[A study of the interaction of boraadamantane with liposomes].

Changes in the surface potential of liposomes obtained from dipalmitoyl phospatidylcholine during their interaction with the new antiviral preparation boraadmantane have been studied. It has been concluded that the saturation of the lipid bilayer of liposomes by boraadmantane occurs at concentrations of the preparation above 10 microg/ml.

[Development and preclinical study of new generation virosomal split influenza vaccine "Grifor"].

New Russian virosomal split vaccine against influenza "Grifor" was developed. The vaccine is represented by mix of highly purified protective external and internal antigens of influenza A (H1N1 and H3N2) and B viruses. Developed technology of manufacture allowed to provide presentation of external antigens of influenza virus in the form of virosomes, and presentation of internal antigens in the form of micelles with maximal preservation of their antigenic activity. Using electron microscopy, electrophoresis in 10% polyacrilamide gel with sodium dodecyl sulfate, and polymerase chain reaction, morphologic and biochemical properties of the vaccine were studied. Preclinical study, including assessment of antigenic characteristics of "Grifor" vaccine compared to vaccine "Vaxigrip" (France), was performed. It was established that administration of the vaccine did not result in death of experimental animals, decrease of body mass, development of pathologic (including inflammatory, dystrophic and necrobiotic) changes in viscera or render adverse effects on blood hematologic and biochemical parameters and on the immune system. The vaccine was not pyrogenic and allergenic, did not have local irritating effects. Obtained results supported the appropriateness of conducting the clinical trials of "Grifor" vaccine on limited number of volunteers.

[New method for measurement of size and concentration of lyposomes].

AIM: To develop new method of determination of size and concentration of lyposomes based on measurement of opacity in dispersed media. MATERIALS AND METHODS: Dispersions of lyposomes from dipalmitoylposphatidylcholine were the object of the study. Opacity spectrum of lyposome suspension was measured with Zenyth 200st spectrophotometer. Mean values of diameter of lyposomes determined by opacity spectrum were compared with the same values measured by electron microscopy (JEM 100-CX, JEOL, Japan) with magnification 58,000 - 100,000. Refraction index was measured with refractometer RPL-3 (Russia). RESULTS: Sizes of lyposomes measured by the new method and by electron microscopy did not differ significantly. Determination of sizes and concentration of lyposomes by the new method did not depend from effect of secondary multiple scattering of light. CONCLUSION: Obtained data allowed to conclude that developed method could be used in practice. Advantages of the new method are usage of common spectrophotometers and photoelectrocolorimeters for deriving of liposomes suspension opacity curve as well as its high validity, which are confirmed by data obtained with electron microscopy.

[Real-time multiplex PCR for rapid detection of enteroviruses, adenoviruses and hepatitis A virus in clinical specimens].

Real-time multiplex polymerase chain reaction (PCR) with internal positive control (IPC) was developed for simultaneous detection of adenoviruses (AV), enteroviruses (EV) and hepatitis A virus (HAV). Primes and probes labeled with different fluorophores (FAM, R6G, ROX, and Cy5) and able to detect up to four viral RNAs (DNAs) with high specificity in a single tube in real-time PCR were designed. Sensitivity and specificity of the method was estimated using cultural strains of 8 serotypes of EV, 5 serotypes of AV and 2 clinical specimens containing HAV. Sensitivity of the method for detection of polioviruses types 1, 2, and 3 (Sabin vaccine strains) was 0.5--1 TCID50 per reaction mixture. Thirty clinical specimens were analyzed by the multiplex PCR with and without IPC, and by mono-specific PCR. Comparison of these methods with cultural one revealed results agreement in 86.7% in case of multiplex PCR with IPC and in 100% in case of multiplex PCR without IPC and mono-specific PCR. This method can be used for rapid diagnostics of enteric viral infections as well as for determination of viral contamination level of water. As intermediate results of the study the methods for quantitative assessment of HAV, AV, and EV nucleic acids were developed which are convenient tools for the control of antiviral therapy effectiveness.

[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.

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.

[Characteristics of Namalwa cells--a substrate for production of human interferon]. / Kharakteristika kletok Namalwa--substrata dlia polucheniia chelovecheskogo interferona.

The properties of lymphoid Namalwa cell line propagated at the USSR Academy of Medical Sciences Research Institute of Viral Preparations for interferon production are described. The scanning and transmissive electron microscopy studies of the cells showed their morphological stability and the absence of microbial contamination. The 46-48-chromosome cells comprised 85% of the population, hypodiploid cells (44-45 chromosomes), 9%, tetraploid and hypertetraploid cells, 3%. Spontaneous aberrations were detected in 3% of the chromosome. Inoculation of the cells into unsuppressed laboratory animals (rabbits, guinea pigs, adult or suckling mice) or chick embryos did not cause the development of any pathological process. Namalwa cells were shown to produce interferon after multiple (up to 4 times) induction with Newcastle disease virus.

Laboratory markers for over-attenuation of mumps vaccine virus.

The properties of mumps vaccine virus (Leningrad-3 strain) gradually changed upon passaging in quail embryo fibroblasts, the substrate normally used for mumps vaccine production in the USSR. Alterations were extremely noticeable in the over-attenuated (38th passage) virus variant, and involved (a) poor, if any, antibody response in guinea-pigs, (b) turbid plaque formation, (c) lack of expression in cell culture of fusion protein and reduced expression of polymerase protein, and (d) enrichment by abnormally small, fusion-protein-deficient virus particles. Two other laboratory strains exhibited a similar trend to over-attenuation, though after variable passage numbers. Due to a good inter-correlation, every test (namely, inoculation of guinea-pigs, plaque assay, protein analysis, or immune electron microscopy) is indicative of mumps vaccine over-attenuation and hence might be valuable in seed virus quality control.

[Use of the zwitterion detergent Sulfobetain-12 for preparation of virosomes and investigation of their immunogenic and protective properties]. / Ispol'zovanie tsvitterionnogo detergenta sul'fobetaina-12 dlia polucheniia virosom i izuchenie ikh immunogennykh i protektivnykh svoistv.

Virosomes were prepared by using the zwitterion detergent sulfobetaine-12. The virosomes included the surface antigens and virus-specific lipids of influenza virus, strain A/PR/8/34. Immunogenic and protective properties of the surface antigens in the micellar form and as a complex with the virosomes were studied. The surface antigens of this complex, like the intact virus, were found to possess the high immunogenic and protective activity in relation to the following infection with the homologous pathogenic virus.

[Electron microscopy study of the nucleocapsid structure of the measles virus isolated from cell cultures with primary and chronic infections]. / Elektronno-mikroskopicheskoe izuchenie struktury nukleokapsidov virusa kori, vydelennykh iz kul'tur kletok pri pervichnoi i khronicheskoi infektsii.

Electron microscopic examination of isolated intracellular measles virus nucleocapsids (NC) revealed a relationship between their structure, cell system, and the type of infection. Acute virus infection of Vero or Japanese quail embryo cells gave rise to the formation of linear NC strands with regularly and tightly stacked turns. Acutely infected L-41 or HEp-2 cells contained heteromorphous viral NC populations which included both typical and loosely packed NC. Persistently infected L-41 and Hep-2 cells predominantly contained NC of the latter type with the appearance of a "strings of beads".

Non-infectious morphologically altered nucleocapsids of measles virus from persistently infected cells.

Persistent measles virus infection of human HEp-2 or L-41 cells was accompanied by pronounced structural and functional changes of isolated intracellular viral nucleocapsids (NCs). The bulk of persistent NCs possessed altered conformation and a "string-of-beads" appearance, contained substantial amounts of subgenomic size RNAs, exhibited reduced transcriptase activity in vitro and lacked infectivity on transfection of susceptible cells. Immunogold staining revealed negligible binding of anti-P protein monoclonal antibodies to the "string-of-beads" type NCs, thus suggesting their non-functional state.

[Characteristics of the ribonucleoprotein of the measles virus persisting in a human cell culture]. / Kharakteristika ribonukleoproteida virusa kori, persistiruiushchego v kul'ture kletok cheloveka.

The properties of virus-specific RNPs recovered from human HEp-2 and L-41 cells chronically infected with measles virus were studied in comparison with those of RNPs formed in acute infection of L-41 cells. The persisting RNP was shown to contain nucleoprotein not differing in the electrophoretic mobility from the same protein of measles virus virions, and RNA in the persisting RNP was found to be insensitive to the action of RN-ase. RNP from chronically infected cells had a changed ultrastructure and conformation as compared with RNP of the original virus and, unlike the latter had no infectivity upon transfection of the sensitive cells by calcium-phosphate precipitation. No differences in relationships of RNP with the cytoskeleton of the infected cells in the acute and chronic infection were observed.

[Physicochemical characteristics of a vaccinal strain of the Leningrad-3 mumps virus (L-3)]. / Fiziko-khimicheskaia kharakteristika vaktsinnogo shtamma virusa parotita Leningrada-3 (L-3).

Purified virions of the vaccine strain Leningrad-3 of mumps virus propagated in Japanese quail embryo cell cultures had a buoyant density 1.18-1.19 g/ml in sucrose gradient, contained 50 S RNA and showed variable sizes in electron microscopy as manifested by heterogeneity of the virus zone in sedimentation analysis. Purified L-3 virus contained 5 major polypeptides with molecular weights of 74,000, 68,000, 58,000, 45,000, and 39,000 daltons. Each polypeptide had an individual oligopeptide composition.

Studies on a temperature-sensitive mutant of fowl plague virus having a mutation in gene 7 coding for the M protein.

A fowl plague virus (FPV) temperature-sensitive mutant, ts 303/1 having a ts mutation in gene 7 coding for the matrix (M) protein has been obtained. The mutant induced synthesis of virus-specific RNA and polypeptides as well as ribonuclear protein (RNP) formation in cells under non-permissive conditions; however, haemagglutinin cleavage was reduced, functionally active haemagglutinin and neuraminidase were absent and virions were not formed. In mutant-infected cells at 36 degrees C haemagglutinin cleavage was also reduced and virions formed had an altered NP:M ratio as well as a decreased haemagglutinin content. A population of virions formed under these conditions was heterogeneous both in morphology and in buoyant density. The data obtained suggest that a mutation in the M proteins of orthomyxoviruses can affect processing of the haemagglutinin and impair final stages of virion morphogenesis.

Accumulation of altered viral nucleocapsids in mumps virus--persistently infected cell cultures.

Two long-term human cell cultures persistently infected with mumps virus accumulated increased amounts of morphologically altered viral nucleocapsids. Alterations involved size, heterogeneity, fine structure and shape. RNA present in intracellular nucleocapsids was predominantly of subgenomic size.

[Characteristics of mumps virus intracellular ribonucleoprotein during chronic infection of cell cultures]. / Kharakteristika vnutrikletochnogo ribonukleoproteida virusa parotita pri khronicheskoi infektsii kletochnykh kul'tur.

Viral nucleocapsids isolated from cell cultures of human origin (HEp-2 and L-41) persistently infected with mumps virus for 4 years were analysed. In persistent infection, nucleocapsids accumulated in the cells in greater amounts than in primary infection. The synthesis of NP protein of nucleocapsids in persistent infection was approximately 10 times lower than that in primary infection. Morphologically, nucleocapsids in primary infection are rigid linear helical structures whereas in persistent infection they are of convoluted shape, fragmented, and partially or completely despiralized and destructuralized.

[Comparative study of the sensitivity of Mycoplasma detection methods in cell cultures]. / Sravnitel'noe izuchenie chuvstvitel'nosti metodov indikatsii mikoplazm v kletochnykh kul'turakh.

The results of a comparative study of the sensitivity of several methods for mycoplasma detection in cell cultures are presented. The most sensitive method was found to be that of electron microscopy detecting mycoplasmal contamination in 100% preparations. Seeding of the material on mycoplasma-elective nutrient medium (0.3% PPLO agar) and the method of autoradiography detect the culture contamination in 90% of the test materials. Staining of cell cultures with orsein and Schiff reagent are less sensitive methods revealing mycoplasmal contamination in 69% and 77.7% of the cultures, respectively.

[Preparation and investigation of some properties of influenza virus nucleoid]. / Poluchenie i izuchenie nekotorykh svoistv nukleoida virusa grippa.

Influenza virus nucleoid ("core") was obtained by the treatment of virions with hydrolytic enzymes without the use of fixing agents. The "core" buoyant density in sucrose density gradient was 1.24 g/cm3. The subvirus particles are completely devoid of surface proteins, glycoproteins, and their polypeptide composition is represented by group P proteins; NP and M proteins. RNA in the core is insensitive to the effect of RNase. Morphological analysis by electron microscopy revealed the presence of spherical compact particles without spikes on their surface.

[Isolation and study of several properties of mycoplasma from Japanese quail embryos]. / Vydelenie i izuchenie nekotorykh svoistv mikoplazmy iz embrionov iaponskogo perepela.

An agent isolated from Japanese quail embryos was found to be pathogenic for chick embryo and quail embryo fibroblast cultures, to cause hemadsorption and hemagglutination of guinea pig erythrocytes. Reproduction of the agent was inhibited by tetracycline and 5-bromodeoxyuridine. The agent incorporated both 3H-thymidine and 3H-uridine and had a buoyant density in sucrose gradient of 1.22 g/ml. Mature mycoplasma forms were determined electron microscopically in gradient fractions in cell sections.