the E1-E2 heterodimer of the VEE virus. The results confirm that human LBP could be a receptor for the VEE virus.

[Late activation of interferon-induced genes IFI-54k and IFI-56k in human RH cells infected with tick-borne encephalitis virus]. / Pozdniaia aktivatsiia interferonindutsiruemykh genov IFI-54k i IFI-56k pri infektsii kletok linii RH cheloveka virusom kleshchevogo éntsefalita.

The genes that were induced and suppressed in human embryonic kidney cell line RH upon the infection with tick-borne encephalitis virus were studied by the method of subtractive hybridization. The expression of interferon-induced genes IFI-54K and IFI-56K in the infected cells was found to increase 50-100-fold.

[Recombinant antibodies to tick-borne encephalitis virus]. / Rekombinantnye antitela k virusu kleshchevogo éntsefalita.

A gene encoding the dimeric form of single-chain antibody fragments (scFv) was designed on the basis of the artificial gene coding monomeric scFv to tick-borne encephalitis glycoprotein E. The sequence encoding histidine oligomer was added to 3'-ends of the genes encoding the monomeric and dimeric forms of scFv antibodies. Escherichia coli strains were constructed for the production of monomeric and dimeric antibodies. These antibodies were purified using Ni-NTA resin. The specificity of the purified monomeric and dimeric antibodies in the binding reaction with tick-borne encephalitis virus was shown by ELISA and Western-blot analysis. The identity of glycoprotein E epitope bound by monomeric and dimeric scFv and parental monoclonal antibodies E6B was confirmed by competitive immunoassay.

Human recombinant laminin-binding protein: isolation, purification, and crystallization.

The mRNA of the precursor of laminin-binding protein (LBP) was isolated from a human embryo kidney cell line and cloned. The determined sequence of the LBP gene showed complete identity with the LBP genes isolated from human lung and large intestine cells. The human LBP was expressed by E. coli cells, and it was purified using Ni-NTA-Sepharose chromatography. The mobility of the homogeneous recombinant human laminin-binding protein on SDS-PAGE was 43 kD. A mixture of eight murine monoclonal antibodies, the MPLR Pool against LBP, reacted with the recombinant LBP in Western blot. The interaction of the antiidiotypical antibodies 10H10 and E6B provided evidence that the epitope binding to protein E of the tick-borne encephalitis (TBE) virus is also preserved on the human recombinant LBP. Enzyme immunoassay confirmed the ability of the recombinant LBP to interact with protein E of TBE virus. The biological activity of the recombinant LBP allowed us to perform X-ray analysis of the spatial arrangement of the LBP molecule using the recombinant protein. For this purpose, crystals of the human LBP were obtained by the standing drop version of the pore diffusion technique. The crystals appropriate for X-ray structural analysis were 0.3 x 0.1 x 0.05 mm in size. The X-ray diffraction field of the crystal extended to 2.5 A.

[Monoclonal anti-idiotypic antibodies, imitating the antigenic determinant of hemagglutination site of Venezuelan equine encephalomyelitis virus glycoprotein E2]. / Monoklonal'nye antiidiotipicheskie antitela, imitiruiushchie antigennuiu determinantu saita gemaggliutinatsii glikoproteina E2 virusa Venesuél'skogo éntsefalomielita loshadei.

A panel of 9 mouse hybridomas producing monoclonal antibodies (MAb-2) to determinants of rat MAb 4H5 (MAb-1) was prepared. MAb-1 blocked hemagglutination caused by different alphaviruses. The specificity of paratopes of MAb-2 was studied by competitive enzyme immunoassay and they were divided into 2 groups: 1) interacting with isotypic and/or allotypic determinants of molecules of MAb 4H5 (5 hybridomas) and 2) specific to idiotypic determinants of MAb 4H5 molecules (4 hybridomas). The antigenic structure of paratopes of 3 antiidiotypic MAb (1C2, 1F9, and 6C8) was specific to the idiotopes localized in the active MAb 4H5 center interacting with the hemagglutination domain of VEE virus glycoprotein E2. These antiidiotypic MAb can agglutinate goose red cells, this indicating that the antigenic structure of their paratopes mimicks the functional determinant of the hemagglutination domain of VEE virus glycoprotein E2.

[Recombinant vaccinia virus expressing Japanese encephalitis virus protein E]. / Rekombinantnyi virus ospovaktsiny, ékspressiruiushchii belok E virusa iaponskogo zéntsefalita.

Recombinant vaccinia virus expressing protein E of Japanese encephalitis virus has been constructed. Polyclonal antibodies to JE virus reacted with recombinant protein E in immunoblotting. Immunochemical analysis of the recombinant protein E with monoclonal antibodies showed that both group specific and receptor domains of the protein were intact.

[Preparation and study of properties of anti-idiotypic antibodies, carrying hemagglutinating paratopes of tick-borne encephalitis virus on their surface]. / Poluchenie i izuchenie svoistv antiidiotipicheskikh antitel, nesushchikh na svoei poverkhnosti gemaggliutiniruiushchie paratopy virusa kleshchevogo éntsefalita.

Rabbit polyclonal and rat monoclonal anti-idiotypical antibodies (AIA) against two hemagglutinating murine monoclonal antibodies to glycoprotein E of tick-borne encephalitis virus (TBE) have been obtained, purified, and studied. All AIA reacted with murine monoclonal antibodies to TBE protein E and did not react with normal murine immunoglobulins in enzyme immunoassay (EIA). Polyclonal AIA to monoclonal E6B and 10H10 antibodies specifically reacted with RH, SPEV, and goose red cells. In addition, polyclonal AIA were capable of agglutinating goose erythrocytes. Mice immunized with purified polyclonal and monoclonal AIA produced antiviral sera, which reacted in EIA with purified TBE virions. These results indicate that AIA carry the TBE virus image and mimic the hemagglutination activity epitopes on glycoprotein E. Interactions of AIA with cell membranes indicates that they fix the cellular receptor needed for adsorption and penetration of TBE virus into the cells.