[Epstein-Barr virus (Herpesviridae: Lymphocryptovirus) types 1 and 2 and other viral markers in patients with nasopharyngeal carcinoma in two geographically and ethnically distinct regions of Russia].

INTRODUCTION: The discovery of two types of Epstein-Barr virus (EBV) (EBV-1 and EBV-2) that have different biological properties stimulated the search for neoplasms associated with each type of the virus. The aim of the work is to study the nature of the association of nasopharyngeal cancer (NPC) with EBV-1 and EBV-2, serological activity for each viral type and the concentration of EBV DNA in the blood plasma of two gender, age and ethnic groups of NPC patients that represent geographically and climatically different regions of Russia,. MATERIALS AND METHODS: In the blood plasma of patients with NPC and other non- EBV associated tumors of oral cavity (OTOCEBV-) from the North Caucasian (NCFD) and Central (CFD) Federal Districts of Russia, the types of EBV and the concentration of viral DNA were determined using respectively «nested¼ and real time PCR; titers of IgG and IgA antibodies to viral capsid antigen (VCA) were measured in indirect immunofluorescence assay. RESULTS: The blood plasma samples testing showed that NPC and OTOCEBV- patients were infected with both types of EBV in approximately equal proportions. In two groups of NPC patients infected with one of the virus types only, EBV-1 or EBV-2, respectively, no statistically significant differences were found between the geometric mean values of IgG and IgA anti-EBV antibody titers and viral DNA concentrations in blood plasma. The distribution of virus types was not affected by either patient gender or ethnogeographic origin. The difference was found only between age groups: EBV-2 dominated in NPC patients up to 60 years, and EBV-1 was prevalent in patients over 60 years. CONCLUSION: The lack of the predominance of one of EBV types in NPC patients that are the representatives of different ethnic groups from geographically and climatically different regions, suggests that none of these factors play an important role in the NPC carcinogenesis. Evidently, each type of EBV, EBV-1 or EBV-2, if the necessary conditions arise, are able to exhibit its oncogenic potential to initiate tumor development.

[Epstein-Barr virus (Herpesviridae: Gammaherpesvirinae: Lymphocryptovirus: Human gammaherpesvirus 4) in Kalmyks and Slavs living in Russia: virus types, LMP1 oncogene variants, and malignancies].

INTRODUCTION: The discovery of the Epstein-Barr virus types (Herpesviridae: Gammaherpesvirinae: Lymphocryptovirus: Human gammaherpesvirus 4) (EBV) - EBV-1 and EBV-2, which have different transforming abilities in vitro, stimulated the study of their prevalence in populations in order to elucidate the relationship with malignant neoplasms.The aims of the work are to study the prevalence of EBV-1 and EBV-2 among representatives of 2 ethnic groups of Russia, Kalmyks and Slavs, sequencing analysis of the LMP1 oncogene in virus isolates, and analysis of the correlation between virus types and the incidence of certain forms of tumors. MATERIALS AND METHODS: DNA samples were isolated from the biological material of oral swabs obtained from ethnic Kalmyks of the Republic of Kalmykia (RK) (n = 50) and Slavs, residents of the Moscow Region (MR) (n = 40). DNA samples were used to amplify EBV DNA, followed by determination of its concentration per 1 cell of washout, amplification of the LMP1 oncogene in viral samples, their sequencing, and determination of LMP1 protein variants. RESULTS: It has been established that with the same burden of EBV among representatives of both ethnic groups in the Kalmyk group, the ratio of persons infected with transforming and non-transforming types of the virus was almost the same (EBV-1 - 51%; and EBV-2 - 49%). Meanwhile, in the group of Slavs the transforming EBV-1 type virus dominated (80.6%). The predominance of EBV-1 type in representatives of the Slavs correlated with increased incidence of certain forms of tumors in the population of the MR when compared with similar values in the population of the RK, where both types of the virus were prevalent. Differences between the compared rates of cancer incidence were not statistically significant. Analysis of viral isolates showed a similar set of LMP1 variants in both ethnic groups. CONCLUSION: In order to establish the influence of EBV types on the incidence of malignant tumors, additional studies involving representatives of various ethnic groups from different geographical regions are needed.

[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.

[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.

EPSTEIN-BARR VIRUS AND NASOPHARYNGEAL CARCINOMA: VIRAL MARKERS FOR DIAGNOSTICS AND ASSESSMENT OF CLINICAL STATUS OF PATIENTS.

The etiological role of the Epstein-Barr virus (EBV) in the development of an undifferentiated histological variant of nasopharyngeal carcinoma (uNPC) found for the first time in regions with a high incidence of this pathology, the Southern provinces of China and the countries of Southeast Asia, and later in the rest of the world, has served as a basis for the widespread use of EBV serological markers for the diagnosis of this form of tumor. In recent years, the use of a test based on the quantitative determination of the EBV DNA concentration in the blood plasma of uNPC patients for early detection and monitoring of the disease has become widespread in endemic regions. In non-endemic regions, such studies virtually have not been carried out, and moreover, the comparative evaluation of the significance of two viral markers, serological and EBV DNA load in the bloodstream of uNPC patients, for diagnostics and evaluation of the therapeutic effect was not investigated. The aim of this study was to compare the clinical value of two serological markers and plasma EBV DNA load in uNPC patients from non-endemic region (Russia). The obtained results indicate that IgA antibodies to the viral capsid antigen (IgA/VCA) and plasma EBV DNA concentration can be successfully used for the diagnosis of uNPC, while IgG/VCA antibodies have no practical significance as an uNPC marker. In addition, it was found that plasma EBV DNA load is more sensitive marker of uNPC than IgA/VCA titers because DNA copy numbers reflect more accurately the effect of the therapy and the clinical state of patients at the stages of remission or relapse. It was shown for the first time that in the non-endemic region the simultaneous evaluation of IgA/VCA antibody levels and the plasma EBV DNA loads are the most effective markers for the diagnostics of uNPC. However, we believe, that it is more practical to use IgA/VCA antibody levels for uNPC screening, and plasma EBV DNA copies - for monitoring of the disease.

Epstein-Barr virus biomarkers for nasopharyngeal carcinoma in non-endemic regions.

The Epstein-Barr virus (EBV) plays a key role in the development of undifferentiated nasopharyngeal carcinoma (uNPC). In uNPC endemic regions EBV-specific antibodies and plasma EBV DNA load are used as markers for the early detection of uNPC and monitoring of the disease. In non-endemic regions, such studies were practically not conducted. The aim of this study was to compare the clinical significance of EBV serological markers and plasma EBV DNA levels for uNPC patients in a non-endemic region, Russia. The results obtained indicate that both viral capsid antigen/immunoglobulin A (VCA/IgA) antibodies and plasma EBV DNA copies can effectively be used for nasopharyngeal carcinoma (NPC) diagnosis. Besides, plasma EBV DNA load was found to be a more sensitive marker of uNPC than VCA/IgA antibody titres, as it reflected the effect of the therapy in stages of remission and relapse of the disease more precisely. Our study, for the first time, demonstrates that the simultaneous use of plasma EBV DNA loads and VCA/IgA antibody levels are indispensable markers for uNPC in non-endemic regions: a serological marker can be more effectively used for NPC screening, but EBV DNA copies are better for monitoring the disease. However, both markers turned out to be practically unsuitable for assessing the clinical status of patients. Serological markers did not correlate with any signs of the tumour process estimated by tumour, node and metastasis (TNM) classification and the plasma EBV DNA loads correlated only with the size of the pathologically altered lymph nodes (N). Additional study is required to confirm these findings.

Diagnostic value of the Epstein-Barr virus serological markers in patients with nasopharyngeal carcinoma in cases of undetectable primary tumor location.

The goal of this work was to describe a method for diagnosis of the non-keratinizing nasopharyngeal carcinoma (nNPC) in cases of the undetectable primary tumor location. The method is based on evaluation of IgG and IgA antibody levels to the capsid (VCA) and early antigens (EA) of the Epstein-Barr virus (EBV). The diagnosis of nNPC is established by a so-called decision rule. The latter was created by mathematical processing of the method of multifactor analysis of the results of anti-EBV antibody testing of 72 patients with clinically and morphologically confirmed nNPC and 72 patients with other head and neck benign tumors (OHNT) not associated with EBV, which were tested as a control group. The diagnostic value of the decision rule which was tested in the group of 77 patients with confirmed nNPC and 231 patients of a control group was high. The numbers of false negative and false positive cases were equal to 5.2% (4/77) and 6.5% (17/231), respectively. Among 32 patients with undetectable primary tumors the decision rule was able to identify 11 cases of nNPC. This diagnosis later was confirmed by morphological and instrumental methods of study. Only in two cases, false negative result was obtained (2/32; 6.3%) indicating that the serological diagnostics of nNPC with the decision rule is highly specific but not exact. Thus, the data obtained allowed us to conclude that the serological testing of EBV specific antibody evaluated by the decision rule can be recommended as an important test supplementing the standard methods of pdNPC diagnostics including cases with undetected primary tumor location.

STRUCTURAL AND FUNCTIONAL CHARACT ERISTICS OF THE LMP1 ONCOGENE IN PATIENTS WITH TUMORS ASSOCIATED AND NOT ASSOCIATED WITH THE EPSTEIN-BARR VIRUS.

Epstein-Barr virus (EBV) - the etiological agent of a number of human benign and malignant tumors including infectious mononucleosis (IM), Burkitt lymphoma (BL), Hodgkin (HL) and non-Hodgkin (NLH) lymphomas, nasopharyngeal carcinoma (NPC), and many other tumors. Latent membrane protein 1 (LMPl) encoded by the gene of the same name (LMP I) is the main oncoprotein of EBV. LMP1 is a transmembrane protein capable of activating many signaling pathways and transcription factors of the cells, which leads to its transformation. Molecular analysis of LMP1 of various clinical origins identified many variants with different types of amino acid mutations that influence its biological activity. Since the role of LMPl in the development of NPC is still not fully understood, it is important to find out how LMPl samples from patients with EBV-associated form of NPC differ from those of patients with other tumors also located in the oral cavity (OTOC), but not associated with this virus. Unlike most investigations conducted in endemic regions, the present work is intended to compare the genetic structure and the transforming activity of LMPl variants from NPC and OTOC patients has been carried out in a non-endemic region of Russia, where NPC is rarely diagnosed. The obtained data show structural and functional similarities of LMP1 variants in the two groups of patients and, accordingly, a genetic relationship of EBV strains persisting in these patients. Our work suggests that in non-endemic regions any EBV strain with any structure of LMP1 may become the etiologic agent of NPC. However, based on modem concepts, the cancer can occur only if EBV-infected persons have a unique pattern of HLA associated with a high sensitivity to the development of NPC combined with exposure to harmful environmental factors (chemical or physical carcinogens) and lifestyle.

[Epstein-Barr Virus LMP1 oncogene variants in cell lines of different origin].

It is well known that the Epstein-Barr virus (EBV) is a widespread infection in the human population. Typically, infection occurs in early childhood without serious consequences for infected people. At the same time, a secondary infection with an additional EBV strain occurs quite often. During the in vitro cultivation of peripheral blood lymphocytes from persons infected with multiple strains of the virus, only one of these strains with higher transforming potential becomes dominant, while the others are eliminated. Under certain conditions, such a highly transforming EBV strain apparently is able to be the etiologic agent of EBVassociated diseases. To find out the range of highly transforming EBV strains prevalent among Russians, cell lines from patients with EBV-associated and non-associated tumors, as well as healthy individuals, were established. The structural analysis of the latent membrane protein 1 gene (LMP1), a key oncogene of the virus, isolated from established cell lines and peripheral blood lymphocytes of blood donors was carried out, and data obtained were compared with the respective data for LMP1 isolates, amplified from cell lines established from African and Japanese patients with Burkitt's lymphoma. The data obtained show a genetic relationship between Russian LMP1 isolates regardless the fact whether they come from patients with tumors or healthy individuals and differ significantly from LMP1 isolates from Burkitt's lymphoma patients. Thus, the results of the study suggest that in nonendemic region for EBV-associated pathology, Russia, any strain of EBV with any structure of LMP1 with concomitant effect of additional factors may become an etiologic agent for EBV-associated neoplasia.

[Molecular biological properties of the Epstein-Barr virus LMP1 gene: structure, function and polymorphism].

Recent studies indicate that the latent membrane protein 1 (LMP1) encoded by the same name gene of the Epstein-Barr virus (EBV) plays an extremely important role in the pathogenesis of a number of malignant neoplasia. Specifically, LMP1 has the ability to transform human B-lymphocytes in vivo and in vitro and rodent fibroblasts (Rat-1) in vitro. The introduction of the latter into athymic mice leads to tumor development. In addition, expression of the oncoprotein has been often found in EBV-associated tumors at the DNA and constantly at the RNA levels. Having pleiotropic effects, LMP1, participates in the transmission and activation of multiple intracellular signals. It is also involved in the inhibition of key tumor suppressors, has significant influence on proliferation, apoptosis and morphological alteration of the infected cells finally resulting in their transformation. General characteristics of EBV and LMP1 gene as well as functional activity of the encoded LMP1 protein and a brief description of human pathologies associated with the virus have been discussed in this review. The questions concerning the polymorphism LMP1 in EBV-associated pathologies have been also analyzed in details.

[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.

Solitary human endogenous retroviruses-K LTRs retain transcriptional activity in vivo, the mode of which is different in different cell types.

Solitary long terminal repeats (LTRs) of human endogenous retroviruses (HERVs), tens of thousands of which are spread all over the genome, contain a variety of potential transcription regulatory elements. Information on transcriptional behavior of individual solitary LTRs, however, is limited. We studied the transcriptional activity of several individual HERV-K LTRs in a variety of tissues and cell lines. The RT-PCR technique targeted at specific amplification of the U3 or U5 regions of individual LTRs together with their unique genomic flanks was used to estimate the content of each region in the transcripts. An unequal abundance of the U3 and U5 regions of the transcripts of the same LTR in different cells and tumors was observed. Each LTR is transcribed differently in different cells or tissues, and transcriptional behavior of different LTRs was different in the same cell line or tissue. The transcriptional status of LTRs varies in response to mitogenic and stress factors and in tumor tissues compared to normal counterparts. The LTRs thus seem to be the subjects of specific transcription regulation. The data obtained indicate that an appreciable fraction of the LTRs retained regulatory potential throughout millions of years of evolution and thus may contribute to the overall transcription regulatory network.

Study on the mechanism of interference between Friend leukemia and Sindbis viruses in tissue culture.

Some mechanisms of interference developing in BALB/c mouse embryo fibroblast culture (MEC) infected with Friend leukemia virus (FLV) and 48 hours later superinfected with Sindbis virus (SV) was studied. In FLV-infected cells the amount of SV antigen formed was 2-3 times lower than in SV monoinfection, as indicated by immunofluorescence and cytofluorimetry. Electron microscopic examination showed that in mixed infection the number of newly formed SV particles decreased markedly (by 90%) despite the presence of compact aggregates of viral nucleocapsids in the cytoplasm. When the cells were initially infected with arbovirus and then superinfected with FLV, formation of virus antigen and virions of both viruses was not disturbed. Pre-treatment of cell monolayer with dactinomycin (0.2 mug/ml) blocked interferon production in MEC culture and inhibited interference between FLV and SV. It is assumed that interference between FLV and SV is associated with known mechanisms of interferon action as well as with disturbance of the stage of SV particles assembly and their release from the cell. Due to incomplete cycle of SV reproduction interrupted at the stage of ribonucleoprotein formation, productive type of its interaction with MEC cells is disturbed.