[Preparation of monoclonal antibodies specific for the major tegument protein pp65 of human herpesvirus type 5].

A panel containing 11 types of monoclonal antibodies (mAb) specific for the recombinant protein pp65 of human herpesvirus type 5 was constructed. Enzyme immunoassay and immunoblotting of the immunochemical properties of mAb indicated that mAb could interact well with both recombinant and native pp65 antigen. Testing the suitability of mAb for an immunocytochemical study established that mAb 5F10 was highly effective in detecting the major tegument protein pp65 in the persistently infected Vero cells. A combination of the immunochemical properties of mAb enables them to be recommended for the immunodiagnosis of human cytomegalovirus infection.

[Herpes simplex virus type 1 and 2 (HSV-1,2) DNA detection by PCR during genital herpes].

The oligonucleotide primers for herpes simplex virus type 1 and 2 DNA detection are developed. In examined group of patients with genital herpes the virus of type 2 and type 1 was detected in 63% and 26% cases, respectively. The mixed infection of both types is revealed in 11% of the patients.

[The study of markers of herpes virus infections in myocardial ischemia].

A group of patients with ischemic heart disease (IHD), who underwent surgical aorta-coronary vascular shunting, was examined in this investigation. Low titers of HSV-1 specific IgG were detected in all patients, the obtained values being consistent with similar data obtained in healthy subjects of the same age. Negative PCR of HSV-I DNA in blood and biopsy results were obtained. None of the patients demonstrated typical clinical pattern of infectious disease caused by herpes simplex virus. These data are evidence of the absence of the HSV-1 correlation with coronary atherosclerosis in patients with the IHD diagnosis. The significance of HCMV specific IgG titers and HCMV DNA detected in blood plasma in 87.7% cases is probably attributed to existence of connection of HCMV infection markers revealing in patient' blood with IHD diagnosis and coronary atherosclerosis. Besides, the HCMV DNA presence in biopsy taken from myocardium or vascular wall with lesion is revealed in 100% cases. The cytomegalovirus markers in tissue lesions with the help of specific antiserums marked to HSMV recombinant proteins are also revealed in 100% cases. This fact indicates the connection between pathological atherosclerotical process in IHD and cytomegalovirus infection.

[Designing of the pp65 recombinant antigen of human cytomegalovirus and investigation of its immunochemical properties].

The primary and secondary structures of the pp65 phosphoprotein of human cytomegalovirus coded by the UL83 gene were studied by the methods of computer-aided analysis. An immunodominant protein fragment with 3 antigenic determinant was detected. The UL83 fragment coding the selected region was amplified and cloned in bacterial expressing vector. The recombinant protein was obtained and purified. On the basis of ELISA findings it was acknowledged as possible to use the pp65 recombinant protein jointly with pp150 and p52 in the diagnosis of antibodies specific to human cytomegalovirus.

[Designing of a liquid serum panel containing or not class IgG antibodies specific to human cytomegalovirus]. / Konstruirovanie zhidkoi paneli syvorotok, soderzhashchikh i ne soderzhashchikh antitela klassa IgG k tsitomegalovirusu cheloveka.

Designing of a liquid serum panel for the determination of class G antibodies specific to human cytomegalovirus (HCMV) is under discussion. Sera were selected by ELISA for antibodies to HCMV and by PCR for CMV DNA. The serum panel comprises samples of positive and negative sera with high and low titers. Sera were stabilized by a stabilization solution. The panel shelf life was evaluated by routine methods and by the "accelerated aging" technique. Sera selected for a standard panel containing or not antibodies to HCMV preserve their properties and stability for as long as 1 year at 4 degrees C.

[Efficacy of therapeutic and prophylactic actions of ultralow doses of antibodies to gamma-interferon in experimental murine model of herpes virus]. / Izuchenie éffektivnosti lechebno-profilakticheskogo deistviia sverkhmalykh doz antitel k gamma-interferonu na éksperimental'noi myshinoi modeli gerpesvirusnoi infektsii.

The process of the disease due to herpes simplex virus types 1 and 2 (HSV-1 and 2) was studied on white uninbred mice weighing 10 to 12 g. The animals were infected intracerebrally or intraperitoneally. Intraperitoneal contamination of the animals with MS strain of HSV-2 was used for the experimental model of the herpes simplex infection. The prophylactic antiherpes action of ultralow doses of the human gamma-interferon antibodies (ULD of anti-IFN-gamma) at a course of its intragastral administration was evaluated. The preparation was shown to have a significant (p < 0.05) protective effect in a dose of 10 LD50, evident from a 10-fold decrease of the HSV-2 accumulation in the brain, a lower percentage of the animal deaths and an increase of the average lifespan of the animals by 3.3 days. The study of the therapeutic action of ULD of anti-IFN-gamma at a course of its intragastral administration showed that the preparation had no significant positive effect on the disease process in the animals infected with HSV-2 in a dose of 10 LD50. However, a positive effect associated with delayed virus replication in the brain was observed in the study on the therapeutic effect of ULD of anti-IFN-gamma after its intragastral administration to the mice infected with a sublethal dose of the virus.

[Study of herpes viruses in the blood of patients with cardiovascular pathology]. / Issledovanie gerpesvirusov v krovi bol'nykh s serdechno-sosudistoi patologiei.

The association between Cytomegalovirus (CMV) and Herpes simplex (HSV-1), on the one hand, and the coronary heart disease and chronic venous insufficiency (CVI), on the other hand, was studied; besides the infection rate by the above viruses in different age groups of donors was examined. The following methods--immune-enzyme analysis and detection of viral DNA by means of the polymerase chain reaction (PCR) as well as immune-fluorescence scanning of atherosclerotic plaques--were made use in the study. The pathogenesis of coronary heart disease was shown to be related with the viral contamination caused by CMV, while there was no such correlation between the former and the CVI patients. Finally, it was established that, with age, the frequency rate of seropositive reactions to CMV and HSV-1 was increasing in virtually healthy people.

[Development and preparation of recombinant gD antigen of the herpes simplex type 1 (HSV-1) virus]. / Razrabotka i poluchenie rekombinantnogo antigena gD virusa prostogo gerpesa 1-go tipa (HSV-1)

The most potent antigen among HSV-1 proteins are glycoproteins gB(UL27) and gD(US6). Multiple amino acid sequence alignment of these proteins shows that gD protein is the most specific for HSV-1. Analysis of gD protein epitopes detected the main antigenic determinants not cross-reactive with antigens of other viruses. Virus was isolated and genome DNA was prepared from morphological elements of a patient with herpes simplex infection. US6 gene fragment was cloned in pUC19 vector. Cloning in bacterial expression vectors helped obtain beta-galactosidase-fused recombinant HSV-1 gD protein with 6-histidines affine target for high-performance chromatography purification. ELISA with a set of HSV-1-positive and negative donor sera and a commercial panel of HSV-1 sera (Vektor-Best) showed that recombinant gD can be used as an antigen to HSV-1-specific IgG.

[Preparation of P52 recombinant antigenic protein from human cytomegalovirus (HCMV)]. / Poluchenie rekombinantnogo antigena belka P52 tsitomegalovirusa cheloveka (HCMV).

Analysis of published reports helped us single out the most potent antigens among HCMV proteins: phosphoproteins pp150(UL32) and p52(UL44). Theoretical computer analysis of p52 epitopes showed the main antigenic determinants not cross-reacting with antigens of other viruses. Virus-containing (strain AD169) material was obtained and genome DNA was isolated. Amplification of a site of gene UL44 coding for unique determinants detected a PCR fragment of required electrophoretic mobility. The fragment was cloned in vector pLBE. The specificity of cloning was confirmed by restriction analysis of theoretical sites. Nucleotide sequence of cloned fragment of UL44 gene was studied by Maxam-Gilbert's method. Cloning in expressing bacterial vectors helped obtain HCMV recombinant protein p52 in the pure form and fused with beta-galactosidase. Enzyme immunoassay with HCMV-positive and negative donor sera and ABBOTT HCMV sera showed that recombinant p52 increased the sensitivity and specificity of a previously obtained recombinant pp150 as an antigen to HCMV-IgG and HCMV-IgM. The sensitivity and specificity is 100% with 98-99% reliability.

[Cloning and sequencing of genes coding the glycoproteins of antigens A and B of oncogenic strain JM of Marek disease virus (MDV)]. / Klonirovanie i sekvenirovanie genov, kodiruiushchikh glikoproteiny A- i B-antigenov onkogennogo shtamma JM virusa bolezni Mareka (MDV).

The genes encoding the precursors of secretory glycoprotein gp57-65 (A antigen) and glycoprotein complex gp100, gp60, gp48 (B antigen) of virulent strain JM of Marek's disease virus (MDV) have been cloned using PCR of viral DNA. Nucleotide sequences of both genes have been determined and amino acid sequence of the precursor peptides predicted, and compared with the corresponding sequences of other MDV strains. The results will be used for developing methods for the diagnosis of Marek's disease and vaccination against it.

[Cloning proviral DNA sequences of the human T-cell leukemia virus (HTLV-1) from the genome of cultured MT-4 cells]. / Klornirovanie provirusnykh DNK-posledovatel'nostei virusa T-kletochnogo leikoza cheloveka tipa 1 (HTLV-1) iz genoma kul'tury kletok MT-4.

MT-4 cell line is a continuous strain of human T lymphocytes expressing defective noninfective subviral HTLV-1 particles. A fragment of sequence encoding the p24 protein and gene for envelope protein (env) have been obtained from genomic DNA of this culture by polymerase chain reaction. Both HTLV-1 fragments were cloned in bacterial vectors, and the nucleotide sequence of these regions was determined. The cloned DNA fragment encoding the p24 has only four point nucleotide exchanges. Analysis of the env gene structure revealed that the sequence had several amino acid exchanges and two deletions (13 bp and 70 bp).

[Cloning and primary structure determination of genes coding for IE2 and PP150 proteins of human cytomegalovirus]. / Klonirovanie i opredelenie pervichnoi struktury genov belkov IE2 i PP150 tsitomegalovirusa cheloveka.

The epidemiological situation with cytomegalovirus infection in Siberia is still to be studied and serological diagnosis of human cytomegalovirus (HCMV) is not satisfactory. Two regions of HCMV genome (strain AD-169) have been examined for obtaining diagnostic reagents by expression cloning. Using polymerase chain reaction, fragments of gene of the immediate early protein (IE2) and of the region encoding for the entire hydrophilic part (1176 bp) of the large phospoprotein gene (pp150) have been obtained. Both fragments were cloned in bacterial vectors. Analysis of nucleotide sequence showed negligible substitutions in comparison with previously reported sequences for these genes.