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In this study we examine the possibility that TiO 2 nanoparticles and their conjugates can penetrate into cultivated cells without any special transfection procedures. Oligonucleotides and their derivates were conjugated with the TiO 2 nanoparticles, which were obtained as colloidal solutions at a concentration of TiO 2 0.3M by TiCl 4 hydrolysis. The electronic microscopy of various cell cultures (KCT, Vero, and MDCK) treated with nanoparticle solutions (20 µg/µl) showed that nanoparticles could enter the cells and accumulate in the vacuoles and phagosomes and form inclusions in cytoplasm. Thus, we demonstrated the penetration of TiO 2 nanoparticles and their oligonucleotide conjugates into intracellular space without any auxiliary operations. Most other researches used electroporation techniques for similar purposes [1, 2, 5]., (© Pleiades Publishing, Ltd. 2009.)

Bicyclic furano[2,3-d]pyrimidine ribonucleosides were synthesized by Pd(0)- and CuI-catalyzed coupling of 5-iodouridine with terminal alkynes. The treatment of the resulting nucleosides with ammonia or methylamine solution in aqueous alcohol resulted in pyrrolo- and N(7)-methylpyrrolo[2,3-d]pyrimidine nucleosides. 5'-O-Triphosphates of bicyclic nucleosides were obtained by the treatment of the nucleosides with POCl3 in the presence of a "proton sponge." The 5'-O-triphosphates are not substrates for HCV RNA-dependent RNA polymerase, but are effective substrates for HCV RNA helicase/NTPase and did not inhibit ATP hydrolysis. Only 3-(beta-D-ribofuranosyl)-6-decyl-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one showed a moderate anti-HCV activity in the HCV replicon system and efficiently inhibited replication of bovine viral diarrhea virus (BVDV) in KCT-cells, other compounds being inactive. None of the compounds were cytotoxic within the tested range of concentrations.

A combinatorial immune library of human single-chain antibody fragments (scFv) was constructed on the base of genes encoding variable domains of heavy and light chains of immunoglobulins cloned from the lymphocytes of four vaccinia virus (VACV) vaccinated donors. The size of the library was 3 x 10(7) independent clones. After the library was enriched with the clones producing scFv against recombinant analogue of variola virus surface protein prA30L, a panel of unique antibodies specific to both prA30L and VACV was selected from the library. A plaque reduction neutralization test was performed for all selected antibodies and two antibodies were shown to be able to neutralize plaque formation of VACV in Vero E6 cells monolayer. Binding specificities of these antibodies were confirmed using ELISA and Western blot analysis. To determine the amino acid sequences of neutralizing antibodies their genes were sequenced.

New 5-azole- and 5-oxime-substituted analogues of 2'-deoxyuridine are synthesized. The analogues with azole ring manifest low toxicities and antiherpetic activities on Vero cell culture, the imidazole derivative being the most active. The inhibitory effects of oximes of 5-formyl-deoxyuridine are comparable with those of the azole-containing nucleoside analogues, although their cytotoxicities are found to be higher; oxime of 5-formyldeoxyuridine is particularly toxic. The nucleoside analogues synthesized exhibit no marked activity on cell cultures infected with various variants of poxvirus. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.

Open reading frame (orf) 129L of ectromelia (EV) and orf A30L of smallpox viruses (SPV) encoding fusion proteins were cloned and expressed in E. coli cells. The recombinant polypeptides (prA30L H pr129L) were purified from cell lysates by Ni-NTA chromatography. Recombinant polypeptides were able to form trimers in buffered saline and they destroyed under treatment with SDS and 2-mercaptoethanol. Reactivity of prA30L, pr129L and orthopoxvirus proteins was analyzed by ELISA and Western blotting with panel of 22 monoclonal antibodies (MAbs) against orthopoxviruses (19 against EV, 2 MAbs against vaccinia virus and 1 Mabs against cowpox virus). This data allowed us to conclude that there are 12 EV-specific epitopes of pr129L and EV fusion proteins, ten orthopox-specific epitopes of EV, VV, CPV fusion proteins, from them 9 orthopox-specific epitopes of prA30L and SPV fusion proteins. Five Mabs, which cross-reacted with orthopox-specific epitopes, were able to neutralize the VV on Vero cells and from them two MAbs has neutralizing activity against smallpox virus. Our findings demonstrate that 129L fusion protein have EV-specific epitopes, that EV 129L and SPV A30L fusion proteins have a several orthopox-specific epitopes to induce a neutralizing antibodies against human pathogenic orthopoxviruses.

The purpose of the study was to investigate, in the Vero cell culture, the antiviral activity of vegetable tritrpens derivatives and ribavirin analogues against the viruses of measles, herpes simple (type 1), cytomegaloviruses and filoviruses. The toxicity and antiviral activity of compounds were determined after coloring of cells with crystal violate. Additionally, the combined action of triterpens' derivatives and ribavirin was investigated. The studied compounds showed relatively low antiviral activity, nonetheless, further research of vegetable triterpens and their derivatives as well as ribavirin analogues would be promising.

A new synthesis of chiral acyclic nucleoside and nucleotide analogues starting from d(-)- or l(+)-riboses was proposed. Antiviral properties of the synthesized compounds towards the pox virus family were evaluated.

A library of human scFv antibodies displayed on the surface of bacteriophages (MRC, Cambridge, England) was panned against the Elstree strain of vaccinia virus (VACV), which resulted in the phage repertoire enriched with clones positive to the strain. Individual clones from the repertoire were screened for binding, independently, to the vaccinia and ectromelia viruses; phage antibodies to the orthopoxviruses were selected. Ten unique antibodies were identified after their Vh- and Vl-genes were sequenced. All selected antibodies were assayed by ELISA for binding to the vaccinia, cowpox and ectromelia viruses. Furthermore, all selected antibodies were assayed for binding with major alastrim strains of the live variola virus. According to the results, the above phage antibodies recognized genus-specific epitopes, some of which differed in their conformation.

An extensive collection of 125 rat hybridomas secreting monoclonal antibodies (Mabs) to ectromelia virus (EV) polypeptide (Poxviridae family, Orhtopoxvirus genus) was set up. A significant portion of Mabs (37 types) recognized epitopes of the 14 kDa polypeptide as well as the 37 and 35 kDa polypeptides. However, a majority of Mabs interacted with conformation-dependent epitopes, which were destroyed in immunoprecipitation. One hundred and thirteen of Mabs cross-interacted with antigenic determinants of vaccinia viruses (VV), cowpox virus (CPV) and smallpox virus (SPV); only 12 of them were found to be specific to EV. The Mabs antigenic activity was tested for 46 types of cross-reactivity Mabs in VV neutralization on Vero cells. Only the 112H12, 113D5, 113F8, 122H9 and 125G9 Mabs, which were specific to the kDa 14 polypeptide (gene A30L EV), had the neutralizing activity. The 122H9 and 125G9 Mabs were able to neutralize SPV. Therefore, it can be assumed that the 14 kDa polypeptide carries, on its surface, cross-reactivity neutralizing epitopes typical of orthopoxviruses.

New phosphorodiamides of modified nucleoside monophosphates were synthesized and their antiviral properties were evaluated.

A representative collection was obtained containing 68 monoclonal antibodies (MAB) to Toxoplasma gondii antigens, which was characterized by the binding with the below fractions of tochizoites in the immune-enzyme assay (IEA) and immunoblotting (IB): membrane (MEM), somatic (water-soluble, SOM) and excretory-secretory (ES). Most of MABs were produced to MEM antigens (43), 6 MABs reacted with the somatic fraction, and 3 MABs reacted with both fractions. Two MABs to ES antigen were detected in the latter group. An analysis of MABs in concurrent IEA and IB revealed the immune-dominant proteins of the MEM and SOM fractions of antibodies to T. gondii tochizoites (p30 and p27, respectively). The presence of 2 non-overlapping antigenic determinants was shown for p30. Further research would detect MABs that could be used in the diagnosis of toxoplasmosis.

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.

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.

The library of human scFv antibodies displayed on the surface of bacteriophages was panned against Vaccinia virus (VACV), strain Elstree. 75% binding with Vaccinia virus. 5 clones were characterized for their binding with VACV and their ability to neutralize VACV in plaque reduction neutralization test (PRNT). Antibodies from the clones were obtained as soluble individual molecules and their binding activities were confirmed in ELISA.

The circulating immune complexes (CIC) that form in the hot just in early Toxoplasma gondii invasion can be present in the blood bed for a while. At the same time, the data on the antigenic composition of CIC in toxoplasmosis are fragmentary and rather contradictory. The investigation used enzyme immunoassay (EIA) to detect specific CIC that contain antigens to T. gondii tachyzoites in the sera of different populations and studied their antigenic composition by immunoblotting after 2-6% polyethylene glycol 6000-induced deposition. Examining 464 sera from groups of individuals with varying-stage T. gondii invasion indicated CIC in each, but showing their different frequency. CIC were virtually present in all sera from children who had been diagnosed as having congenital toxoplasmosis. In groups of seropositive pregnant women, CIC detection rates were noticeably higher in the samples showing both IgG and IgM antibodies (40.2 and 66.4%, respectively). CIC were also revealed in 9.8% of the seronegative blood donors; however, immunoblotting failed to confirm that they had no components that specifically reacted with T. gondii tachyzoite antigen antibodies. There were some differences in the composition of CIC in the serum yielding positive results in both EIA and immunoblotting. The serum CIC from pregnant women that exhibited only IgG antibodies contained mainly T. gondii antigens having molecular weights of 67 and 30 kD. The serum CIC from children with congenital toxoplasmosis and from pregnant women with serologically detected IgM antibodies to Toxoplasma antigens were found to contain 55-58-, 48-, 44-, 38-, 30-, and 26-kD components. The same molecular weight proteins were detected by electrophoretic studies of Toxoplasma excretory-secretory antigen (ESA). Comparing the findings suggests that in acute toxoplasmosis, the circulating complexes mainly contain ESA of the tachyzoites which appear in human blood just at the onset of invasion. Thus, this study demonstrates that specifically CIC are detectable in the sera of individuals infected with T. gondii and their antigenic composition varies with the stage of disease. In the authors' opinion, the detection of specific CIC and the determination of their antigenic composition may be serve an additional test in diagnosing acute toxoplasmosis.

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.

A set of oligo-1,3-thiazolecarboxamide derivatives able to interact with the minor groove of nucleic acids was synthesized. These oligopeptides contained different numbers of thiazole units presenting dimethylaminopropyl or EDTA moieties on the C-terminus, and aminohexanoyl or EDTA moieties on the N-terminus. The inhibition of such compounds on HIV-1 reverse transcriptase activity was evaluated using different model template primer duplexes: DNA x DNA, RNA x DNA, DNA x RNA and RNA x RNA. The biological properties of the thiazolecarboxamide derivatives were compared to those of distamycin, another minor groove binder which contains three pyrrole rings. Similar to distamycin, the thiazole containing oligopeptides were good inhibitors of the reverse transcription reaction in the presence of DNA x DNA. But in contrast to distamycin, the oligothiazolide derivatives were able to inhibit reverse transcription in the presence of RNA x DNA or DNA x RNA template primers. Both distamycin and oligothiazolecarboxamides had low affinity for RNA x RNA duplexes. The inhibition obtained with the newly synthesized thiazolecarboxamides showed that these compounds were more powerful and versatile inhibitors of the RT-dependent polymerization than the natural minor groove binder distamycin.

The major proteins of T. gondii excretory and secretory antigens (ESA) obtained during cultivation of tachyzoites by using cultured Vero cells were shown to have molecular weights of 79, 70, 57, 48, 36, and 29 kD. ESA and somatic antigen immunoblotting demonstrated that there were noticeable differences in the immunoactive proteins of these antigens. The antibodies of the sera of patients with toxoplasmosis mainly interacted with ESA proteins having molecular weights of 79, 70, 57, 48, 36, and 29 kD, 57-kD ESA protein antibodies being present on the immunoblots of all the tested sera. When ESA was used as an antigen during enzyme immunoassay, it showed a high sensitivity in the detection of IgM antigens in congenital toxoplasmosis. At the same time, ESA identified the antibodies of this class in the sera of healthy donors much infrequently than somatic antigen (p < 0.05).

Whether purified antigens of Lamblia intestinalis trophozoites can be used to detect these antibodies by immunoassay. The drugs of immunodominant Lamblia antigens were prepared by anion-exchange chromatography of solubilized trophozoite components and they are mainly presented by proteins having molecular weights of 70, 56, and 49 kD. Immunoassay using these antigens revealed antibodies to Lamblia trophozoite antigens in sera of 87.6% of patients with lambliasis (its diagnosis was established on the basis of microscopic data on the duodenal content) and only in 16.2% of clinically healthy blood donors. Twenty six sera from patients with trichomoniasis having high levels of antibodies to trichomonad antigens were studied to evaluate the specificity of this method for detection of antibodies. It has been found that the proportion of subjects in this group who have also antibodies to Lamblia antigens does not greatly differ from that of healthy blood donors (19.2 and 16.2, respectively).

Hybridomas producing monoclonal antibodies (MAb) to Marburg virus proteins are prepared. Positive hybridomas were selected by solid-phase enzyme immunoassay (EIA) by specificity of their immunoglobulin reaction with Marburg virus antigen. Virus proteins reacting with MAbs were identified by immunoblotting. Out of 20 examined hybridoma antibodies, 5 reacted with protein VP35, 5 with VP40, 3 with NP, 1 with protein complex VP35-VP40, MAb 7H10 detected 2 proteins (VP40 and NP), and 5 MAbs did not bind virus proteins in this assay. Marburg virus antigen adsorbed on the surface of plates were detected by indirect EIA with biotin-treated MAbs (PEIA-MAb) and indirect EIA (IEIA-MAb). The sensitivity of both methods differed with different hybridoma antibodies and was the maximum with MAb 5F1 specific to Marburg virus nucleoprotein: 5-10 and 1-2 ng/ml for the direct and indirect methods, respectively. Purified MAbs 7C4, 7D8, and 5F1 were used as antigen captures in EIA for detecting immunoglobulins to Marburg virus in a serum from convalescent after Marburg fever. The results recommend the above MAbs for use in test systems for the diagnosis of the disease and detecting virus antigen.

The protein composition and immunochemical properties of Toxoplasma gondii tachyzoites cultured on Vero cells and on mice were studied. Despite the fact that the main components of both preparations were shown to be proteins with molecular weights of 47, 34, 24, and 22 kDa, Toxoplasma-infected human sera antibodies interact mainly with the antigens of 66, 62, 57, 42, 38, 37, 36, 31, and 24 kDa. Comparing efficiency of enzyme immunoassay using the antigens of the tachyzoites obtained in different culture systems showed that the preparation of cultured Vero cells is similar to those of peritoneal exudates from infected mice and may be successfully used for the detection of antitoxoplasma antibodies in the sera of infected subjects.

Seroprevalence to Toxocara antigens was studied among Novosibirsk inhabitants and a focus of toxocariasis was found in the southern regions of west Siberia. Analyzing the sera of opisthorchiasis patients revealed antibodies to Toxocara antigens, with high titers (1:800 or more) in 1.80% of cases. Immunoblotting of these sera and those from patients with toxocariasis and opisthorchiasis, which were seropositive in one of the above helminths showed that there were mixed human infections with Toxocara and Opisthorchis in this area.

Marburg virus was shown to survive for up to 4-5 days on contaminated surfaces. In aerosol it was not stable, the specific rate of its inactivation being 0.05 min-1. This brought the authors to a conclusion that a relatively close contact is needed for virus transmission from man to man, although the possibility of aerosol transmission of the infection may be appreciably increased in case of the hemorrhagic syndrome with a high level of viremia.

Nucleotide sequences of the genes 5 and 6 of the Marburg virus, Popp strain, were determined. ORFs encoding polypeptides VP30 (281 a.a., MW 32,640) and VP24 (253 a.a., MW 28,621) were found. The putative transcription start and stop signals for viral RNA-dependent RNA polymerase were revealed for both genes. Overlapping of genes 5 and 6 was shown. The deduced amino acid sequences of VP30 and VP24 proteins displayed significant homology with the analogous proteins of another filovirus, the Ebola virus (33% and 37%, respectively). The VP24 appeared to have a hydrophobic amino acid composition; content of hydrophobic amino acids was 40.7%. Model of VP24 location in the virion was suggested.

DNA of the variola major virus strain India-1967 in the region of HindIII M, L, I fragments has been sequenced. Analysis of this sequence of 18029 bp revealed 19 potential open reading frames (ORFs). Four proposed proteins (L2R, H9R, L5L, L6R) contain metal-binding domains. Comparison of the variola virus (VAR) and vaccinia virus strain Copenhagen (COP) sequences show that the main differences are between proteins L1R and I5R. L1R contains 6 additional amino acid residues on the C-terminus. The protein I5R of VAR contains three Ca2+ binding domains but this COP has deletions in 2 of the 3 established domains. Possible functions of the predicted viral polypeptides are discussed.

HindIII and XhoI genome fragments of variola major virus strain India-1967 were inserted into the bacterial plasmids and cosmid. Sequencing and computer analysis of the region of HindIII M, L, and I DNA fragments of the virus studied have been carried out.

A comparative study on the features of the pathogenesis of Marburg disease after parenteral and aerosol infection of green monkeys with a virus prepared from native culture suspension and that after lyophilization was carried out. The changes in the dynamics of the clotting time, the activity of serum aminotransferases, the duration of prefebrile period and survival time were analysed in different cases. No lethality was observed in animals infected with small doses of aerosol preparations.

Aminotransferase levels in the peripheral blood serum of African green monkeys were studied after aerosol infection with lyophilized virus preparation. A correlation between aminotransferase activity and other symptoms of hemorrhagic fever was shown. Considerable variations in the features of aminotransferase activity in severe and mild cases of the disease were observed.

Decamethoxin is shown to be able to increase membrane permeability of Pseudomonas aeruginosa, Escherichia coli and Micrococcus lysodeikticus, that is confirmed by a loss of compounds with the absorption maximum at 260 nm by cells. Parallel with this the number of viable individuals has fallen and activity of dehydrogenases has been inhibited. The aspartate and alanine aminotransferase activity was not inhibited by decamethoxin and even increased. Decamethoxin lysed the protoplasts of the tested microorganisms. At high decamethoxin concentrations (over 500 micrograms/ml for P. aeruginosa and over 200 mu/ml--for E. coli) the outflow of components from the cells of gram-negative bacteria ceased, that may be associated with the coagulation changes in the cytoplasm. A loss of the low-molecular components by M. lysodeikticus cells and lysis of protoplasts proceeded less intensely than the same processes in the gram-negative microorganisms, that is explained by a less resistance of M. lysodeikticus to decamethoxin and earlier coagulation of the cytoplasm preventing lysis.

Fifty-seven temperature-sensitive (ts) mutants of a neurotropic variant of vaccinia virus were obtained. A comparison of the effectiveness of 6 different mutagens showed that in in vivo experiments nitrosomethylurea induced 10%, nitrosoguanidine 8%, and bromodeoxyuridine 2.5% ts mutants. In vitro, at the same degree of inactivation, UV irradiation, hydroxylamine and nitrous acid induced 2.5% ts mutants. Out of 49 mutants studied, 27 had a DNA+ phenotype, 19 a DNA+/-and 3 a DNA- phenotype. Under permissive conditions, two thirds of all the mutants tested formed thermolabile virions. In experiments on genetic recombination, the recombination frequency between different pairs was from 2.8 to 57%. Seven of the mutants were plotted on the virus genetic map in a certain sequence.

The possibility of using nitrosomethylurea for production of one-hit temperature-sensitive (ts) mutants of vaccinia virus suitable for genetic and biochemical studies was explored. The effect of nitrosomethylurea on replicating DNA of the virus produced 26 ts mutants, the effectiveness of mutagenesis being 10%. In recombination experiments with 12 of them it was shown that all the mutants under study had single ts defects of the genome. All ts mutants were capable of synthesizing DNA under nonpermissive conditions; 16 ts mutants had thermolabile virions.