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The development of nasopharyngeal carcinoma (NPC) and its unique geographic distribution have long been attributed to a combination of dietary intake of salt-preserved fish, inherited susceptibility, and early-life infection with the Epstein-Barr virus (EBV). New findings from our large, rigorously designed, population-based case-control study of NPC in southern China have enabled substantial revision of this causal model. Here, we briefly summarize these results and provide an updated model of the etiology of NPC. Our new research identifies two EBV genetic variants that may be causally involved in the majority of NPC in southern China, and suggests the rise of modern environmental co-factors accompanying cultural and economic transformation in NPC-endemic regions. These discoveries can be translated directly into clinical and public health advances, including improvement of indoor air quality and oral health, development of an EBV vaccine, enhanced screening strategies, and improved risk prediction. Greater understanding of the roles of environmental, genetic, and viral risk factors can reveal the extent to which these agents act independently or jointly on NPC development. The history of NPC research demonstrates how epidemiology can shed light on the interplay of genes, environment, and infections in carcinogenesis, and how this knowledge can be harnessed for cancer prevention and control.

Background Adenovirus is a gene transfer vehicle used in anticancer biotherapy. Oncolytic adenoviruses are prepared so that the recombinant viruses possess more potent cytotoxicity to target cells. E1B is an anti-apoptotic protein encoded by adenovirus. In the present study we tested whether the potential antitumor activity of BLU recombinant adenovirus of tumour suppressor BLU could be enhanced by silencing E1B with siRNA. Methods The shuttle plasmid of BLU was constructed as described, and was assembled to be recombinant adenovirus. siRNAs against adenoviral E1B were designed by locating oligo?nucleotides downstream of AA dinucleotides within the genomic DNA sequence and chemically synthesised. Their ability to silence the expression of E1B was tested by transfection and anti-E1B Western blotting. The potential of BLU to inhibit in vitro and in vivo tumour cell growth was compared between siRNA transfected cells and controls. Results Up to 6 strains of siRNAs were designed and synthesised. They silenced the expression of E1B to different extents, and remarkably reduced the cell viability and formation of xenograted tumours in nude mice. Conclusion In addition to knockout of the E1B coding cassette, siRNA mediated E1B also serves as an approach to prepare oncolytic adenovirus. Acknowledgements Supported by grants from UICC (ICRETT Fellowship), Guangdong Commission of Health and Family Plan (Grant No. 2014A276).

In this study we have raised the question of whether DNA can be transferred from one cell to another by phagocytosis of apoptotic bodies. We have used integrated copies of the Epstein-Barr virus (EBV) as a marker to follow the fate and expression pattern of apoptotic DNA in the phagocytotic host. Apoptosis was induced in EBV-carrying cell lines by irradiation before cultivation with either human fibroblasts, macrophages, or bovine aortic endothelial cells. Analysis of the expression pattern of EBV-encoded genes was performed by immunofluorescent staining as well as in situ hybridization. Cocultivation of apoptotic bodies from lymphoid cell lines containing integrated but not episomal copies of EBV resulted in expression of the EBV-encoded genes EBER and EBNA1 in the recipient cells at a high frequency. Fluorescence in situ hybridization analysis showed uptake of human chromatin as well as integrated EBV-DNA into the nuclei of bovine aortic endothelial cells. These data show that DNA may be rescued and reused from apoptotic bodies by somatic cells. In addition, our findings suggest that apoptotic bodies derived from EBV-carrying B lymphocytes may serve as the source of viral transfer to cells that lack receptors for the EBV virus in vivo.

High-grade malignant nonHodgkin's lymphomas--five lymphoblastic, three pleomorphic, and two immunoblastic--developed in 10/25 cynomolgus monkeys (Macaca fascicularis) followed for up to 746 d after infection with simian immunodeficiency virus, strain SIVsm. These lymphomas were shown to be associated with an Epstein-Barr (EB)-like cynomolgus B-lymphotropic herpesvirus (CBLV) by electron microscopy, by Southern blot hybridization with probes against human EBV, and by the expression of antigens corresponding to EBV-associated nuclear antigens (EBNAs) involved in human B cells transformation. Southern blot demonstration of immunoglobulin gene rearrangements and homogeneous EBV episomes indicated that all the lymphomas were CBLV-associated monoclonal B cell proliferations. Our findings suggest that these tumors correspond to the EBV-associated malignant lymphomas in acquired immunodeficiency syndrome with respect to clinical, morphological, phenotypic, and genotypic characteristics. The particular susceptibility of SIVsm immunodeficient cynomolgus monkeys for CBLV-associated lymphomagenesis appears therefore a useful model for EBV-associated lymphomas in humans.

Sixty-one human nasopharyngeal carcinomas (NPC) were examined by allelotype analysis for the purposes of detecting potential association between loss of heterozygosity (LOH), clinicopathological parameters and Epstein-Barr virus (EBV) infection. LOH was performed using 257 polymorphic markers on 22 chromosomes. High frequency LOH (or = 60%) was observed on 12 chromosome arms including 1p, 2p, 2q, 3p, 3q, 5q, 9p. 9q, 11q, 13q, 14q and 17q, with the highest LOH frequency of 91% on 3p. Seventy-three loci presented LOH frequencyor = 30%; most of these loci clustered on 1p36 p34, 2p25-p24, 3p14-p21, 3p24-p26, 5q11-q14, 5q31-q33, 9p21-p23, 9q33-q34, 11q23-q25, 13q12 q14, 13q31-q33, 14q13-q11, 14q32 and 19q13. On 1p36-p34, 2p25-p24, 5q13-q11, 5q31-q33 and 19q13 are reported for the first time. LOH was correlated with specific clinicopathological parameters including tumor T-stage, N-stage, TNM-stage, tumor differentiation and serum antibody titers of IgA against virus capsid antigens (VCA) and early antigen (EA) of EBV in NPC (LOH frequencyor = 30%). Significantly high LOH frequency was observed on 9p21 (56%) and 19q13 (50%) in NPC with stage T3+T4, while significantly higher LOH frequency was observed on 12p11 (65%) in NPC with stage T1 + T2. Significantly higher LOHfrequency on 19q13 was also observed in NPC with advanced TNM-stage (III+IV). High fractional allelic loss (FAL) value and high antibody titers of EBV IgA/VCA and/or IgA/EA were significantly correlated with T3 + T4-stage, distant lymph node metastasis and advanced TNM-stage of NPC. We also found that NPC patients with high titers of IgA/VCA and IgA/EA showed high LOH frequency on 16q (48%) and 19q13 (48%). These results suggest that LOH on 9p21, l6q and 19q13 may be responsible for the aggressiveness and progression of NPC; there may be an interaction between allelic loss and EBV infection in the etiology of NPC. High frequency of LOH on 4q21 and 14q11-q12 were alsofound to be correlated with WHO type III NPC histopathology, suggesting that LOH on these regions may cause poor NPC differentiation. Our data also may be useful for the development of a NPC molecular staging system, a system which may augment the use of clinical pathological features in the diagnosis and prognosis of this disease.

Three types of virus-host cell interactions have been described in cells latently infected with EBV: EBNA 1 expression in type I Burkitt's lymphoma cell lines (BL), EBNA 1, LMP1 and 2 expression in most nasopharyngeal carcinomas (NPC) and EBNA 1-6 with LMP 1 and 2 expression in group III BL-lines as well as lymphoblastoid cell lines (LCL). Two group I BL lines that express only EBNA 1 were found to initiate their EBNA 1 mRNA transcription from a promoter in the Bam HI Q-fragment. They use a sequence at +210 bp relative to the Fp transcription initiation site in group I BL cell lines. The Fp promoter-region seems to be activated in the lytic cycle. LCLs initiate their transcription from one of several upstream sites, usually the Cp promoter or, less frequently, one of several Wp-promoters. Using RNA-reverse transcription polymerase chain reaction (RT-PCR), we have now shown that EBV carrying cells that do not express EBNA 2-6 always splice their EBNA mRNA at the Q-exon, while EBNA 2-6 positive cells use either the Cp or one of the Wp promotors. When EBNA 2-6 are downregulated by somatic cell hybridisation between EBNA 1-6 positive B-cell lines and non B-cells of hematopoetic, epithelial or fibroblastic origin that express the phenotype of the non-B cell parent, the parental usage of Cp/Wp is switched off, and the Q-exon is activated. NPC cells show the same pattern of promoter usage as the hybrids with non-B phenotype. Group III BL cells use both promoter regions. Thus, the virus can use two alternative programs, depending on the cell phenotype. The "EBNA-1 only" program is activated from the Q-promoter. In cells with an immunoblastic (LCL or BL group III) phenotype, the upstream Cp/Wp promoters generate a 100 kb. long pre-mRNA, from which all the EBNAs are spliced. As a rule, only one of the two programs is used for each phenotype, except for the BL group III cells that began as group I but subsequently developed into a more LCL-like cell. Such cells used both promoter regions, with or without activation of the lytic cycle.

Development of the methods permitting cloning of identical sequences between two sources of DNA can be very useful for many purposes, including isolation of disease genes. Here we describe a new method called CIS (cloning of identical sequences). A combination of digestion with MvnI, treatment with mung bean nuclease, UDG (uracil-DNA glycosylase) and PCR with 5'-methyl-dCTP and dUTP was used to isolate identical sequences between two micro-cell hybrid lines (MCH). In a control experiment, mouse MCH903.1 and MCH939.2 containing human chromosome 3 from different individuals, were compared using the CIS procedure. Only background fluorescence in-situ hybridization (FISH) was achieved. In another experiment, mouse MCH903.1, containing complete human chromosome 3, and rat MCH429.11, containing a part of human 3q from the same chromosome were compared. The experiment showed that the original MCH429.11 and the DNA purified using the CIS procedure had identical FISH patterns to human metaphase chromosomes, thus demonstrating the efficiency of CIS.

Two isolates of the EBV-LMP1 gene were compared for their ability to induce phenotypic changes in a non-tumorigenic human keratinocyte line, Rhek-1, immortalized with an adenovirus 12-SV40 hybrid virus. One isolate, designated B-LMP1, was derived from B95-8, a B-cell line of marmoset origin, that carries a viral strain from a mononucleosis patient. The other, designated C-LMP1, originated from a nude mouse passaged Chinese NPC tumor, CAO. Both types of transfectants were less serum dependent than the non-transfected and the vector-transfected controls. The ability to grow on low serum increased with increasing LMP1 expression. All transfectants were more highly clonable than the non-transfected or vector-transfected controls. Clonability in soft agarose increased with increasing LMP1 expression. Nine of 24 C-LMP1 transfectants produced tumors in SCID mice. Seven of them grew invasively into the surrounding tissue. Only one of 12 B-LMP1 transfected Rhek-1 clones was tumorigenic. It did not grow invasively. All tumorigenic transfectants expressed LMP1 at high or moderate levels. All tumors were found to express LMP1. Transfectants with low LMP1 expression did not produce tumors. The untransfected Rhek-1 cells and six vector control clones failed to produce tumors.

Epstein-Barr virus infection (EBV) was discovered 25 years ago in tumour cells from Burkitt's lymphoma. EBV causes infectious mononucleosis and contributes to the pathogenesis of Burkitt's lymphoma and nasopharyngeal cancer. Atypical courses of the primary infection are attracting increasing attention and may present problems of diagnosis at the same time as no clear diagnostic or laboratory-based definition of the reactivation of this herpes virus is available. The article reports on the clinical course, the diagnosis, and the possibilities of treatment.

Epstein-Barr virus (EBV) will infect at least every third cell if exposed in vitro to an extensively purified B cell population from human peripheral blood. About 10% of such infected cells will be driven into immunoglobulin synthesis and secretion, as judged by the indirect protein A plaque assay. The appearance of EB nuclear antigen, de novo DNA synthesis, and immunoglobulin secretion are linked phenomena accompanying infection as judged by viral dilution experiments, which yield kinetics of a one-hit order. Induction of immunoglobulin secretion in B cells by EBV requires de novo synthesis of DNA, and consequently, nontransforming EBV (P3HR1) will not induce immunoglobulin secretion and will also specifically block such induction from subsequently added EBV. The termination of immunoglobulin induction by EBV in short-term cultures appears to be T cell dependent.

Eleven biopsies from 9 patients with Burkitt's lymphoma were tested for their sensitivity to the cell multiplication inhibitory activity of interferon. Three were resistant to interferon while 8 were sensitive to various degrees. Different biopsies from the same patient did not differ in interferon sensitivity. These results indicate that Burkitt's lymphoma cells might be resistant to interferon already in vivo as previously shown for some derived cell lines tested in vitro. The results imply an inverse relationship between patient survival and interferon sensitivity of the tumor cells.

In addition to the spontaneous expression of markers of the early and late phases of the viral cycle [Epstein-Barr virus (EBV), early viral cycle antigens (EA), and viral capsid antigen (VCA)] by a Panel of hybrid cell lines derived by fusion of human hematopoietic cell lines, the induction of these markers by three chemical inducers [5-iodo-2'-deoxyuridine, the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA), and sodium butyrate] were analyzed. A variant of the prototype producer of lytic EBV, P3HR-1 (PICAT) was observed to show consistent low spontaneous and induced production of EA and VCA as compared to the P3HR-1 line from which it was derived. An "autohybrid" (PICATPO-1) between these two lines showed a low production of VCA. Hybrids between Raji and P3HR-1 sublines (RUD-PICAT-1 and RUDPUT-2) showed reduced expression of EBV antigens. Both hybrids were capable of VCA expression, in contrast to the Raji parent that expressed EA but not VCA. A new Raji-Daudi hybrid (DITRUD-1) expressed spontaneous and induced EA and VCA at levels intermediate between its two parents. A different type of hybrid was derived from a Daudi subline and the K562 leukemia cell line (DUTKO-1) and was found to be capable of spontaneous as well as induced EA and VCA expression. Both DUTKO-1 and DITRUD-1 were similar to Daudi in their induction profile in respect to a very low response to TPA.

Shedding of Epstein-Barr virus (EBV) into saliva was studied in 31 patients with verified acute infectious mononucleosis. The patients had been randomized for intravenous treatment with acyclovir (ACV) at 10 mg/kg body weight at 8 h intervals for 7 days, or placebo, in a double-blind trial. EBV in centrifuged throat washings was detected by transformation of umbilical cord lymphocytes and by immunofluorescence staining for EBV-associated nuclear antigen in fixed cell smears. Saliva samples were obtained before and during treatment, and after 4 weeks and 6 months, respectively. ACV effectively but transiently interrupted EBV production (P less than 0.001), but virus shedding resumed at the initial level within 3 weeks of cessation of the treatment. Initially, 93.5% of the patients had detectable EBV in the saliva compared with 83% in the 4th week and 58% after 6 months.

Quantitative aspects of Epstein-Barr virus infection and transformation of human neonatal B-lymphocytes have been investigated. 72 to 90% B-cells were obtained with enrichment. Of the B-cells, 19 to 97% showed nuclear antigen (EBNA) 2 days after infection. A difference between different B-cell donors in susceptibility to infection was noted. Analysis of the virus dose-response curves obtained with twofold virus dilutions showed that one virus particle is sufficient to induce EBNA in a cell. Of the infected cells, 50 to 95% multiplied in microtitre wells containing a human fibroblast feeder layer, while only a small proportion established growing colonies in soft agarose, that could be picked up and subcultured.

The study of antigenic markers and antibodies directed against them has occupied a central position in all Epstein-Barr virus (EBV) research, ever since the discovery of the virus. Detailed seroepidemiologic studies on the distribution and pathogenic significance of virus infections, retrospective and prognostic studies on patients with EBV-related diseases, and tracing of early events during primary viral infection of normal cells in vitro are only some of the areas that were largely or entirely based on antigen-antibody tests. Serology played a primary role in establishing the relationship between the virus and the three EBV-related diseases: infectious mononucleosis (IM), Burkitt’s lymphoma (BL), and nasopharyngeal carcinoma (NPC). Furthermore, the majority of viral infectivity assays are based on antigen induction in appropriate target cells.

Leukocyte adhesion to cells is mediated by the cell-surface glycoprotein complex CD11a-c/CD18 and, in some cases, the glycoprotein gp84. The process is associated with leukocyte activation and modulates lymphocyte proliferation and maturation. Epstein-Barr virus (EBV)-transformed normal B lymphocytes give rise to lymphoblastoid cell lines (LCLs) which grow as large clusters mediated by adhesion molecules. In contrast, newly explanted EBV-infected Burkitt lymphoma (BL) cells usually grow as single cells or as loose clusters. We now report that EBV positive- and EBV-negative BL lines lack the adhesive protein complex, or have only low levels of it, whereas LCLs, representing various stages of B-lymphocyte development, contain considerably higher amounts, as measured by immunofluorescence flow cytometry and immunoprecipitation. The level of gp84 expression is of the same magnitude in both types of cells.

A substantial proportion of the lymphomas in HIV-carriers are EBV positive. Together with the fact that there are multiple signs of EBV-activation in AIDS-patients and patients with ARC or PGL, this suggests that these virus carrying tumors develop as results of the immunosuppression, and that EBV has an important role in their pathogenesis.

Both oral and intravenous acyclovir administration for seven days in the early stages of infectious mononucleosis caused an inhibition of oropharyngeal Epstein-Barr virus (EBV) replication. Minimal effect on clinical symptoms was observed. Development of normal cellular and humoral EBV-specific immunity was seen in all patients. The combination of intravenous acyclovir and prednisolone treatment for 10 days in 11 patients with fulminant mononucleosis caused transient cessation of virus shedding in all patients. A dramatic clinical effect on pharyngeal symptoms and on fever was seen in nine of 11 patients within 72 hours. Treatment with chemotherapy or irradiation is recommended in EBV-associated B cell lymphomas seen in immunosuppressed, transplanted, and human immunodeficiency virus-I seropositive patients. No effect of acyclovir has been reported, but such therapy may be considered in the early stage when EBV induces a polyclonal B cell activation. Acyclovir treatment is effective in the EBV-genome positive hairy leukoplakia in human immunodeficiency virus-seropositive patients. No effect of antiviral therapy has been reported in the X-linked lymphoproliferative syndrome. Prophylactic use of immunoglobulin or acyclovir has been suggested in susceptible children.