[Oncolytic potential of recombinant influenza A virus vectors on a model of malignant glioma in vivo].

Malignant glioma is the most frequently occurring primary brain tumor. Despite significant progress in the diagnostics and treatment of neoplastic diseases the prognosis for patients with III-IV grade gliomas, remains extremely unfavorable. Rapidly developing area in oncology is the employment of therapeutic viruses with natural or genetically engineered oncolytic activity. In the present study we demonstrated the oncolytic potential of a recombinant influenza A virus vector with impaired interferon antagonism function of NS1 protein in treatment of malignant glioma. Recombinant influenza A virus (HA-DS-GFP) expressing green fluorescent protein from the NS1 open reading frame was used as a model vector. HA-DS-GFP virus has shown infectivity towards glioma cells both in vitro, and in vivo (experimental glioma model in rats). Intratumoral inoculation of HA-DS-GFP resulted in a substantial inhibition or complete regression of tumor growth. Our data demonstrate that recombinant influenza vectors have promising potential in therapy of malignant gliomas.

[New monoclonal kits for the diagnosis of the adenoviral infection].

Diagnostic properties of new monoclonal antibodies (MAbs) to hexon adenovirus antigen (AB) monoclonal ELISA kit for early diagnosis of adenoviral infection were tested. Developed ELISA kit and FITC-conjugate of new monocional antibodies for immunofluorescent analysis were used for detection of different types of adenoviruses in clinical materials. The availability of their use in clinical and epidemiological practice was validated.

[Molecular-biological properties of the rubella virus strains isolated in St. Petersburg].

In the surveillance of rubella in the northwest region of Russia samples of nasopharyngeal swabs from 37 patients with rubella, which were treated in the 442nd district military hospital named after Z.P. Solovyov in autumn 2007 were screened for the rubella virus using RK-13 cell line, 22 strains of rubella virus were isolated. Gene sequencing of E1 region of rubella virus isolates was carried out. Rubella virus strains isolated in St. Petersburg during the 2007 outbreak belonged to rubella virus genotype 1E. The morphogenesis of RK-13 cells with formation of replication complexes and enveloped virions of rubella virus was shown.

[Effect of Ingavirin on the ultrastructure of the morphogenesis of adenovirus infection in vivo].

The goal of this study was to evaluate the effect of Ingavirin on the morphological features of the foci of adenovirus hepatitis in Syrian hamsters by electron microscopy. The use of the drug was shown to cause a substantial reduction in the rate of destructive processes and inflammatory reactions in the liver, by normalizing its structure at the levels of both tissue and individual hepatocytes. After administration of Ingavirin, the morphogenesis of adenovirus infection in the infected hepatocytes did not differ from that in the controls; however, the infected cells were fewer. The proportion of morphologically inadequate virions in the presence of Ingavirin increased from 35 to 46%. The findings suggest that Ingavirin is an effective drug that has antiviral, anti-inflammatory, and cytoprotective activities in the focus of adenovirus tissue involvement.

[In vitro and in vivo effects of ingavirin on the ultrastructure and infectivity of influenza virus].

The aim of this investigation was to study the effect of ingavirin on the structure and properties of influenza virions forming in its presence. The infectious activity of the virus and the morphology of the virions were analyzed by titration in cell culture and electron microscopy, respectively. The use of ingavirin was shown to reduce the proportion of morphologically intact virions and to increase that of filamentous and giant particles. No defects of surface glycoproteins were observed. The effect of the drug did not depend on the chosen model of virus replication and it was similarly shown in both cultured human cells and laboratory animals. In MDCK and A549 cells and in the mouse lungs, viral infectious activity was decreased by 1-2 orders of magnitude in relation to a model. The findings suggest that Ingavirin is able to impair the processes of viral morphogenesis, which in turn leads to a reduction in the infectivity of progeny virions.

[Effect of antiviral drugs with various mechanisms of action on morphogenesis of infection caused by extremely pathogenic influenza virus strains in animals].

The effect of meglumine salt of acridonoacetic acid (cycloferon) on the in vivo morphogenesis of influenza infection caused by viruses of different origin (avian, swine and human) and variable susceptibility to antivirals (rimantadine and oseltamivir) has been studied. The administration of cycloferon results in stimulation of the immune response, restriction of the foci of post-influenza pneumonia, and normalization of the structure of respiratory zones independently of the susceptibility or resistance of infectious virus to the drugs. Among virions formed in the lungs of cycloferon-treated mice, prevalence of irregular-shaped virions with defects of surface glycoproteins was observed. The data obtained suggest that cycloferon is a drug with the complex mechanism of activity.

[Experimental study of Ingavirin antiviral activity against human adenovirus].

Antiviral properties of Ingavirin were investigated in the Hep-2 cell culture with respect to the human respiratory tract virus (type 5 adenovirus). In concentrations of Ingavirin of 1000, 100 and 10 mcg/ml the generated posterity showed lower infective capacity (by 250, 100 and 10 times respectively). The electron microscopy of the infected cells confirmed the Ingavirin ability to disturb the adenovirus normal morphogenesis.

Clinical and immunological efficiency of anaferon (pediatric formulation) in calicivirus infection in children.

Therapy with anaferon (pediatric formulation) during the acute period of calicivirus infection shortened the duration of the main symptoms of the disease and period of virus release. Changes in the immunological status included increased production of IgA and IgM and activation of IFN-alpha synthesis.

DNA-polycation complexes: effect of polycation structure on physico-chemical and biological properties.

The purpose of the study was to investigate the influence of cationic polymer structure on the formation of DNA-polycation complexes and their transfection activity. Primary, tertiary, and quaternary polyamines with molecular masses ranging from 8000 to 200,000 were investigated. DNA-cationic polymer interaction was characterized by low gradient viscometry, dynamic light scattering, circular dichroism, UV spectrometry, flow birefringence, DNA electrophoresis, and electron microscopy. Transfection activity of the complexes was evaluated by the expression of reporter gene (beta-galactosidase) and using synthetic FITC-labelled oligonucleotides. Complex formation was found to be dependent on the structure and molecular weight of the polymer and the ionic strength of the solution. Secondary DNA structure in complexes was not disrupted, and DNA was protected from protonation. Cell lines of different origin were used for testing of transfection activity of the complexes. The sensitivity of the cells to transfection was established to be highly dependent on the cell line. DNA-polycation complexes are non-toxic according to MTT. Polyallylamine, and polydimethylaminoethylmethacrylate were found to be the most promising polycations for gene delivery. Transfection efficacy of their complexes with DNA to T-98G cells reaches up to 90-100%. It was found that optimal molecular mass of polydimethylaminoethylmethacrylate is in the range of 8000-50,000 Da.

[Ultrastructural changes in the cells of human embryo lung fibroblasts in the reproduction of the coronavirus HCoV/SPb/01/03].

The reproduction of the new coronavirus HCoV/SPb/01/03 in the cultured human embryo lung fibroblasts (HELFBs) was electron microscopically studied. The virus was shown to replicate in the cultured HELFBs, by using for this a cell membrane system and causing profound changes in its morphology. After 24 hours of cell infection, there were mature and defective HCoVISPb/01/03 virions were detected in the vacuoles near the peripheral cisterns of the Golgi apparatus. Some of the vacuoles contained folded membranous structures along with virions.

Direct antiviral effect of cycloferon (10-carboxymethyl-9-acridanone) against adenovirus type 6 in vitro.

Adenoviruses represent a broad group of human pathogens that currently have no specific and safe drugs for treatment. We demonstrated direct (non IFN-mediated) antiviral activity of cycloferon (10-carboxymethyl-9-acridanone, CMA), a potent interferon inducer, against adenovirus type 6 (Ad6) in Hep-2 cells. Virus production and details of morphogenesis were studied by ELISA with antibodies to the Ad6 hexon protein, and transmission electron microscopy, respectively. Immunoenzyme assay revealed that CMA does not inhibit viral protein synthesis but instead strongly reduces the ability of the virus to generate infectious progeny virus in a dose dependent manner. Ultrastructural study shows that CMA alters the structure of intranuclear virus-specific inclusions. We suggest that CMA suppresses the late stages of viral cycle in the infected cell.

[Development of new approaches to standartization of enzyme immunoassay test systems for detection of antibodies to respiratory syncytial virus using electron microscopy and monoclonal antibodies]. / Razrabotka novykh podkhodov k standartizatsii immunofermentnykh test-sistem dlia detektsii antitel k respiratorno-sintsitial'nomu virusu s ispol'zovaniem élektronnoi mikroskopii i monoklonal'nykh antitel.

Respiratory syncytial virus (RSV), strain Long, was purified through 20-60% sucrose gradient. The virions from different sucrose density zones were tested by ELISA for reactivity with monoclonal antibodies (MAB) to F- (MAB 9C5) and N- (MAB 8B10) proteins of RSV. Comparative study of the same patterns of RSV by electron microscopy after negative staining showed a close relationship between the virion morphology and MAB binding in ELISA. MAB 9C5 were highly reactive with the surface domains of both mature RSV virions and "empty" virion envelopes without formed inner nucleocapsid structures. MAB 8B10 reacted well only with mature virions with completely assembled nucleocapsids. These MAB failed to reorganize the N-protein epitope of immature and destroyed virions, which indicated a conformation dependence of the 8B10 binding site. For practical purposes, MAB tests can be used to determine the RSV patterns, which can be used in ELISA for serologic diagnosis of RSV infection. Testing with these MAB demonstrate the stability of RSV to extreme exposures (lyophilization, storage, heating), which is important for creation of sensitive ELISA test systems and their standardization.

[Development of immunoenzyme assay for subtype-specific detection of antibodies to influenza viruses A (H1N1) and A (H3N2)]. / Razrabotka immunofermentnykh test-sistem dlia podtipospetsificheskoi detektsii antitel k virusam grippa A (H1N1) i A (H3N2).

Conditions were developed for obtaining surface viral glycoprotein (GP) fraction intended for solid phase sensitization with the aim of constructing enzyme immunoassay test systems (EIATS) for detection of subtypical IgG and IgG to influenza A (H1N1) and A (H3N2) viruses. New variants of test systems were compared with the traditional methods for serological diagnosis of influenza. GP-based EIATS more often diagnosed influenza than EIATS based on purified whole-virion (WV) suspensions, hemagglutination inhibition and neutralization tests. Evaluation of conversions of subtypical IgG showed that the results coincided with the findings of neutralization and hemagglutination inhibition tests in 83-90% (EIATS-GP) and 50% (EIATS-WV). Cross-detection of antibodies to both virus subtypes in EIATS-GP and EIATS-WV was observed in 4 and 31% cases, respectively.

[Seroepidemiological study of coronavirus infection in children and adults in St. Petersburg]. / Seroépidemiologicheskoe izuchenie koronavirusnoi infektsii u detei i vzroslykh Sankt-Peterburga.

As the result of prolonged (17 years) observations of patients with acute respiratory infections hospitalized in basic departments of clinics of the Research Institute of Influenza, coronavirus infection was found to be the cause of respiratory diseases, on the average, in 12% of cases (in some years in 6.8% to 28.6% of cases). The analysis of extensive morbidity rates among different age groups of the population showed that children were affected by coronavirus infection 5-7 times more often than adults. Three year cycles of this infection were established. The periods of coronaviruses activation were accompanied by their detection in patient material by electron-microscopy, a sharp increase of immune response of patients as well as in the number of nosocomial infections and the proportion of the monoinfection of the coronavirus nature. Coronaviruses played the leading role among other viruses in the etiology of hospital respiratory infections. Mucosal antibodies to coronaviruses in the secretions of the nasal cavity proved to be more important than serum antibodies not only in protection from infection, but also in the pattern of clinical manifestations of the disease.

Role of the C-terminal fragment of human transthyretin in abnormal fibrillogenesis.

Polypeptide chain fragments of recombinant transthyretin (TTR) with leucine-55 substituted by proline (L55P), which are involved in abnormal fibrillogenesis of this protein, were studied. No fibrils were produced in purified preparations of TTR(L55P) under the optimum conditions for fibrillogenesis but in absence of protease inhibitors. The ability of TTR for fibrillogenesis was lost because of a limited proteolysis resulting in detachment of the TTR polypeptide chain C-terminal fragment of approximately 18 amino acid residues in length. This proteolysis seemed to occur with involvement of a bacterial serine endopeptidase sohB (EC 3.4.21), which was identified in TTR preparations by the MALDI-TOF method. The presence of the C-terminal fragment of the TTR polypeptide chain seems to be crucial for production of abnormal fibrils.