[Improvement of erythropoietin bioassay.]

The relevance of bioassay standardization results from the lack of consistent national regulatory requirements for evaluation of recombinant human erythropoietin quality and the need to harmonize these requirements with international ones. Precision studies were carried out in 6 experiments on Balb/C mice. The factors that can influence the accuracy of the method were altered during the experiments. Each experiment included three levels: 20, 40 and 80 IU/ml, and 8 replicates for the reference and test samples. The trueness was estimated by bias relative to the reference value at 5 levels: 10, 20, 40, 80 and 160 IU/ml, and 4 replicates for the reference and test samples at each level. The test samples were prepared by a series of independent dilutions of the reference standard. Reticulocyte count was performed using a flow cytometer. 5 µmol acridine orange solution was used as a dye. Experimental study of accuracy and optimization of erythropoietin bioassay procedure helped to obtain two validation characteristics (trueness and precision). It was shown that logarithms of erythropoiesis registered values could reasonably be used in statistical calculations of erythropoietin specific activity and evaluation of the method's validation parameters. The theoretically and experimentally justified test procedure includes three levels of doses: 20, 40 and 80 IU/ml, and 8 animals for each level, which is consistent with the international requirements for accuracy. According to the results of experimental studies, the trueness is characterized by a bias of no more than 9 % and does not exceed the range of the calculated activity (80-125 %). Statistical processing of the test results by the parallel-line method makes it possible to check the assumption of equivalence of the test and reference samples and to calculate the test sample activity. The confidence limit of the calculated activity for intra-laboratory precision of 5.6 % is equal to 76-131 % which complies with the proposed range (64-156 %, P=0.95).

[EXPERIENCE OF STUDY AND POSSIBLE WAYS OF ELIMINATION OF FALSE POSITIVE AND FALSE NEGATIVE RESULTS DURING EXECUTION OF POLYMERASE CHAIN REACTION ON AN EXAMPLE OF JUNIN VIRUS RNA DETECTION].

AIM: Experience of study and possible ways of elimination of false positive and false negative results during execution of polymerase chain reaction on an example of Junin virus RNA detection. MATERIALSS AND METHODS: Junin virus--causative agent of Argentine hemorrhagic fever (AHF) strain XJpR37/5787 was obtained from the State collection of pathogenicity group I causative agents of the 48th Central Research Institute. Reagent kit for detection of Junin virus RNA by RT-PCR was developed in the Institute and consists of 4 sets: for isolation of RNA, execution of reverse-transcription reaction, execution of PCR and electrophoretic detection of PCR products. RT-PCR was carried out by a standard technique. Continuous cell cultures of African green monkey Vero B, GMK-AH-1(D) were obtained from the museum of cell culture department of the Centre. RESULTS: An experimental study of the effect of various factors of impact on the sample under investigation ("thawing-freezing", presence of formaldehyde, heparin) on the obtaining of false negative results during Junin virus RNA detection by using RT-PCR was studied. Addition of 0.01% heparin to the samples was shown to completely inhibit PCR. Addition of 0.05% formaldehyde significantly reduces sensitivity of the method. A possibility of reduction of analysis timeframe from 15 to 5 days was shown during detection of the causative agent in samples with low concentration of the latter by growing the samples and subsequent analysis of the material obtained by using RT-PCR. CONCLUSION: During detection of causative agent by using RT-PCR false negative results could appear in the presence of formaldehyde and heparin in the sample. A possibility of elimination of false negative PCR results due to concentration of the causative agent in the sample under investigation at a level below sensitivity threshold was shown on the example of Junin virus RNA detection by using growing of the pathogen in appropriate accumulation system with subsequent analysis of the material obtained using PCR.

[The estimation method of compounds opiate activity based on universal three-dimensional model of the nonselective opiate pharmacophore].

Created by means alternative strategy of structural similarity search universal three-dimensional model of the nonselective opiate pharmacophore and the estimation method of agonistic and antagonistic properties of opiate receptors ligands based on its were described. The examples of the present method use are given for opiate activity estimation of compounds essentially distinguished on the structure from opiates and traditional opioids.

[Production of 70 kDa recombinant human heat shock protein in baculovirus expression system and assessment of its antiviral activity].

AIM: To obtain human recombinant 70 kDa heat shock protein (Hsp70) in baculovirus expression system and to study its antiviral activity. MATERIALS AND METHODS: Baculovirus expression system was used to obtain recombinant HSP70. Plasmid pFastBacHTb-Hsp70 containing sequence coding HSP70 gene with insertion of 6 histidine residues in protein reading frame was constructed. Competent cells MAX Efficiency DH 10 Bac were transfected with pFastBacHTb-Hsp70 plasmid with following extraction of recombinant bacmid Bac-Hsp70. In order to obtain baculovirus expressing HSP70, Sf-9 cells were transfected with Bac-Hsp70 bacmid. Hsp70 extraction and purification was performed with column metal-chelating affinity chromatography using Ni2+ ions. Protective efficacy of recombinant human HSP70 was estimated using model of Venezuelan equine encephalitis (VEE) in mice. RESULTS: Recombinant bacmid Bac-Hsp70 was constructed based on Bac-to-Bac expression system. Baculovirus expressing human HSP70 have been produced after transfection of Sf-9 cells with Bac-Hsp70 bacmid. Cultivation of recombinant baculovirus in Sf-9 cells and application of metal-chelating affinity chromatography allowed to extract purified fraction of HSP70. Experiments on mice infected with VEE virus demonstrated significant protection from death after administration of HSP70 in dose 15 mcg/mice. CONCLUSION: Application of baculovirus expression system and insect cell line for accumulation of recombinant baculoviruses in combination with Ni(2+)-mediated metal-chelating affinity chromatography allowed to obtain highly purified human recombinant HSP70 with marked antiviral activity.

[Using reverse genetics method for developing recombinant strains of influenza viruses acceptable for use as live attenuated vaccines].

Perspectives of using reverse genetics methods for constructing of recombinant influenza virus strains acceptable for use as live attenuated vaccines are discussed. Using of attenuated NS-vectors of influenza virus opens possibilities for the development of recombinant vaccines with optimal ratio of immunogenicity and safety. Reverse genetics is applicable for development of effective vaccines against new pathogens such as highly pathogenic avian influenza A/H5N1.

Structural organization of the genome of SARS-associated coronavirus (Strain SoD) isolated on the territory of the Russian Federation.

Complete nucleotide cDNA sequence (29715 nucleotides) of SARS-associated coronavirus (strain SoD) isolated for the first time in the territory of the Russian Federation was determined. Phylogenetic analysis revealed maximum similarity between strain SoD genome and Frankfurt 1 strain genome. Three nucleotide substitutions determining two amino acid substitutions were detected.

[Isolation and identification of the infective agent of severe acute respiratory syndrome (SARS) from a patient with atypical pneumonia]. / Vydelenie i identifikatsiia vozbuditelia tiazhelogo ostrogo respiratornogo sindroma (TORS) ot bol'nogo atipichnoi pnevmoniei.

The virological, morphological, molecular biological and immunochemical study of the infective agent isolated from the patient with the symptoms of atypical pneumonia, hospitalized in the infectious department of the clinical hospital in Blagoveshchensk, was carried out. Thus the fact of the appearance of SARS virus on the territory of Russia was proved. The isolated infective agent, identified as coronavirus strain CoD, was partly characterized and deposited to the virus collection of the Center of Special Laboratory Diagnostics and Treatment of Quarantine and Exotic Infectious Diseases.

[Isolation and identification of Crimean-Congo hemorrhagic fever virus in the Stavropol territory]. / Vydelenie i identifikatsiia virusa Krymskoi-Kongo gemorragicheskoi likhoradki v Stavropol'skom krae.

The results of the isolation and identification of the causative agent of a haemorrhagic fever outbreak in the Stavropol Territory are presented. The virus isolated from blood of haemorrhagic fever patients by virological methods was identified in serological and molecular tests as Crimean haemorrhagic fever virus. This epidemiological analysis testify to increased activity of the natural focus of Crimean-Congo haemorrhagic fever in this area due to a number of natural and other factors leading to intensification of its epidemic realization.

[The laboratory diagnosis of an outbreak of hemorrhagic fever at Oblivskaya village, Rostov Province: proof of the etiological role of the Crimean-Congo hemorrhagic fever virus]. / Laboratornaia diagnostike vspyshki gemorragicheskoi likhoradki v stanitse Oblivskoi Rostovskoi oblasti: dokazatel'stva étiologicheskoi roli virusa krymskoi-kongo gemorragicheskoi likhoradki.

The results of the molecular biological detection of the etiologic agent of hemorrhagic fever in Rostov Province are presented. The role of the causative agents of Astrakhan rickettsial fever, hemorrhagic fever with the renal syndrome, Q fever, leptospirosis and listeriosis has been excluded by means of such immunochemical reactions as the direct and indirect immunofluorescent tests, the solid-phase immunoenzyme assay, the complement fixation test and the agglutination test. The relationship between the cases of hemorrhagic fever in the focus of the outbreak and Crimean-Congo hemorrhagic fever virus has been demonstrated due to the use of the polymerase chain reaction with preliminary reverse transcription.

[Crimean-Congo hemorrhagic fever in Rostov Province: the epidemiological characteristics of an outbreak]. / Krymskaia-Kongo gemorragicheskaia likhoradka v Rostovskoi oblasti: épidemiologicheskie osobennosti vspyshki.

The results of the epidemiological analysis of the outbreak of hemorrhagic fever which was caused by Crimean-Congo hemorrhagic fever virus and occurred during the period of July 3-19, 1999, in the Oblivskaya district of Rostov Province are presented. The specific epidemiological features of the outbreak have been determined. The possible versions of the appearance of the focus of infection and the role of Ixodes ticks in the circulation of the infective agent are discussed.

[Use of aluminum foil baths for embedding biological materials in epoxide resins]. / Ispol'zovanie vannochek iz aliuminievoi fol'gi dlia zalivki biologicheskogo materiala v époksidnye smoly

The baths intended for embedding the biological material into epoxide resins are made of aluminium foil, 0.1 mm thick, cut in the form of rectangles (13 X 18 mm). The rectangular foil plates are placed on a soft microporous rubber separator 30--40 mm thick and by means of a form with the base equal to 5 X 10 mm the baths are pressed down by 4 mm deep. The baths are stuck to the paper stripes by rubber cement to ensure easy handling and numeration. In the process of embedding and polymerization the paper stripes having the baths are placed in the exsiccator with P2O5 and thermostate on special aluminium stands.