Ridostin induces transcription of a wide spectrum of interferon genes in human cells.

The effects of Ridostin on the transcription of IFN family genes in human fibroblasts and lymphocytes were studied by quantitative real-time PCR. The degree of gene induction by Ridostin was most pronounced in fibroblasts, and was significantly higher than the induction by Kagocel: transcription of IFN-ß, oligoadenylate synthetase, and double-stranded RNA-dependent protein kinase genes increased by about 2000, 100, and 20 times, respectively. In lymphocytes, Ridostin also activated a wide variety of IFN family genes, including genes of IFN-ß, IFN-γ, and IFN-dependent enzymes, but this induction was less pronounced than in the fibroblasts. It was shown that gene response in lymphocyte from a child with cancer is reduced in comparison with that of adult healthy participant. Ridostin, and even more so Reaferon up-regulated activities of ß-actin, glycerophosphate dehydrogenase, and ß2-microglobulin genes, thus making impossible or limiting their use as constitutive stable reference genes (standards) in PCR-assays of IFN and their inductors.

Identification of alpha interferon-induced genes associated with antiviral activity in Daudi cells and characterization of IFIT3 as a novel antiviral gene.

A novel assay was developed for Daudi cells in which the antiviral (AV) and antiproliferative (AP) activities of interferon (IFN) can be measured simultaneously. Using this novel assay, conditions allowing IFN AV protection but no growth inhibition were identified and selected. Daudi cells were treated under these conditions, and gene expression microarray analyses were performed. The results of the analysis identified 25 genes associated with IFN-α AV activity. Upregulation of 23 IFN-induced genes was confirmed by using reverse transcription-PCR. Of 25 gene products, 17 were detected by Western blotting at 24 h. Of the 25 genes, 10 have not been previously linked to AV activity of IFN-α. The most upregulated gene was IFIT3 (for IFN-induced protein with tetratricopeptide repeats 3). The results from antibody neutralizing experiments suggested an association of the identified genes with IFN-α AV activity. This association was strengthened by results from IFIT3-small interfering RNA transfection experiments showing decreased expression of IFIT3 and a reduction in the AV activity induced by IFN-α. Overexpression of IFIT3 resulted in a decrease of virus titer. Transcription of AV genes after the treatment of cells with higher concentrations of IFN having an AP effect on Daudi cells suggested pleiotropic functions of identified gene products.

Comparative effects of stabilizing additives on the rates of heat inactivation of recombinant human interferon alpha-2b in solution.

This study was aimed at the comparative evaluation of stabilizing additives used for the protection of the antiviral activity of interferon-alpha2b against thermal inactivation, at 60 degrees C. The comparative effects of amino acids, polyhydric alcohols, saccharides and nonionic surfactants were studied. All were effective. Representing the thermal inactivation process with first order kinetics, a maximal prolongation of antiviral activity half-life of 39-fold was achieved with the most effective procedure. Inactivation rate constants varied from (53.3+/-4.6)x10(-3) to (2.5+/-0.3)x10(-3) min(-1). Human serum albumin, nonionic surfactants and monosaccharides increased half-life values by 5-39-, 5-23-, 4-20-fold, respectively. Amino acids, polyhydric alcohols and disaccharides increased t(1/2) values by 4-11-, 2-8- and 3-8-fold, respectively. These data provide useful information for the selection of stabilizing additives for IFN-alpha2b formulations.

Virucidal effect of ozone treatment of laboratory animal viruses.

An ozonization method was used to inactivate the viral pathogens of laboratory animals. Ozone at a concentration of over 100 ppm with high humidity was highly virucidal against 4 RNA viruses: HVJ, Theiler's murine encephalomyelitis virus (TMEV), Reo type 3 virus (RV) and murine hepatitis virus (MHV). For the ozone tests, 0.1 ml of a virus suspension in deionized water or saline and was placed in 35-mm dishes. The titer of 10(6) plaque-forming units of TMEV in a liquid-phase, which was highly stable against physical treatments, was reduced within 1 hr to a level of 0 by 300 ppm of ozone at 80% humidity and 22-25 degrees C. HVJ and MHV were more susceptible than TMEV to the ozone treatment. RV was the most resistant of the 4 viruses. The ozonization method may be a good way to disinfect not only for the laboratory animal RNA-viruses (both of enveloped and unenveloped viruses) but also animal rooms, clean rooms and even safety cabinets.

Inhibition of Theiler's virus-induced demyelination in vivo by tumor necrosis factor alpha.

Employing a murine model of multiple sclerosis which utilizes intracranial injection of Theiler's virus murine encephalomyelitis (TMEV) into SJL/J mice, we tested the potential role of tumor necrosis factor alpha (TNF-alpha) in ameliorating CNS demyelination. Infection with TMEV caused early grey matter inflammation (7 days post-infection) in the brain and spinal cord followed by chronic demyelination (35 days post-infection) in the spinal cord. Administration of recombinant human or mouse TNF-alpha starting 12 h prior to infection and then three times weekly had minimal effect on development of grey matter inflammation in the spinal cord. In contrast, TNF-alpha dramatically reduced demyelination present in spinal cord on days 14 and 35 after TMEV infection (P less than 0.01) when compared to controls. CNS virus titers of TMEV were not modified by TNF-alpha administration as measured on days 7, 14, and 35 following infection. In vivo administration of TNF-alpha inhibits TMEV-induced demyelination in susceptible SJL/J mice without affecting virus replication in the CNS.

Recombinant interferons alpha and gamma: comparative antiviral activity and synergistic interaction in encephalomyocarditis virus infection of mice.

The antiviral properties of 2 recombinant DNA-produced interferons, a human hybrid interferon alpha that is active in mice and a murine interferon gamma, were examined in the treatment of mice infected with encephalomyocarditis virus. A single dose of interferon alpha induced a protective state in mice more rapidly than did interferon gamma, but the activity of the latter was more long lasting. When interferon and virus were administered 6 h apart, either intraperitoneally or intravenously, interferons alpha and gamma were equally effective. However, this was not the case when the routes of treatment and infection were different. Interferon alpha showed somewhat reduced activity when the route of administration (intravenous) was different from the route of virus challenge (intraperitoneal) while interferon gamma showed very little activity when tested in this manner. When interferons alpha and gamma were administered in combination to mice, a marked synergistic antiviral effect was observed.

[Interferon-inducing and antiviral activity of dsRNA of yeast origin]. / Interferonindutsiruiushchaia i protivovirusnaia aktivnost' dsRNK drozhzhevogo proiskhozhdeniia.

The paper describes derivation of double-stranded RNA from K plasmid of Sachcaromyces cerevisiae yeast by means of electrophoresis in agar gel and by differential salting out with 4 M lithium chloride. Studies in vitro and in vivo demonstrated a high interferon-inducing and antiviral activity of dsRNA preparations from the yeast plasmid.

Theiler's virus-specified polypeptides made in BHK-21 cells.

This study was initiated to determine whether the Theiler's murine encephalomyelitis viruses (TMEV) use the same strategy of gene expression as other picornaviruses. The precursor-product relationships apparent from pulse-chase experiments indicated that the virus-specified polypeptides of the GDVII strain are generated by post-translational cleavages. Pactamycin mapping experiments also showed a gene order for GDVII virus similar to that derived for other picornaviruses by this technique. Finally, six other TMEV induced species of polypeptides similar to those of GDVII virus, but there were minor differences in some of their electrophoretic mobilities.