[Detection of rubella virus RNA in clinical material by real time polymerase chain reaction method].

AIM: Development of a reagent kit for detection of rubella virus RNA in clinical material by PCR-RT. MATERIALS AND METHODS: During development and determination of analytical specificity and sensitivity DNA and RNA of 33 different microorganisms including 4 rubella strains were used. Comparison of analytical sensitivity of virological and molecular-biological methods was performed by using rubella virus strains Wistar RA 27/3, M-33, "Orlov", Judith. Evaluation of diagnostic informativity of rubella virus RNAisolation in various clinical material by PCR-RT method was performed in comparison with determination of virus specific serum antibodies by enzyme immunoassay. RESULTS: A reagent kit for the detection of rubella virus RNA in clinical material by PCR-RT was developed. Analytical specificity was 100%, analytical sensitivity - 400 virus RNA copies per ml. Analytical sensitivity of the developed technique exceeds analytical sensitivity of the Vero E6 cell culture infection method in studies of rubella virus strains Wistar RA 27/3 and "Orlov" by 11g and 31g, and for M-33 and Judith strains is analogous. Diagnostic specificity is 100%. Diagnostic specificity for testing samples obtained within 5 days of rash onset: for peripheral blood sera - 20.9%, saliva - 92.5%, nasopharyngeal swabs - 70.1%, saliva and nasopharyngeal swabs - 97%. Positive and negative predictive values of the results were shown depending on the type of clinical material tested. CONCLUSION: Application of reagent kit will allow to increase rubella diagnostics effectiveness at the early stages of infectious process development, timely and qualitatively perform differential diagnostics of exanthema diseases, support tactics of anti-epidemic regime.

[In vitro activity of Russian ribavirin on the models of Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Tahyna and Dhori viruses].

The high activity of ribavirin made by effective biotechnology in Russia was established in in vitro experiments using the models Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Tahyna and Dhori viruses, which suggests that it is promising in using the drug in the treatment of infection with these viruses.

[ELISA and PCR test systems used to detect Crimean-Congo hemorrhagic fever virus].

Studying the sensitivity and specificity of enzyme-linked immunosorbent assay (ELISA) for the indication of Crimean-Congo hemorrhagic fever (CCHF) virus antigens and those of reverse transcription and polymerase chain reaction (RT-PCR) for the detection of CCHF virus RNA, and those of a intercerebral infection method in newborn albino mice systems for the determination of viral infectious activity established that the sensitivity of ELISA was 1-2 orders of magnitude less than that of RP-PCR. The latter proved to be better in studying the sera sampled from patients with CCHF. The results of studying the samples of H. marginatum ticks, the CCHF virus vectors by ELISA and RT-PCR were similar.

[Morphological and immunohistochemical changes in tissues of the lung, myocardium and kidney in experimental West Nile fever].

An experimental infection of mice with West Nile virus (WNV) showed pronounced dystrophic changes in tissues of the kidneys and myocardium as well as expression of WNV antigens in cells of the lungs, kidneys and myocardium, which can denote tropism of WNV to tissues of the lungs, kidneys and myocardium.

[Pathomorphology of West Nile fever]. / Patomorfologiia likhoradki zapadnogo Nila.

8 patients, aged from 61 to 70 years, died from serous meningoencephalitis during the outburst of the West Nile fever in Volgograd in 1999-2001, were studied morphologically. Serous meningoencephalitis with necrotic vasculitis is a characteristic feature of this fever with degenerative changes and destructive foci in the brain. Proteinic and fatty inflammatory and lymphomacrophageal reactions were observed in the liver, serous productive myocarditis and cardiomyocytic necrosis dystrophy. Desquamative pneumonia, intracapillary nephritis were also seen. Immunohistochemical reaction against virus of this fever was positive in the vascular endothelium of parenchymal organs, hepatocytes, neurons of the brain.

[Analysis of new variants of West Nile fever virus]. / Analiz novykh variantov virusa likhoradki Zapadnogo Nila.

The complete nucleotide sequences for 6 strains of the West Nile fever virus were determined. For the first time the complete nucleotide sequences of the Indian isolate and Krsn190 strain, that is the most far phylogenetically from all isolates known at present time were established. The scheme for separation of virus variants into 4 groups and criteria for determination the group to which the isolate belongs are suggested.

[The study of immune structure of Astrakhan region population to West Nile virus in 1998 and 1999]. / Izuchenie immunostruktury naseleniia Astrakhanskoi oblasti k virusu Zapadnogo Nila v 1998 i 1999 gg.

Immunostructure of the Astrakhan Region population to West Nile fever (WNF) was studied in the preepidemic period (1998) and after the outbreak (1999). Among the sera obtained in 1998, 63 (26.3%) were positive in neutralization reaction, 84 (27.1%) in enzyme immunoassay IgG and 20 (7.8%) in HAIR. IgM-antibodies were found in none of 142 samples. Overall number of donors having antibodies to WNF virus by three reactions reached 97 (31.6%). In the sera obtained in 1999, virus neutralizing antibodies were detected in 72(44.4%) cases, specific IgM antibodies detected by EIA_in 5(3.1%), IgG_in 44(27.1%), antihemagglutinins_in 11 (6.9%). The number of positive findings in 4 reactions in 1999 was 81(50.0%). The results of examination of the sera collected for two years (1998 and 1999) were the following: of 402 samples examined in NR positive were 135(33.6%), of 304 five (1.6%) were IgM positive, 128(27.1%) of 472 were IgG positive, and 31(7.4%) of 417 responded in HAIR. Overall index of humoral immunity for 2 years was 37.9% (in males and females 39.8 and 32.8%, respectively. In persons aged 20-29 years_36.9%, 50-59 years_42.9%. Thus, by 2-year results, antibodies to WNF virus occurred in 51.9% rural citizens and 35.0% city population.

[Specific antibodies in convalescents after West Nile fever]. / Pokazateli spetsificheskikh antitel u rekonvalestsentov, perenesshikh likhoradku Zapadnogo Nila.

Blood serum samples were obtained from patients with West Nile fever (WNF) from the city of Volgograd and its region within the disease weeks 1-3 (July-October 1999), month 6-7 (May_June 2000) and month 12-18 (May-June 2001) after the disease onset. In the sera of 40 patients examined 6-7 months after the disease onset IgM-antibodies to WNF virus were absent (dilution 1:50), IgG ones were detected in 35 patients (87.5%). 18 months after the disease IgG-antibodies were identified in 23 of 26 convalescents (88.5%). The serum samples obtained in autumn 1999 and May-June 2000 contained antihemagglutinins to WNF virus for 6-7 months in all 18 patients. In May-June 2000 the sera were positive in 38 convalescents who had WNF in 1999.

[Virus-like particles as a source for obtaining antigens for diagnosing rubella]. / Virusopodobnye chastitsy kak istochnikh polucheniia antigenov dlia diagnostiki krasnukhi.

Rubella diagnostic agents for hemagglutination inhibition (HI) and enzyme immunoassay (EIA) based on rubella virus-like particles (RVLP) have been developed. Noninfectious RVLPs containing three structural E1, E2, and C proteins were expressed in transfected CHO24S cell culture. HI titer in culture medium was 1:256. Tween-80 treatment and ether increased HI titer 4-6-fold and rendered the antigen higher stability. Immunogenic properties of RVLPs were similar to the native rubella virus in HI test with international reference human rubella serum and sera from convalescents after rubella. Serum of mice immunized with RVLP reacted similarly with RVLP antigens and native virus. Antigen for EIA from RVLP was prepared by concentrating RVLP from culture fluid and partial purification by ultracentrifugation. The results of human sera testing by HI and EIA with RVLP and native virus antigens coincided. RVLP are identical to antigenic structure of the virus, are stable and easily purified, and can therefore be used for commercial production of HI and EIA antigens.

[Epidemic outbreak of meningitis and meningoencephalitis, caused by West Nile virus, in the Krasnodar territory and Volgograd region (preliminary report)]. / Epidemicheskie vxpyshki meningita i meningoéntsefalita v Krasnodarskom krae i Volgogradskoi oblasti, vyzvannye virusom Zapadnogo Nila (Predvaritel'noe soobshchenie).

Sera from 102 inpatients from the Volgograd region (64) and Krasnodar region (38) were tested for antibodies to West Nile (WN) virus in hemagglutination inhibition (HI) test and for IgM and IgG antibodies in enzyme immunoassay (EIA). Diseases etiologically associated with WN virus were diagnosed in 81 patients: in 50 out of 64 in the Volgograd region and in 31 out of 38 in the Krasnodar region, which makes 79.4%. Specificity of antibodies to WN virus was confirmed in HI and EIA with WN antigens, related flaviviruses (Japanese encephalitis and yellow fever), and Sindbis alfavirus. A considerable number and the incidence of WN infection suggest that an epidemic caused by WN virus occurred in the Krasnodar and Volgograd regions in summer 1999.

[Preparation and use of monoclonal antibodies to the Inkoo virus (Bunyaviridae, Bunyavirus) for differentiating between California serogroup viruses]. / Poluchenie i ispol'zovanie monoklonal'nykh antitel k virusu Inko (Bunyaviridae, Bunyavirus) dlia differentsiatsii virusov Kaliforniiskoi serogruppy.

Five types of monoclonal antibodies (Mab) to Inkoo virus were obtained. The direction of Mabs to virus proteins was determined and reactions of Mabs with other California group viruses studied by the fluorescent antibody method.

[Use of 3,3',5,5'-tetramethylbenzidine as substrate in immunoenzyme analysis]. / Ispol'zovanie 3,3',5,5'-tetrametilbenzidina v kachestve substrata pri immunofermentnom analize.

The possibility of using 3,3",5,5'-tetramethylbenzidine and its derivatives in solid-phase enzyme immunoassay and in dot enzyme immunoassay on membrane filters has been demonstrated in experiments with arboviruses. All the tested substrates were fit for specific indication of viral antigens and provided an adequate sensitivity of analyses in comparison with the routinely used substrates, ortho-phenylenediamine and ortho-toluidine. An important advantage of the tested substrates is the absence of carcinogenicity.

[The rapid detection of arboviruses by dot immunoenzyme analysis on membrane filters]. / Ekspress-indikatsiia arbovirusov metodom tochechnogo immunofermentnogo analiza na membrannykh fil'trakh.

A direct and an indirect variants of dot enzyme immunoassay on membrane filters (dot-EIA) for detection of arbovirus antigens and antibodies were developed. Both variants had similar sensitivity and specificity as compared with the solid-phase enzyme immunoassay usually used for this purpose. The advantages of dot-EIA consist in the possibility of specific detection of antigens in unpurified biological specimens by direct adsorption of the antigen on the membranes, economy aspects, and shortening of the time of analysis.

[Significance of viruses of antigen complex of California encephalitis in pathology]. / Znachenie virusov antigennogo kompleksa kaliforniiskogo éntsefalita v patologii.

A study was undertaken to examine 320 patients with seasonal fevers occurred in June to September. These included fever of unknown etiology, acute respiratory virus diseases, pneumonias, bronchitis, enterovirus diseases, and serous meningitis. Serological tests revealed that in 20 (6.3%) of them the viruses of the complex in California encephalitis (Tyagin's virus or antigenically related virus) contributed to the etiology of the disease. Major clinical symptoms of the disease were defined in this group of patients. The disease appeared as neuroinfections (serous meningitis), influenza-like conditions (fever, symptoms of intoxication), occasionally, infiltrative changes in the lung. Thus, the viruses of the antigen complex of California encephalitis makes an etiological contribution to infectious abnormalities.

[Indirect immunoenzyme method for the laboratory diagnosis of Lassa and Ebola hemorrhagic fevers]. / Nepriamoi immunofermentnyi metod dlia laboratornoi diagnostiki gemorragicheskikh likhoradok Lassa i Ebola.

Conditions for performing solid-phase indirect enzyme-immunoassay (SPEIA) for the detection of Lassa and Ebola virus antigens and antibodies to them using horseradish peroxidase-labeled antispecific globulins were developed. The method is highly sensitive, specific, and reproducible. By this method, antigens of Lassa and Ebola viruses could be detected in tissue culture fluid of the infected cell cultures and in animal organ suspensions. Detection of antibodies to Lassa and Ebola viruses in human convalescent sera and in normal donors by means of SPEIA opens possibilities for its use in large-scale diagnostic and seroepidemiological surveys.