[Protective activity of aqueous extracts from higher mushrooms against Herpes simplex virus type-2 on albino mice model].

Toxicity and antiviral activity of aqueous extracts from higher mushrooms such as Lentinula edodes (Berk.) Pegler (shiitake), Pleurotus ostreatus (Jacq.) P. Kumm. (oyster), Inonotus obliquus (Ach. ex Pers.) Pilát (chaga), Hydnellum compactum (Pers.) P. Karst. (compact tooth) were studied. In doses of 0.8 to 4.0 mg (dry weight) per mouse administered orally or intraperitoneally the extracts showed no acute toxicity. When the dose of the chaga extract was increased to 20 mg per mouse, a half of the animals died. Intraperitoneal administration of the aqueous extracts in a dose of 0.4-2 mg per mouse prior to the contamination by a single LD50 of Herpes simplex type 2 provided 100-percent survival of the animals exposed to the Lentinula edodes or Pleurotus ostreatus extracts and 90-percent survival of the animals exposed to the Inonotus obliquus or Hydnellum compactum extracts.

[In vitro cytotoxicity of nitroxyl radicals with respect to tumor and diploid human cells and estimation of their antiviral activity].

Thirty nine water soluble nitroxyl radicals of various classes, belonging to piperidine, pyrrolidine and imidazolidine series were synthesized. Twenty seven of them were cytotoxic in vitro with respect to the tumor cell culture A431. The CC50 of the most active nitroxyl radicals with respect to cells SW480 and A431 was within 0.16-2.5 mM at the selectivity index of 3.91-7.81 in relation to cytotoxicity of the compounds for the cells of the normal L68 phenotype and tumor cells. The tests on the antiviral activity showed that 16 out of 22 nitroxyl radicals had antiviral activity in Vero cell culture with respect to the West Nile virus and Herpes simplex virus of type II respectively. The EC50 ranged within 0.09-3.45 mM. Some of the nitroxyl radicals had only antiviral activity, but a number of the compounds had both cytotoxic properties and antiviral activity.

[Preparation of monoclonal antibodies specific for the major tegument protein pp65 of human herpesvirus type 5].

A panel containing 11 types of monoclonal antibodies (mAb) specific for the recombinant protein pp65 of human herpesvirus type 5 was constructed. Enzyme immunoassay and immunoblotting of the immunochemical properties of mAb indicated that mAb could interact well with both recombinant and native pp65 antigen. Testing the suitability of mAb for an immunocytochemical study established that mAb 5F10 was highly effective in detecting the major tegument protein pp65 in the persistently infected Vero cells. A combination of the immunochemical properties of mAb enables them to be recommended for the immunodiagnosis of human cytomegalovirus infection.

[Antigenic differences in wild-type and guinea pig-adapted Ebola virus strains].

The splenocytes isolated from the mice immunized with wild-type or guinea pig-adapted Ebola virus strains were used to obtain hybridoma collections. Investigation of the monoclonal antibodies (mAb) obtained to one of the strains to another revealed antigenic interstrain differences in nucleoprotein and VP40. It is interesting that the differences were found in the hydridoma collection obtained against the wild-type strain. The mAbs produced by hydridomas to the adapted strain were found to equally well the antigens of both strains.

[Antiviral activity of aqueous extracts and polysaccharide fractions from mycelium and fruit bodies of higher fungi].

Sixty preparations of basidiomycetes (Ganoderma, Lentinus, Pleurotus, Laetiporus, Polyporus, Inonotus, Flammulina, Grifola, Trametes) were investigated with respect to their toxicity for Vero cells and antiviral activity. The antiviral activity was estimated with the use of the West Nile virus and type 2 Herpes simplex. It was shown that 11 preparations of Ganoderma, Lentinus and Pleurotus completely inhibited the infective activity in doses not lower than 1000 TCD50 (the West Nile virus) and 100 PPU (type 2 Herpes simplex). The antiviral activity of the preparations was likely due to the content of polysaccharides or their derivatives in the composition. It increased with increasing of the quantity of the total polysaccharide fraction or its concentration.

[The avidicity index of specific IgG in the diagnosis of diseases caused by west Nile and tickborne encephalitis viruses].

The significant antigenic crossovers between West Nile virus (WNV) and tick-borne encephalitis virus (TBEV) make the immunological diagnosis of these diseases difficult. The avidicity index of virus-specific class G immunoglobulins (IgG) was used as a criterion for the differentiation of an immune response to WNV or TBEV in patients and convalescents. The panels of the sera sampled from patients with tick-borne encephalitis and convalescents in the Novosibirsk and Tomsk Regions and in the Primorye Territory and from those with West Nile fever and convalescents in the Volgograd Region. The determination of the avidicity index could establish that in the convalescents' sera, the avidicity index of virus-specific IgG was much higher than that in the patients' sera in the acute phase of infection. In relation to heteroantigen, the avidicity index and the positivity coefficient were substantially less than those in the reaction with homoantigen. The findings have indicated that the determination of the value of the avidicity index of virus-specific IgG and the positivity coefficient makes it possible to differentiate West Nile fever and tick-borne encephalitis with confidence on the basis of solid-phase enzyme immunoassay in determining virus-specific IgG in the sera of patients and convalescents in different regions of Russia.

Mapping of two dominant sites of VP35 of Marburg virus.

Five types of anti-VP35 monoclonal antibodies (MAbs), four immune sera against Marburg virus (MBGV), and 11 overlapping recombinant VP35 fragments were used to map the epitopes for VP35 of MBGV. The purified full-size recombinant VP35 was highly immunogenic and retained the B-cell epitopes that were identical to those of the viral VP35. Two major sites on VP35 and a set of truncated VP35 fragments were found by use of an enzyme immunoassay and immunoblot. Site I was located in a region between amino acids 1 and 174 of the VP35 sequence, and only polyclonal antibodies (PAbs) against MBGV recognized epitopes at this site. Site II was mapped by use of anti-VP35 MAbs to the region between amino acid residues 167 and 278 of VP35. Amino acids 252-278 of VP35 might be involved in the formation of the epitopes for MAbs. B-cell epitopes were not found on the C-terminus of VP35 by use of PAbs or MAbs.

[Comparative study of the morphology and antigenic properties of recombinant analogs of a Marburg virus nucleoprotein]. / Sravnitel'noe izuchenie morfologii i antigennykh svoistv rekombinantnykh nalogov nukleoproteina virusa Marburg.

The full-length gene for Marburg virus (MV) nucleoprotein (NP) was cloned in prokaryotic pQE32 under the control of the T5 promoter and in eukaryotic pTM1 under the control of the promoter for T7 RNA polymerase. Recombinant NP was synthesized in Escherichia coli and in human kidney cell line 293 cotransfected with recombinant vaccinia virus vTF7-3 expressing T7 RNA polymerase. On evidence of electron microscopy with immune detection, recombinant NP formed tubules of two types in E. coli and of a single type in cell line 293. ELISA and immunoblotting with polyclonal and monoclonal antibodies revealed common antigenic determinants in recombinant NP and natural MV NP.

[Analysis of antigenic determinant profiles of the Ebola virus VP35 protein N-terminal region using its short recombinant fragments]. / Vyiavlenie antigennykh determinant na N-kontse belka VP35 virusa Ebola s pomoshch'iu korotkikh rekombinantnykh fragmentov étogo belka.

cDNA of fragments of gene VP35 of the Ebola virus (EV) were expressed in vector pQE30 for the purpose of isolation of recombinant fragments of protein VP35. Five short affinity-purified fragments of the EV VP35 protein were analyzed, by using the methods of IEA and immunoblotting, with polyclonal antiviral sera (PAS) against EV and with hybrid monoclonal antibodies (Mabs) IC6 and 6F7 specific to EV VP35 protein. All fragments of protein VP35 with an intact N-terminal region and removed C-terminal region were found to interact effectively with PAS and with Mabs IC6 and 6F7. Rec86N, the smallest of the above fragments, comprised the initial 86 amino acid residues of the VP35 N-terminal region. A removal of 36 amino acid residues from the N-terminal region of Rec310N, the largest recombinant fragment, resulted in a loss of interaction with Mabs IC6 and 6F7, while the interaction with polyclonal antibodies remained intact. The obtained results show that the initial 86 amino acid residues of the N-terminal region of EV VP35 are of the key importance in forming the antigenic structure of VP35 and that they contain multiple B-cell epitopes. Finally, the initial 36 amino acids of VP35 predetermine the shaping-up of two antigenic determinants for Mabs IC6 and 6F7.

[Monoclonal antibodies in human hepatitis A virus immunoenzyme diagnosis]. / Monoklonal'nye antitela v immunofermentnoi diagnostike virusa hepatita A cheloveka.

Antigen and antibody detection in EIA is a good tool in diagnosing HAV infections, especially in their differentiation from other hepatitides. Commercial kits containing polyclonal antibodies and murine MAbs to identify HAV are now available. Rat MAbs have not been assayed so far. Peroxidase-labelled rat MAbs and purified rat MAbs as antigen capture were used to modify commercial "VectoHep A-IgM" and "VectorHep A-Ag" kits. The results obtained with modified "VectoHep A-IgM" and "VectorHep A-Ag" kits with labelled rat MAbs suggest that labelled rat MAbs can increase the sensitivity and specificity of EIA. MAbs used as antigen capture and labelled antibodies permit at least an 8-fold increase in sensitivity as compared to polyclonal antibodies. The modified "VectorHep A-Ag" kit with labelled rat MAbs provided 100% sensitivity and specificity in EIA. The modified "VectorHep A-Ag" kit also allowed the authors to determine viral antigens in the cell lysate, homogenates of the infected monkey liver, stools from patients, and sewage water samples. The rat MAbs modified kits can be recommended for using in epidemiological and clinical studies of HAV infections.

[Detection of antiviral activity of monoclonal antibodies, specific to Marburg virus proteins]. / Vyiavlenie protivovirusnoi aktivnosti monoklonal'nykh antitel, spetsifichnykh k belkam virusa Marburg.

Monoclonal antibodies (MAbs) specific to Marburg virus (MBG), Popp strain, have been previously produced and characterized by indirect ELISA. Protein specificity of MAbs was determined by immunoblotting with SDS-PAGE proteins of MBG: one to NP, four to VP40, and protein specificity of one antibody was not detected. The effect of MAb binding to protein epitopes on viral functions was investigated in vitro and in vivo. None of antibodies neutralized the virus in the neutralization test in vitro, but MAb 5G9.G11 and 5G8.H5 specific to MBG VP40 protein were active in antibody-dependent complement mediated lysis of virus-infected cells. In vivo these antibodies (5G9.G11 and 5G8.H5) protected guinea pigs from lethal MBG infection after passive inoculation. Studies of biological activity and analysis of epitope specificity of MAb-antiVP40 by competitive ELISA showed that 2 of 7 epitopes of VP40 protein of MBG induce the production of protective antibodies. Hence, MAbs 5G9.G11 and 5G8.H5 reacting with MBG VP40 protein caused lysis of virus infected cells in the presence of the complement in vitro and protected guinea pigs from MBG infection by passive inoculation.

[Monoclonal antibodies to Ebola virus: isolation, characteristics, and study of cross reactivity with Marburg virus]. / Monoklonal'nye antitela k virusu Ebola: poluchenie, kharakteristika i izuchenie perekrestnoi reaktivnosti s virusom Marburg.

Thirteen hybridoma strains producing monoclonal antibodies (Mabs) to Ebola virus were prepared by fusion of NS-O mouse myeloma cells with splenocytes of BALB/c mice immunized with purified and inactivated Ebola virus (Mayinga strain). Mabs directed against viral proteins were selected and tested by ELISA. Protein specificity of 13 Mabs was determined by immunoblotting with SDS-PAGE proteins of Ebola virus. Of these, 11 hybridoma Mabs reacted with 116 kDa protein (NP) and 2 with Ebola virus VP35. Antigenic cross-reactivity between Ebola and Marburg viruses was examined in ELISA and immunoblotting with polyclonal and monoclonal antibodies. In ELISA, polyclonal antibodies of immune sera to Ebola or Marburg viruses reacted with heterologous filoviruses, and two anti-NP Ebola antibodies (Mabs 7E1 and 6G8) cross-reacted with both viruses. Target proteins for cross-reactivity, Ebola NP (116 kDa) and Marburg NP (96 kDa), and VP35 of both filoviruses were detected by immunoblotting with polyclonal and monoclonal antibodies (6G8) to Ebola virus.

[Antigenic structure of Ebola virus VP35 protein]. / Issledovanie antigennoi struktury belka VP35 virusa Ebola.

Antigenic structure of Ebola virus (EV) (strain Mayinga) nucleocapsid protein VP35 was analyzed using monoclonal antibodies to EV VP35 and polyclonal antibodies to EV. EV protein VP35 was shown to have antigenic sites inducing the production of antibodies in animals. For better characterization of protein VP35 antigenic structure. EV gene encoding the full-length VP35 was cloned in vector pQE31 as a recombinant fusion protein (rec.VP35). The antigenic and immunogenic properties of rec.VP35 and EV VP35 were compared by ELISA and Western blot analysis with polyclonal and monoclonal antibodies. Antibodies of positive sera and VP35 MAbs cross reacted with the analyzed antigens. The topography of epitopes on EV VP35 and rec.VP35 was studied using MAbs and polyclonal antibodies to rec.VP35 in a competitive antibody binding assay. Two epitopes of one site were identified on these proteins. These epitopes are present on infectious virion protein VP35 and are stable during physicochemical exposures.

[Comparative study of the antigenic and immunogenic properties of native and recombinant Marburg virus VP35 proteins]. / Sravnitel'noe issledovanie antigennykh i immunogennykh svoistv prirodnogo i rekombinantnogo belkov VP35 virusa Marburg.

Antigenic structure of Marburg virus (MBG) VP35 was compared with that of the recombinant VP35 (f35) expressed in a prokaryotic system. For this purpose, a gene encoding the full-length VP35 was cloned in vector pQE31(QIAGEN) and expressed at about 70 mg/liter culture fluid in Escherichia coli JM103 as a recombinant fusion protein f35. BALB/c mice were immunized with soluble f35 or purified inactivated virions of MBG. Antibodies cross-reacting with VP35 and f35 antigens were detected by ELISA and Western Blot analysis in immune sera. Serum from a convalescent after Marburg disease and polyclonal antibodies from animals immunized with MBG recognized f35 and the MBG VP35. VP35 and its recombinant analog induced the production of specific antiviral antibodies in mice, which cross-reacted with the studied antigens. Competitive EIA showed that VP35 and f35 cross-inhibit the antigenic reactivity with polyclonal antibodies of immune sera. Antigenic structure of f35 protein corresponded to antigenic structure of MBG VP35 protein.