Persistence of Epstein-Barr viral nuclear antigen (EBNA) in cells entering the EB viral cycle.

It was shown by double immunofluorescence studies that Epstein-Barr viral nuclear antigen (EBNA) was preserved in EBV-infected cells after they had entered the productive viral cycle, as signalled by the appearance of the early antigen (EA)complex. A nuclear component of the EA comples could be clearly distinguished from EBNA with regard to antigenic specificity.     

Relationship between Epstein-Barr virus (EBV) DNA and the EBV-determined nuclear antigen (EBNA) in Burkitt lymphoma biopsies and other lymphoproliferative malignancies

Twenty-six of 27 African Burkitt lymphomas with histologically confirmed diagnosis contained relatively large amounts of EBV DNA (10–101 viral genomes per cell), as determined by nucleic acid hybridization. Twenty-five of the 26 EBV DNA-positive lymphomas contained the EBV-determined nuclear antigen, EBNA, in the majority of the nuclei. Technical reasons may have accounted for the apparent EBNA-negativity of one EBV DNA-positive biopsy. Four African lymphoma biopsies, one with a definite diagnosis of Burkitt's lymphoma and three with a questionable diagnosis of the same disease, were all EBV DNA- and EBNA-negative. The same was true for a collection of Swedish cases of Hodgkin's disease, lymphocytic lymphoma, chronic lymphatic leukemia and some other lymphoproliferative malignancies. Thus, there is excellent agreement between the presence of EBV DNA and of EBNA in tumor biopsies. The EBNA antigen test therefore appears a relatively simple way of testing for the presence of the virus genome, provided it is carried out with appropriate controls. Several of the EBV-genome and EBNA-negative cases came from patients with high serum titers of EBV antibodies. It is concluded that the virus does not really travel along with malignant lymphomas as a passenger in the seropositive patients. In comparison with other lymphomas, African Burkitt's lymphoma of the high endemic areas is unique in that the tumors (with rare exceptions) represent the proliferation of an EBV-genome carrying clone. These findings stress the necessity to distinguish between EBV-seropositive status and evidence for EBV-genome-carrying neoplastic cells.     

Discordant Epstein-Barr virus nuclear antigen (EBNA) antibody patterns in nasopharyngeal carcinoma

Epstein-Barr virus nuclear antigen (EBNA) preparations from three sources were tested with sera from normal individuals and patients with Hodgkin's disease, breast carcinoma, Burkitt's lymphoma, and American and Chinese nasopharyngeal carcinoma. Individual sera with discordant antibody patterns were noted in all groups. Sera from both NPC groups gave significantly higher anti-EBNA titers on cell lines converted with P3HR-1 or B95-8 virus compared with anti-EBNA titers on Raji cells. Anti-EBNA titers of Chinese NPC sera showed no correlation among the three EBNA sources, while all other groups had highly correlated titers. Cross-absorption experiments present evidence for more than one antigenic determinant on EBNA. These results suggest an additional parameter for distinguishing Chinese NPC from other EBV-related disorders.     

The detection of the Epstein-Barr virus (EBV) nuclear antigen (EBNA) by anticomplement immunofluorescence. Immunoglobulin class of antibodies and role of complement.

The Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) was detected by anti-complement immunofluorescence (ACIF) in Raji lymphoblastoid cell line, and the mechanism of the reaction explored, using Ig fractions of anti-EBNA sera and purified components of complement. Following fractionation of serum from 13 donors, anti-EBNA antibodies were always found to be associated with IgG, but were also detectable in the IgM fraction of four sera. Sequential addition of functionally pure complement components in the ACIF reaction showed that the classical sequence of complement activation in involved. Anti-EBNA antibody reactions in Raji cell nuclei can also be detected by anti-Ig immunofluorescence with a low level of sensitivity. The same pattern of granular fluorescence was observed when C3 (beta1A/beta1C), or C4 or IgG anti-EBNA antibodies were revealed with the corresponding flurescein-conjugated reagents. Blocking of the ACIF reaction was achieved with Fab 2 anti-EBNA antibody fragments, which therefore provided an appropriate specificity control for the detection of EBNA.     

Study of Epstein-Barr virus-determined nuclear antigen (EBNA) by chromatography on fixed cell nuclei.

Low ionic strength (50 to 100 mM NaCl) and pH 6.0 were found to be optimal conditions for in vitro conversion of Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA)-negative nuclei to EBNA-positive nuclei by addition of the complement-fixing (CF) antigen extracted from Raji cells. In vitro conversion of nuclei to EBNA-positively was sensitive to DNase but not to RNase treatment. This suggests that nuclear DNA is a specific target substance to which EBV-CF antigen binds. If nuclei were fixed with methanol/acetic acid and subsequently treated with 0.6 M NaCl, EBNA could be eluted from in vitro-converted Ramos nuclei with 0.3 and 0.4 M NaCl. The same conditions were also found to be optimal for the adsorption and elution of EBV-CF antigen in DNA-cellulose chromatography. This indicates that the DNA-binding properties of EBNA antigen can be studied by "chromatography" on fixed nuclei followed by the ACIF test. The obvious advantages of this method over chromatography on DNA-cellulose are its simplicity, the possibility of testing many samples in one experiment and, especially, the use of minimal amounts of material. Significant differences in elution patterns for EBNA were found when nuclei derived from different cell lines (Ramos, Raji, and P3HR-1) were converted in vitro to EBNA-positivity. EBNA is eluted from in vitro-converted nuclei of EBV genome-positive P3HR-1 cells at an almost 0.1 M higher concentration of NaCl than is necesssary for a similar degree of elution from nuclei of EBV genome-negative Ramos cells.     

Antibody response against the Epstein-Barr virus-coded nuclear antigen2 (EBNA2) in different groups of individuals

Specific antibody responses against the 2 major subcomponents of EBNA, EBNA1 and EBNA2 were evaluated, in order to study whether this serological study was beneficial compared to classical EBV serology. During this investigation, 491 sera, obtained from blood donors and patients with Burkitt's lymphoma (BL), nasopharyngeal carcinoma (NPC), infectious mononucleosis (IM), Hodgkin's disease, renal transplantation, rheumatoid arthritis and Human Immunodeficiency Virus (HIV) infection, were tested. While the anti-EBNA1 response followed the classical anti-EBNA/Raji response (99% of anti-EBNA/Raji-positive sera also recognize EBNA1), the anti-EBNA2 response was much less frequent and did not correlate with either anti-EBNA/Raji or anti-EA antibodies. In a control population, 8% of individuals had antiEBNA2 antibodies at titers greater than or equal to 10. The percentage was 45% in NPC and 38% in EBV-associated BL; thus, although not detected in all patients with EBV-associated tumors, anti-EBNA2 serology might be a useful marker in BL and NPC. No antibody was detected in the early course of IM, but in rheumatoid arthritis and in HIV-infected patients, the percentage of positive individuals reached 54 and 68, respectively. Seroconversion to EBNA2 was noted in a few cases, including renal transplant recipients, AIDS patients, and complicated IM. This suggests that in these situations, EBNA 2 serology might represent a useful marker related to modulation of the immune status or EBV reactivation.     

胃癌组织中EB病毒的检测及其增殖期基因的表达

目的；探讨EB病毒(Epsteirt-Barr virus．EBV)感染与胃癌发生的关系及其增殖期基因在EBV阳性胃癌发生中的作用。方法：应用聚合酶链反应（PCR）-Southern杂交检测185例胃瘟组织和相应癌旁组织中特异性EBVDNA片段．PER阳性标本用原位杂交(ISH)技术检测石蜡切片组织中EBV犏码小RNA1(EBER1）的表达．以确定EBV阳性胃癌一再应用RT-PCR和Southern杂交技术检测EBV增殖期基因(即刻早期基困BZLF1、BRLF1，早期基因BARF1、BHRF1．晚期基冈BcLF1、BLLF1)的表达．结果：13例胃癌组织EBV阳性(7．03％)，癌旁组织未检测到EBV感染．胃癌和癌旁组织中EBV阳性纺有显著性差异(X^2=11.0769．P=0．0009)。EBV阳性和阴性胃癌在年龄、病理类型、临床分期、淋巴结转移和发生部位的差别均无显著性(P=0．973，0．141，0．259，0．586，0．062)．但性别之间的差异有显著性，男性EBV阳性率高于女性（X^2=52317．P=0．021)。增殖期基因中即刻早期基因BZLF1有6例表达阳性．而BRLF1均为阴性；早期基因中有6铡BARF1表达阳性．2例BHRF1表达阳性；晚期基因BcLF1有7倒表达阳性．而BLLF1均为阴性。结论：EBV感染与胃癌的发生有一定的相关性．部分EBV阳性胃癌组织中存在EBV增殖性感染．早期基因BARF1和BHRF1在EBV阳性胃癌发生过程中可能有重要作用。     

Three Epstein-Barr virus (EBV)-determined nuclear antigens induced by the BamHI E region of EBV DNA

In our previous study (Takada et al., 1986a), we showed that the BamHI E fragment of Epstein-Barr virus (EBV) DNA induces a nuclear antigen that is detected by human antisera against EBV-determined nuclear antigen (EBNA), when transfected into baby hamster kidney (BHK) cells. The present study shows that the sub-fragment containing the central open reading frame BERF2b of the BamHI E fragment (Baer et al., 1984) is responsible for nuclear antigen induction. In addition, 2 fragments corresponding to 2 other open reading frames of the BamHI E, BERFI and BERF4 also induce nuclear antigens upon transfection into BHK cells. These 3 antigens, designated RF2b, RFI and RF4 antigens, were serologically classified as EBNA and antigenically distinct. In immunoblotting analysis of latently EBV-infected BJ-B95-8 cells, 3 high-molecular-weight polypeptides (136, 142 and 147 kDa) were identified by anti-EBNA sera. Immunoblotting analysis of transfected BHK cells indicated that the RF2b antigen is 145 kDa in its native form and antigenically related to the 147-kDa protein of BJ-B95-8 cells. Although RFI and RF4 antigens were not detected by immunoblotting, reactivities of sera with RFI and RF4 antigens in the immunofluorescence test were correlated with those of sera with the 136- and 142-kDa polypeptides of BJ-B95-8 cells, respectively. The results suggest that 3 high-molecular-weight proteins of latently EBV-infected cells are encoded by 3 open reading frames of the BamHI E DNA fragment.     

Chemical carcinogen Epstein-Barr virus (EBV) synergism: EBV genome amplification and site-specific mutation during transformation

When treated with chemical carcinogens, peripheral blood lymphocytes (PBLs) from normal adults or patients with acquired immunodeficiency syndrome (AIDS) were more readily transformed than non-treated PBLs into immortalized lymphoblastoid cell lines (LCLs) by the Epstein-Barr virus (EBV, FF41-1). Chemical carcinogens also enhanced spontaneous transformation in both PBL populations. Co-cultivation of lethally ultraviolet (UV)-irradiated uninfected PBLs with EBV-infected PBLs resulted in a dose-related enhancement of transformation, providing evidence that enhancement is in part mediated by carcinogen-induced diffusible trans-acting factors. Analysis of the EcoRI J region of resident EBV genomes showed that LCLs established from carcinogen-treated PBLs frequently had EBV genome amplification higher than that seen in controls. Carcinogen-induced EBV genome amplification was shown to be dose-related. The organization of the terminal region of the EBV genome was analyzed in LCLs derived from carcinogen-treated PBLs and compared to that of FF41-1 and B95-8. LCLs established from N-methyl-N'-nitroso-nitrosoguanidine (MNNG)- and aflatoxin B1-treated PBLs frequently had 2 circular forms of the EBV genome and several linear forms varying by numbers of terminal repeats (TRs) at the 3' and 5' end. This is in contrast to the single episomal circular and linear form of the EBV genome found in the parent FF41-1 and single episomal circular form in control FF41-1-carrying LCLs. Linear forms of the EBV genome were only found in FF41-1-transformed LCLs derived from carcinogen-treated PBLs and were associated with the production of unusually large amounts of infectious EBV. These results demonstrate that chemical carcinogen-mediated enhancement of transformation is accompanied by quantitative and qualitative alterations in the physical structure, organization and expression of the EBV genome which may in turn be the result of a carcinogen-induced cellular SOS response.     

Detection of Epstein-Barr virus (EBV)-associated nuclear antigen in human lymphoblastoid cell lines by means of an 125-I-IgG-binding assay

The elution at low pH of human EBV-positive ¹²⁵I-IgG bound to preparations of EBV-carrying non-producer lymphoblastoid cell lines has been investigated. Specific binding was demonstrated. A number of approaches were used to confirm the specificity. The stoichiometry of binding and elution was studied. The sub-cellular location of the EBV-associated antigen (s) detected by this method was shown to be predominantly nuclear. This confirms other studies demonstrating the presence of a nuclear antigen in non-producer lymphoblastoid cell lines carrying the EBV genome.     

Solubilisation of Epstein-Barr virus (EBV)-associated nuclear antigen from Raji cells and chromatin by treatment with various molarities of NaCl.

The solubilisation of Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) by treatment with various molarities of NaCl was investigated using the 125I--IgG absorption assay. Ninety percent of the antigenic activity detected using the 125I--IgG absorption assay was insoluble at 0.15 M NaCl. It could be rendered soluble by treatment with 2.0 M NaCl, but reprecipitated upon return to 0.15 M NaCl. EBNA was partially extracted from Raji chromatin by treatment with 0.35 M NaCl. The efficiency of extraction was increased by homogenisation in 2.0 M NaCl followed by dialysis to 0.35 M NaCl. The data demonstrate the close association of EBNA with Raji chromatin and suggest that it may be a chromatin-associated non-histone protein.     

Serum and salivary IgA antibodies against a defined epitope of the Epstein-Barr virus nuclear antigen (EBNA) are elevated in nasopharyngeal carcinoma.

The Epstein-Barr virus nuclear antigen I (EBNA I) is the only latent EBV antigen consistently expressed in malignant tissues of the nasopharynx. A 20-amino-acid synthetic peptide, p107 contains a major epitope of EBNA I. We tested sera from 210 patients with nasopharyngeal carcinoma (NPC) and from 128 normal individuals (NHS) for IgA antibodies to p107 using an enzyme-linked immunosorbent assay (ELISA). Whereas 191/210 (91%) of NPC patients had IgA antibodies to p107, only 17/128 (13.3%) of NHS had such antibodies and only 6/57 (10.5%) of sera from patients with malignancies other than NPC had IgA-p107 reactivity. Thirty-nine salivary samples from 46 NPC patients (84.8%) also contained IgA-p107 antibodies whereas only 3/42 (7.1%) of normal saliva samples were IgA-p107 positive. The results suggest that IgA antibodies to EBNA I may become a useful, easily measurable, marker for NPC.     

Column separation of viral capsid antigen (VCA)-positive cells from VCA-negative cells in an Epstein-Barr virus (EBV)-producing lymphoid line,.

Virus producing, VCA (viral capsid antigen)-positive cells could be selectively removed from Epstein-Barr virus (EBV)-carrying, virus-producer P3HR-1 cultures by two different methods of column passage. In the first, the virus-producing cells were covered with human EBV antibody and subsequently passaged through anti-human-Ig columns. In the second methods, untreated P3HR-1 cells were allowed to pass through columns of EBV receptor-positive cell lines or, as controls, EBV receptor-positive cell lines or, as controls, EBV receptor-negative cells. The majority of the VCA-positive cells were selectively removed by both techniques. The second method involves the attachment of EBV-producer cells, known to accumulate viral envelope material in their plasma membrane, to EBV receptors. In view of recent evidence indicating an association between EBV and complement receptors, human and mouse complement were tested for their ability to block this attachment. Fresh mouse and human complement regularly exerted blocking activity, whereas heat-inactivated human serum did not block. Heat-inactivated mouse serum did block occasionally, but the effect was more irregular than with fresh mouse serum. Trypsin treatment of the EBV-receptor colum abolished its ability to retain VCA-positive cells.     

Studies on Epstein-Barr virus-related antigens. III. Purification of the virus-determined nuclear antigen (EBNA) from non-producer Raji cells.

The purification of Epstein-Barr virus-determined nuclear antigen (EBNA) was attempted on the basis of its biochemical and physicochemical properties and its immunological specificity, assayed by the indirect single radial immunodiffusion test and anti-complement immunofluorescence absorption test. When non-producer Raji cell extract was subjected to 20--60% saturated ammonium sulfate fractionation, followed by DNA-cellulose chromatography and immunoadsorbent chromatography, EBNA was purified more than 3,000 times with a 8% yield. Such purified protein was composed of three polypeptides with molecular weights of 100,000 +/- 5,000, 70,000 +/- 5,000 and 50,000 daltons, respectively, on SDS-polyacrylamide gel electrophoresis. Similar purification was achieved by heating the extract at 70 degrees C for 10 min, instead of ammonium sulfate fractionation, followed by DNA-cellulose chromatography and immunoadsorbent chromatography. This final preparation consisted almost exclusively of 100,000 +/- 5,000 daltons polypeptide, 50,000 polypeptide, and the 70,000 +/- 5,000 polypeptide passing through the adsorbent column. These findings suggest that EBNA is probably a molecular complex of three smaller subunits of heat-stable 100,000 +/- 5,000 and 50,000 daltons and heat-labile 70,000 +/- 5,000 daltons polypeptides, respectively.     

富硒米提取液对EB病毒转化脐血B淋巴细胞及EBV早期抗原表达的影响

背景与目的:硒是人体必需的微量元素,具有抗氧化、抗衰老和抑制肿瘤细胞增殖的作用,为进一步探讨硒在肿瘤防治中的作用,我们培育出富含有机硒的大米,现研究富硒米对EB病毒激发脐血B淋巴细胞转化及对Raji细胞EB病毒早期抗原表达的影响。方法:①用富硒米及普通米提取液以1∶4、1∶8的浓度分别加到制备好的EB病毒液中,加入脐血单个核细胞进行淋巴母细胞转化培养实验,计算细胞转化抑制率;②Raji细胞经正丁酸及巴豆油联合诱导的同时加富硒米提取液共同培养,以间接荧光法,经伊文氏蓝复染观察计算EBV-EA抗原阳性率和抑制率。结果:含硒0.11μg/ml(1∶8稀释)的富硒米提取液显著抑制EB病毒对脐血B淋巴细胞的转化,抑制率为83.4%,明显高于普通米提取液的抑制率61.3%(P<0.05)。富硒米提取液对Raji细胞EB病毒早期抗原(earlyantigen,EA)表达有明显抑制作用,0.016、0.078、0.388μg/ml硒浓度组对EA抗原抑制率分别为2.85%、12.88%、20.75%。结论:富硒米提取液对EB病毒转化脐血B淋巴细胞和对Raji细胞EB病毒早期抗原均有明显的抑制作用。     

Detection and expression of Epstein-Barr Virus (EBV) DNA in tissues from penile tumors in Brazil

Epstein-Barr Virus (EBV) is prevalent in all human populations and high titers of antibody correlate with specific malignancies such as Burkitt's lymphoma, nasopharyngeal carcinoma, and Hodgkin's disease. Our study detected EBV DNA in 20 of 21 penile tumor samples using PCR reaction. Expression of EBV protein LMP-1 was identified in tumor cells from two EBV PCR-positive tumors. Our findings indicate that EBV can be implicated in rising and/or progression of penile tumors independently of the histological type.