[Problems of ante mortem diagnostics of prion diseases].

The review presents the state-of-the-art on the problem of diagnosis of prion diseases (PD) in humans and animals with a brief description of their etiology and pathogenesis. We pointed out that understanding the nature of the etio logical agent of PD determined their zoonotic potential and led to the development of highly specific immunological diagnostic methods aimed at identifying the infectious isoform of prion protein (PrPd) as the only marker of the disease. In this regard, we briefly summarize the results of studies, including our own, concerning the conversion of normal prion protein molecules (PrPc) to PrPd, the production of monoclonal antibodies and their application as immunodiagnostic reagents for the post-mortem detection of PrPd in various formats of immunoassay. We also emphasize the issues related to the development of methods for ante mortem diagnostics of PD. In this regard, a method for amplifying amino acid sequences using quacking-induced conversion of PrPc to PrPd in real time (RTQuIC) described in details. The results of recent studies on the assessment of the sensitivity, specificity and reproducibility of this method, carried out in various laboratories around the world, are presented. The data obtained indicate that RT-QuIC is currently the most promising laboratory assay for detecting PrPd in biological material at the preclinical stage of the disease. The significant contribution of US scientists to the introduction of this method into clinical practice on the model of diagnosis of chronic wasting disease of wild Cervidae (CWD) is noted. The possible further spread of CWD in the population of moose and deer in the territories bordering with Russia, as well as the established fact of alimentary transmission of CWD to macaques, indicate the threat of the appearance of PD in our country. In conclusion, the importance of developing new hypersensitive and/or selective components of known methods for PrPd identification from the point of view of assessing the risks of creating artificial infectious prion proteins in vivo or in vitro, primarily new pathogenic isoforms ("strains") and synthetic prions, was outlined.

[Methods for diagnosis of prion diseases].

The epidemic of bovine spongiform encephalopathy (BSE) in the United Kingdom, its related occurrence of a new type of Creutzfeld-Jacob disease and proven cases of this type of the disease transmitted by blood transfusion initiated intensive studies to develop a inexpensive, prompt, and sensitive method for the early lifetime diagnosis of prion diseases. This would permit initiation of the timely treatment of the patients and prevention of contamination of foodstuffs. However, despite significant progress made in this direction, this objective has not yet been achieved. The present review highlights the currently available methods for the diagnosis of transmissible spongiform encephalopathies, as well as the latest developments in the ultrasensitive detection of these diseases, which is based on the misfolded prion protein complex.

[Development of a diagnostic test system on the basis of sandwich ELISA for the detection of avian influenza A virus].

A panel of hybridomas producing monoclonal antibodies (MAbs) to nucleocapsid protein (NP) of avian influenza A virus was obtained. On the basis of 2 MAbs, the authors designed an antigen-bound ELISA (sandwich ELISA), in which NP3 MAbs were used as antigen-bound antibodies and NP MAbs conjugated with horse radish peroxidase as antigen detection antibodies. The specificity of the test system to avian influenza virus was determined. The developed test system was ascertained to specifically detect influenza A virus of all study subtypes and to yield no cross reactions with other tested virus pathogens. The sensitivity of the sandwich ELISA was 30 ng/ml of NP in the urine-treated virus preparations. The assay was tested on experimental H5N1-infected mice. The findings positively correlated with the results of postmortem studies and with the virus isolation method in the chick embryos. The developed test system may be used to detect avian influenza A virus as an alternative or supplement to other diagnostic techniques.

Isolation and characterization of full-length recombinant cattle PrPC protein.

Full-length Bos taurus PrPC protein was obtained in the eu- and prokaryotic expression systems. Immunoblotting and indirect enzyme immunoassay demonstrated high specificity and antigenic activity of full-length proteins in the reactions with monoclonal antibodies (anti-SAF-32 and VRQ-84). Membrane location of recombinant PrPC protein in insect cells was shown by immunofluorescent analysis.

[Use of recombinant protein E2 of the classical swine fever virus for immunization of animals]. / Primenenie rekombinantnogo belka E2 virusa klassicheskoi chumy svinei dlia immunizatsii zhivotnykh.

Recombinant major surface glycoprotein E2 from virulent Shimen strain of classical swine fever virus (CSFV) has been tested for immunogenicity in animal immunization experiments. Immunization of 3-month-old piglets with 200 micrograms of recombinant protein protected the animals from lethal challenge with virulent CSFV strain. CSFV-specific antibody detection test based on competitive ELISA has been developed using the recombinant E2 protein. The test can evaluate specific antibody levels after subunit vaccination with recombinant E2 after immunization with live vaccine based on attenuated CSFV strain.

[Synthesis and immunochemical properties of the recombinant major surface glycoprotein E2 of the classical swine fever virus]. / Sintez i immunokhimicheskie svoistva rekombinantnogo glavnogo poverkhnostnogo glikoproteina E2 virusa klassicheskoi chumy svinei.

Recombinant E2 protein from vaccine strain of classical swine fever virus (CSFV) and from SCFV virulent strain Shimen was synthesized in SF-21 and High-Five cell culture with baculovirus as the expressing vector. For secretion, hydrophobic C-terminal transmembrane domain was removed and N-terminal signal polypeptide of 38 amino acids was added. Maximum accumulation of recombinant products in SF-21 cells was observed after 48 h and in medium 96 h after infection with recombinant baculovirus. In High-Five cells and in culture medium the maximum accumulation of E2 was observed after 96 h. The level of E2 expression is 5-10 micrograms/106 cells. The products of expression were purified by affinity chromatography and their specificity confirmed in immunochemical tests with a series of reference monoclonal antibodies. The product can be used for detecting antibodies to SCFV by competitive enzyme immunoassay.

[Activation of viral reproduction in a mixed infection with human immunodeficiency virus and herpes viruses]. / Aktivatsiia reproduktsii virusov pri smeshannoi infektsii virusom immunodefitsita cheloveka i gerpes-virusami.

Mutual activation of reproduction of type 1 HIV and herpes simplex types 1 and 2 viruses (HSV) was observed in simultaneous infection of continuous T-cellular lymphoblastoid lines (CEM, 119, Hut-78, MT-4, Jurkat-tat) and U-937 monocytic line. Syncytium formation and cytodestructive pattern of reproduction of viruses of both families in these cell lines necessitated the use of enzyme immunoassay (EIA) to detect the antigens of these viruses in order to assess the level of reproduction. The concentration of HIV antigens in EIA increased in mixed infection by 1.4 to 2.1 times in different cultures in comparison with the culture infected with HIV-1 alone, and concentrations of HSV-1 and HSV-2 increased by 1.3-1.8 times in mixed infection, in comparison with reproduction in lymphoblastoid cultures infected with HSV alone. EIA was alone used to examine the production of IgG and IgM antibodies to Epstein-Barr virus, another representative of Herpesviridae family, in the blood sera of patients with immunodeficiency states in whose sera antibodies to proteins produced by gag HIV gene (p15/17, p24, p55) were detected. Increased concentration of IgG antibodies were revealed in 36% of these patients, whereas in healthy donors the sera with elevated concentrations of IgG to Epstein-Barr virus were far less incident (12%). A hypothesis about mutual activation of HIV and herpes viruses is put forward.

[In vitro immunization of human lymphocytes with Epstein-Barr virus capsid antigen]. / Immunizatsiia in vitro limfotsitov cheloveka kapsidnymi antigenami virusa Epshteina-Barr.

Conditions for in vitro immunization of human lymphocytes from adult peripheral blood, tonsils and cord blood with Epstein-Barr Virus (EBV) capsid antigens have been studied. Pokeweed mitogen and B cell growth factor from Namalva cell line were shown to induce a significant production of specific antibodies by human lymphocytes stimulated with EBV. This effect made it possible to generate primary immune response in vitro using lymphocytes from EBV seronegative donors.

[Mitochondrial proteinase of yeast splitting the products of mitochondrial translation]. / Mitokhondrial'naia proteinaza drozhzhei, osushchestvliaiashchaia rasshcheplenie produktov mitokhondrial'noitransliatsii.

It has been shown that mitochondria of the yeast Saccharomyces cerevisiae contain proteinase, which is bound to the inner mitochondrial membrane and catalyzes the hydrolysis of mitochondrial translation products in vitro. The efficiency of proteolysis depends on the state of mitochondria: e.g. the degradation of completely formed organelles corresponding to stationary cells, is twice as low as compared to the "young" organelles typical for the beginning of a logarithmic phase of growth. The proteolysis of mitochondrial translation products can occur not only in mitochondria, but also in "inside out" submitochondrial particles. In order to prove the absence of concomitant vacuolar proteinases in preparations of mitochondria and submitochondrial particles, the specific antisera against proteinases A and B have been used. The activity of mitochondrial proteinase is completely inhibited by the natural peptide inhibitors antipain and chymostatin. Of special importance is the fact that another natural peptide inhibitor--leupeptin, having no effect on the activities of vacuolar proteinases, significantly decreases the rate of hydrolysis of mitochondrial translation products. The role of yeast mitochondrial proteinase in regulation of mitochondrial formation is discussed.

[Proteolysis of products of mitochondrial protein synthesis in isolated mitochondria of Saccharomyces cerevisiae yeasts]. / Proteoliz produktov mitokhondrial'nogo belkovogo sinteza v izolirovannykh mitokhondriiakh drozhzhei Saccharomyces cerevisiae.

Products of mitochondrial protein synthesis were specifically labeled with 3H-leucine in the presence of cycloheximide at the end of the exponential phase of yeast aerobic growth on glucose. The mitochondria isolated from these cells lost 37-40% of the label from the protein fraction during 60 min incubation at 35 degrees, which was accompanied by the accumulation of 3H-leucine in TCA-soluble fraction. This process was suppressed by phenyl-methyl sulfonyl fluoride and p-chloromercuriphenyl sulfonate, the inhibitors of proteases, and could thus be considered as the proteolysis of the products of mitochondrial protein synthesis. The proteolysis was ATP dependent and was stimulated by puromycine which is known to induce the removal of incomplete polypeptides from mitochondrial ribosomes. A body of indirect evidence allows a suggestion to be made that the observed proteolysis can hardly be due to the action of cytoplasmic proteinases.

[Lability of the products of mitochondrial protein synthesis in Saccharomyces cerevisiae yeasts]. / Labil'nost' produktov mitokhondrial'nogo belkovogo sinteza v drozhzhakh Saccharomyces cerevisiae

Estimation of the rate of degradation of the products of mitochondrial protein synthesis in S. cerevisiae cells is reported. The method developed for this purpose is based on pulse incorporation of a labeled amino acid in the presence of an inhibitor of cytoplasmic protein synthesis and allows one to monitor postincorporation of the label. The label incorporated is shown to be rapidly released from mitochondria. Its content is decreased 2-fold during 20-30 min at the beginning and 50-60 min at the end of the exponential phase of growth. The label is detected in cytosol proteins and the TCA-soluble fraction of mitochondria, which is indicative of possible proteolysis of mitochondrial membrane proteins. Since release of the label does not undergo inhibition by specific inhibitors of yeast cell proteinases (pepstatin and phenylmethylsulfonyl fluoride), it may be assumed that these proteinases are either not involved in the digestion of the products of mitochondrial protein synthesis or do not represent a rate-limiting step of the process.

[Degradation of intracellular proteins at different stages of growth of Saccharomyces cerevisiae]. / Degradatsiia vnutrikletochnykh belkov na raznykh fazakh rosta drozhzhei Saccharomyces cerevisiae.

The rate of degradation of intracellular proteins at different growth stages of Saccharomyces cerevisiae yeast was determined. It has been demonstrated that the rate of degradation of intracellular proteins increases 2--3-fold at the late exponential phase. The increase was accompanied by corresponding changes in the activities of yeast proteinases A and B. In the presence of specific yeast proteinase inhibitors (pepstatin and phenylmethylsulfonyl fluoride) the rate of protein degradation in vivo decreased. The intermediate products of cell protein degradation have been found. These TCA-insoluble products could be extracted by various solvent systems. Their subsequent brakdown was suppressed by specific proteinase inhibitors.