When Epstein-Barr virus persistently infects B-cell lines, it frequently integrates.

In this study we used Gardella gel analysis of intact DNA, Southern blotting of digested DNA, and fluorescence in situ hybridization to provide complementary and unequivocal information on the state of the Epstein-Barr virus (EBV) genome in persistently infected cells. The fluorescence in situ hybridization technique allowed us to directly visualize both integrated and episomal EBV DNA at the single-cell level. We show here that circularization of the EBV genome is rarely detected upon infecting activated normal B cells. The virus can persist upon infection of a different proliferating B-cell target, EBV-negative Burkitt's lymphoma tumor cell lines. Analysis of 16 such lines reveal again, that the virus infrequently persists as covalently closed episomes; rather, the virus preferentially persists by integrating into the host DNA (10 of 16 clones). The integrated virus is linear and usually intact, although 3 of 10 isolates have deletions from the left-hand end including the latent origin of replication. At the level of our analysis, no obvious relationship was seen between the integration sites. These studies provide, for the first time, a reproducible in vitro model system to study integration by EBV.     

Localization polymorphism of EBV DNA genomes in the chromosomes of Burkitt lymphoma cell lines

The localization of Epstein-Barr virus (EBV) genomes in nuclei of the human lymphoblastoïd cell lines Raji, Jijoye, P₃HR-1, Daudi and Ramos was investigated by in situ hybridization with biotinylated EBV DNA probes. We found that all sites of hybridization were associated with the chromosomes. Only some of these sites were present on both chromatids and these had a non-random distribution; these sites could represent EBV sequences integrated at specific points on the chromosomes. The total mean site number corresponded with the number of viral DNA copies estimated in the different cell lines by other techniques, but the copy number was highly variable from cell to cell in a given line.     

Differentiation-associated expression of the Epstein-Barr virus BZLF1 transactivator protein in oral hairy leukoplakia.

The BZLF1 protein of Epstein-Barr virus (EBV) is a key immediate-early protein which has been shown to disrupt virus latency in EBV-infected B cells. We have generated a monoclonal antibody, BZ1, to BZLF1 which reacts in immunohistology, immunoblotting, and immunoprecipitation and which recognizes both the active, dimeric form and the inactive, monomeric form of the protein. Biopsies of oral hairy leukoplakia, an AIDS-associated lesion characterized by high-level EBV replication, were examined by immunohistochemistry using the BZ1 monoclonal antibody. A differentiation-associated pattern of BZLF1 expression was observed, BZ1 reacting with nuclei of the upper spinous layer of the lesion. This finding suggests that the BZLF1 promoter may be regulated by the degree of squamous differentiation. A comparison of in situ hybridization to EBV DNA and viral capsid antigen staining with BZ1 reactivity suggested that BZLF1 expression precedes rampant virus replication. The inability to detect EBV in the lower epithelial layers of oral hairy leukoplakia raises questions concerning the nature of EBV latency and persistence in stratified squamous epithelium.     

Analysis of Epstein-Barr viral genomes in lymphoid malignancy using Southern blotting, polymerase chain reaction and in situ hybridization

109 malignant lymphomas were surveyed by Southern blot analysis and polymerase chain reaction (PCR) for Epstein-Barr virus (EBV) DNA and compared with 16 examples of non-neoplastic lymphadenopathy and 4 normal thymuses. In specimens positive by the method of Southern and PCR, in situ hybridization studies were performed on formalin-fixed, paraffin-embedded sections. By Southern blot analysis, two of seven Hodgkin's disease samples (29%) (one of mixed cellularity and the other of lymphocyte predominance type), three of 56 B-cell lymphomas (5.6%) and five of 46 T-cell lymphomas (11%) demonstrated EBV DNA. However, the 16 examples of lymphadenitis and the 4 normal thymuses showed no EBV DNA. With PCR, EBV DNA was identified in one B-cell lymphoma, nine T-cell lymphomas, ten lymphadenitis specimens and two of the normal thymus, in addition to the positive specimens determined by the Southern blotting method. These results indicate that the presence of EBV DNA is not related to lymphoid malignancy, but enhancement of the DNA is demonstrated in some neoplastic conditions. By in situ hybridization, EBV genomes were not detected in all PCR-positive cases, but only in those positive by Southern blot analysis.     

Spontaneous reduction in Epstein-Barr virus (EBV) DNA copy number in EBV-infected epithelial cell lines.

We found that spontaneous and 12-0-tetradecanoylphorbol-13-acetate-induced Epstein-Barr virus (EBV) reactivation occurred in short-term (ST)-cultured EBV-infected epithelial cell lines GT38 and GT39 after their establishment; however, it diminished in the long-term (LT)-cultured cells passaged for more than 2 years from ST-cultured cells. We hypothesized that the EBV reactivation may be related to the EBV DNA copy number in the cells. A higher level of EBV DNA content was detected in ST-cultured cells than in LT-cultured cells by Southern hybridization using an EBV DNA XhoI probe. Fluorescence in situ hybridization using EBV DNA BamHI W fragments showed that ST-cultured cells contained a higher EBV DNA copy number than that of LT-cultured cells. EBV DNA-negative cells were detected in small proportions in LT-cultured cells, but were undetected in ST-cultured cells. These results demonstrate that EBV genomes are not maintained stably in the cell lines, and some of them are lost in continuous passages of the cells. We discuss the mechanisms of reduction of EBV reactivation and EBV DNA in the cell lines.     

Epstein-Barr virus and oral squamous cell carcinoma in patients without HIV infection: viral detection by polymerase chain reaction.

In order to test the hypothesis that Epstein-Barr virus (EBV) may be a cofactor for oral squamous cell carcinoma (OSCC) the authors evaluated tumour cells from OSCC of 108 patients without HIV infection, for the presence of EBV DNA by polymerase chain reaction. The sequences of oligonucleotides used in the amplification and hybridization included a set for the DNA polymerase region. The amplification was detected using an ELISA assay with peroxidase. EBV DNA was detected in 17.59% of the tumours. Inhibition studies showed that the ability to detect EBV DNA was not affected by the pathological material, suggesting that the negative PCR results in these samples were not caused by PCR inhibitors in the biopsy. Results revealed that 63.1% of the tumours (12 cases) were DNA positive affecting the lateral margin of the tongue, and were statistically significant (p < 0.001; chi 2). In the pool of tumours with EBV DNA only 26.3% (5 of 19 cases) were well differentiated OSCCs whereas the remaining 73.7% (14 of 19 cases) were moderately and poorly differentiated OSCCs, with a statistical significance of p = 0.08; chi 2. This study suggests a relationship between OSCC and EBV.     

The prototypical Epstein-Barr virus-transformed lymphoblastoid cell line IB4 is an unusual variant containing integrated but no episomal viral DNA.

IB4 is a prototype, latently Epstein-Barr virus (EBV)-infected, lymphoblastoid cell line. We show here that IB4 contains only integrated EBV genomes. Episomal EBV DNA is not detected by Gardella gel analysis or in situ hybridization. Restriction enzyme mapping indicates that the EBV genomes first circularized and then integrated into and deleted part of the BamHI C fragment. IB4 is therefore the only lymphoblastoid cell line described to date that lacks episomal EBV and has integrated EBV genomes with joined ends. Thus, the detection of joined EBV termini on Southern blots is not as reliable as the Gardella gel system for detecting episomal EBV DNA, and IB4 is not an ideal prototype cell line for the study of latent infection by EBV.     

Biotinylation of denatured double-stranded DNA after transamination of cytidines: molecular biology applications.

Transamination at 100 degrees C of cytosines in denatured double-strand DNA is a rapid and reliable method to obtain DNA molecules containing N4-aminoethylcytosine (4aeC), which can be quantitatively conjugated to biotinyl-N-hydroxysuccinimide ester (BHS) at 37 degrees C, yielding chemically labelled probes for molecular hybridization. The adopted transamination reaction temperature allows for a ten-fold reduction of the time required for labelling at 42 degrees C, and probes obtained by this procedure are equally effective for general use in molecular biology. Dot-blots with 1-5 pg of target lambda DNA were detected by streptavidin-acid phosphatase complex after hybridization with its homologous sequences. Chemically biotinylated mouse satellite DNA has been used in combination with avidin-horseradish peroxidase to detect metaphase and interphase centromeres via in situ hybridization. Moreover probes labelled with differentially spaced linker arms were prepared by this method.     

Computer assisted signal co-localization for simultaneous detection of antigen by immunohistochemistry and DNA by non-isotopic in situ hybridization

A method has been developed to co-localize signals for antigen and DNA using a desktop microcomputer system (computer assisted signal co-localization). Antigens were detected by standard immunohistochemical methods and DNA was detected by non-isotopic in situ hybridization (NISH). Using this method, NISH signals can be precisely located in cells with well-preserved morphology captured by computer. The removal of the first immunohistochemical reaction products and reagents eliminates possible interference with hybridization and non-specific binding to the probe; therefore the sensitivity of the original NISH method remains. The captured NISH signals can be converted to any other colour which contrasts with the immunostaining. We have used detection of Epstein-Barr virus (EBV) and keratins as a model system. This method is straightforward, and with necessary modifications, will be applicable to any type of combined immunohistochemistry and in situ hybridization technique for simultaneous detection of antigen and nucleic acids or two types of nucleic acids in the same cells.     

Specific sites for EBV association in the Namalwa Burkitt lymphoma cell line and in a lymphoblastoid line transformed in vitro with EBV

Localization of Epstein-Barr virus (EBV) DNA was studied by in situ hybridization on chromosomes from the Namalwa Burkitt lymphoma cell line and from a lymphoblastoid cell line transformed in vitro (ATL9/g). The five chromosome bands 1p32, 1q31, 5q21, 13q21, and 16p13 showed the presence of EBV DNA in both of the lines. Grain deposition at the site on chromosome 1q of the Burkitt line was particularly intense. It was also found that EBV DNA in the lymphoblastoid cell line co-localized with a stable achromatic gap at 1p32 whose presence seems to confer a proliferative advantage on the cells.     

Use of whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization

Summary We report a general procedure which allows the application of whole cosmid cloned genomic sequences for non-radioactive in situ hybridization. The presence of highly repetitive sequences, like Alu and Kpn fragments, is eliminated through competition hybridization with Cot-1 DNA. The method has been tested and optimized with several randomly chosen cosmids of the human thyroglobulin (Tg) gene (8q24). At present, the procedure can be performed with three of the four tested individual cosmids. In cases where a single clone does not result in a specific signal, a larger fragment may be required, which can be accomplished by using two (partially overlapping) cosmids of the same region. The advantages and further potentialities of such a hybridization approach are discussed.     

Primary gastric lymphoma is rarely associated with Epstein-Barr virus

Recently, the association of Epstein-Barr virus (EBV) with undifferentiated lymphoepithelioma-like carcinoma and adenocarcinoma of the stomach has been described. In this study of 55 primary gastric lymphomas, most of them belonging to the group of MALT lymphomas, the question of possible EBV involvement has been addressed using in-situ hybridization (ISH) and blot techniques. EBV DNA and/or DNA sequences were found in only two of 24 centroblastic and B-immunoblastic lymphomas and in one anaplastic large cell lymphoma of null cell phenotype. In a further centroblastic lymphoma, a few positive nontumorous (bystander) cells were identified by ISH.     

Detection of virus-specific RNA in simian sarcoma-leukemia virus-infected cells in in situ hybridization to viral complementary DNA.

An in situ molecular hybridization system which will detect retrovirus RNA in the cytoplasm of individual virus-infected cells has been developed. The technique was applied to cells infected with simian sarcoma-leukemia virus, where the virus-specific RNA was detected by hybridization to simian sarcoma-leukemia virus 3H-labeled complementary DNA. The system is useful for detecting viral RNA-containing cells in the presence of an excess of virus-negative cells and for determining which type of cell in a heterogenous population is expressing viral RNA.     

Reassociation Kinetics for Epstein-Barr Virus DNA: Nonhomology to Mammalian DNA and Homology of Viral DNA in Various Diseases

The number of Epstein-Barr virus (EBV) genomes per cell in established leukocytic lines and tissue specimens has been evaluated by measuring DNA-DNA reassociation kinetics with hydroxyapatite chromatography. Under the proper conditions, this method is sufficiently sensitive to detect EBV DNA in the amount of 0.1 genome per cell. All the samples tested that have been suspected to be without EBV DNA by cRNA hybridization proved negative by this more sensitive specific analysis. These included Hela and Hep2 cells, a negative case of Burkitt's lymphoma, two negative cases of nasopharyngeal carcinoma, and two established human leukocytic lines. Homology tests conducted with single-strand-specific nuclease S1 indicated that the viral DNA from a nasopharyngeal carcinoma and infectious mononucleosis were more than 90% homologous to EBV DNA.     

Identification of EBV-DNA in lymph nodes from patients with lymphadenopathy and lymphomas associated with AIDS

Fourteen examples of non-Hodgkin's lymphoma (NHL) and four of Hodgkin's disease in patients with AIDS as well as lymph nodes exhibiting changes related to the lymphadenopathy syndrome (LAS) from 11 HIV-positive individuals were studied for the presence of Epstein-Barr virus (EBV) genome both by in situ DNA hybridization and blotting techniques. Both methods were performed using formalin-fixed paraffin-embedded material. All the NHLs were of high malignancy and all but one were of the B-cell type. Of the four examples of Hodgkin's disease, two were lymphocytic predominant, one of mixed cellularity and one of the nodular sclerosing variety. The lymph nodes of patients with LAS were mostly stage I with marked follicular hyperplasia. In 7 of the 14 NHLs the presence of EBV-DNA was clearly demonstrated by dot-blotting and by in situ hybridization. All lymph nodes from the patients with LAS and AIDS-related Hodgkin's disease were negative for EBV by dot-blot and in situ hybridization assays. We conclude that EBV plays a role in the development of AIDS-related lymphomas, but the fact that half these lymphomas are EBV-negative suggests that other mechanisms such as polyclonal stimulation of B-cells by HIV products may also be important.     

Chromosomal localization of the Epstein-Barr virus (EBV) genome in Bloom's syndrome B-lymphoblastoid cell lines transformed with EBV

The localization of the Epstein-Barr virus (EBV) genome in chromosomes of human B-lymphoblastoid cell lines (LCLs) transformed with EBV, and the effect of EBV DNA on the level of sister chromatid exchange (SCE) in Bloom's syndrome (BS) B-LCLs, were examined with chromosomal in situ hybridization techniques using a ³H-EBV DNA probe. EBV DNA was detected in chromosomes 1–5 and 13–15 at specific G band regions in BS as well as in normal B-LCLs, regardless of SCE. Several chromosomal sites (1p31, 1q31, 4q22–24, 5q21, 13q21, 14q21) carrying EBV DNA seemed to be very characteristic in normal as well as in BS B-LCLs. There was no statistically significant difference in silver grain counts due to EBV DNA and their distribution in different chromosomes or groups among normal and BS B-LCLs with normal and high SCE. These findings strongly indicate that EBV infection did not introduce a correcting factor for BS SCE.     

Epithelial cell retention of transcriptionally active, P3HR-1-derived heterogeneous Epstein-Barr virus DNA with concurrent loss of parental virus

Deleted, rearranged, heterogeneous (het) Epstein-Barr virus (EBV) DNA with the distinctive capability of disrupting EBV latency has been reported in biopsy samples of EBV-associated tumors whose onset in immunocompetent hosts is characteristically preceded by an antibody response indicative of EBV reactivation. Using the EBV P3HR-1 strain, we have reproduced in long-term culture of SVK epithelial cells an unusual pattern of infection previously observed in a subset of tumor biopsy samples: the persistence of het DNA in the absence of the parental helper virus. Fluorescence in situ hybridization (FISH) of infected cell subclones indicated the retention of het DNA in an integrated form. Incorporation of an intact het DNA molecule was confirmed by PCR, using primers that framed junctions of the four rearranged EBV DNA segments comprising P3HR-1-derived het DNA. Structural analysis of EBV terminal repeats revealed a banding pattern consistent with the integration of het DNA as a concatemer. Linkage of concatemeric monomers was defined at a nucleotide level, and that junctional sequence was detected in cell-free P3HR-1 virion DNA, confirming that subgenomic het DNA was packaged into infectious particles in a concatemeric configuration. Stable integration into cells having lost the standard viral genome allowed the unambiguous designation of het DNA as the source for viral gene products potentially encoded by both. Continuous expression of the latency-to-lytic switch protein Zta and detection of the BALF4 gene product gB, known to expand the target cell range of standard virus when incorporated at augmented levels into infectious progeny, add to a presumption of het DNA-enhanced pathogenesis in diseases of EBV reactivation.