[Epstein-Barr virus (EBV) in Russia: infection of the population and analysis of the LMP1 gene variants in patients with EBV-associated pathologies and healthy individuals].

The Epstein-Barr virus, widespread herpesvirus among the population of the planet, is also the etiologic agent for a number of malignancies. One of the oncoproteins encoded by the virus, the latent membrane protein 1 (LMP1I), through activation of the complex signaling pathways is involved in the processes of cell immortalization and transformation. The goal of this work was to study the level of the EBV infection in Russian population and LMP1 polymorphism in patients with benign and malignant EBV-associated diseases and healthy virus carriers. Studies have shown that by the age of 5-9 years the percentage of the infected persons and the level of antibody titers reaches almost the maximum values. With the age, virus specific antibody titers are decreased (with a high percentage of infected persons) and increased again in groups of older persons. The analysis of the nucleotide sequences of the gene LMP1 translated in amino acid (aa) sequences unexpectedly revealed the dominance a low divergent variant LMP1 B95.8A not only in healthy individuals but also in patients with all forms of EBV-associated diseases. Highly divergent variants Ch1 and Med +, containing a deletion of 10 aa, and characterized by elevated transforming activity more often were detected in the tumor tissue samples than in the blood samples/mouth washes of the same patients. Detection of highly transforming variant LMP1 Ch1 in blood samples of healthy individuals indicates that this analog of Chinese variant Cao may persist in any population and is not necessarily associated with the occurrence of the EBV-associated disorders.

[The influence of point mutations in the Epstein-Barr virus LMP1 oncogene on the cell cytoskeleton and activation of inducible form of NO synthase].

One of the latent proteins encoded by the Epstein-Barr virus (EBV), the latent membrane protein 1 (LMP1), plays a key role in developing of EBV-associated human malignancies. Polymorphism of LMP1 protein is its characteristic feature. Some specific mutations in LMP1 genome have previously been detected in different geographic regions, however, the influence of these mutations on functional activity of LMP1 was not still determined. In this study we demonstrated for the first time the significance of individual point mutations among common ones observed in LMP1 and their combination on activation of the inducible form of nitric oxide synthase (iNOS). In addition, the influence of above mutations localized in the CTAR regions of the LMP1 molecule has also been investigated on structural components of the fibroblasts of the Rat1cell line.

[COX-2 as an early diagnostic marker of virus-associated human malignant neoplasms].

The review analyzes recent data and current ideas on the enzyme cyclooxygenase 2 (COX-2) as a possible biomarker of virus-associated human malignant neoplasm. Possible mechanisms of COX-2 activation in the cells infected with oncogenic human viruses, such as hepatitis B virus, Epstein-Barr virus, and human papillomavirus are considered in detail.

[LMP1 gene polymorphism in patients with Epstein-Barr virus-negative gastric carcinomas in Russia].

The investigation was undertaken to study the molecular characteristics of Epstein-Barr virus (EBV) LMP1 gene samples amplified from the tumor and intact tissues of patients with EBV-negative forms of gastric carcinoma (GC). The genetic structure of these samples determined by their sequencing was compared with that of the gene samples isolated from the cells of oropharyngeal washing specimens from the same patients with GC, as well as peripheral blood lymphocytes of patients with infectious mononucleosis (IM) and blood donors. The findings suggest that the samples of tumor tissue LMP1 from patients with GC have higher divergence than those from patients with IM and blood donors although no specific variants of the gene for GC were found. Comparison of LMP1 sequences from tumor tissue and cells of oropharyngeal washing specimens from the same patients with EBV-negative GC revealed the common LMP1 variant in 2 cases while they differed in 3 cases. The findings are an initial step in studying the role of EBV in the carcinogenesis of EBV-negative GC that is likely to be established by investigations on representative clinical material, by applying the up-to-date technologies.

[Ancient variants of the Epstein-Barr virus (Herpesviridae, Lymphocryptovirus, HHV-4): hypotheses and facts.]

INTRODUCTION: Molecular studies have shown that viruses appeared in the early stages of the evolution of life. For millions of years, viruses have evolved by changing old and acquiring new sequences in their RNA or DNA. It is assumed that most viruses have common ancestors. Such an ancestor, an ancient strain, probably existed for Epstein-Barr virus (EBV) as well. AIM: To find out whether ancient strains of EBV persist in modern Russian ethnic groups today. MATERIAL AND METHODS: The object of the study was the EBV LMP1oncogene, which is most suitable for molecular genetic analysis. LMP1 was amplified from the oral cavity washings obtained from representatives of two ancient ethnic groups of Russia - Tatars and Slavs. The LMP1 amplicons were sequenced in both directions; their nucleotide sequences translated into amino acid (LMP1) were evaluated using the classification suggested by Edwards et al. 1999. To establish genetic relationships between LMP1 variants, a phylogenetic tree was constructed by the neighbor-joining method using the MEGA software package. RESULTS AND DISCUSSION: Analysis of LMP1 sequences from washings of the Slavs oral cavity demonstrated the presence of LMP1 variants with varying degrees of transforming potential: B98.5/A, China1, Med-, and NC. The analysis of LMP1 sequences from washings of Tatar oral cavity also made it possible to identify oncoprotein variants such as B95.8/A, China1, Med-, as well as a group of variants out of classifications (LMP1-OK). An important finding was the identification of 7 variants of LMP1 from Tatars, designated as LMP1-TatK, that contained two unique deletions of 5 aa in codons 312-316 and 382-386, which were absent in the LMP1 variants from Slavs and from previously examined cancer patients and healthy individuals, as well as in sequences from open computer databases. The uniqueness of the LMP1-TatK variant is confirmed both by phylogenetic analysis of LMP1 sequences of Tatar origin and by the analysis of 11 aa repeats and 5 aa insertions in the C-terminal region of the oncoprotein. The morbidity and mortality rates from neoplasms, including EBV-associated pathologies, did not differ significantly between two studied ethnic groups infected with different EBV strains. CONCLUSION: The data obtained allowed us to suggest that: 1) LMP1-TatK could be refered to an evolutionarily ancient EBV strain that persists among Tatars and; 2) LMP1-TatK belongs to the so-called "Volga" EBV virus strain, the common strain among the population of the Volga region. Extended studies of EBV isolates from residents of this region may probably shed the light on the origin of LMP1-TatK.

[HERV-K-associated carcinogenesis: co-expression of viral and cellular proteins in the development of human germ-cell tumors].

To elucidate the role of some viral and cellular proteins in the occurrence and development of HERV-K-associated germ-cell tumors (GCT), reverse-transcription polymerase chain reaction using specific primers has been employed to study the transcription of the protein Rec HERV-K and the possible interaction of the protein Rec(cORF), that has transforming properties, and the cellular protein PLZF, that is a negative regulator of cell division, in human GCT tissues, in the testicular parenchyma adjacent to a tumor, and in the normal testicular tissues. It was shown that there was expression of Rec(cORF) of mRNA, rather than cellular PLZF in all malignant GCT tissues, this led to the conclusion that no interaction occured between the Rec HERV-K and PLZF proteins in the GCT cells. At the same time co-expression of Rec and PLZF protein was first revealed at the level of transcription in the testicular parenchyma adjacent to a tumor that exhibited carcinoma in situ cells. By taking into account that the protein Rec HERV-K has transforming activity and it is presumed to be Implicated in the development of GCT, the authors discuss a possible role in the Rec HERV-K/HTDV and cellular PLZF interaction in the pathogenesis of GST at the early stages of its genesis.

[Diagnostics of nasopharyngeal carcinoma with Epstein-Barr virus (Herpesviridae, Lymphocryptovirus, HHV-4) serological and molecular markers in cases of undetected primary tumor location.]

INTRODUCTION: The reasons of late diagnosis of nasopharyngeal carcinoma (NPC) are the long asymptomatic course of the pathological process, the anatomical structure of the nasopharynx, often small, visually and endoscopically undetectable tumor and other factors. It is proved that the Epstein-Barr virus (EBV) is an etiological agent in the most common undifferentiated non-keratinizing histological type of NPC (uNPC). OBJECTIVES: The aim of the work was to assess the significance of diagnostic markers of EBV (titers of humoral antibodies to the virus and the concentration of viral DNA in plasma) for the diagnosis of uNPC in a group of patients with metastatic lesions of the cervical lymph nodes without an identified localization of the primary tumor focus. MATERIAL AND METHODS: The material for the study was blood plasma of 83 patients with metastatic lesions of the cervical lymph nodes and not established localization of the primary tumor. Plasma samples were tested for the anti-EBV IgG and IgA antibody content and titers and the concentration of viral DNA. RESULTS AND DISCUSSION: The data obtained indicate that the parallel testing of blood plasma for EBV-specific antibodies and viral load is a useful tool for preliminary screening of uNPC patients. The final diagnosis is confirmed by the data of subsequent morphological and instrumental studies. Several examples also show that the concentration of viral DNA in the blood plasma of patients with uNPC reflects the effect of the therapy and the prognosis of the disease: remission, stabilization of the tumor process, relapse or metastasis. CONCLUSION: Although the titers of virus-specific antibodies are found to reflect clinical manifestations of the disease less accurately than the plasma concentrations of viral DNA, serological markers are extremely important for the preliminary diagnostics of uNPC in cases of undetected primary tumor location. They are also useful for primary screening of this neoplasm among individuals at risk.

[Mutations of the Epstein-Barr virus LMP1 gene mutations in Russian patients with lymphoid pathology and healthy individuals].

Epstein-Barr virus (EBV) is an etiological agent of a number of benign and malignant human diseases, such as infectious mononucleosis (IM), Hodgkin's lymphoma (HL), and non-Hodgkin's lymphoma (NHL). EBV latent membrane protein 1 (LMP1) gene (recognized as a viral oncoprotein) of various clinical and geographical origin was found to have different types of amino acid mutations affecting its biological activity. Since there was no information on the strain differences in LMP1 of EBV persisting in Russia, the authors made a sequence analysis of LMP1 samples amplified from the biological materials of Russian patients with IM, HL, and NHL and healthy individuals. The studies have shown that LMP1 variants of Russian origin are a mixed heterogeneous group containing both the earlier characterized and presumably new genetic variants. Among the point amino avid substitutions, the mutations S366T, F106Y, 185L, and E328Q associated with the enhanced transforming activity of a LMP1 molecule and its reduced cytotoxicity. There was no specific association between the certain Russian variants of LMP1 and the specific forms of the disease (IM, HL, and NHL).

[The distribution of HLA genes in Russian patients with germ cell tumors].

The correlation between DRB1, DQA1, and DQB1 genes of HLA class II, and the development of germ cell tumors (GCTs), as well as serological response to HERV-K proteins were investigated. Genomic DNA prepared from 99 GST patients was subjected to HLA typing by polymerase chain reaction (PCR) using the set of sequence specific primers (PCR-SSP). This set of primers made it possible to detect 14 specificities of DRB 1 locus, 12 alleles and groups of alleles of DQB 1 locus, and 8 alleles of DQA1 locus. Alongside with the definition of the occurrence of HLA markers in the total group of patients, the frequency of the occurrence of HLA-DR-DQ alleles was calculated in: 1) patients with different morphological forms of GSTs (seminomas and non-seminomas); 2) GCT patients producing or non-producing antibodies to Gag and/or Env HERV-K proteins. The comparison group consisted of 300 Moscow blood donors. The study did not reveal statistically significant differences in the frequency of the occurrence of DRB1, DQA1, and DQB1 alleles between the total group of GCT patients, its subgroup, and the control group. Thus, the data obtained demonstrated the absence of a strict correlation between the distribution of HLA class II alleles and GCT occurrence in the Russian population, as well as the ability of GCT patients to develop an antibody to HERV-K proteins, though more numerous observations are required to confirm this conclusion.

[Antibodies to stuctural proteins of endogenous retrovirus of the HERV-K/HTDV family as markers of human germ cell tumors].

Human germ cell tumors (GCT) have been found to be closely associated with the expression of HERV-K/HTDV proviruses and most patients with GCT produce antibodies to the major HERV-K/HTDV Gag and Env proteins. The findings have shown a strong association of the level of HERV-K/HTDV antibodies with the clinical course of the disease and therapy success, which makes it possible to confirm the fact that viral protein antibodies may be used as an additional marker of GCT.

[LMP1 gene variants of Epstein-Barr virus in patients with some lymphoproliferative malignancies].

The samples of tumor biopsy, blood, and saliva from 10 patients with Hodgkin's disease, 10 patients with non-Hodgkin's lymphoma, and the blood samples of 20 donors were tested by polymerase chain reaction (PCR) for standard (wild) B95-8 and Cao-like (deleted) variants of the LMP1 gene. The paraffin sections of most PCR-tested tumors were also investigated by immunohistochemistry using the monoclonal antibodies S12 or 7D7 to detect the expression of the standard or Cao-like variants of LMP1 protein, respectively. It is suggested that Eptein-Barr virus (EBV) that contains the above deletion is not crucial for the development of the study lymphoproliferative malignancies. The fact that in some cases there is the Cao-like variant of LMP1 in the tumor biopsy specimen and its standard variant LMP1-B95-8 in the biological fluids of the same patient is very likely to suggest that the patient is infected with both types of the virus or there is genetic mutation(s) of EBV during viral carcinogenesis preceding or accompanying the development of a tumor.

[A new view of approaches to diagnostics and treatment of Kaposi's sarcoma].

The paper contains literature data and the results of author's own observations, devoted to the clinical manifestations, ethiology and pathogenesis of Kaposi's sarcoma. An algorithm of diagnostics of the disease and methods of treatment are offered, which are developed taking into account the type of the disease, its form, the extent of the pathologic process and immunological parameters.

[Antibodies to the human T-cell leukemia virus in the blood serum of the inhabitants of Sakhalin and Iuzhno-Kuril'sk]. / Antitela k virusu T-kletochnogo leikoza cheloveka v syvorotke krovi u zhitelei Sakhalina i Iuzhno-Kuril'ska.

Two immunological tests were used in testing of 808 blood serum samples from inhabitants of Sakhalin and of the city of Yuzhno-Kurilsk for antibodies against HTLV-1 etiologically associated with human T-cell leukemia. Antibodies to HTLV-1 were detected in 4.2% of Nivkhs, 1.2% of Oroks and 1.5% of Russians. The highest level of virus-carriers reaching 6.0% was detected in Nivkhs from the middle part of Sakhalin. The antibodies were detected more often in persons of old age and in women.

[Serological examination of the population of the Far East for the presence of antibodies to human T cell leukemia virus HTLV-1]. / Serologicheskoe obsledovanie zhitelei Dal'nego Vostoka na nalichie antitel k virusu T-kletochnogo leikoza cheloveka HTLV-1.

Antibodies to HTLV-1 were detected in sera from 1298 healthy adult persons living in the Far East. It is shown that virus infectivity of the Nigidalts was 8%, Nivkhs--2%, Udegeits--2.9%, Oroches--2.4%, Russians--0.9%. However, the incidence of acute leukemia in these regions is low. It is suggested that these regions are not endemic areas for HTLV-1-associated haemoblastoses.

[Human acute lymphoblastic leukemia associated with HTLV-I virus and its infection in healthy populations from various regions of the USSR]. / Ostryi limfoblastnyi leikoz cheloveka, assotsiirovannyi s virusom HTLV-I, i infitsirovannost' im zdorovogo naseleniia nekotorykh regionov SSSR.

Patients with various forms of hemoblastoses (328) and clinically healthy persons (530) were examined for antibodies to HTLV-I. The presence of antibodies was detected in 3 out of 40 patients with acute lymphoblastic leukemia and only in 1 of 70 patients with lymphosarcoma. These data indicate that sporadic cases of T-cell leukemia associated with HTLV-I were detected in the USSR. Out of 530 healthy persons only 4 contained antibodies to HTLV-I antigens.

[The role of region pX in the life cycle of HTLV-I and in carcinogenesis]. / Oblast' pX HTLV-I v zhiznennom tsikle virusa i kantserogeneze.

The review considers the molecular biological organization of genome region pX of the human T-cell leukemia virus type I (HTLV-I) along with the structure and functions of regulatory proteins Tax, Rex, and poorly studied Rof and Tof. Tax functions are described, including transcriptional trans-activation, trans-repression, and effects on apoptosis and the cell cycle. The roles of Tax and Rex in controlling gene expression and replication of HTLV-I are discussed.

[Expression of the Epstein-Barr virus terminal repeat protein 1 in Escherichia coli cells]. / Ekspressiia belka terminal'nogo povtora 1 virusa Epshteina-Barr v kletkakh Escherichia coli.

A pMAL-vector-based plasmid clone with synthetic tac-promotor effectively expressing full-length terminal repeats protein 1 (TP1) of Epstein-Barr virus (EBV) in E. coli was constructed. It is important that the N-terminal region of recombinant TP1 was represented by a maltose-binding protein. The latter can be used to separate TP1 from bacterial lysate by affinity chromatography. Moreover, after treatment with the proteolytic factor Xa, full-length TP1 can be recovered in a discrete form. On the basis of the pATH tryptophan-regulated vector, several plasmid clones expressing different fragments of N- and C-terminal regions of TP1 were also constructed. This collection of recombinant proteins could be used as an important tool for obtaining corresponding antisera and for immunological mapping of the TP1 molecule.

[Differential expression of recombinant hybrid proteins containing amino- and carboxy-terminal regions of the exterior large glycoprotein (gp46) of the human T-cell leukemia virus (HTLV-I) in Escherichia coli cells]. / Differentsial'naia ékspressiia rekombinantnykh gibridnykh belkov, soderzhashchikh amino- i karboksiterminal'nuiu oblasti naruzhnogo bol'shogo glikoproteida (gp46) virusa T-kletochnogo leikoza cheloveka (HTLV-I) b kletkakh Escherichia coli.

Plasmid clones capable of expressing a recombinant fusion proteins containing the anthranilate synthase of E. coli (TrpE) and different regions of gp46 HTLV-I were constructed on the basis of pATH-vectors. A high extent of TrpE-gp46 proteolytic degradation took place independently of the bacterial La-protease. Fusion proteins containing an N-terminal part of gp46 were more stable and could be purified in preparative quantities but were less antigenic. On the contrary, a TrpE-gp46 protein encoded by the TaqI-TaqI DNA fragment and containing only 35 C-terminal amino acids was still susceptible to degradation but possessed good serologic reactivity. Some of the recombinant proteins obtained can be useful for diagnostics and for preparing monoclonal or polyclonal antibodies.

[Immunoreactivity of synthetic peptides, corresponding to B-cellular epitopes of human type I T-lymphotrophic virus structural proteins]. / Immunoreaktivnost' sinteticheskikh peptidov, sootvetstvuiushchikh B-kletochnym épitopam strukturnykh belkov T-limfotropnogo virusa cheloveka I tipa.

The immunoreactivity of 25 synthetic peptides corresponding to amino acid fragments of the HTLV-I structural proteins p19 gag, gp46 and gp21 env were studied in enzyme linked immunosorbent assay using a serum panel of 70 reference positive specimens with anti-HTLV-I antibodies. The location of the synthetic peptides containing the B-cell epitopes of HTLV-I was established. Anti-HTLV-I antibodies effectively recognized these peptides. The significance of some amino acids for forming the HTLV-I antigenic determinants was estimated. The synthetic peptides with amino acid sequences 100-130 p19 gag and 176-201 gp46 env were found to have most immunoreactivity (90-99% recognition by sera of HTLV-I infected patients) and mimic the immunodominant B-cell epitopes of HTLV-I structural proteins.

[Cross reactivity and amino acid structural homology of immunodominant peptide fragments of structural viruses from the human retroviruses HTLV-I, HTLV-II, HIV-1, and HIV-2]. / Perekrestnaia reaktivnost' i gomologiia aminokislotnoi struktury immunodominantnykh peptidnykh fragmentov strukturnykh belkov retrovirusov cheloveka HTLV-1, HTLV-II, HIV-1 i HIV-2.

Twelve synthetic peptides corresponding to 9 immunodominant regions of structural proteins of human retroviruses HTLV-I, HIV-1, and HIV-2 were studied in enzyme-linked immunosorbent assay for cross reactivity with heterotypical for each peptide anti-retroviral antibodies. The search of amino acid homologies was carried using the special computer program followed by the correspondence analysis of the discovered homologies and immunodominant fragments. It was found that peptides 100-130 p19 gag HTLV-I, 376-392 gp21 env HTLV-I, 381-400 gp21 env HTLV-I, 306-328 gp120 env HIV-1, 495-516 gp120 env HIV-1, 584-612 gp41 env HIV-1, and 581-603 gp36 env HIV-2 have type-specific reactivity and also cross react with 3-54% human sera containing antibodies against heterotypical retroviruses. On the other hand, peptides 120-130 p19 gag HTLV-I, 176-201 gp46 env HTLV-I, 291-312 gp46 env HTLV-I, 330-363 p24 gag HIV-1, and 602-624 gp41 env HIV-1 have shown no cross reactive properties; they may be effectively used for type-specific and differential serodiagnosis of human retroviral infections.