[Influence of siRNA complexes on the reproduction of influenza A virus (Orthomyxoviridae: Alphainfluenzavirus) in vivo].

INTRODUCTION: Influenza is one of the most pressing global health problems. Despite the wide range of available anti-influenza drugs, the viral drug resistance is an increasing concern and requires the search for new approaches to overcome it. A promising solution is the development of drugs with action that is based on the inhibition of the activity of cellular genes through RNA interference. AIM: Evaluation in vivo of the preventive potential of miRNAs directed to the cellular genes FLT4, Nup98 and Nup205 against influenza infection. MATERIALS AND METHODS: The A/California/7/09 strain of influenza virus (H1N1) and BALB/c mice were used in the study. The administration of siRNA and experimental infection of animals were performed intranasally. The results of the experiment were analyzed using molecular genetic and virological methods. RESULTS: The use of siRNA complexes Nup98.1 and Nup205.1 led to a significant decrease in viral reproduction and concentration of viral RNA on the 3rd day after infection. When two siRNA complexes (Nup98.1 and Nup205.1) were administered simultaneously, a significant decrease in viral titer and concentration of viral RNA was also noted compared with the control groups. CONCLUSIONS: The use of siRNAs in vivo can lead to an antiviral effect when the activity of single or several cellular genes is suppressed. The results indicate that the use of siRNAs targeting the cellular genes whose expression products are involved in viral reproduction is one of the promising methods for the prevention and treatment of not only influenza, but also other respiratory infections.

[A PERSPECTIVE CULTURAL MODEL FOR CONTROL OF BIOLOGICAL ACTIVITY OF HUMAN INTERFERONS].

AIM: Study species specificity of human lymphocyte interferon alpha in vitro in cell cultures of swine origin for expansion of cell line spectrum for interferon titration and control of newly created interferons and interferon-like preparations in vivo in mini-pig model. MATERIALS AND METHODS: Cell cultures of various species origin were used: Vero (monkey kidney), MDBK (bull kidney), HEK 293T (human embryo kidney), PK-15 (swine kidney), SPEV(swine embryo kidney), PTP (swine testicles), MDCK (canine kidney), RK-13 (rabbit kidney). Human lymphocyte interferon alpha (hINF-alpha) from Biomed company (1000 IU/ml), established in MDBK cells, was tested. Vesicular stomatitis virus (Indiana strain) was used. Human plasma was obtained from heparin-treated venous blood in the process of human peripheral blood lymphocyte isolation in medium for lymphocyte separation (Ficoll with a density of 1.077 g/cm3). RESULTS: Vesicular stomatitis virus, adapted to Vero cells, was established to have the least active reproduction in Vero and MDBK and reproduces more actively in cell of swine origin by 0.25 - 0.75 lg TCD50. At the same time, virus, adapted to cells of swine origin, reproduces more actively by 2 - 3 lg TCD50 in both cells of swine origin and Vero and MDBK. CONCLUSION: A possibility of titration of hINF-alpha in cells of swine origin was shown for both 100 doses of the indicator virus and low virus doses (5 and 10). This allows to determine low titers of hINF-alpha in blood plasma as one of the important indicators of interferon status--sera hINF-alpha.

[Monoclonal antibodies to rubella virus glycoprotein E1].

AIM: To obtain monoclonal antibodies (MCAs) to glycoprotein E1 of rubella virus, to assess their immunochemical characteristics and ability to use fluorescent MCA for rapid identification of rubella virus. MATERIALS AND METHODS: Rubella virus strain C-74 (Moscow), vaccine strains "Orlov" (Saint-Petersburg), Wistar RA 27/3 (USA) as well as strain Judith (Germany) were used. Viral antigens were obtained using diploid cells L-68 and cell lines VNK-21-F and Vero E6. MCAs were produced by conventional method and their isotype was determined: Immunoblotting, immunoenzume assay (IEA), hemagglutination inhibition assay (HIA) and immunofluorescence assay (IFA) were performed. RESULTS: Five monoclonal antibodies--Kh-252.1, Kh-347.2, Kh-183.3, Kh-214.4, Kh-187.5--to antigens of rubella virus strain C-74 were obtained. Isotypes of these antibodies were determined and their reactivity with native and denaturated antigens of other strains ("Orlov", Wistar RA 27/3, Judith) was characterized. IEA showed that all MCAs interacted with rubella virus glycoprotein E1 at high titers ranging from 1/1600 to 1/200,000. Immunoblotting demonstrated that 4 MCAs (Kh-252.1, Kh-347.2, Kh-183.3, Kh-214.4) had aforementioned feature. MCAs inhibited hemagglutinating activity of Judith strain in titer from 1/16 to 1/1024 in HIA. FITC conjugate of MCA Kh-347.2 (most sensitive variant) allowed to detect rubella virus in infected Vero E6 cells after 24 hours since infection, whereas FITC conjugates of 3 MCAs (Kh-183.3, Kh-214.4, Kh-187.5)--after 72 hours since infection. CONCLUSION: Use of FITC conjugates of MCAs is a perspective tool for identification of rubella virus glycoprotein E1 in infected cell cultures and nasopharyngeal swabs.

[Experimental study of antiviral activity of spore-forming bacterium Bacillus pumilus "Pashkov"].

AIM: To study antiviral activity of metabolites of spore-forming strain "Pashkov" of B. pumilus on the model of enterovirus infection in vitro. MATERIALS AND METHODS: B. pumilus strain "Pashkov" isolated from environment and identified by common methods. Cell cultures: Vero-6, Vero-ECC, and Vero-E6. Enteroviruses: type 1 poliovirus, Coxsackie B virus (1-6), ECHO-3, and ECHO-6 viruses. Unfectious activity of viruses was evaluated according to their cytopathogenic effect on Vero- E6 cell line by method of serial dilutions. Cultural fluid (CF) for the study was obtained by centrifugation and sterilizing filtration of B. pumilus strain "Pashkov" biomass produced by cultivation during 72 hours on optimized nutrient medium. Cytotoxicity of CF (chronic and acute) and maximal tolerated dose were measured by effect on viability of Vero-E6 cells, which was assessed by trypan blue exclusion test of cell viability. For measurement of antiviral activity (AV-activity), two treatment schedules--therapeutic and prophylactic--were used. RESULTS: The most sensitive cell lines were Vero-ECC and Vero-E6. Assessment of AV-activity showed that protective effect was observed for all dilutions of CF and lasted for 7 days from time of infection by used doses of virus. CF does not have acute and chronic cytotoxicity. CF studied in vitro with Vero-E6 cells infected with 4 types of enteroviruses provided protection against viruses and had prophylactic effect. Degree of effect of CF depended from type of enterovirus, dose used and CF dilution. CONCLUSION: For the first time effective antiviral activity of CF, which have low cytotoxicity for Vero-E6 cell culture in vitro and is produced by strain "Pashkov" of B. pumilus, was demonstrated. Obtained data open perspectives for development of medications against enterovirus infections.

[Expression of receptors for human alpha- and gamma-interferons on the surface of peripheral blood mononuclear cells in viral infections]. / Ekspressiia retseptorov dlia chelovecheskikh alpha- i gamma-interferonov na poverkhnosti mononuklearnykh kletok perifericheskoi krovi pri virusnykh infektsiiakh.

Expression of receptors on peripheral blood mononuclears of donors and patients with viral infections during therapy was studied using FITC-labeled monoclonal antiidiotypical antibodies with the "internal image" of human alpha- and gamma-interferons and monoclonal antibodies to these interferons. Activation of the immune system caused by infection modifies the expression of interferon receptors and leads to appearance of membrane-bound interferons, mainly gamma-interferon.

[The protective effect of monoclonal anti-idiotypic antibodies in experimental herpes infection]. / Protektivnyi éffekt monoklonal'nykh antiidiotipicheskikh antitel pri éksperimental'noi gerpeticheskoi infektsii.

The protective effect of monoclonal anti-idiotypic antibodies m(anti-ID)Abs, simulating the biologic effects of human alpha interferons, were studied in male guinea pigs with genital herpes simplex. Therapeutic effect of m(anti-ID)Abs daily applied as liquid dosage form and as suppositories+ was assessed. Experiments demonstrated that m(anti-ID)Abs in liquid dosage form to a different measure decreased the severity of urogenital infection in guinea pigs and that m(anti-ID)Abs-25 completely prevented the development of grave infection.

[The isolation and characteristics of monoclonal antibodies to different proteins of the herpes simplex virus types 1 and 2]. / Poluchenie i kharakteristika monoklonal'nykh antitel k razlichnym belkam virusa gerpesa prostogo tipov 1 i 2.

The authors prepared 156 mouse hybridomas producing monoclonal (MCA) antibodies to type- and group-specific antigenic determinants of HSV-1 and HSV-2. Seven of them were studied at length by western blot and radioimmunoprecipitation methods. The cell lines 1H97 and 2H141 were shown to produce immunoglobulins of G2 beta and M class, respectively, and were directed against group-specific antigenic determinants of the major nucleocapsid protein p150. The cell lines 1H38 and 1H110 produced immunoglobulins of M and G2 beta, respectively, and were directed against type-specific antigens of HSV-1 glycoprotein gB. At the same time, the presence of group-specific antigenic determinants on glycoprotein gB molecule was indicated by MCA 1H188 belonging to immunoglobulins of G2 alpha class. Two cell lines, 2H208 and 1H225, produced immunoglobulins G2 alpha directed against type-specific antigenic determinants of HSV-2 glycoprotein gD and group-specific antigenic determinants of HSV-1 gD, respectively. The results of immunoelectron microscopy indicated that MCA 1H110 and 2H208 were directed against virus envelope proteins.

[The antiproliferative activity of monoclonal anti-idiotypic antibodies imitating the biological effects of human alpha-interferons]. / Antiproliferativnaia aktivnost' monoklonal'nykh antiidiotipicheskikh antitel, imitiruiushchikh biologicheskie éffekty chelovecheskikh interferonov al'fa.

The experimental data from the study of antiproliferative activity (AP-activity) of monoclonal antiidiotypic antibody (mono-Ai-At) imitating biological effects of human alpha interferons (h-IF-alpha) are presented. The mono-Ai-At present in the ascitic and culture fluids (the culture fluid was obtained from cultivation of hybrid cells immobilized in alginate-gelatine gel) were shown to inhibit propagation of IF-sensitive continuous cells from normal and tumor tissues. The AP-activity of mono-Ai-At was found to be within the range of their antiviral activity. It is concluded that mono-Ai-At may be used as a new experimental model for investigation of h-IF-alpha biological effects.

[Monoclonal antibodies to Chlamydia psittaci: their isolation, characteristics and use]. / Monoklonal'nye antitela k Chlamydia psittaci: poluchenie, kharakteristika i primenenie.

A panel of 22 hybridomas producing monoclonal antibodies (McAb) to C. psittaci was obtained. 15 hybridomas produced IgG1 antibodies, 4 hybridomas produced IgM antibodies and 3 hybridomas produced IgG2b, IgG3 or IgA antibodies. IgG1 antibodies and 2 IgM antibodies did not bind complement in the complement fixation test. All McAb were reactive in the enzyme immunoassay and the indirect immunofluorescence test and did not precipitate specific antigens. Peroxidase conjugates on the basis of McAb effectively detected Chlamydia antigen, prepared from the crude suspension of chick embryo yolk sack infected with different strains of C. psittaci and C. trachomatis, in different modifications of EIA.

[Monoclonal anti-idiotypic antibodies simulating the biological effects of human alpha-interferons]. / Monoklonal'nye antiidiotipicheskie antitela, imitiruiushchie biologicheskie éffekty chelovecheskikh al'fa-interferonov.

The method of somatic hybridization was used to generate a panel of hybridomas producing monoclonal anti-idiotypic antibodies (mono-Ai-Ab) imitating biological effects of human alpha-interferons (hIF-alpha). Induction of syngeneic anti-idiotypic antibodies in BALB/c mice was achieved with monoclonal antibodies (MCA) IF-39 capable of neutralizing three kinds of hIF-alpha (lymphoblastoid, leukocyte, genetic-engineering). The screening of mono-Ai-Ab was done by determinations of antiviral activity (AV-activity) of supernatants from growing hybrid cell cultures caused by the cytopathic effect of 10-100 doses of mouse encephalomyocarditis virus (MEMC) by a micromethod in Vero cells grown in 96-well plates. Mono-Ai-Ab were found to neutralize MCA IF-39 and not to bind with immunosorbent of staphylococcal reagent containing protein A and BALB/c mouse immunoglobulins. It was shown that mono-Ai-Ab possessed AV-activity against MEMC and vesicular stomatitis viruses and were not inferior in this activity to commercial preparations of leukocyte IF-alpha. Mono-Ai-Ab had tissue species-specificity triggering the mechanism of AV-activity in human and simian cells as well as bovine kidney cells (MDVK line) imitating hIF-alpha in this effect.

[Monoclonal antibodies to natural human gamma-type interferon]. / Monoklonal'nye antitela k prirodnomu chelovecheskomu interferonu tipa gamma.

Nine hybridomas producing monoclonal antibodies (MCA) to natural human gamma interferon (IF-gamma) were generated. BALB/c mice were immunized with nonpurified IF-gamma preparation synthesized by lymphoid cells of the peripheral blood of donors in response to induction with staphylococcal enterotoxin A. For the first time somatic hybridization was done with the use of a medium with a high content of HEPES which maintained hybridomas viable for a long period of time. Out of 9 hybridomas, two (-gamma 6.1 and gamma-8.1) were shown to produce MCA with a high binding activity in the enzyme immunoassay. The same MCA effectively neutralized the biological activity of natural IF-gamma.

[The protective effect of hemagglutinin-specific monoclonal antibodies in experimental influenza infection]. / Protektivnyi éffekt gemaggliutininspetsifichnykh monoklonal'nykh antitel pri éksperimental'noi grippoznoi infektsii.

Experiments in mice showed a high protective effect of monoclonal antibodies (MCA) to influenza A/Krasnodar/101/59 (H2N2) virus hemagglutinin, possessing neutralizing activity in ovo. A 100% protective effect was observed upon intranasal administration of MCA Kp/101-3 48 hours before infection, and 90% effect upon administration of MCA 96 hours before infection. A 100% therapeutic effect was observed upon intranasal administration of MCA Kp/101-3 less than 24 hours postinfection and 70% therapeutic effect was achieved by administration of these MCA 48-72 hours postinfection.

[2-site competitive immunoenzyme analysis based on monoclonal antibodies for the detection and titration of antihemagglutinating measles antibodies]. / Dvukhsaitovyi konkurentnyi immunofermentnyi analiz na osnove monoklonal'nykh antitel dlia vyiavleniia i titrovaniia antigemaggliutiniruiushchikh korevykh antitel.

Using monoclonal antibodies (MCA) to measles virus (strain Leningrad-16) hemagglutinin, a competitive enzyme immunoassay (cEIA) was developed for the detection and titration of antihemagglutinating antibodies in human sera. Serum samples from children, collected in measles foci, were tested in cEIA, SP-EIA, HI, and PHA tests. The results evidence a high correlation of antihemagglutinin titres in cEIA and HI tests, the correlation coefficient of cEIA and PHA being 97.7%. The cEIA based on MCA to measles virus hemagglutinin is highly specific for the detection and titration of measles antihemagglutinins, it does not involve the use of simian erythrocytes. A significant advantage of this test system consists in the possibility of using unpurified antigen prepared from the infected cell lysate.

[The development and use of an immunoenzyme test system for detecting and the species identification of the monkey pox virus]. / Razrabotka i primenenie test-sistemy IFA dlia vyiavleniia i vidovoi identifikatsii virusa ospy obez'ian.

An enzyme immunoassay (EIA) system for the species-specific diagnosis of monkeypox, based on the use of monoclonal antibodies (McAb) to monkeypox virus, has been developed. Immunoglobulins, isolated from McAb-containing cultural and immune ascitic fluids, have been conjugated with horse-radish peroxidase and used as detector antibodies. For immunosorption, rabbit polyclonal antibodies to the vaccine virus have been used. The specificity and sensitivity of the EIA system thus obtained have been tested on animals and humans having monkeypox and confirmed by traditional diagnostic methods (the isolation of the virus on chick embryo chorioallantoic membranes and in cell culture).

Further-attenuated measles vaccine: virus passages affect viral surface protein expression, immunogenicity and histopathology pattern in vivo.

Measles vaccine viruses Leningrad-16 (L-16) and Moscow-5 (M-5, an L-16-derived clonal variant), at passage levels used for vaccination and after ten further low-multiplicity passages on quail embryo (QE) cells, were compared for (1) immunogenicity, (2) histopathological lesions induced in vivo and (3) surface protein expression within infected cells and on the virion surface. At the 10th passage, viruses evoked a poorer neutralizing antibody response in guinea pigs, induced an earlier appearance of more pronounced pathological lesions and replicated faster in Vero cells than the original viruses. H protein expression increased 1.8-2.3-fold after 10 passages of the L-16 variant, but remained virtually unaltered for the M-5 variant. F protein expression of both 10th-passage variants was 0.5-0.8 that of the original virus variants. A similar two-fold decrease in F protein expression was noted after a single virus passage in guinea pigs. The data implicate the loss of F protein as a cause of reduced immunogenicity of further attenuated measles vaccines.

[Preparation and properties of monoclonal antibodies to influenza virus A/Krasnodar/101/59 (H2N2)]. / Poluchenie i svoistva monoklonal'nykh antitel k virusu grippa A/krasnodar/101/59 (H2N2).

Somatic hybridization produced a set of 6 mouse hybridomas producing monoclonal antibodies of G isotype to influenza A/Krasnodar/101/59 (H2N2) virus. MCA were characterized by solid phase enzyme-immunoassay, hemagglutination-inhibition test, and indirect immunofluorescence technique. According to the results of radioimmunoprecipitation, all 6 hybridomas produced MCA to hemagglutinin of influenza A/Krasnodar/101/59 (H2N2) virus.