Key words EB virus; infection; tumor

Epstein-Barr virus (Epstein  Barr virus, EBV) is a gamma herpes virus subfamily members of one human a specificity addicted lymphocytic herpes virus. It is mainly spread through human saliva, respiratory tract is the largest venue of the Epstein-Barr virus latent. Early phase of infection in developing countries, about 3 to 5-year-old has reached its peak, more than 80% of children under five years of age EB virus serology positive. Serological survey of children 3 to 5 years old EB virus VCA  lgG antibody positive rate of 90% or more, most of early childhood infections, no obvious symptoms, or cause mild pharyngitis and upper respiratory tract infections; while in developed countries, due to the health condition better, only 40% to 50% of children under five years of age EB virus serology positive, the infection often deferred to the youth [1], 15 to 20-year-old to peak [2]. According to the results of the survey, more than 90% of the world’s population, the EB virus latent infection or become the EB virus carriers [3]. More important is the Epstein-Barr virus infection with a growing number of human malignancies, in situ hybridization proved EB virus carrying high copy can be transformed lymphocytes, tumor cells can give growth advantage, making it the advantage cell populations, showing the transformation characteristics. The EB virus led to 1% of the global cancer, accounting for 5.6% of all infectious cancer [4]. Classification of carcinogenic factors according to the International Agency for Research on Cancer, EB virus is listed in the first group of carcinogenic factor [5].

    1 EBV infection and disease relationship

    Clinical EB virus infection often caused by septic multiple organ damage, liver damage, the most common, accounting for 25%; accounted for 4.1% of the heart; blood system accounted for 2.5%; kidney, brain damage found. Now clear: the Epstein-Barr virus in addition to nasopharyngeal carcinoma (in nasopharyngeal carcinoma NPC), a variety of lymphoma [6], and lung cancer [7], gastric cancer [8], cervical cancer [9], oral gland tumors, kidney tumors, thymoma, such as the occurrence of a variety of tumors.

    The carcinogenic effects of EB virus needs synergy with the conduct of the cancer-promoting factors, the adult prevalence of EB virus latent infection occur EB virus-associated tumors, but only a few people, and ethnic, regional and gender differences in the occurrence of these tumors exist. NPC is the most common tumors of southern China, the Chinese in Southeast Asia and North Africa also have a higher incidence of about more than 80,000 new cases occur each year. According to WHO statistics, about 80% of the NPC in China [10]. Burkitt’s lymphoma is a common malignancy of children in Africa, occurred before the age of 15, a high mortality rate [11]. NPC has the incidence of Burkitt lymphoma geographical limitations, the occurrence of these diseases there are other synergistic factors. The synergy of the EB virus and tumor promoters, and other factors worthy of further study.

    In 1987 Begin et al [12] reported the first case with the EB virus-like carcinoma of the lung lymph;, Lung [7] bronchial exfoliated cells material EB virus detection. The discovery of the Epstein-Barr virus infection and lung cancer related; wax block specimens of the Beijing all types of lung cancer 87 cases, domestic Lei et al [13] EB virus detection, results showed that 59.8% of lung cancer in the Epstein-Barr virus infection rate of detection of lung cancer tissue EB virus infection copy number was significantly higher than the adjacent lung tissue, and put forward related to the biological characteristics of the EB virus infection and lung cancer cells and the development of lung cancer; FENG Xin Chu et al [14] found that IgG antibodies in the serum of patients with lung cancer and cancer organizations increased EB virus was positively correlated description of the occurrence of blood cells and cancer organizations EB virus and lung cancer’s progress there is a certain relationship [14]; but Jiaxin Shan [15] EBER  1 molecular hybridization method to check Shenyang City, 58 cases of lung cancer, the results were negative, showing that the Epstein-Barr virus infection marked regional difference.

    Known for about 10% of normal gastric adenocarcinoma [8], more than 80% of the stomach lymphatic epithelioid carcinoma [16] and 35% of the residue of the incidence of gastric cancer and EB virus infection [17], and it said this type of gastric EB virus gastric cancer (Epstein  Barr virus associated gastric carcinoma, EBVaGC). Global annual increase of nearly 900,000 cases of gastric cancer patients EBVaGC absolute numbers far exceed other EB virus-related cancer cases. The study found the EB virus EBVaGC cancerous there is a certain relationship: first of all EBVaGC cancer have EB virus expression is detected in the adjacent normal mucosa and highly proliferative cells less than EB virus expression [18]; Secondly, EB virus exists in the form of additional volume within the nucleus of the infection, i.e. the linear double-stranded DNA is formed in the free cyclic molecule, and the end of the EB virus was consistent with the number of repeat sequences, suggesting that a single tumor lines infected with EB virus cancer cells monoclonal hyperplasia due; Third, there is evidence that similar to other EB virus-associated tumors, clinical diagnosis before EBVaGC the presence of serum in patients with high titers of anti-viral antibodies, suggesting EBVaGC occurred before there is an active EB virus infection.

    As early as 1993, Landers et al [19] in 18 cases of cervical cancer tissue found eight cases of Epstein-Barr virus infection; same year, such as Se Thoe [20] using the same method detected 63 cases of cervical cancer tissue EB virus DNAs; Guo eup, etc. [21] found that in 2003, 56 cases of cervical cancer patients with tumors in the EB virus infection, the experiment also found that Epstein-Barr virus-positive rate of cervical cancer is significantly higher than the normal cervix group, the results support the EB virus infection may be associated with cervical related to the pathogenesis of cancer.

    Recent studies have found that the individual EB virus encoded protein could lymphocyte eternal life, and play a major role in EB virus-related lymphoma. Such as lymphoma, Hodgkin’s lymphoma receiving immunosuppressive treatment of organ transplant patients, people with AIDS, central nervous system lymphoma, subcutaneous panniculitis-like T-cell lymphoma [6].

    Of 2 EB virus biological traits

    The EB virus Round diameter of 180nm, the basic structure of the three parts of the nuclear-like material, capsid and envelope. Nuclear-like material for the diameter of 45nm dense, mainly double-stranded linear DNA, its length varies with the different strains, an average of 17.5 × 104 bp molecular weight of the 108D. Capsid icosahedral three-dimensional symmetry, is composed of 162 shell particles. EB virus is assembled in the nucleus sac membrane, cytoplasm Golgi endomembrane system, wrapped in the cell transport, sprouting from the cell membrane to release viral particles acquire the virus through the nucleation mode. Capsular component on which the membrane glycoproteins of the virus-encoded by the nuclear membrane of the infected cell, identify the EB viral receptors on lymphocytes, and cell fusion, and other functions. In addition there is a layer between the capsular capsid protein envelope. EB virus in cells in the life cycle can be divided into the incubation period, the immediate early, early, viral DNA replication and late. The majority of virus infected B lymphocytes in a latent state. The linear DNA of the virus at this time cyclized to form the episome (episome), separated from the cell chromosome, the results lead to the conversion of the infected cells the ability to obtain immortalized. EB virus only in the B-lymphocyte proliferation, to convert it to long-term subculture.

    The virus-infected cells can produce a variety of antigens, to stimulate the body to produce the corresponding antibodies. EB virus antigen components: Epstein-Barr virus nuclear antigen (EBV  specified nuclear antigens, EBNA) including EBNA1, 2,3 A, EBNA4 (3B), EBNA6 (3C) and EBNA5 (leader protein, LP) 6. Early antigens (early antigen, EA) is divided into two categories: one widely present in the nucleus and cytoplasm called diffuse early antigen (EA  D); another type of most of the in nuclear cytoplasm near, called localized early antigen (EA  R). EA  D and EA  R is not constituted by a single antigen, but is constituted by a variety of antigen complexes. Viral late antigen composition of the capsid of the virus nucleocapsid antigen (viral capsid antigens, VCA), lymphocyte recognition the virus target antigen (viral target antigen LYDMA), and viral late membrane antigen (membrane Antigens, MA). MA including small RNA molecules EBER in latent membrane protein (latent membrane proteins, LMP1,  2A and  2B), and encoding the BamHI-A fragment of the Barts and not translated into protein. LMP1 molecular size of 60 to 66 kD, is a transmembrane protein consisting of 386 amino acid residues, including a 24 amino acid residues consisting of the N-terminus and a close contact with the signal transduction by the 200 amino acid residues composed carboxy-terminal cytoplasmic domain and six transmembrane hydrophobic domains. Transmembrane region of the cell membrane polymerization, of great significance for the LMP1 exerts its biological effects; EBER (The genome of EBV encodes two Epstein  Barr virus  associated small RNAs EBER) with poly (A) tail, of EBER1 (166 bases) and EBER2 (172 bases). Has proven anti-MA antibodies can neutralize the EB virus, but the humoral immune system only to prevent exogenous virus infection, but it can not destroy the virus latent infection. The virus in this state only the expression of the viral proteins of about about 10, EBNA1 and, 2,3 A, the 3B, LP, 3c, LMP1, LMP2A was, LMP2B.

    The 3 EB virus pathogenicity

    EB virus by the salivary glands of infected lymphocytes via the contact of the cells on the first oropharyngeal epithelial cells infection, and to be active in these cells replication of new viral particles may be secreted out of the pass through the next film or infection host B lymphocytes spread to other epithelial cells. EB virus can stimulate the proliferation of B cells also formed a strong T cell antigen for EB virus, acquired immune response to the Epstein-Barr virus infection in the host immune activity preserved. Since the normal immune surveillance function, latent infection of EB virus is controlled in a relatively quiescent state, the virus persistence precisely latent. EB virus infection primarily by the virus and the virus receptor cell surface CD21 to complete. CD21 also called the CR2 or C3dR, and which itself is the complement fragment C3d receptor. The CD21 molecule on the surface of B lymphocytes, while the majority of the surface of the epithelial cells is not found. When the CD21 molecule artificially inserted into the surface of the epidermal cells, these cells may be EB virus, generating infectious viral particles. And enter the blood circulation and causing systemic infection.

    Of infected B lymphocytes can be two kinds of fate: (1) due to the dissolution of the infection has been eliminated, the release of a large number of virus particles and infect neighboring epithelial cells and B lymphocytes, the spread of infection; (2) become transformed lymphocytes and begin to proliferate. Dissolved infected and transformed lymphocytes expressing viral antigens on their cell surface and therefore may be EB virus-specific cytotoxic T lymphocytes (cytotoxic T cells, CTL) will be clear. But part of the transformation of lymphocytes may be due to virus antigen expression and shift behavior type 0 latently infected as retained memory B lymphocytes. Such memory B cells may be due to the opportunity of antigen stimulation is activated, activation of EB virus can be further infection of surrounding epithelial cells and B-lymphocytes, or by a secondary CTL response was rapidly cleared. Epithelial cells infected almost all dissolved infection, generated at this time EB virus excretion in saliva and become a source of infection. Latent infection in memory B lymphocytes EB virus is activated, depending on the struggle between the expression of viral and host defense mechanisms. During immunosuppressive, immune dysfunction, activation of the formation of the recurrence of EB virus latent infection, often accompanied by obvious clinical disorders, including lymphoma and other tumors occur. EB virus latent infection activates three possible ways: (1) fast reaction in the form, then entering dissolved infected state; (2) delayed reaction in the form lymphoblastoid cell into a cell-like morphology; (3) not entering dissolved infected state, virus latent infection only a small amount of virus replication. EB virus-related tumors can be detected in the EB virus latent infection status of the gene product, and the the serological examination antibody levels, illustrate the the EB virus dissolution infection.

    EB virus the solubility infection of cells, BV  DNA linear form. Latent infection, BV  DNA connected by the end of the genome cyclization presence of loose bodies, most Burkitt’s lymphoma and nasopharyngeal carcinoma can be detected; EBV  DNA can also be integrated into the cell chromosome, its integration with the chromosome abnormalities closely related, can lead to cell growth and tumor formation related to abnormal gene expression, and promote the malignant transformation of cells [22]. Its possible mechanisms: on the one hand, tumor cell chromosomal instability, DNA recombination occurs frequently, EB virus thus integrated into the DNA has been subject to the site of injury; the other hand, EB virus integration can be increased chromosomal instability, causing deeper gene level changes. It has been confirmed that EB genomic integration can affect some oncogenes and tumor suppressor genes function [23  24].

    3.1 EBNA EBNA  1 binding to oriP on the EB virus (Epstein-Barr virus DNA replication origin P) of the EB virus DNA in the nucleus to maintain. EBNA  1 also activates other EBNA gene transcription. University of Toronto researchers have identified EBNA1 binding to specific proteins in the cell and interfere with cell growth regulation and cell immortalization, ultimately, to the risk of certain cancers, EBNA  1 for the maintenance of Epstein-Barr virus latent state must. EBNA  2 can activate the LMP  1, LMP  2 and CD21, CD23 and of C  fgr gene expression. The CD23 molecule is one of the B cell growth regulator [24], while the C  fgr a Src original gene family, which encodes a protein kinase, may regulate important regulator of B cell growth substances. EBNA3 for CTL main target, to activate CD21 gene expression, Epstein-Barr virus latent protein to activate some of the B-cell gene expression.

    3.2 EBER EBER abound in the incubation period of the nucleus, is the transformation of cells in vitro are indispensable. It is generally a lot of expression in EB virus-associated cancer cells, malignant tumor apoptosis resistance. Studies reveal the EBER antiviral activity with IFN-related double-stranded RNA-dependent protein kinase (PKR) combine to inhibit the kinase activity [25]. Seen in slices to large lymphocyte karyotype irregular cells were EBER positive, suggesting that the cell activation and transformation has occurred, indicating that the cells of these cells is not a latent state. If it is in the disease state, EBER the number of positive cells will be increased, one slice of at least greater than 3 to 5. Thus, the latent state and disease state EB virus infection on the morphology and number of EBER-positive cells are different.

    Some chemical factors (such as TPA, butyric acid, etc.), and anti-IgM antibodies and other substances to stimulate the EB virus immediate early protein BZLF1 gene expression, it plays a key role in the process of EB virus latent cycle into the breeding cycle . The their product ZEBRA raised including the expression of immediate early gene including its own gene. The immediate-early gene product of further activation EB virus early gene, including the EB virus DNA polymerase and TK kinase to lay the foundation for the replication of viral DNA, the expression of the late structural proteins.

    3.3 LMP  1 LMP  1 is now widely considered to be tumorigenic gene of EB virus, it can promote the proliferation, inhibition of differentiation, causing the cells to malignant transformation. With the in-depth research of the LMP1 molecular structure, its mediated signal transduction pathways and biological functions of breakthrough has been made. LMP1 EB virus-associated cancer therapy may become molecular targets.

    LMP  1 to play the most important role on the B cells immortalized. Now known, CIM0 ligand IL  4 B cell activation of B lymphocytes with EB virus activation mechanism is very similar, can recognized as LMP  1 analog mediated CIM0, and cell activation of the signal transduction system is formed. LMP1 as capable of the no ligand sustained activation of membrane proteins analog of CD40 (TNF receptor), raised tumor necrosis factor receptor family members to be mediated regulation of apoptosis. CD40 on the regulation of cell growth is affected by it with the binding of a ligand, and LMP  1 can continue to stimulate cell growth signal transmission, so that the growth of the cells to lose control. 2 region in the carboxyl terminal cytoplasmic LMP  1: CTAR1 (Carboxy Terminal activating Region 1, 194 ~ 231AA), by TRAF2 (tumor necrosis factor receptor associated factor 2) mediated about 25% NF  κB activation, and directly raised by TRAF3 EGFR expression; another located 351 to 386 CTAR2 role with TRADD (tumor necrosis factor associated dead domain) mediated 75% of NF  KB activation, activation of NF  κB regulator p53, CyclinD1 of MMP9 downstream gene expression [26], in the lymphocytes immortalized on even CTARI with CTAR2 important transformation effector site (transformation effector site, TES) was named the active expression that the activation of NF  κB The most important [27]. Activation of NF-κB by regulating the expression of apoptosis-related proteins regulate apoptosis, LMP1 regulate intracellular signaling pathways, apoptosis-related molecules in the trans-activation of cell-mediated mitochondrial pathway and death receptor pathway two cells apoptotic pathway, in order to achieve its regulation of apoptosis; LMP  1 expression in epidermal cells can change the morphology of cells, can inhibit programmed cell death in B lymphocytes. Can also enhance the oncogene bcl  2 expression. LMP  1 has a dual biological effects promote apoptosis and inhibition of apoptosis.

    The variation of the EB virus the LMP1 gene of (BNLF1 gene) is generally considered to be an important oncogene in tumorigenesis. LMP  1 exon 1 encoding the polypeptide of (N-terminal region) and exon 3 encoded polypeptide (C-terminal region) is very important in the process of tumorigenesis transmembrane and membrane within the functional domains. Lin Su leisure, etc. [27] found that: the Guangzhou area nasopharyngeal EB virus LMP  1 gene sequence variation type Xho Ⅰ  loss/de1 LMP1 dominated; Xho Ⅰ restriction sites lost in its N  terminal region is likely to C  terminal region of 30bp deletion based on the type of sequence variation may occur; 4 on behalf of the the NPC during carcinogenesis EB virus evolution within the host phase. Studies have pointed out that the NPC patients may be more carcinogenic EB virus variant (for example of Xho Ⅰ  loss / del  LPM1) secretion into the saliva, re-infection of other individuals, the result that the normal population of the endemic areas carry may be more carcinogenic EB virus variant ratio compared to non-endemic areas has changed, which will lead to the EB virus endemic areas and non-endemic areas, people carrying the so-called regional differences [28].

    Vidy et al [29] used the pLNSX pLNSX  LMP1 and pLNSX  LMP1TTRADD plasmid E  Cadherin reporter gene were co-transfected into 293 cells to detect LMP1 E  Cadherin promoter activity. The results showed: inhibition of E  Cadherin promoter activation may be EBV  LMP1 the down E  Cadherin protein expression, and promote the CNE2 cell migration mechanism one. TRADD binding sites is the LMP1 cause the CNE2 cell migration, phenotypic changes and E  Cadherin expression inhibit functional areas, suggesting that LMP  1 may be directly involved in the development of the NPC invasion and metastasis. It also found that keratinocyte cell line BHEK1 LMP1 in vitro transformation of not only the morphological changes, and access to the malignant phenotype in vivo tumorigenicity LMP1 positive expression of differentiated squamous cell carcinoma cell lines SCC12 was significantly inhibited, and also enables kidney epithelial cells (MDCK) to generate morphological transformation and invasive growth [30].

    The diagnosis and treatment of of 4 EB virus infection

    Mainly in situ PCR and RT  PCR technology, fluorescence quantitative polymerase chain reaction (FQ  PCR), enzyme-linked immunosorbent assay [31], detected EBV  DNA as well as EBNAs, LMPs, and EBERs or other transcripts.

    In situ hybridization with the target genes of EBV  DNA BamHI  W, EBERl, a small number of Pol  1 and LMP  2 fragment, it may search formalin-fixed, paraffin-embedded NPC specimens, in tumor cells in situ detect the presence of EB virus, positioning accuracy, specificity. Expensive but the probe; NPC smaller organizations often choose to avoid wasting reagents; sometimes NPC tissue fixation poor organization nucleic acid denaturation, dispersion, and effective point of integration to reduce staining may appear weak positive mark, even false negative . Immunohistochemical detection of commonly Epstein-Barr virus-encoded LMP  1 membrane protein, it can not detect the quantity of virus positioning and transcription. However, immunohistochemical methods with respect to in situ hybridization step is simple, easy to operate, high detection sensitivity, and cheap, you can put it as an EB virus screening means, pending immunohistochemical staining and then the original The in situ hybridization to eliminate false positives may. The combination of the two will reduce the chance of false positives and false negatives, so as to improve the accuracy of detection.

    NPC show dynamic changes in free circulating EBV  DNA copy number in peripheral blood as systemic chemotherapy cycle progression, the expression level of the detection of patients with NPC EBV  DNA sensitivity to determine the residual disease recurrence, metastasis, to determine the treatment or change as a comprehensive treatment program in a timely manner, to confirm and determine the efficacy, prognosis have important guiding significance, has become an important NPC diagnosis and treatment of serum molecular markers. Lo et al [32] found by quantitative PCR technology: EBV  DNA expression can be detected in 96% of patients with NPC plasma, significantly higher expression levels of late than early, in January after the end of radiotherapy, the tumor was inhibited by EBV  DNA remained low recurrence increased again. Analysis of the dynamic characteristics of EBV  DNA metabolism: EBV  DNA biological half-life of 3.8 days during radiotherapy. To [33] confirmed: the level of expression of EBV  DNA in the plasma of patients with complete surgical resection of NPC, follow-up of 6.7 days decreased to 0, bit half-life of 139min, the plasma is able to detect the level of expression of EBV  DNA, you must Most clinical recurrence can speculate: detection of NPC patients the plasma EBV  DNA in the expression level of one hand to the efficacy and outcome of allergic reactions; on the other hand also able to determine the clinical discharge, sensitivity and short-term effect of chemotherapy. Makitie et al [34] pointed out: detection in peripheral blood of patients with NPC EBV  DNA copy number with positron emission tomography (PET) examination of monitoring local recurrence or distant metastasis has consistency. After treatment for NPC patients detect residual disease, to evaluate the efficacy determine recurrence, metastasis and prognosis provides a new noninvasive sensitive method. Lin et al [35]: NPCs pre-treatment plasma EBV  DNA expression rate was 94.95%, while health and cure are 0, and the treatment of former plasma EBV  DNA expression levels ≥ 1 500 copies / ml Overall survival and relapse-free survival rate was significantly lower than <1500 copies / ml plasma one week after the end of radiotherapy can detect to EBV  DNA expression overall survival and recurrence-free survival rate was significantly lower than that can not be detected EBV  DNA Expresser.

    EBV infection prevention, two kinds of vaccine will be available, one for China with the pox virus vectors expressing EBV gp320 and HBsAg antigen, focusing on using the in NPC high incidence; another for the the virus gp320 film protein purification are British students patients in a small-scale vaccination, in order to observe whether the vaccine can reduce the incidence of infectious mononucleosis syndrome.

    EB virus with a variety of diseases, EB virus as a promising virus-specific immunotherapy target. Molecular targeted therapy in patients with peripheral blood or tumor tissue EBV  DNA is a good idea to treat Epstein-Barr virus infection.