[The interaction between nerve cells and carbon nanotube networks made by CVD process investigation].

In this research we investigate neuroblastoma cells cultivated on single-walled carbon nanotubes networks made by CVD method on silicon substrates. The complex analysis of grown cells made by atomic force, electron microscopy and Raman spectroscopy was carried out and the effect of nanotube growth process on proliferation factor was investigated. It is shown that despite of a weak decrease in proliferation, cell morphology remains unchanged and no physical or chemical interaction between carbon nanotubes and cells is observed. The results of the research can be used to investigate the interaction between conductive nanomaterials and cells for the development of neural replacement implants. Also they can be useful in bio-electronic interface investigation of signal propagation in neurons.

[Reproduction of the metapneumovirus in different cell lines].

The reproduction of the metapneumovirus was comparatively studied in 19 human and animal cell lines. The most sensitive transplanted cell lines were found to be human Chang Conjunctiva (clone 1-5C4) and animal cell lines of feline kidney CRFK.

[Changes in the reproduction of tick-borne encephalitis virus in cell cultures].

The currently used tick-borne encephalitis virus vaccines are based on the inactivation of tick-borne encephalitis virus (TBEV) of Far Eastern or West European genetic types from the primary cultures of chick embryo fibroblasts. Since the WHO recommends that vaccines should be designed using continuous cell cultures rather than chick embryos as a substrate, this investigation has compared the infection of continuous monolayer SPEV, Vero E6, and vaccine line Vero (B) cell cultures with TBEV strains of the Siberian and Far Eastern genetic types dominating in the endemic regions of Russia. After cell infection with Far Eastern (Sofyin and 205 strains) or Siberian (Aina, 2530, 2689, and 2703 strains) TBEV genetic types, the viable TBEV titers reached 2.8 Ig CPD50 for Vero (B) cells, 5.5 Ig CPD50 for Vero E6 cells, and up to 9 Ig CPD50 for SPEV cells. The quantitative scores of TBEV E antigen in enzyme immunoassay (EIA) and genome equivalents by reverse-transcription polymerase chain reaction (PCR), followed by real-time PCR, permitted one to estimate as high as 108 virions in 1 ml of culture fluid, which corresponded to those of the microscopic observations of CPD for SPEV cells and substantially exceeded the values for Vero E6 cells, and for Vero (B) cells in particular. The data of TBEV strain titration, EIA, and realtime reverse-transcription PCR suggest that the Russian vaccine Vero (B) cell line defined as meeting the WHO requirements, as well as Vero E6 cells may be used to design tick-borne encephalitis vaccine.

[Cultivation of continuous cell lines on the substrate of carbon nanotubes and the effect of electric stimulation on cell proliferation].

It was demonstrated that the three studied samples of carbon nanotubes of domestic production fixed on the substrate surface did not have toxic effect and could be used for cell cultivation. A biocompatible conductive coating based on carbon nanotubes and bovine serum albumin was developed. The efficacy of the coating for growing in vitro cell cultures was tested. A device was developed for electric stimulation of the cells. Local electric potential was applied to the cells using nanoscale electrodes. The results of human embryonic fibroblast cultivation in a pulsed electric field on conductive nanocomposite substrates were presented. An 26% increase in the proliferative activity of cells was observed at potentials up to 100 mV.

[Analysis of the cell tissue culture contamination with the bovine viral diarrhea virus and mycoplasmas].

Different cell tissue cultures and commercial fetal calf sera (FTS) used in biological and virological research were screened for the bovine viral diarrhea virus (BVDV, Pestivirus genus, Flaviviridae family) and mycoplasma contamination. BVDV was detected using RT-PCR and Indirect immunofluorescence (with monoclonal antibodies) methods in 33% cases of the studied cell lines and in > 60% cases of FCS. BVDV was shown to present and reproduce in high spectra of human cell lines, as well as in monkey, pig, rabbit, goat, dog, and cat cells at high levels (up to 100-1000 genome-equivalent copies per cell) and reached up to 10(3)-10(7) genome-equivalent copies per serum ml. The molecular mechanisms of the long virus persistence without definite signs of destruction should be studied.

[Comparative estimation of the indicators of interferon, immune, and cytokine states in the comprehensive study of patients with herpesvirus infections].

The paper provides the data of a comparative analysis of the indicators of immune and interferon states and cytokine profile and the results of virological studies in patients with different (acute and chronic) forms of mixed herpesvirus infection (with virus simplex herpes types 1 and 2, Epstein-Barr virus, human herpesvirus type 6, and others). Pronounced changes were found in immune responses in such patients. There were decreases in IFN-alpha and IFN-gamma values in 36 and 13%, respectively; 51% of the subjects showed a reduction in both IFN-alpha and IFN-gamma along with the high titers of antibodies to viruses of the Herpesviridae family and their infectious activity. There were changes in the cytokine profile, activation of IFN-alpha, IL-2, IL-8, IL-10, and IL-18 gene expression, and suppression of IL-2 gene transcription in the majority of the patients. Determination of IFN susceptibility revealed that 86% of the subjects responded to IFN-alpha therapy and only 11% of cases did to IFN-gamma one.

[Comparative immunogenicity studies of cultural and peptide influenza vaccines].

AIM: Comparative immunogenicity studies of experimental vaccines based onA/Aichi/2/68 neuraminidase peptide fragments (NA) and influenza virus A and B strains produced in MDCK cell culture. MATERIALS AND METHODS: Anti-hemagglutinin and virus neutralizing activity of mice sera was determined in MN and HI reactions in accordance with the WHO recommendations. RESULTS: Sera against peptides 136-147 and 154-164 from variable sites, as well as against peptide 314-328 from conservative region of the heavy chain of A/ Aichi/2/68 influenza virus NA showed distinct anti-hemagglutinin and neutralizing activity against homologous influenzavirus. Anti-(314-328) serum was also active in HI and MN reactions against other strains of the H3N2 subtype. Combined administration of peptide sample with an immunomodulator (Immunomax) increased the immunogenicity to the level of the cultural samples based on influenza A virus. CONCLUSION: The results show higher immunogenicity of cultural vaccines based on influenza virus in comparison to peptide samples. A possibility of peptide vaccine immunogenicity increase was demonstrated by combined administration with the immunomodulator.

[Influenza virus reproduction in the MDCK cells adapted to growth in serum-free Hybris-2 medium].

Whether the MDCK cell line might adapt to grow in serum-free Hybris-2 medium and influenza viruses might be reproduced in the adapted cells was studied. Seventeen passages using the Hybris-2 medium yielded cells adapted to growth in this medium (MDCK-BS). The reproduction of influenza A (H1N1 and H3N2) and B viruses versus the cells cultured in Eagle's medium was studied. The laboratory strain of influenza A/Aichi/1/68 (H3N2) and the strain B/Ohio/01/05 of influenza B in equal titers were shown to be reproduced in both control cells on Eagle's medium and MDCK-BS cells adapted to growth in the Hybris-2 medium. The reproduction of the strains A/Brisbane/10/07 (H3N2) and A/Solomon Islands/3/06 (H1N1) was less active in the MDCK cells. Each strain of influenza viruses displayed varying infective activities. The developed serum-free Hybris-2 medium may be used for cultivation of monolayer continuous MDCK cells and for their reproduction of influenza A and B viruses.

[Vitaherpavac is the first Russian herpes simplex virus vaccine obtained on the Vero B continuous cell line].

Vitaherpavac, a dry inactivated herpes simplex virus (HSV) culture vaccine, has been obtained, by using the Vero B continuous cell line as a substrate for accumulation of herpes simplex virus types 1 (US strain) and 2 (VN strain). Vitaherpavac and the similar vaccine Herpovax made by the Research Institute of Vaccines and Sera, Saint Petersburg (for which preparation a primary trypsinized chick embryo cell culture used as a substrate for accumulation of HSV types 1 and 2), underwent comparative clinical trials. The tolerability and therapeutic effectiveness of the vaccine were tested in patients diagnosed as having chronic frequently recurring herpes. The trials have yielded positive results that suggest that it is expedient to introduce of the new vaccine Vitaherpavac into practice to treat chronic recurrent herpetic infection of various localizations. Vitaherpavac has been registered in the Russian Federation and permitted for medical application.

[The spectrum of vetebrates' cell lines sensitive to highly pathogenic influenza A/tern/SA/61 (H5N3) and A/duck/Novosibirsk/56/05 (H5N1) viruses].

The reproduction of highly pathogenic avian influenza (HPAI) A/tern/SA/61 H5N3 and A/ducklNovosibirsk/56/05 H5NI viruses was comparatively studied in 16 human and animal cell lines. The strain A/duck/Novosibirsk/56/05 was shown to have a wider range of hosts. The most sensitive transplanted cell lines were found to be feline fibroblasts (CC-81), primarily trypsin-treated cells of chick embryonic fibroblasts (CEF), the kidney of dogs (MDCK), pigs (SPEV), monkeys (Vero), the human conjunctiva (1-5C-4), and, to a lesser extent, the feline kidney (CRFK). Unlike the strain A/tern/South Africa/61, that A/duck/Novosibirsk/56105 replicated in the polecat brain cells (Mpf).

[Peptide mapping of the monoclonal antibodies against the heavy chain hemagglutinin from influenza virus H3N2].

Interaction of the synthetic peptides corresponding to the regions 122-133, 136-147, 154-164 and 314-328 of the virus A/Aichi/2/68 hemagglutinin heavy chain with monoclonal antibodies specific for this hemagglutinin was assayed in a variety of tests, e.g., ELISA, competition RIA, hemagglutinin-inhibition and virus-neutralization assays. The monoclonal antibody 152 reacted with the area 136-147 (epitope A), three monoclonal antibodies 3, 19 and 63 reacted exclusively with the area (154-164) Glu (epitope B). Mapping of two monoclonal antibodies IV A1 and IV G6 specific for the influenza virus A/Dunedin/ 4/73 hemagglutinin heavy chain and cross-reacting with a number of the H3 subtype viruses was carried out. The specificity of the interaction of the conservative peptide H3(314-328) with monoclonal antibodies IV A1 and IV G6 was confirmed by competition RIA and by competition hemagglutinin inhibition and virus neutralization.

[Activity of virion RNA-polymerase in influenza A and B virus variants with different pathogenicities for mice]. / Aktivnost' virionnoi RNK-polimerazy u variantov virusa grippa A i B, razlichaiushchikhsia po priznaku patogennosti dlia myshei.

RNA polymerase activities in parental strains of influenza A and B viruses nonpathogenic for mice and their pathogenic variants have been studied. The parental strains are A/seal/Massachusetts 1/80, A/USSR 05/81, A/Philippines 2/82, B/Singapore 222/79. The RNA polymerase activity has been also studied in recombinant strains obtained by crossing various parental strains, one of which is pathogenic for mice (AR/PR 8/34), and having different degrees of pathogenicity. The nonpathogenic viruses had low transcriptase activity. RNA polymerase activity in pathogenic variants is shown to be 1.5-3 times higher than that in the parental strains. All the recombinants, whatever their pathogenicity, had approximately the same transcriptase activities which were 1.5-2 times higher than those registered in parental nonpathogenic strains.

[Pseudosuspended cultured cells for obtaining influenza A and B viral antigens]. / Psevdosuspenzionnoe kul'tirirovanie kletok dlia polucheniia antigenov virusov grippa A i B.

Conditions for pseudosuspension culturing of continuous cells of the kidneys of dogs (MDCK), green marmosets (CV-1), and swine (SPEV) on two types of microcarriers (Cytogel-3 and cytolar-2) under semiproduction conditions (50 liter bioreactor) were developed on a model of reassortants of influenza viruses A and B. To standardize the conditions, native sera were replaced by growth-stimulating proteins isolated from the sera of various animals (cattle, northern deer, swine). A better adhesive capacity and more active cell proliferation on porous microcarrier cytogel-3 were observed. The highest proliferative activity of cells was observed when porcine growth-stimulating proteins were used. Influenza A virus reassortant (H3N1) was actively reproduced in MDCK cells. The reproduction of influenza B virus reassortant was similarly higher in MDCK cells, in comparison with SPEV cells; at the same time, no differences in hemagglutinin titers in the two cell cultures were observed. The pseudosuspension method of cell culturing is recommended for the preparation of influenza antigens.

[The effect of different factors on the reproduction of influenza viruses and reassortants in cell cultures]. / Vliianie razlichnykh faktorov na reproduktsiiu virusov grippa i reasortantov v kletochnykh kul'turakh.

The influence of the maintenance medium, polyethylene glycol (PEG), DEAE-dextran, and low temperature on reproduction of influenza A, B, and C viruses and their reassortants in diploid and continuous cell cultures was determined. Lowering of pH in the maintenance medium to 6.5 was found to decrease reproduction of influenza A (H1N1) and A (H3N2) viruses and increase that of influenza B viruses. Treatment of cells with PEG solution increased the yield of influenza B and C but not A viruses. However, influenza A virus strains proved to be capable of producing infectious progeny in nonpermissive cell lines treated with PEG. Addition of DEAE-dextran to the medium exerted no effect on the infectivity of influenza A and B reassortants. Moreover, infection of MDCK cells after a "cold shock" led to an increase in hemagglutinin titres in influenza A reassortants.

[Variability of influenza A2 viruses in nature]. / Izmenchivost' virusov grippa A2 v estestrennykh usloviiakh

A comparative study of variability of the properties of influenza A2 viruses isolated in different epidemic years (1964-1965, 1968-1969, 1972-1973) showed that in the course of influenza virus evolution changes occurred not only in the antigenic properties of surface proteins (hemagglutinins and neuraminidase) but in a number of other properties as well. In contrast to previously circulating strains, influenza A2 virus strains isolated in the epidemic of 1971-1972 were found to have higher reproducing and neuraminidase activities, greatly increased thermostability and the capacity to induce viremia regularly in white mice.

[A comparative study of monoclonal and monospecific antibodies in determining the immunodominant antigenic sites of influenza virus A (H3N2) hemagglutinin]. / Sravnitel'noe issledovanie monoklonal'nykh i monospetsificheskikh antitel v opredelenii immunodominantnykh antigennykh saitov gemaggliutinina virusa grippa A (H3N2).

Comparative studies of monospecific (MSA) and monoclonal (MCA) antibodies showed MSA to detect three non-overlapping immunodominant sites on the surface of hemagglutinin (HA) molecule whereas MCA established more subtle differences in HA antigenic structure on the level of epitopes with different immunological significance. The activity of MSA and MCA differed in various tests. While MCA were more active in HI and EIA tests, MSA had a higher neutralizing activity, reducing the infectious virus titre by 5.0-7.5 Ig. Similar reduction of the virus biologic activity was observed only with two MCA whereas the other 20 MCA had a poor neutralizing effect (the virus titre reduction not more than by 2.5 Ig). The employment of MSA and MCA gives most complete information on antigenic restructuring of influenza virus HA at the site level and more subtle structures, epitopes, in the process of evolutionary variability.

[The selection of cold-adapted variants of the influenza viruses H1N1 and H3N2 and their antigenic and genetic characteristics]. / Selektsiia kholdoadaptirovannykh variantov virusov grippa H1N1 i H3N2, ikh antigennaia i geneticheskaia kharakteristika.

As a result of serial passages (42 passages) at low temperatures (26 degrees--28 degrees C) of two influenza H1N1 and H3N2 virus strains stable cold-adapted (ca) variants were produced. Investigations of them showed the ca A/USSR/03/84 (H1N1) variant to have ts-mutations in genes 1, 2, 4, 5, 7, and 8 and the ca A/USSR/215/79 (H3N2) to have ts-mutations in genes 1, 3, 4, 5, 7 and 8. These ca-variants may be recommended as attenuation donors to be used in recombination experiments with epidemic influenza viruses in order to obtain attenuated reassortant candidate vaccine strains.

[The characteristics of the evolutionary variability of influenza A (H1N1) viruses]. / Osobennosti évoliutsionnoi izmenchivosti virusov grippa A (H1N1).

Studies of the antigenic structure of hemagglutinins of influenza A (H1N1) viruses isolated in 1978-1988 using monospecific and monoclonal antibodies demonstrated the strains of the H1N1 subtype to be highly apt to antigenic drift. The evolutional variability of that period was peculiar and characterized by antigenic drift in various directions. In those years, the variants were regularly isolated which had retained the determinants of viruses of 1933-1957 circulation period in their hemagglutinin structure. The variants containing in their hemagglutinin 2 antigenic sites common with A/USSR/090/77 virus and antigenic groupings characterizing the strain specificity of each isolate, were epidemically active. At the same time, epidemically important variants were dominant whose properties were markedly different from those of previously known viruses. Their hemagglutinin contained 2 basically new antigenic determinants. This direction of evolutional development of influenza A (H1N1) virus is the most prospective epidemically.

[Functional activity of monoclonal and monospecific antibodies in the interaction with antigenic sites of influenza virus hemagglutinin]. / Funktsional'naia aktivnost' monoklonal'nykh i monospetsificheskikh antitel pri vzaimodeistvii s antigennymi saitami gemaggliutinina virusa grippa.

Monospecific antibodies to three nonoverlapping sites of influenza A/Singapore/1/57 (H2N2) virus isolated from hyperimmune serum by specific adsorption were characterized by capacity to inhibit virus hemagglutination, immunochemically interact with hemagglutinin, and neutralize the virus (6-7.5 lg). Monoclonal antibodies to the same virus hemagglutinin displayed an even higher activity than monospecific antibodies in serological and immunochemical tests and were characterized by a rather low (1.0-4.5 lg) or null activity in biological neutralization of the virus.

[Replacement of native sera with growth-stimulating proteins during culturing of mammalian cells]. / Zamena nativnykh syvorotok roststimuliruiushchimi belkami pri kul'tivirovanii kletok mlekopitaiushchikh.

The authors recommend to replace nonstandard native animal sera by growth-stimulating proteins isolated from the sera of cattle, northern deer, pigs, and sheep by fractionation with polyethylene glycol aqueous solutions (mol. m 6000 dalton). Growth-stimulating proteins are conducive to active proliferation of primary and continuous cell cultures at various methods of culturing. Growth-stimulating proteins were for the first time added to nutrient media during culturing of mammalian cells on microcarriers.