[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 (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.

[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.

[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.

[Functional analysis of Epstein-Barr virus latent membrane proteins (LMP1) in patients with limphoproliferative disorders].

Latent membrane protein1 (LMP1) encoded by the LMP1 gene of the Epstein-Barr virus (EBV) is a transmembrane protein, which can activate a number of cellular signal cascades and transcriptional factors leading to cell transformation. In the present study the sequencing of full-length LMP1 variants isolated from Russian patients with Hodgkin's lymphoma (HL), non-Hodgkin's lymphomae (NHL) and infectious mononucleosis (IM) has been carried out. The phylogenetic analysis of obtained sequences revealed dominance of the LMP1 variants belonging to proteins of low-divergent group LMP1-B95.8b characterized by minimal set of mutations. Investigation of biological properties in the Russian representatives of this group revealed that expression of studied LMP1 variants in embryonic kidney 293 cells was accompanied by insignificant increase in transcriptional factor NF-kappaB activation and had minor influence on activation of transcriptional factor AP-1. It was also detected that all investigated low-divergent LMP1 variants expressed in Rat-1 cells induced activation of inducible NO-synthase (iNOS) and intracellular production of nitric oxide (NO). At the same time the level of NO accumulation was lower than that induced by the low-transforming prototype variant LMP1-B95.8. The data obtained indicate that the LMP1 variants, which are the most common among Russian patients with EBV-associated lymphoproliferative diseases, are characterized by minimum number of mutations and rather low ability to activate basic cellular signaling pathways regardless the nature of pathological process, benign (IM) or malignant (HL, NHL). It is suggested that in addition to the modest activation of NF-kappaB and iNOS induction by LMP1 other factors are involved in the cell transformation process.

[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.

[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.

[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.

[Search for HHV-8 associated diseases reservoir and spread paths of HHV-8 in Russia]. / Poiski HHV-8-assotsiirovannykh zabolevanii, rezervuara i putei rasprostraneniia HHV-8 v Rossii.

Summarized in the paper are study results of human herpesvirus type 8 (HHV-8) and of its association with Kaposi's sarcoma (KS). The data obtained denotes that the share of individuals producing the antibodies to HHV-8 in a majority of studied patients was low and ranged form 0 to 5.5%, which is indicative of a low degree of the virus spread in population. At the same time, a high share of persons with antibodies to HHV-8 was detected among HIV-infected homosexuals (71.4%), kidney recipients (26.0%) and among AIDS-KS patients (78.6%). It was also unexpectedly high among patients with T- and B-cell lymphomas (50%), encephalopathy (27.3%) and with stomach cancer (41.8%): the appropriate parameters were 7-12-fold higher versus healthy subjects. The HHV-8 markers, i.e. virus specific antibodies and/or nucleotide sequences of the virus, were detected in blood serum and ejaculate of a significant number of patients with different pathologies of the prostate. Such detection of viral markers in the above categories of patients is suggestive of that sexual contacts with such patients are decisive for the HHV-8 spread in population.

[Antibodies to herpesvirus type 8 in Kaposi's sarcoma patients and controls in Russia]. / Antitela k gerpesvirusu tipa 8 u bol'nykh sarkomoi Kaposhi i lits kontrol'nykh grupp v Rossii.

The purpose of the present study was to investigate the HH-8 seroprevalence among patients with Kaposi's sarcoma (KS), melanoma and gastric carcinoma (GC) as well as among renal recipients and blood donors. The obtained data revealed a high percentage of seropositive KS patients, which ranged from 83.6% in the classical disease type to 68.8% and to 71.4% in the immunosuppressive and AIDS-associated disease types, respectively. On the whole, the positive humoral response to HHV-8 reliably correlated with the positive findings if the viral genetic information in tumor tissue samplings. An unexpectedly high percentage of seropositive persons was found among the GC patients (41.8%) and among the renal recipients (26%), which is apparently predetermined by the immunosuppressive condition of such patients. Seroprevalence was found only in 4% of blood donors. Thus, the obtained data make it possible to conclude that KS cases, as diagnosed in Russia, are tensely associated with HHV-8 in spite of a low virus spread among the healthy population. Patients with pathology concomitant with a pronounced immunosuppression are characterized by a high prevalence of HHV-8 and belong to the category of persons with a KS risk.

[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.

[Structural and functional features of Epstein-Barr virus LMP-1 gene in patients with anaplastic carcinoma of the nasopharynx in Russia]. / Strukturnye i funktsional'nye osobennosti gena LMP-1 virusa Epshteina-Barr u bol'nykh nedifferentsirovannym rakom nosoglotki v Rossii.

Epstein-Barr virus (EBV) is known to be closely associated with the development of anaplastic nasopharyngeal carcinoma (NPC) in some malignancy endemic regions in South-East Asia. LMP1 gene is one of the EBV latent genes, which encodes a latent membrane protein. LMP1 gene is thought to be a classical oncogene since it morphologically transforms cells in vitro and induces tumors in experimental animals in vivo. LMP1 is one of a few genes which is expressed in NPC tissues. It was first shown that C-terminus of LMP1 gene obtained from NPC patients in South-East Asia contained a deletion of 30 base pairs (bp). However, this deleted LMP1 gene was then found in the EBV isolates persisting among healthy virus carriers and patients with other EBV-associated abnormalities from both NPC endemic and non-endemic regions. The aim of this investigation was to accomplish a molecular biological analysis of EBV LMP1 genes obtained from Russian NPC patients. To this end, the authors isolated and sequenced the LMP1 clones amplified from the tumor tissues from 7 NPC patients at the N. N. Blokhin Russian Cancer Research Center and primary blood lymphocytes (PBL) from 6 healthy donors. As a result, the authors could not find the deletion of the above-mentioned 30 bp in NPC LMP1 clones, but could in one healthy donor (PBL-2). A functional analysis revealed no significant differences between LMP1 variants with or without 30 bp deletion in their capacity to activate NF kappa B and jun/AP-1 transcription factors. Nevertheless, Russian NPC-derived LMP1 variants as compared with those from PBLs featured some specific amino acid exchanges. These data indicate that the 30 bp deletion of LMP1 gene is not a factor that predisposes to NPC in Russia.

[Expression of endogenous retroviruses of the HERV-K/HTDV family in patient with germinogenic tongue tumors]. / Ekspressiia éndogennykh retrovirusov semeistva HERV-K/HTDV u bol'nykh germinogennym opukholiami iaichka.

Transcription of HERV-K/HTDV proviruses in various morphological forms of GCTs and in normal testicular parenchyma and placenta was studied by RT-PCR with specific primers discriminating type 1 proviruses from type 2 ones. The results indicate that transcription of type 2 HERV-K/HTDV proviruses takes place and mRNA of protein cORF, coded for only by type 2 proviruses, is synthesized in all malignant germinogenic tumors. In normal testicular tissue and placenta type 1 HERV-K/HTDV proviruses are expressed. No expression of HERV-K/HTDV proviruses was detected in nongerminogenic testicular tumors. Since cORF protein possesses transforming activity, its possible role in the pathogenesis of GCTs is discussed. Generation of humoral immune response to structural HERV-K/HTDV proteins in various morphological forms of GCTs confirmed the possibility of using antibodies to structural HERV-K/HTDV proteins as additional markers of GCTs.

[PCR diagnosis of sequences of a novel human herpes virus type 8 in patients with Kaposi sarcoma in Russia]. / PTsR-diagnostika posledovatel'nostei novogo gerpesvirusa cheloveka 8-go tipa u bol'nykh sarkomoi Kaposhi v Rossii.

Associations of a new human herpesvirus type 8 (HHV-8) with different forms of Kaposi's sarcoma (KS) in Russia have been studied. Search for this virus genetic information has been carried out in biopsy specimens of benign and malignant tumors other than KS, and probable sites of HHV-8 latency in human body have been checked. HHV-8 sequences were detected by polymerase chain reaction (PCR). HHV-8 sequences were most often detected in idiopathic (80.6%), AIDS-associated (80%), and immunosuppressive (100%) KS. The results indicate a selective association of HHV-8 with KS. No probable sites of the virus latency were detected in peripheral blood cells of patients with KS and in the prostate of patients with chronic prostatitis. The only exception was the husband of a patient with KS: HHV-8 sequences were detected in his prostatic secretion by nested PCR.

[Molecular biological and clinical-morphological studies of gastric tumors associated with Epstein-Barr virus]. / Molekuliarno-biologicheskoe i kliniko-morfologicheskoe issledovaniia opukholei zheludka, assotsiirovannykh s virusom Epshteina-Barr.

The paper presents the results of the studies of gastric cancer (GC) associated with Epstein-Barr virus (EBV) among the patients residing in 4 geographical regions. In situ hybridization (ISH) techniques revealed that 49(11.4%) of the 430 examinees were EBV positive (EBV+), the virus-specific marker mRNA-1 of EBV, EBER-1) was found to be present in 80-100) of tumor cells. The proportion of EBV(+)-associated GC cases in different geographic regions ranged from 7.3 to 15%. These tumors were predominant in males (15%) as opposite to females (5.5%). Histological types most common among EBV+ tumors and their location in the stomach are also described. Serological findings indicated that the increased anti-EDV antibody response in 70% of GC cases coincided with the presence of the viral genetic information detected by ISH. In contrast to a humoral response to EBV, a humoral response to Helicobacter pylori was equal both in patients with EBV(+)- and EBV(-)-associated gastric tumors. Further molecular biological analysis of EBV isolates from virus positive and virus negative GC may answer the question whether there are really the so-called tumor and non-tumor variants of EBV.